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TAKENAKA ShinjiGraduate School of Agricultural Science / Department of AgrobioscienceProfessor
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■ Paper- ABSTRACT Royal jelly (RJ) is known to contain 10‐hydroxydecanoic acid (10HDAA), which has been shown to have immune activation properties, including the promotion of M cell differentiation. However, the natural concentration of 10HDAA in RJ is relatively low. To enhance the functional use of RJ as an immunostimulatory food ingredient, this study aimed to increase its 10HDAA content using bacteria capable of converting 10‐hydroxy‐2‐decenoic acid (10H2DA) to 10HDAA in RJ. A lactic acid bacterium, Lactobacillus panisapium, was isolated from the digestive tract of queen bees and demonstrated a high capacity to convert 10H2DA to 10HDAA. Using the isolated strain, fermented RJ (fRJ) with a fivefold increase in 10HDAA content was produced compared to raw RJ. Preliminary evaluations of fRJ's immune‐stimulating effects revealed significant benefits, including enhanced M cell differentiation, activation of macrophage phagocytic ability, and increased immunoglobulin (Ig) A secretion in individuals with reduced salivary IgA levels. Safety assessments confirmed that fRJ is safe for consumption. In summary, fRJ enriched with 10HDAA was produced and demonstrated potential as an immune‐stimulating food.Last, Wiley, Feb. 2025, Food Science & Nutrition, 13(2) (2), e70041[Refereed]Scientific journal
- Abstract Background Fermented katsuobushi, a traditional Japanese seasoning, is produced from skipjack tuna through smoking, drying and fermentation by xerophilic Aspergillus molds, primarily Aspergillus chevalieri and Aspergillus pseudoglaucus. In this study, we characterized lipolytic enzymes (cLip_1 to cLip_5 and pLip_1 to pLip_3) to clarify their roles in lipid hydrolysis during katsuobushi production under low water activity. Results The enzymes showed significant diversity in their activity, stability and substrate specificity, and in the hydrolysis profiles of their reactions with fish oil. Phylogenetic analyses revealed that cLip_5 showed a high identity with pLip_2 (94%) and these enzymes formed a phylogenetic cluster with filamentous fungal lipases. Purified recombinant enzymes (rcLip_1, rcLip_2, rcLip_4 and rcLip_5) and wild‐type enzymes (cLip_3 and pLip_3) showed varying substrate preferences toward p‐nitrophenyl esters. The addition of glycerol to reduce the water activity in the reaction mixture led to increased activities of rcLip_1 and rcLip_4, but it did not affect the activity of the other three enzymes. Among the tested six enzymes, cLip_5 showed the highest hydrolytic activity toward fish oil. The cLip_5 and pLip_2 gene transcript levels were moderately high in strains MK86 and MK88, respectively. Conclusion cLip_5 and its homolog pLip_2 were identified as the most promising enzymes for katsuobushi fermentation, because of their high hydrolytic activities toward fish oil and adaptability to low water activity conditions. These findings support the selection of optimal Aspergillus strains as starter cultures to potentially shorten the fermentation time and improve the quality and shelf life of katsuobushi. © 2025 Society of Chemical Industry.Wiley, Feb. 2025, Journal of the Science of Food and Agriculture, English[Refereed]Scientific journal
- Elsevier BV, Nov. 2023, Biocatalysis and Agricultural Biotechnology, 102943 - 102943, Co-authored internationally[Refereed]Scientific journal
- Corresponding, Springer Science and Business Media LLC, Aug. 2023, Archives of Microbiology, 205(9) (9), 310, English[Refereed]Scientific journal
- Elsevier BV, May 2023, Process Biochemistry, 130, 534 - 544, English[Refereed]Scientific journal
- Elsevier BV, Apr. 2023, Enzyme and Microbial Technology, 110240 - 110240, English[Refereed]Scientific journal
- Springer Science and Business Media LLC, Oct. 2022, 3 Biotech, 12(10) (10), English[Refereed]Scientific journal
- Lead, Wiley, Nov. 2021, Journal of Basic Microbiology, 62(2) (2), 174 - 184, English[Refereed]Scientific journal
- This study focused on the applying endo-xylanases to reduce the use of bleaching agent, coupled with the production of xylooligosaccharides (XOs), in mulberry paper making. Paper mulberry pulp (PMP) was consecutively prepared from paper mulberry bark by traditional NaOH-treatment, and two types of thermostable endo-xylanase from Streptomyces thermovulgaris TISTR1948 (wild-type and recombinant endo-xylanases) were employed in the biobleaching of PMP. This process was optimized to achieve maximum XOs yields and the highest PMP quality. The optimal condition was an enzyme dosage of 125 U/g PMP at 12 h of reaction time, both in a 500 mL laboratory bottle and a 150 L reactor. The mixture obtained from the reactor was separated as liquid of XOs derived from PMP (PMP-XOs) and solid biobleached PMP. The PMP-XOs from wild-type endo-xylanase were composed of 31.6% xylopentaose (X5), 30.9% xylohexaose and higher-degree XOs (X ≥ 6), and 11.7% xylobiose (X2), whereas 76.6% of X5 and 8.6% of X2 were the main products from recombinant endo-xylanase. The PMP-XOs derived from both endo-xylanase types exhibited high antioxidant activities, reducing power, phenolic contents, and prebiotic efficacy. In addition, the application of both endo-xylanases enhanced the brightness of PMP by 5.1% and 3.5%, and reduced the kappa number by 9.1% and 3.6%, respectively. Biobleached PMP was subsequently subjected to the NaOCl bleaching step to produce the mulberry paper. This approach could reduce NaOCl consumption by 20–25%, making it an environmentally friendly alternative. The production of valuable prebiotics, such as PMP-XOs, further enhances the economic viability of this approach. Graphic Abstract: [Figure not available: see fulltext.].Springer Science and Business Media B.V., Oct. 2021, Waste and Biomass Valorization, 12(10) (10), 5347 - 5360, EnglishScientific journal
- Lead, Elsevier BV, Sep. 2021, International Journal of Food Microbiology, 353, 109299 - 109299[Refereed]Scientific journal
- We describe the effect of enzyme (protease M) addition on reducing turbidity and self-flocculating of solid particles (mainly starch) in the drainage water after boiling a type of wheat-flour noodle (DWBWN) without the addition of flocculants to decrease waste-water-treatment costs to reuse the content of such drainage water. Reduction in turbidity and flocculation/sedimentation of solid particles in DWBWN are highly dependent on enzyme addition/concentration and pH level. We conducted experiments to determine the effect of adding protease to DWBWN. A decrease in turbidity of over 90% was observed in protease-added DWBWN. A decrease in total carbon (TC) of approximately 40% in the clear upper portion of DWBNW was also observed after protease addition and sedimentation. This TC was almost the same as with the supernatant of stock DWBWN without protease addition after centrifuging. Adding protease to DWBWN decreased the protein content, strengthened the negative surface electron charge, and decreased the isoelectric point of the solid particles. To determine the dissociation of phosphate ions in relation to reduction in turbidity and flocculation/sedimentation of solid particles, these processes were observed at the same pH level of a specific dissociate form of phosphorylated (mono-ester phosphates) starch in solid particles. This suggest that the strong negative zeta-potential of solid particles formed in DWBWN reduce turbidity, and flocculation/sedimentation of solid particles depends on the electrostatic properties.ELSEVIER, Jun. 2021, WATER RESOURCES AND INDUSTRY, 25, EnglishScientific journal
- BACKGROUND: Cocoon waste, solid waste from the silk industry, is currently utilized as low-cost spun silk yarn. In this study, the enzymatic valorization process of yellow cocoon waste to produce antioxidative sericin hydrolysate and fibroin film was demonstrated. RESULTS: The enzymatic process involving thermostable alkaline protease from Bacillus halodurans SE5 (protease_SE5) was superior to the conventional high temperature and high pressure and 0.5% Na2CO3 processes. Protease_SE5 produced sericin hydrolysate with high peptide concentration (0.335 mg mL–1) and provided degummed fibroin with a complete structure. Sericin hydrolysate from protease_SE5-assisted hydrolysis showed remarkable radical scavenging activities on ABTS, DPPH and FRAP assays with 602, 3.90 and 24.8 μmol Trolox equivalents (TE) g−1 protein, respectively. After ultrafiltration and size-exclusion chromatography, two active fractions (F1 and F2) were obtained from sericin hydrolysate with ABTS radical scavenging activity of 2120 and 3289 μmol TE g−1 protein, respectively. Identification of bioactive peptides by de novo sequencing was conducted, and seven candidate peptides were identified with high content of key antioxidative amino acids (His, Phe, Trp, Tyr and Arg) in the sequences. Moreover, bioactivities of several of the peptides were predicted by bioinformatics databases. Besides, the mechanical properties of enzyme-derived fibroin film were also improved, with tensile strength and elongation at break of 62.1 MPa and 3.6% in the dry state, and 8.5 MPa and 140% in the wet state. CONCLUSION: This enzymatic process could be an effective way for valorizing cocoon waste. The value-added products from agricultural waste have promising applications in food, pharmaceutical and biomedical materials. © 2020 Society of Chemical Industry (SCI).John Wiley and Sons Ltd, Apr. 2021, Journal of Chemical Technology and Biotechnology, 96(4) (4), 953 - 962, EnglishScientific journal
- A soup stock made from katsuobushi is an important element of, and the basic seasoning responsible for the taste of, traditional Japanese cuisine. Fermented and ripened katsuobushi, called karebushi, is manufactured via a repeated molding process on the katsuobushi surface. Our aim was to characterize the surface Aspergillus community and their enzymes involved in the fermentation and ripening. Five dominant Aspergillus species isolated from the karebushi surface were identified-A. amstelodami, A. chevalieri, A. pseudoglaucus, A. ruber, and A. sydowii. Analyses were performed on final molding stage-samples from different manufacturers, and 1st to 4th molding stage-samples from the same manufacturer. The composition ratios of the five Aspergillus spp. varied according to the manufacturer of the karebushi. A. amstelodami and A. chevalieri tended to be detected as dominant species when the water content of the karebushi fillet was >15% and the fat content was >3.5%, respectively. In samples from a given manufacturer, the dominant species in the final molding stage tended to be A. chevalieri and A. pseudoglaucus. Mixed molds were cultured by solid-state fermentation using katsuobushi powder medium at two different water activity (aw) levels. Crude extracts from each culture showed lipase, aminopeptidase, carboxypeptidase, and protease activities. Notably, the crude extracts cultivated at 0.85 aw showed higher protease activity toward hemoglobin and lipase activity toward p-nitrophenyl palmitate than those at 0.95 aw. These hydrolytic enzymes are probably involved in decolorization of katsuobushi and lipid degradation during the long fermentative and ripening period. In addition, mixed cultures could transform 2,6-dimethoxyphenol into 1,2,3-trimethoxybenzene, previously reported as an attractive and mild flavor component. Our results may help promote the use of desirable Aspergillus spp. as starter cultures for manufacturers to stabilize and improve the quality of fermented and ripened karebushi.Aug. 2020, International journal of food microbiology, 327, 108654 - 108654, English, International magazineScientific journal
- Blastochloris tepida is a newly described thermophilic purple bacterium containing bacteriochlorophyll b. Using purified light-harvesting 1 reaction center (LH1-RC) core complexes from Blc. tepida, we compared the biochemical, spectroscopic, and thermal denaturation properties of these complexes with those of its mesophilic counterpart, Blc. viridis. Besides their growth temperature optima, a striking difference between the two species was seen in the carotenoid composition of their LH1-RC complexes. The more thermostable Blc. tepida complex contained more carotenoids with longer conjugation lengths (n > 9), such as lycopenes (n = 11), and had a total carotenoid content significantly higher than that of the Blc. viridis complex, irrespective of the light intensity used for growth. The thermostability of LH1-RCs from both Blc. tepida and Blc. viridis decreased significantly in cells grown in the presence of diphenylamine, a compound that inhibits the formation of highly conjugated carotenoids. In contrast to the thermophilic purple bacterium Thermochromatium tepidum, where Ca2+ is essential for LH1-RC thermostability, Ca2+ neither was present in nor had any effect on the thermostability of the Blc. tepida LH1 RC. These results point to a mechanism that carotenoids with elongated conjugations enhance hydrophobic interactions with proteins in the Blc. tepida LH1-RC, thereby allowing the complexes to withstand thermal denaturation. This conclusion is bolstered by a structural model of the Blc. tepida LH 1-RC and is the first example of photocomplex thermostability being linked to a carotenoid-based mechanism.AMER CHEMICAL SOC, Jun. 2020, BIOCHEMISTRY, 59(25) (25), 2351 - 2358, EnglishScientific journal
- Elsevier BV, Oct. 2019, Process Biochemistry, 85, 156 - 163Scientific journal
- The light-harvesting 1 reaction center (LH1-RC) complex in the purple sulfur bacterium Thiorhodovibrio (Trv.) strain 970 cells exhibits its LH1 Q(y) transition at 973 nm, the lowest-energy Q(y) absorption among purple bacteria containing bacteriochlorophyll a (BChl a). Here we characterize the origin of this extremely red-shifted Qy transition. Growth of Try. strain 970 did not occur in cultures free of Ca2+, and elemental analysis of Ca2+-grown cells confirmed that purified Try. strain 970 LH1-RC complexes contained Ca2+. The LH1 Qy band of Try. strain 970 was blue-shifted from 959 to 875 nm upon Ca2+ depletion, but the original spectral properties were restored upon Ca2+ reconstitution, which also occurs with the thermophilic purple bacterium Thermochromatium (Tch.) tepidum. The amino acid sequences of the LH1 alpha- and beta-polypeptides from Try. strain 970 closely resemble those of Tch. tepidum; however, Ca2+ binding in the Try. strain 970 LH1-RC occurred more selectively than in Tch. tepidum LH1-RC and with a reduced affinity. Ultraviolet resonance Raman analysis indicated that the number of hydrogen-bonding interactions between BChl a and LH1 proteins of Try. strain 970 was significantly greater than for Tch. tepidum and that Ca2+ was indispensable for maintaining these bonds. Furthermore, perfusion-induced Fourier transform infrared analyses detected Ca2+-induced conformational changes in the binding site closely related to the unique spectral properties of Try. strain 970. Collectively, our results reveal an ecological strategy employed by Try. strain 970 of integrating Ca2+ into its LH1-RC complex to extend its light-harvesting capacity to regions of the near-infrared spectrum unused by other purple bacteria.AMER CHEMICAL SOC, Jun. 2019, BIOCHEMISTRY, 58(25) (25), 2844 - 2852, English[Refereed]Scientific journal
- Elsevier BV, Apr. 2019, Process Biochemistry, 79, 74 - 80Scientific journal
- © 2018 Elsevier Ltd A combination of isoelectronic precipitation and electrolyzed water treatment (IP-EWT) process was use for simultaneous recovery of protein and phosphorus compounds from heat-stabilized defatted rice bran (HSDFRB). The highest protein content (65.1 w/w%) recovery ratio (over 50%) of protein concentrate were attained by two-stage protein extraction with IP-EWT. Analysis of amino-acid composition demonstrated excellent amino acid value (76.6%) in the protein fraction than those of commercial cereals such as rice (61%), wheat (39%), and corn (31%). In addition, according to SDS-PAGE and immunoblotting analysis, neither rice allergenic protein nor heavy metals were detected in either HSDFRB or protein fractions extracted by IP-EWT. These results suggest that as an eco-friendly process, this combined (IP-EWT) process is suitable for practical recovery of highly concentrated and safe protein from HSDFRB without using enzymes or chemicals such as organic solvents, buffering agents, and surfactants.Jan. 2019, LWT, 99, 262 - 267[Refereed]Scientific journal
- Bacillus halodurans SE5 was newly isolated and grew well on a medium containing crude sericin extract from cocoon. Thermostable alkaline serine protease (protease_SE5) capable of decomposing sericin extract was purified to homogeneity from culture supernatant with a final yield of 25% and 2.01 x 10(4) U/mg. Among the six natural proteins tested, protease_SE5 showed the highest activity toward sericin. The sericin degumming, bio-bleaching coupled with sericin hydrolysate production from yellow cocoon by protease_SE5 and commercial Alcalase were demonstrated in the presence or absence of dithiothreitol (DTT). The addition of DTT enhanced the efficacy of both proteases. However, without DTT, the protease_SE5 had higher degumming ability and produced sericin hydrolysate 4-times higher than commercial enzyme based on the soluble protein concentration. SDS-PAGE and size exclusion chromatography analysis revealed that the maximal concentration of oli-gopeptides was observed with the hydrolysate prepared by protease_SE5 and showed higher antioxidant activity than those from Alcalase. The appreciable radical scavenging activities of the crude peptide (1.36 +/- 0.07 mM) on ABTS, DPPH, and FRAP assay were 1568 +/- 78, 3.6 +/- 1.6, and 13.6 +/- 0.4 mu mol TE/L, respectively. Protease_SE5 has potential application for one step degumming and preparation of bioactive peptides from yellow cocoon.ELSEVIER SCI LTD, Jan. 2019, Process Biochemistry, 78, 63 - 70, English[Refereed]Scientific journal
- Background Bradyrhizobium diazoefficiens USDA110 nodulates soybeans for nitrogen fixation. It accumulates poly-3-hydroxybutyrate (PHB), which is of physiological importance as a carbon/energy source for survival during starvation, infection, and nitrogen fixation conditions. PHB accumulation is orchestrated by not only the enzymes for PHB synthesis but also PHB-binding phasin pBMC, Oct. 2018, BMC Microbiology, 18, English[Refereed]Scientific journal
- Sep. 2018, Journal of the science of food and agriculture, English[Refereed]Scientific journal
- Aug. 2018, Biopharmaceutics & drug disposition[Refereed]
- The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium tepidum exhibits a largely red-shifted LH1 Q(y) absorption at 915 nm due to binding of Ca2+, resulting in an "uphill" energy transfer from LH1 to the reaction center (RC). In a recent study, we developed a heterologous expression system (strain TS2) to construct a functional hybrid LH1-RC with LH1 from Tch. tepidum and the RC from Rhodobacter sphaeroides [Nagashima, K. V. P., et al. (2017) Proc. Natl. Acad. Sci. U.S. A. 114, 10906]. Here, we present detailed characterizations of the hybrid LH1-RC from strain TS2. Effects of metal cations on the phototrophic growth of strain TS2 revealed that Ca2+ is an indispensable element for its growth, which is also true for Tch. tepidum but not for Rba. sphaeroides. The thermal stability of the TS2 LH1-RC was strongly dependent on Ca2+ in a manner similar to that of the native Tch. tepidum, but interactions between the heterologous LH1 and RC became relatively weaker in strain TS2. A Fourier transform infrared analysis demonstrated that the Ca2+-binding site of TS2 LH1 was similar but not identical to that of Tch. tepidum. Steady-state and time-resolved fluorescence measurements revealed that the uphill energy transfer rate from LH1 to the RC was related to the energy gap in an order of Rba. sphaeroides, Tch. tepidum, and strain TS2; however, the quantum yields of LH1 fluorescence did not exhibit such a correlation. On the basis of these findings, we discuss the roles of Ca2+, interactions between LH1 and the RC from different species, and the uphill energy transfer mechanisms.AMER CHEMICAL SOC, Jul. 2018, Biochemistry, 57(30) (30), 4496 - 4503, English[Refereed]Scientific journal
- An integrated process for xylooligosaccharides (XOs) and bioethanol production from corncob was investigated. XOs were produced by a consecutive process of KOH treatment and hydrolysis by an in-house thermostable endo-xylanase from Streptomyces thermovulgaris. XO yields of 0.15 g/gKOH-treated corncob (22.13 g/L) and 0.52 g/graw corncob of cellulose-rich corncob (CRC) were obtained. After 96 h of enzymatic hydrolysis, CRC hydrolysate contained 62.16, 51.21, 10.03 and 0.92 g/L of total sugar, glucose, xylose and arabinose, respectively. Bioethanol production by separate hydrolysis and fermentation (SHF) using CRC hydrolysate, and by simultaneous saccharification and fermentation (SSF) using CRC was studied at 40 °C for thermotolerant Candida glabrata. SHF showed an ethanol yield of 0.28 g/gCRC (21.92 g/L) and ethanol productivity of 0.304 g/L/h with 93% theoretical yield. Surprisingly, by SSF, those parameters were 0.27 g/gCRC (31.32 g/L), 0.33 g/L/h and 89%, respectively. This integrated process might be a new cost-effective approach for corncob valorization.Elsevier Ltd, May 2018, Bioresource Technology, 256, 399 - 407, English[Refereed]Scientific journal
- The new amylolytic oleaginous red yeast, Sporidiobolus pararoseus KX709872, produced both α-amylase (540 ± 0.09 mU/mL) and amyloglucosidase (23 ± 0.00 mU/mL) and showed good ability to directly convert rice residue from canteen waste to biomass and lipids. Effects of medium composition and cultivation conditions on growth and lipid accumulation for strain KX709872 were investigated under shaking flask and upscaling levels. At C : N ratio of 25 : 1, pH 5.45, 22.36°C, and 199.40 rpm for 7 days, volumetric production of biomass and lipids, lipid content, and lipid productivity reached 17.69 ± 0.44, 8.35 ± 0.19 g/L, 49.48 ± 0.41% (w/w), and 1.67 ± 0.11 g/L/day, respectively. Production of lipids was also implemented in 5.0-L stirred tank bioreactor with 2.5 L of optimized medium at 300 rpm and 3.0 vvm for 5 days. Volumetric production of biomass and lipids, lipid content, and lipid productivity were 16.33 ± 0.49, 8.75 ± 0.13 g/L, 56.61 ± 0.04% (w/w), and 2.19 ± 0.03 g/L/day, respectively. Meanwhile, the fatty acids of lipids from strain KX709872 had high oleic acid content (60−62%) which was similar to those of vegetable oils, indicating that these lipids are promising as an alternative biodiesel feedstock. Moreover, the biodiesel derived from lipids of strain KX709872 had properties satisfying the criteria of ASTM D6751 and EN 14214 standards.Taylor and Francis Inc., Apr. 2018, Preparative Biochemistry and Biotechnology, 48(4) (4), 361 - 371, English[Refereed]Scientific journal
- Background: The conjugative plasmid, pLS20, isolated from Bacillus subtilis natto, has an outstanding capacity for rapid self-transfer. In addition, it can function as a helper plasmid, mediating the mobilization of an independently replicating co-resident plasmid. Results: In this study, the oriT sequence of pLS20cat (oriT LS20) was eliminated to obtain the plasmid, pLS20catoriT. This resulted in the complete loss of the conjugative transfer of the plasmid but still allowed it to mobilize a co-resident mobilizable plasmid. Moreover, pLS20catoriT was able to mobilize longer DNA segments, up to 113kb of chromosomal DNA containing oriT LS20, after mixing the liquid cultures of the donor and recipient for only 15min. Conclusions: The chromosomal DNA mobilization mediated by pLS20catoriT will allow us to develop a novel genetic tool for the rapid, easy, and repetitive mobilization of longer DNA segments into a recipient chromosome.BioMed Central Ltd., Jan. 2018, Microbial Cell Factories, 17(1) (1), 13, English[Refereed]Scientific journal
- 2018, Biochemistry, in press, EnglishBiochemical and spectroscopic characterizations of a hybrid light harvesting reaction center core complex[Refereed]Scientific journal
- Objectives: A bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential. Results: Halotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis strain no. 16 was characterized. Multiple alignments with previously reported non-halotolerant proteases, including subtilisin Carlsberg, indicated that Apr_No16 has eight acidic or polar amino acid residues that are replaced by nonpolar amino acids in non-halotolerant proteases. Those residues were hypothesized to be one of the primary contributors to salt adaptation. An eightfold mutant substituted with Ala residues exhibited 1.2- and 1.8-fold greater halotolerance at 12.5% (w/v) NaCl than Apr_No16 and Carlsberg, respectively. Amino acid substitution notably shifted the theoretical pI of the eightfold mutant, from 6.33 to 9.23, compared with Apr_No16. The resulting protein better tolerated high salt conditions. Conclusions: Changing the pI of a bacterial serine protease may be an effective strategy to improve the enzyme’s halotolerance.Springer Netherlands, Jan. 2018, Biotechnology Letters, 40(1) (1), 189 - 196, English[Refereed]Scientific journal
- CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6-hydroxylation, progesterone 6-/16-hydroxylation, estrone 11-hydroxylation and estradiol 6-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16-hydroxylase activity and CYP2C9.12 showed higher estrone 11-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids.WILEY, Nov. 2017, BIOPHARMACEUTICS & DRUG DISPOSITION, 38(8) (8), 486 - 493, English[Refereed]Scientific journal
- To predict the amino acid residues playing important roles in acetyl-CoA and substrate binding and to study the acetyl group transfer mechanism of Chryseobacterium sp. 5-3B N-acetyltransferase (5-3B NatA). A 3-dimensional homology model of 5-3B NatA was constructed to compare the theoretical structure of this compound with the structures of previously reported proteins belonging to the bacterial GCN5 N-acetyltransferase family. Homology modeling of the 5-3B NatA structure and a characterization of the enzyme's kinetic parameters identified the essential amino acid residues involved in binding and acetyl-group transfer. (126)Leu, (132)Leu, and (135)Lys were implicated in the binding of phosphopantothenic acid, and (100)Tyr and (131)Lys in that of adenosyl biphosphate. The data supported the participation of (83)Glu and (133)Tyr in catalyzing acetyl-group transfer to l-2-phenylglycine. 5-3B NatA catalyzes the enantioselective N-acetylation of l-2-phenylglycine via a ternary complex comprising the enzyme, acetyl-CoA, and the substrate.SPRINGER, Nov. 2017, BIOTECHNOLOGY LETTERS, 39(11) (11), 1699 - 1707, English[Refereed]Scientific journal
- Background: Bacillus subtilis is able to utilize at least three inositol stereoisomers as carbon sources, myo-, scyllo-, and D-chiro-inositol (MI, SI, and DCI, respectively). NAD(+)-dependent SI dehydrogenase responsible for SI catabolism is encoded by iolX. Even in the absence of functional iolX, the presence of SI or MI in the growth medium was found to induce the transcription of iolX through an unknown mechanism. Results: Immediately upstream of iolX, there is an operon that encodes two genes, yisR and iolQ (formerly known as degA), each of which could encode a transcriptional regulator. Here we performed an inactivation analysis of yisR and iolQ and found that iolQ encodes a repressor of the iolX transcription. The coding sequence of iolQ was expressed in Escherichia coli and the gene product was purified as a His-tagged fusion protein, which bound to two sites within the iolX promoter region in vitro. Conclusions: IolQ is a transcriptional repressor of iolX. Genetic evidences allowed us to speculate that SI and MI might possibly be the intracellular inducers, however they failed to antagonize DNA binding of IolQ in in vitro experiments.BIOMED CENTRAL LTD, Jul. 2017, BMC MICROBIOLOGY, 17(1) (1), 154, English[Refereed]Scientific journal
- Bacillus subtilis genes iolG, iolW, iolX, ntdC, yfiI, yrbE, yteT, and yulF belong to the Gfo/Idh/MocA family. The functions of iolG, iolW, iolX, and ntdC are known; however, the functions of the others are unknown. We previously reported the B. subtilis cell factory simultaneously overexpressing iolG and iolW to achieve bioconversion of myo-inositol (MI) into scyllo-inositol (SI). YulF shares a significant similarity with IolW, the NADP(+)-dependent SI dehydrogenase. Transcriptional abundance of yulF did not correlate to that of iol genes involved in inositol metabolism. However, when yulF was overexpressed instead of iolW in the B. subtilis cell factory, SI was produced from MI, suggesting a similar function to iolW. In addition, we demonstrated that recombinant His(6)-tagged YulF converted scyllo-inosose into SI in an NADPH-dependent manner. We have thus identified yulF encoding an additional NADP(+)-dependent SI dehydrogenase, which we propose to rename iolU.TAYLOR & FRANCIS LTD, May 2017, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 81(5) (5), 1026 - 1032, English[Refereed]Scientific journal
- Background: A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer's disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inositol stereoisomers, including MI and SI. Previously, we reported a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. The strain was constructed by deleting all genes related to inositol metabolism and overexpressing key enzymes, IolG and IolW. By using this strain, 10 g/l of MI initially included in the medium was completely converted into SI within 48 h of cultivation in a rich medium containing 2% (w/v) Bacto soytone. Results: When the initial concentration of MI was increased to 50 g/l, conversion was limited to 15.1g/l of SI. Therefore, overexpression systems of IolT and PntAB, the main transporter of MI in B. subtilis and the membrane-integral nicotinamide nucleotide transhydrogenase in Escherichia coli respectively, were additionally introduced into the B. subtilis cell factory, but the conversion efficiency hardly improved. We systematically determined the amount of Bacto soytone necessary for ultimate conversion, which was 4% (w/v). As a result, the conversion of SI reached to 27.6 g/l within 48 h of cultivation. Conclusions: The B. subtilis cell factory was improved to yield a SI production rate of 27.6 g/l/48 h by simultaneous overexpression of IolT and PntAB, and by addition of 4% (w/v) Bacto soytone in the conversion medium. The concentration of SI was increased even in the stationary phase perhaps due to nutrients in the Bacto soytone that contribute to the conversion process. Thus, MI conversion to SI may be further optimized via identification and control of these unknown nutrients.BIOMED CENTRAL LTD, Apr. 2017, MICROBIAL CELL FACTORIES, 16(1) (1), 67, English[Refereed]Scientific journal
- A lactic acid producing bacterium, Lactobacillus rhamnosus M-23, newly isolated from a rice washing drainage storage tank was found to produce L-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. This strain has the ability to utilize the non-sterilized rice washing drainage and rice bran as a source of carbohydrate, saccharifying enzymes and nutrients for lactic acid production. Observation of extracellular protease activity in SSF culture broth showed that a higher protease activity was present in strain M-23 than in other isolated lactic acid producing bacteria (LABs). To investigate the structural changes of solid particles of rice washing drainage throughout LAB cultivation, scanning electron microscopic (SEM) observation and Fourier transform infrared-spectroscopy (FT-IR) analysis were performed. The results of the SEM observation showed that the surface material could be removed from solid particles of rice washing drainage treated by culture broth (supernatant) of strain M-23, thus exposing the crystal structure of the starch particle surface. The results of the FT-IR analysis revealed that the specific transmittance decrease of the C-C and C-O stretching and OH- group of the solid particles of the rice washing drainage were highly correlated with the produced lactic acid concentration and extracellular protease activity, respectively. These results demonstrate the high lactic acid producing ability of strain M-23 from a non-sterilized mixture of rice washing drainage and rice bran under the SSF condition due to the removal of proteinaceous material and exposure of the starch particle surface by extra cellular protease. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Feb. 2017, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 123(2) (2), 245 - 251, English[Refereed]Scientific journal
- BACKGROUNDAspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorises red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme. RESULTSThe genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repenseMK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%. PepA_MK82 and its recombinant had similar characteristics in terms of their optimal pH, thermostability, and decolorisation activity. The recombinant was also able to decolorise flaked, dried bonito and to bleach a blood-stained cloth. CONCLUSIONGiven its ability to hydrolyse and decolorise red-pigmented proteins, recombinant PepA_MK8 can be exploited in the food industry and as a stain-removal agent in laundry applications. (c) 2016 Society of Chemical IndustryWILEY-BLACKWELL, Jan. 2017, JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, 97(1) (1), 95 - 101, English[Refereed]Scientific journal
- Background: In Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5'-nucleotidases that hydrolyze the phosphate group of 5'-nucleotides. In Bacillus subtilis, genes encoding 5'-nucleotidase have remained to be identified. Results: We found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His(6)-tagged proteins. YcsE exhibited significant 5'-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5'-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth on solid medium. In the presence of diamide, only mutants lacking yktC exhibited enhanced growth defects, whereas the other mutants without ycsE or yqeG did not. Conclusions: Accordingly, in B. subtilis, at least YcsE and YktC acted as major 5'-nucleotidases and the four minor enzymes might function when the intracellular concentrations of substrates are sufficiently high. In addition, YktC is involved in resistance to oxidative stress caused by diamide, while YqeG is necessary for normal colony formation on solid medium.BIOMED CENTRAL LTD, Oct. 2016, BMC MICROBIOLOGY, 16(1) (1), 249, English[Refereed]Scientific journal
- A crude xylanase preparation from Streptomyces thermovulgaris TISTR1948 was able to hydrolyze KOH-treated corncob and to produce bioactive xylooligosaccharides (XOs). A thermostable cellulase-free endoxylanase from strain TISTR1948 was purified 15.0-fold from the crude preparation, with a recovery yield of 13.0%. On SDS-PAGE, the purified enzyme had an apparent molecular mass of 46.2 kDa. The N-terminal and internal amino acid sequences were determined and the cloned xylanase gene were sequenced. The 1434-bp gene encodes a protein with a predicted molecular mass of 46,976 Da. The deduced amino acid sequence had a high degree of identity with the sequences of GH 10 xylanases from Streptomyces spp. The purified xylanase was highly stable within a pH range of 4.0-11.5 and was thermostable within a temperature range of 50-70 degrees C. The activity of the enzyme reached a maximum at 65 degrees C; the enzyme's half-life was 90 min at 70 degrees C. Enzymatic activity was enhanced in the presence of metal ions, Ca2+, Co2+, and Mn2+ but almost completely inhibited by Hg2+, Pb2+, and SDS. The K-m and V-max values of the enzyme with beechwood xylan as the substrate were 37.6 mu M and 303 U/mg, respectively. The crude, partially purified, and purified xylanases were assayed for XO production from KOH-treated corncob. The main component of the XO products was xylobiose, with very little xylose and arabinose. An in vitro evaluation of XOs from the purified xylanase showed that they enhanced the growth of probiotic Lactobacillus plantarum TISTR1465. (C) 2016 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Jul. 2016, JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 129, 61 - 68, English[Refereed]Scientific journal
- We modified GntR regulation in Bacillus subtilis to devise transient induction systems. GntR is the repressor antagonized by gluconate to induce transcription of the gntRKPZ operon for gluconate catabolism. On the other hand, the gnt operon is repressed by glucose via carbon catabolite repression involving CcpA/P-ser-HPr, which binds to two cre sites: one located in the gnt promoter region and the other within the gntR coding region. We initiated gntKPZ encoding of enzymes for gluconate catabolism expressed independently from the operon; this allowed constitutive degradation of gluconate. Both cre sites were mutated to abolish catabolite repression. The mutated gnt promoter was set up to drive the expression of the lacZ reporter under the control of GntR. Even in the presence of glucose, lacZ was induced upon the addition of gluconate and shut down again as gluconate was consumed. Thus, modified GntR regulation enables artificial transient induction. This will allow us to design a flexible metabolic engineering system with genes expressed only temporarily as desired.MICROBIOL RES FOUNDATION, 2016, JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, 62(6) (6), 277 - 285, English[Refereed]Scientific journal
- We evaluated the metabolism of several herbicides and progesterone by two P450 proteins (CYP1A9 and CYP1C1) from Japanese eel (Anguilla japonica). Expression vectors harboring CYP1A9 and CYP1C1 sequences were introduced into Escherichia coli. E. coli membrane fractions were incubated with each substrate, and the metabolites were analyzed. CYP1A9 and CYP1C1 deethylated 7-ethoxycoumarin and phenacetin, and demethylated chlorotoluron, diuron, and linuron. CYP1C1 specifically hydroxlyated progesterone at the 6 beta and 16 alpha positions. Five amino acids of CYP1A9 related to substrate binding were selected for mutation analyses [CYP1A9(F128A), CYP1A9(F229A), CYP1A9(F263A), CYP1A9(V387A), and CYP1A9(I391A)]. Two variants, CYP1A9(F229A) and CYP1A9(F128A), changed the ratio of 16a hydroxyprogesterone to 613 hydroxyprogesterone. Among all the variants, CYP1A9(F263A) showed the highest activity towards substrates used. CYP1A9(V387A) and CYP1A9(I391A) showed higher activities than that of CYP1A9 toward progesterone. The substrate specificity of CYP1A9 may be altered by replacing an amino acid related to substrate binding. (C) 2015 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Sep. 2015, ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY, 40(2) (2), 360 - 368, English[Refereed]Scientific journal
- We evaluated the metabolism of several herbicides and progesterone by two P450 proteins (CYP1A9 and CYP1C1) from Japanese eel (Anguilla japonica). Expression vectors harboring CYP1A9 and CYP1C1 sequences were introduced into Escherichia coli. E. coli membrane fractions were incubated with each substrate, and the metabolites were analyzed. CYP1A9 and CYP1C1 deethylated 7-ethoxycoumarin and phenacetin, and demethylated chlorotoluron, diuron, and linuron. CYP1C1 specifically hydroxlyated progesterone at the 6 beta and 16 alpha positions. Five amino acids of CYP1A9 related to substrate binding were selected for mutation analyses [CYP1A9(F128A), CYP1A9(F229A), CYP1A9(F263A), CYP1A9(V387A), and CYP1A9(I391A)]. Two variants, CYP1A9(F229A) and CYP1A9(F128A), changed the ratio of 16a hydroxyprogesterone to 613 hydroxyprogesterone. Among all the variants, CYP1A9(F263A) showed the highest activity towards substrates used. CYP1A9(V387A) and CYP1A9(I391A) showed higher activities than that of CYP1A9 toward progesterone. The substrate specificity of CYP1A9 may be altered by replacing an amino acid related to substrate binding. (C) 2015 Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Sep. 2015, ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY, 40(2) (2), 360 - 368, English[Refereed]Scientific journal
- Two amylases, amylase I and amylase II from Bacillus subtilis strain FP-133, were purified to homogeneity and characterized. Their stabilities toward temperature, pH, and organic solvents, and their substrate specificities toward polysaccharides and oligosaccharides were similar. Under moderately high salt conditions, both amylases were more stable than commercial B. licheniformis amylase, and amylase I retained higher amylase activity than amylase II. The N-terminal amino acid sequence, genomic southern blot analysis, and MALDI-TOFF-MS analysis indicated that the halotolerant amylase I was produced by limited carboxy-terminal truncation of the amylase II peptide. The deduced amino acid sequence of amylase II was >95% identical to that of previously reported B. subtilis -amylases, but their carboxy-terminal truncation points differed. Three recombinant amylases full-length amylase corresponding to amylase II, an artificially truncated amylase corresponding to amylase I, and an amylase with a larger artificial C-terminal truncation were expressed in B. subtilis. The artificially truncated recombinant amylases had the same high amylase activity as amylase I under moderately high salt conditions. Sequence comparisons indicated that an increased ratio of Asp/Glu residues in the enzyme may be one factor responsible for increasing halotolerance.WILEY-BLACKWELL, Jun. 2015, JOURNAL OF BASIC MICROBIOLOGY, 55(6) (6), 780 - 789, English[Refereed]Scientific journal
- Background: The two-component regulatory system, involving the histidine sensor kinase DegS and response regulator DegU, plays an important role to control various cell processes in the transition phase of Bacillus subtilis. The degU32 allele in strain 1A95 is characterized by the accumulation of phosphorylated form of DegU (DegU-P). Results: Growing 1A95 cells elevated the pH of soytone-based medium more than the parental strain 168 after the onset of the transition phase. The rocG gene encodes a catabolic glutamate dehydrogenase that catalyzes one of the main ammonia-releasing reactions. Inactivation of rocG abolished 1A95-mediated increases in the pH of growth media. Thus, transcription of the rocG locus was examined, and a novel 3.7-kb transcript covering sivA, rocG, and rocA was found in 1A95 but not 168 cells. Increased intracellular fructose 1,6-bisphosphate (FBP) levels are known to activate the HPr kinase HPrK, and to induce formation of the P-Ser-HPr/CcpA complex, which binds to catabolite responsive elements (cre) and exerts CcpA-dependent catabolite repression. A putative cre found within the intergenic region between sivA and rocG, and inactivation of ccpA led to creation of the 3.7-kb transcript in 168 cells. Analyses of intermediates in central carbon metabolism revealed that intracellular FBP levels were lowered earlier in 1A95 than in 168 cells. A genome wide transcriptome analysis comparing 1A95 and 168 cells suggested similar events occurring in other catabolite repressive loci involving induction of lctE encoding lactate dehydrogenase. Conclusions: Under physiological conditions the 3.7-kb rocG transcript may be tightly controlled by a roadblock mechanism involving P-Ser-HPr/CcpA in 168 cells, while in 1A95 cells abolished repression of the 3.7-kb transcript. Accumulation of DegU-P in 1A95 affects central carbon metabolism involving lctE enhanced by unknown mechanisms, downregulates FBP levels earlier, and inactivates HPrK to allow the 3.7-kb transcription, and thus similar events may occur in other catabolite repressive loci.BIOMED CENTRAL LTD, Feb. 2015, BMC MICROBIOLOGY, 15, 43, English[Refereed]Scientific journal
- Phytases comprise a group of phosphatases that trim inorganic phosphates from phytic acid (IP6). In this study, we aimed to achieve the efficient secretion of phytase by Bacillus subtilis. B. subtilis laboratory standard strain 168 and its derivatives exhibit no phytase activity, whereas a natto starter secretes phytase actively. The natto phytase gene was cloned into strain RIK1285, a protease-defective derivative of 168, to construct a random library of its N-terminal fusions with 173 different signal peptides (SPs) identified in the 168 genome. The library was screened to assess the efficiency of phytase secretion based on clear zones around colonies on plates, which appeared when IP6 was hydrolyzed. The pbp SP enhanced the secretion of the natto phytase most efficiently, i.e. twice that of the original SP. Thus, the secreted natto phytase was purified and found to remove up to 3 phosphates from IP6.TAYLOR & FRANCIS LTD, 2015, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 79(11) (11), 1906 - 1914, English[Refereed]Scientific journal
- N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of L-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for L-2-phenylglycine and its chloro-and hydroxyl-derivatives. The K-m and V-max values for L-2-phenylglycine were 0.145 +/- 0.026 mM and 43.6 +/- 2.39 mu mol . min(-1) . mg protein(-1), respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to L-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).AMER SOC MICROBIOLOGY, Mar. 2014, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 80(5) (5), 1770 - 1776, English[Refereed]Scientific journal
- Bacillus subtilis is used industrially for the production of secreted enzymes. The most characteristic feature of the secreted enzymes is variation in the N-terminal signal peptides that is recognized by secretion machinery, which is one of the determinants of efficiency and must be customized in each case. Culturing cellulolytic B. subtilis to secrete heterologous cellulases combined with customized signal peptides would be beneficial for producing biocommodities from cellulosic biomass. Four Clostridium thermocellum genes, encoding endoglucanases (celA and celB) and exoglucanases (celK and celS) were cloned to construct random libraries of combinations with 173 different signal peptides originating from the B. subtilis genome. The libraries were successfully screened to identify the signal peptides most efficient in secretion of each of the four cellulases, which were theoretically unpredictable. The secreted cellulases were assayed on carboxymethyl cellulose, phosphoric acid swollen cellulose, and microcrystalline cellulose to determine the possible effects of the signal peptides on substrate specificity. The customized signal peptides for CelA, CelB, and CelS did not affect enzyme performance but those for CelK might influence its substrate specificity.MICROBIOL RES FOUNDATION, 2014, JOURNAL OF GENERAL AND APPLIED MICROBIOLOGY, 60(5) (5), 175 - 182, English[Refereed]Scientific journal
- Background: Bradyrhizobium japonicum USDA110, a soybean symbiont, is capable of accumulating a large amount of poly-beta-hydroxybutyrate (PHB) as an intracellular carbon storage polymer during free-living growth. Within the genome of USDA110, there are a number of genes annotated as paralogs of proteins involved in PHB metabolism, including its biosynthesis, degradation, and stabilization of its granules. They include two phbA paralogs encoding 3-ketoacyl-CoA thiolase, two phbB paralogs encoding acetoacetylCoA reductase, five phbC paralogs encoding PHB synthase, two phaZ paralogs encoding PHB depolymerase, at least four phaP phasin paralogs for stabilization of PHB granules, and one phaR encoding a putative transcriptional repressor to control phaP expression. Results: Quantitative reverse-transcriptase PCR analyses of RNA samples prepared from cells grown using three different media revealed that PHB accumulation was related neither to redundancy nor expression levels of the phbA, phbB, phbC, and phaZ paralogs for PHB-synthesis and degradation. On the other hand, at least three of the phaP paralogs, involved in the growth and stabilization of PHB granules, were induced under PHB accumulating conditions. Moreover, the most prominently induced phasin exhibited the highest affinity to PHB in vitro; it was able to displace PhaR previously bound to PHB. Conclusions: These results suggest that PHB accumulation in free-living B. japonicum USDA110 may not be achieved by controlling production and degradation of PHB. In contrast, it is achieved by stabilizing granules autonomously produced in an environment of excess carbon sources together with restricted nitrogen sources.BIOMED CENTRAL LTD, Dec. 2013, BMC MICROBIOLOGY, 13, 290, English[Refereed]Scientific journal
- Background: Bacillus subtilis 168 possesses an efficient pathway to metabolize some of the stereoisomers of inositol, including myo-inositol (MI) and scyllo-inositol (SI). Previously we reported a prototype of a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. However, it wasted half of initial 1.0% (w/v) MI, and the conversion was limited to produce only 0.4% (w/v) SI. To achieve a more efficient SI production, we attempted additional modifications. Results: All "useless" genes involved in MI and SI metabolism were deleted. Although no elevation in SI production was observed in the deletion strain, it did result in no wastage of MI anymore. Thus additionally, overexpression of the key enzymes, IolG and IolW, was appended to demonstrate that simultaneous overexpression of them enabled complete conversion of all MI into SI. Conclusions: The B. subtilis cell factory was improved to yield an SI production rate of 10 g/L/48 h at least. The improved conversion was achieved only in the presence of enriched nutrition in the form of 2% (w/v) Bacto soytone in the medium, which may be due to the increasing demand for regeneration of cofactors.BIOMED CENTRAL LTD, Dec. 2013, MICROBIAL CELL FACTORIES, 12, 124, English[Refereed]Scientific journal
- The demand for d-2-phenylglycine used to synthesize semisynthetic antibiotics and pesticides is increasing. We have isolated a Chryseobacterium sp. that selectively transformed the l-form of racemic d,l-2-phenylglycine to (2S)-2-acetylamide-2-phenylacetic acid with a molar yield of 50 % and an enantiomer excess of > 99.5 % under optimal culture conditions, consequently resulting in 99 % pure d-2-phenylglycine remaining in the culture. The enantioselective N-acetylation was catalyzed by an acetyl-CoA-dependent N-acetyltransferase whose synthesis was induced by l-2-phenylglycine. The enzyme differed from previously reported bacterial arylamine N-acetyltransferases in molecular mass and substrate specificity. The relative activity ratio of the enzyme with the substrates l-2-phenylglycine, d-2-phenylglycine, 2-(2-chlorophenyl)glycine, and 5-aminosalicylic acid (a good substrate of arylamine N-acetyltransferase) was 100:0:56.9:5.49, respectively. The biotransformation by the N-acetyltransferase-producing bacterium reported here could constitute a new preparative route for the enzymatic resolution of d,l-2-phenylglycine.SPRINGER, Jul. 2013, BIOTECHNOLOGY LETTERS, 35(7) (7), 1053 - 1059, English[Refereed]Scientific journal
- Background Katsuobushi is a dried, smoked and fermented bonito used in Japanese cuisine. During the fermentation process with several Aspergillus species, the colour of Katsuobushi gradually changes from a dark reddish-brown derived from haem proteins to pale pink. The change in colour gives Katsuobushi a higher ranking and price. This study aimed to elucidate the mechanism of decolourisation of Katsuobushi. Results A decolourising factor from the culture supernatant of Aspergillus (Eurotium) repens strain MK82 was purified to homogeneity. The purification was monitored by measuring the decolourising activity using equine myoglobin and bovine haemoglobin as substrates. It was found that the decolourising factor had protease activity towards myoglobin and haemoglobin. Complete inhibition of the enzyme by the inhibitor pepstatin A and the internal amino acid sequence classified the protein as an aspartic protease. The enzyme limitedly hydrolysed myoglobin between 1-Met and 2-Gly, 43-Lys and 44-Phe, and 70-Leu and 71-Thr. The purified enzyme decolourised blood of Katsuwonus pelamis (bonito) and a slice of dried bonito. Conclusion It is proposed that aspartic protease plays a role in the decolourisation of Katsuobushi by the hydrolysis of haem proteins that allows the released haem to aggregate in the dried bonito. (C) 2012 Society of Chemical IndustryWILEY-BLACKWELL, Apr. 2013, JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, 93(6) (6), 1349 - 1355, English[Refereed]Scientific journal
- Background: The agrichemical 4-aminopyridine is used as a bird repellent in crop fields and has an epileptogenic action in a variety of animals, including man and mouse. 4-Aminopyridine is biodegraded in the environment through an unknown mechanism. Results: A 4-aminopyridine-degrading enrichment culture utilized 4-aminopyridine as a carbon, nitrogen, and energy source, generating 4-amino-3-hydroxypyridine, 3,4-dihydroxypyridine, and formate as intermediates. 4-Amino-3-hydroxypyridine could not be further metabolized and probably accumulated as a dead-end product in the culture. Biodegradability tests and partial sequence analysis of the enrichment culture indicated that 4-aminopyridine was mainly degraded via 3,4-dihydroxypyridine and that the metabolite is probably cleaved by 3-hydroxy-4-pyridone dioxygenase. Seven culturable predominant bacterial strains (strains 4AP-A to 4AP-G) were isolated on nutrient agar plates. Changes in the bacterial populations of 4-aminopyridine, 3,4-dihydroxypyridine, or formate/ammonium chloride enrichment cultures were monitored by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Sequence analysis of the 16S rRNA gene fragments derived from predominant DGGE bands indicated that Pseudomonas nitroreducens 4AP-A and Enterobacter sp. 4AP-G were predominant in the three tested enrichment cultures and that the unculturable strains Hyphomicrobium sp. 4AP-Y and Elizabethkingia sp. 4AP-Z were predominant in 4-aminopyridine and formate/ammonium chloride enrichment cultures and in the 3,4-dihydroxypyridine enrichment culture, respectively. Among the culturable strains, strain 4AP-A could utilize 3,4-dihydroxypyridine as a growth substrate. Although we could not isolate strain 4AP-Y on several media, PCR-DGGE analysis and microscopy indicated that the unique bi-polar filamentous bacterial cells gradually became more dominant with increasing 4-aminopyridine concentration in the medium. Conclusions: Hyphomicrobium sp. 4AP-Y, P. nitroreducens 4AP-A, and Elizabethkingia sp. 4AP-Z probably play important roles in 4-aminopyridine degradation in crop fields. In the enrichment culture, 3,4-dihydroxypyridine and its metabolites including formate might be shared as growth substrates and maintain the enrichment culture, including these indispensable strains.BIOMED CENTRAL LTD, Mar. 2013, BMC MICROBIOLOGY, 13, 62, English[Refereed]Scientific journal
- Geobacillus kaustophilus HTA426, a thermophilic Bacillus-related species, utilizes some inositol stereoisomers, including myo-, D-chiro- and scyllo-inositols (MI, DCI and SI), as sole carbon sources. Within its genome are three paralogous genes that possibly encode inositol dehydrogenase. These genes are located in tandem within a large gene cluster containing an almost complete set of iol genes homologous to genes involved in inositol catabolism in Bacillus subtilis. Each of the three plausible inositol dehydrogenases was purified as a His(6)-tag fusion. The enzymes exhibited thermophilic activity, each with its own characteristic specificity for the inositol stereoisomers and cofactors. Northern blot and primer extension analyses revealed that the three enzymes were encoded by the same 5 kb polycistronic transcript and were induced simultaneously in the presence of MI. HTA426 was subjected to ethyl methanesulfonate (EMS) mutagenesis to isolate a mutant strain, PS8, which was not able to utilize MI. In PS8, inositol dehydrogenase activity was abolished along with the 5 kb transcript, suggesting that any of the three enzymes supports MI-dependent growth. Analysis of metabolites in HTA426 cells grown in the presence of MI revealed that substantial amounts of DCI and SI appeared intracellularly during the stationary phase, while only MI was present in PS8 cells, suggesting that interconversion of inositol stereoisomers may involve these three enzymes.SOC GENERAL MICROBIOLOGY, Aug. 2012, MICROBIOLOGY-SGM, 158(Pt 8) (Pt 8), 1942 - 1952, English[Refereed]Scientific journal
- Jun. 2012, Nanomedicine (London, England), 7(6) (6), 855 - 865[Refereed]
- Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in . The recombinant protease, over-expressed in , was inactive but addition of acetone to crude cell extracts restored the activity and removed many proteins. We purified the active, acetone-treated protease to homogeneity in a single chromatography step with 57% recovery. The recombinant protease partially hydrolyzed eggshell membrane and produced more soluble peptides and proteins than commercial elastase, alpha-chymotrypsin, and collagenase. The soluble peptides produced from hydrolyzed eggshell membrane inhibited angiotensin-I-converting enzyme activity. The degradation of eggshell membrane by the recombinant elastase could be applied to the production of soluble bioactive peptides.SPRINGER, May 2012, BIOTECHNOLOGY LETTERS, 34(5) (5), 949 - 955, English[Refereed]Scientific journal
- Background: A stereoisomer of inositol, scyllo-inositol, is known as a promising therapeutic agent for Alzheimer's disease, since it prevents the accumulation of beta-amyloid deposits, a hallmark of the disease. However, this compound is relatively rare in nature, whereas another stereoisomer of inositol, myo-inositol, is abundantly available. Results: Bacillus subtilis possesses a unique inositol metabolism involving both stereoisomers. We manipulated the inositol metabolism in B. subtilis to permit the possible bioconversion from myo-inositol to scyllo-inositol. Within 48 h of cultivation, the engineered strain was able to convert almost half of 10 g/L myo-inositol to scyllo-inositol that accumulated in the culture medium. Conclusions: The engineered B. subtilis serves as a prototype of cell factory enabling a novel and inexpensive supply of scyllo-inositol.BIOMED CENTRAL LTD, Sep. 2011, MICROBIAL CELL FACTORIES, 10, 69, English[Refereed]Scientific journal
- Burkholderia sp. strain AK-5 converts 4-aminophenol to maleylacetic acid via 1,2,4-trihydroxybenzene, which is unstable in vitro and non-enzymatically auto-oxidized to 2-hydroxy-1,4-benzoquinone. Crude extract of strain AK-5 retarded the auto-oxidation and reduced the substrate analogue, 2,6-dimethoxy-1,4-benzoquinone, in the presence of NADH. The two enzymes responsible were purified to homogeneity. The deduced amino acid sequence of the enzyme that inhibited the auto-oxidation showed a high level of identity to sequences of iron-containing superoxide dismutases (Fe-SODs) and contained a conserved metal-ion-binding site; the purified enzyme showed superoxide dismutase activity and contained 1 mol of Fe per mol of enzyme, identifying it as Fe-SOD. Among three type SODs tested, Fe-SOD purified here inhibited the auto-oxidation most efficiently. The other purified enzyme showed a broad substrate specificity toward benzoquinones, including 2-hydroxy-1,4-benzoquinone, converting them to the corresponding 1,4-benzenediols; the enzyme was identified as 2-hydroxy-1,4-benzoquinone reductase. The deduced amino acid sequence did not show a high level of identity to that of benzoquinone reductases from bacteria and fungi that degrade chlorinated phenols or nitrophenols. The indirect role of Fe-SOD in 1,2,4-trihydroxybenzene metabolism is probably to scavenge and detoxify reactive species that promote the auto-oxidation of 1,2,4-trihydroxybenzene in vivo. The direct role of benzoquinone reductase would be to convert the auto-oxidation product back to 1,2,4-trihydroxybenzene. These two enzymes together with 1,2,4-trihydroxybenzene 1,2-dioxygenase convert 1,2,4-trihydroxybenzene to maleylacetic acid.SPRINGER, Feb. 2011, BIODEGRADATION, 22(1) (1), 1 - 11, English[Refereed]Scientific journal
- Burkholderia sp. strain AK-5 converts 4-aminophenol to maleylacetic acid via 1,2,4-trihydroxybenzene, which is unstable in vitro and non-enzymatically auto-oxidized to 2-hydroxy-1,4-benzoquinone. Crude extract of strain AK-5 retarded the auto-oxidation and reduced the substrate analogue, 2,6-dimethoxy-1,4-benzoquinone, in the presence of NADH. The two enzymes responsible were purified to homogeneity. The deduced amino acid sequence of the enzyme that inhibited the auto-oxidation showed a high level of identity to sequences of iron-containing superoxide dismutases (Fe-SODs) and contained a conserved metal-ion-binding site; the purified enzyme showed superoxide dismutase activity and contained 1 mol of Fe per mol of enzyme, identifying it as Fe-SOD. Among three type SODs tested, Fe-SOD purified here inhibited the auto-oxidation most efficiently. The other purified enzyme showed a broad substrate specificity toward benzoquinones, including 2-hydroxy-1,4-benzoquinone, converting them to the corresponding 1,4-benzenediols; the enzyme was identified as 2-hydroxy-1,4-benzoquinone reductase. The deduced amino acid sequence did not show a high level of identity to that of benzoquinone reductases from bacteria and fungi that degrade chlorinated phenols or nitrophenols. The indirect role of Fe-SOD in 1,2,4-trihydroxybenzene metabolism is probably to scavenge and detoxify reactive species that promote the auto-oxidation of 1,2,4-trihydroxybenzene in vivo. The direct role of benzoquinone reductase would be to convert the auto-oxidation product back to 1,2,4-trihydroxybenzene. These two enzymes together with 1,2,4-trihydroxybenzene 1,2-dioxygenase convert 1,2,4-trihydroxybenzene to maleylacetic acid.SPRINGER, Feb. 2011, BIODEGRADATION, 22(1) (1), 1 - 11, English[Refereed]Scientific journal
- We cloned the genes encoding the two distinct extracellular halotolerant proteases of Bacillus subtilis FP-133 Expro-I and Expro-II, which were classified as alkaline serine and neutral proteases respectively. Three-dimensional modeling suggested that acidic and polar amino acid residues located on the surface stabilize protein structure in the presence of relatively high NaCl concentrations.TAYLOR & FRANCIS LTD, Jan. 2011, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 75(1) (1), 148 - 151, English[Refereed]Scientific journal
- The 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d produces a deaminase that catalyzes the deamination of 2-amino-5-carboxymuconic 6-semialdehyde. A gene encoding the deaminase, ahdB, was cloned and expressed in Escherichia coli; ahdB is located downstream from the previously reported genes encoding 4-amino-3-hydroxybenzoate2,3-dioxygenase ( ahdA) and a LysR-type regulator. The deduced amino acid sequence of ahdB shows 30-33% identity to those of previously reported 2-aminomuconate deaminases. We identified a region (RAGDFLXVSG) conserved in AhdB and three other deaminases. The recombinant enzyme AhdB was purified to homogeneity. After a coupled enzyme assay with purified AhdA, AhdB, and the substrate 4-amino-3-hydroxybenzoate, the final product, formed by the action of AhdA, AhdB, and by nonenzymatic decarboxylation, was identified by HPLC, MS, and H-1-nuclear magnetic resonance analyses as 2-hydroxymuconic 6-semialdehyde.OXFORD UNIV PRESS, Sep. 2009, FEMS MICROBIOLOGY LETTERS, 298(1) (1), 93 - 98, English[Refereed]Scientific journal
- Phlebia radiata strain BP-12-2, isolated from forest soil, decolorized fungal melanin and produced an extracellular laccase in culture. The enzyme was purified to homogeneity and characterized. It showed no absorption hands in the ultraviolet above 300 nm or visible regions. It differed from the reported laccases in substrate specificity, kinetic parameters, and NH2 terminal amino acid sequences.TAYLOR & FRANCIS LTD, Apr. 2009, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73(4) (4), 939 - 942, English[Refereed]Scientific journal
- A bacterial strain, MEA isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH4)(2)SO4, as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the PI position and Lys at the P2 position. it also preferred hydrophobic amino acid residues at the PI I and 1321 positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan. (c) 2009, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Apr. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 107(4) (4), 373 - 378, English[Refereed]Scientific journal
- Bacillus cereus strain 10-L-2 synthesizes two arylamine N-acetyltransferases (Nat-a and Nat-b) with broad substrate specificities toward aniline and its derivatives. In southern blot analysis using probes encoding the NH2-terminus of Nat-b and a conserved region of N-acetyltransferases, digested total DNA of strain 10-L-2 showed one positive band. We cloned and sequenced the gene encoding Nat-b. The NH2-terminal amino acid sequence predicted from the open reading frame (768 base pairs) corresponded to that of purified Nat-b. The cloned Nat-b gene was expressed in Escherichia coli. The expressed enzyme (BcNAT) from the recombinant strain was partially purified and characterized. Nat-b from strain 10-L-2 and BcNAT from the recombinant strain were slightly different from each others in substrate specificity and thermo-stability. We examined the biotransformations of 2-aminophenols and phenylenediamines by the whole cells of the recombinant strain. The cells converted these compounds into their corresponding acetanilides. Only one amino group of phenylenediamines was acetylated. The cells utilized 4-nitroacetanilide as an acetyl donor instead of acetyl-CoA. 4-Aminoacetanilide was produced and 4-nitroaniline was released almost stoichiometrically. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.SOC BIOSCIENCE BIOENGINEERING JAPAN, Jan. 2009, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 107(1) (1), 27 - 32, English[Refereed]Scientific journal
- A newly isolated strain, MS-2-5, identified as Bacillus halodurans, produced five alkaline and thermotolerant amylases. The five amylases, named amylases A-E, were separated from each other, and purified to homogeneity. The molecular masses of these amylases were different from each other, and estimated to be 90, 85, 70, 65, and 58 kDa. These amylases showed the maximal activities at 60-65 degrees C and pH 10.5-11. A predominant product by each enzyme reaction was maltotetraose. These amylases were classified as an alpha-amylase by anomeric form analysis of the reaction products. Internal amino acid sequence analyses of the purified enzymes suggested that these enzymes were produced from a single polypeptide by proteolytic degradation. The gene, named amyA, was cloned and expressed in the T7 promoter systems of Escherichia coli. To increase yield and productivity of recombinant enzyme, cultivation conditions were examined. The maximal amount of enzyme was produced when an E. coli transformant carrying amyA was cultivated at 25 degrees C in Luria-Bertani medium supplemented with 1.0% D-glucose, 1.0% D-sorbitol, 0.1% MgSO4 center dot 7H(2)O, and 2.0% yeast extract. The yield of the transformant increased 104-fold as, compared with that of the parent strain MS-2-5. (c) 2008 Elsevier Inc. All rights reserved.ELSEVIER SCIENCE INC, Oct. 2008, ENZYME AND MICROBIAL TECHNOLOGY, 43(4-5) (4-5), 321 - 328, English[Refereed]Scientific journal
- Ceriporiopsis sp. strain MD-1, isolated from forest soil, produced several extracellular enzymes that decolorized human hair melanin. Among them, three enzymes (E1, E2-1, and E2-2) were purified to homogeneity and characterized. The enzymes required hydrogen peroxide in their enzyme reactions and, typical of other fungal peroxidases, oxidized various phenol compounds such as guaiacol, but not 3,4-dimethoxybenzyl alcohol. The spectra of the three enzymes showed an absorption maximum at 406 urn, indicating that they were heme proteins. However, the A(406)/A(280) values of the enzymes were below 0.4, which was lower than those of other peroxidases. E2-1 and E2-2 were similar to each other in their molecular and catalytic properties, and they possibly represent products of posttranslational modifications and/or allelic variants of the same gene, mdcA. The corresponding cDNA was cloned and sequenced; the deduced amino acid sequence showed high identities to the manganese peroxidases from other microorganisms. The specific activities and K-m values of E2-1 and E2-2 for synthetic and human hair melanins were much higher than those of Phanerochaete chrysosporium manganese peroxidase and lignin peroxidase.AMER SOC MICROBIOLOGY, Aug. 2008, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 74(16) (16), 5106 - 5112, English[Refereed]Scientific journal
- Bacillus pumilus X-6-9, isolated from soil and subsequently identified, produced xylooligosaccharides with long chains from xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increase in the production of the xylooligosaccharides was established when compared with the original culture conditions of B. pumilus X-6-19. The addition of D-glucose to the culture of the mutant strain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase but not xylanase. For these reasons, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharides with long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized. The hydrolyzates generated by the purified xylanase contained xylobiose, xylotriose, xylotetraose, and xylopentaose but not xylose. © 2008 Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology.Jul. 2008, Chinese Journal of Biotechnology, 24(7) (7), 1221 - 1227, English[Refereed]Scientific journal
- Analysis and Application of Inducible and Constitutive Expressions of Catabolic Genes Participating in the Degradation of Organic Chemical PollutantsIn the biodegradation of environmental pollutants, the mode of gene expression is an important subject in order to practice its performance effectively on the site. The aniline-assimilating bacteria Frateuria sp. ANA-18 and Rhodococcus sp. AN-18 were found to have inducible and constitutive expression systems of catabolic genes, respectively. The mechanism and application to a practical use of the two gene expression systems are described.RESEARCH JOURNAL BIOTECHNOLOGY, 2008, RESEARCH JOURNAL OF BIOTECHNOLOGY, 148 - 154, English[Refereed]Scientific journal
- A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 1011, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding a-amylase I was cloned and named amyI. Production of Amyl with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.TAYLOR & FRANCIS LTD, Oct. 2007, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 71(10) (10), 2393 - 2401, English[Refereed]Scientific journal
- A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 1011, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding a-amylase I was cloned and named amyI. Production of Amyl with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.TAYLOR & FRANCIS LTD, Oct. 2007, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 71(10) (10), 2393 - 2401, English[Refereed]Scientific journal
- Lactobacillus casei TISTR 1500 was isolated from soil of a dairy wastewater treatment plant and selected as the most active azo dye degrader of 19 isolates. Growing cells and freely suspended cells of this strain completely degraded methyl orange, thereby decolorizing the medium. The strain stoichiometric ally converted methyl orange to N,N-dimethyl-p-phenylenediamine and 4-aminobenzenesulfonic acid, which were identified by HPLC, GC, and GC-MS analyses. The enzyme activity responsible for the cleavage of the azo bond of methyl orange was localized to the cytoplasm of cells grown on modified MRS medium containing methyl orange. The effect of sugars, oligosaccharides, organic acids, metal ions, pHs, oxygen and temperatures on methyl orange decolorization by freely suspended cells was investigated. The optimal conditions for the decolorization of methyl orange by the Lactobacillus casei TISTR 1500 are incubation at 35 degrees C and pH 6 with sucrose provided as the energy source. (C) 2006 Elsevier Ltd. All rights reserved.PERGAMON-ELSEVIER SCIENCE LTD, Mar. 2007, WATER RESEARCH, 41(5) (5), 985 - 992, English[Refereed]Scientific journal
- A thermotolerant bacterium, identified as Bacillus licheniformis, completely utilized 0.1% (w/v) NH4NO3 at 30 and 50 degrees C under aerobic condition. The addition of 0.5 mM Fe2+ to the NH4NO3 medium markedly promoted the utilization of NH4+ and NO3-. At 50 degrees C, of total nitrogen originally provided, 24% was taken up into the cells and 20% remained in the culture supernatant. Residual nitrogen (56%) was probably removed into the atmosphere. The cell extracts contained enzymes involved in denitrification. GC-MS demonstrated that NH415 NO3 had been converted to (N2O)-N-15. These results indicate that the strain has denitrification ability under aerobic condition.SPRINGER, Mar. 2007, BIOTECHNOLOGY LETTERS, 29(3) (3), 385 - 390, English[Refereed]Scientific journal
- Pseudomonas sp. strain 7-6, isolated from active sludge obtained from a wastewater facility, utilized a quaternary ammonium surfactant, n-dodecyltrimethylammonium chloride (DTAC), as its sole carbon, nitrogen, and energy source. When initially grown in the presence of 10 mM DTAC medium, the isolate was unable to degrade DTAC. The strain was cultivated in gradually increasing concentrations of the surfactant until continuous exposure led to high tolerance and biodegradation of the compound. Based on the identification of five metabolites by gas chromatography-mass spectrometry analysis, two possible pathways for DTAC metabolism were proposed. In pathway 1, DTAC is converted to lauric acid via n-dodecanal with the release of trimethylamine; in pathway 2, DTAC is converted to lauric acid via n-dodecyldimethylamine and then n-dodecanal with the release of dimethylamine. Among the identified metabolites, the strain precultivated on DTAC medium could utilize n-dodecanal and lauric acid as sole carbon sources and trimethylamine and dimethylamine as sole nitrogen sources, but it could not efficiently utilize n-dodecyidimethylamine. These results indicated pathway I is the main pathway for the degradation of DTAC.AMER SOC MICROBIOLOGY, Mar. 2007, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 73(6) (6), 1797 - 1802, English[Refereed]Scientific journal
- Eighteen bacterial stock cultures were examined for their ability to utilize NH4+ and NO3- simultaneously in a medium containing NH4NO3 with shaking using a test tube capped with a cotton stopper. Pseudomonas aeruginosa NBRC 12689 utilized I mg/ml of NH4NO3 most rapidly of the cultures tested. The bacterium could completely utilize 5 mg/ml of NH4NO3 within 3 d, 6 mg/ml of NH4Cl within 3 d, and 20 mg/ml of NaNO3 within 2 d under optimum conditions. The addition of Fe2+ to the NH4NO3 medium markedly promoted the utilization of the two ions. When the Pseudomonas strain utilized 5 mg/ml of NH4NO3 completely, the total nitrogen in the culture including its cells decreased to 41% of that of the NH4NO3 originally provided. GC-MS analysis showed that the removed nitrogen was probably denitrified. When the bacterium was incubated in the NH4NO3 medium with shaking in a vial sealed with a rubber stopper, N-2 accumulated, but not N2O at the final phase of cultivation. On the other hand, both N-2 and N2O were detected in the NaNO3 medium. We concluded that the bacterium removed NH4+ from NH4NO3 as a nitrogen source for its cell components, together with the denitrification of NO3- under controlled shaking conditions. In addition, NH4+ promoted the cell growth of the bacterium and denitrification to N-2, preventing the accumulation of N2O.SOC BIOSCIENCE BIOENGINEERING JAPAN, Feb. 2007, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 103(2) (2), 185 - 191, English[Refereed]Scientific journal
- A bacterium, strain 10-L-2, that was isolated from soil and identified as Bacillus cereus grew well on medium containing 4-phenylenediamine and Polypepton. Strain 10-L-2 converted a wide variety of anilines, including 4-phenylenediamine, to their corresponding acetanilides. Growing cells acetylated a single amino group of 4-phenylenediamine to form 4-aminoacetanilide with a 97% molar yield, as shown by mass spectrometry and HPLC. Cell extracts exhibited arylamine N-acetyltransferase (NAT) activity toward 4-phenylenediamine. Two NATs, namely, NAT-a and NAT-b, were separated by DE52 column chromatography and were further purified and characterized. The subunit molecular masses of NAT-a and NAT-b were 31.0 and 27.5 kDa, respectively, as determined by SDS-PAGE analysis. The two enzymes had similar pH- and thermo-stabilities and were similarly affected by pH, temperature, and several reagents. The enzymes showed peak activity toward 5-aminosalicylic acid of the substrates tested, but they differed in substrate specificity. Only NAT-a had activity toward sulfamethazine. Although other wild-type bacterial cultures also synthesize NAT, the ability of strain 10-L-2 to convert and detoxify 4-phenylenediamine is much higher. This report provides the first evidence of two NATs in a eubacterium.SOC BIOSCIENCE BIOENGINEERING JAPAN, Feb. 2007, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 103(2) (2), 147 - 154, English[Refereed]Scientific journal
- Bordetella sp. strain 10d metabolizes 4-amino-3-hydroxybenzoic acid via 2-hydroxymuconic 6-semialdehyde. Cell extracts from 4-amino-3-hydroxybenzoate-grown cells showed high NAD(+)-dependent 2-hydroxymuconic 6-semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase activities, but no 2-hydroxymuconic 6-semialdehyde hydrolase activity. These enzymes involved in 4-amino-3-hydroxybenzoate metabolism were purified and characterized. When 2-hydroxymuconic 6-semialdehyde was used as substrate in a reaction mixture containing NAD(+) and cell extracts from 4-amino-3-hydroxybenzoate-grown cells, 4-oxalocrotonic acid, 2-oxopent-4-enoic acid, and 4-hydroxy-2oxovaleric acid were identified as intermediates, and pyruvic acid was identified as the final product. A complete pathway for the metabolism of 4-amino-3-hydroxybenzoic acid in strain 10d is proposed. Strain 10d metabolized 2-hydroxymuconic 6-semialdehyde derived from 4-amino-3-hydroxybenzoic acid via a dehydrogenative route, not via a hydrolytic route. This proposed metabolic pathway differs considerably from the modified meta-cleavage pathway of 2-aminophenol and those previously reported for methyl- and chloroderivatives.TAYLOR & FRANCIS LTD, Nov. 2006, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70(11) (11), 2653 - 2661, English[Refereed]Scientific journal
- A bacterial isolate, strain PDa-1, grew well on basal medium supplemented with 2-phenylenediamine, sucrose, and ammonium nitrate and completely transformed 2-phenylenediamine. The isolate was identified as Bacillus cereus. The product formed from 2-phenylenediamine was identified by EI-MS and NMR as 2-aminoacetanilide; whole cells converted 2-phenylenediamine to the product with a 76% molar yield. Whole cells also showed a broad substrate specificity toward 20 of 26 tested arylamines with substituent groups of various size and positions. Especially 2-aminobenzoic acid, 4-aminosalicylic acid, 5-aminosalicylic acid, and 2-aminofluorene were converted completely to the corresponding product with an aminoacetyl group. Cell extracts of strain PDa-1 had a high arylamine N-acetyltransferase activity. The partially purified enzyme converted 2-phenylenediamine to 2-aminoacetanilide. Strain PDa-1 constitutively expressed the enzyme in the absence of 2-phenylenediamine. Effects of 2-phenylenediamine and 2-aminoacetanilide on growth indicated that this enzyme probably plays a role in the detoxification of toxic arylamines in this strain.SOC BIOSCIENCE BIOENGINEERING JAPAN, Jul. 2006, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 102(1) (1), 21 - 27, English[Refereed]Scientific journal
- Bacillus subtilis strain FP-133, isolated from a fermented fish paste, synthesized two novel halotolerant extracellular proteases (expro-I and expro-II), showing activity and stability at concentrations of 0-20% (w/v) NaCl. Each protease was purified to homogeneity and characterized. The purified expro-I was a non-alkaline serine protease with an optimum pH of 7.5, although most serine proteases from Bacillus strains act at the alkaline side. The molecular mass of expro-I was 29 kDa. The purified expro-II was a metalloprotease with a molecular mass of 34 kDa. It was activated by Fe2+, which has never been reported as a bacterial protease activator. At a concentration of 7.5% (w/v) NaCl, both proteases preferred animal proteins to vegetable proteins as natural substrates. In addition, under saline conditions, expro-I and II showed high catalytic activity toward gelatin and casein respectively.TAYLOR & FRANCIS LTD, Feb. 2006, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70(2) (2), 433 - 440, English[Refereed]Scientific journal
- The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg 2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90°C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA. initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC. © 2006 Biochemical Society.Jan. 2006, Biochemical Journal, 393(1) (1), 219 - 226, English[Refereed]Scientific journal
- A halotolerant strain FP-133, able to grow at concentrations of 0-12.5% (w/v) NaCl, was isolated from a fish paste and identified as Bacillus subtilis. B. subtilis strain FP-133 produced an intracellular protease which showed catalytic activity under saline conditions. The enzyme was purified to homogeneity 143-fold with a yield of 0.9%. The purified enzyme showed an optimum activity at a concentration of 5% (w/v) NaCl. After storage in 7.5% (w/v) NaCl at 4 degrees C for 24 h, the enzyme kept 100% of its activity. The molecular mass of the protease was determined to be 59 kDa by gel filtration; the protein consisted of four subunits each with a molecular mass of 14 kDa. The enzyme showed aminopeptidase activity. It acted on L-leucyl-p-nitroanilide, L-leucyl-p-naphthylamide, and oligopeptides containing glycine, L-histidine, or L-leucine. The K-m and V-max values for L-leucyl-p-nitroanilide were 18 mu m and 2.2 mM/h mg, respectively. The enzyme was activated by Fe2+, Fe3+, and Ni2+ in synergism with Mg2+.WILEY-V C H VERLAG GMBH, 2006, JOURNAL OF BASIC MICROBIOLOGY, 46(4) (4), 294 - 304, English[Refereed]Scientific journal
- 2006, Bioscience, 70(2) (2), 433 - 440, EnglishConstitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production[Refereed]Scientific journal
- Pseudomonas sp. strain AP-3 grows on benzoate, phydroxybenzoate, protocatechuate, and 2-aminophenol as sole carbon and energy source. This strain converted benzoate and p-hydroxybenzoate to catechol and protocatechuate respectively, which were metabolized via the ortho-cleavage pathway. The enzymes responsible for these reactions were shown to be inducible. In contrast, strain AP-3 constitutively expresses the enzymes involved in the metabolism of 2-aminophenol.TAYLOR & FRANCIS LTD, May 2005, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 69(5) (5), 1033 - 1035, English[Refereed]Scientific journal
- 2005, Journal of Bioscience and Bioengineering, 98(2) (2), 71 - 76, EnglishConstitutive synthesis of enzymes involved in 2-aminophenol metabolism and inducible synthesis of enzymes involved in benzoate[Refereed]Scientific journal
- The Biochemical Society, 2005, Biochemical Journal, 393(1) (1), 219 - 226, English[Refereed]Scientific journal
- A catechol 1,2-dioxygenase (CD) was found, which was synthesized constitutively in the aniline-assimilating bacterium Rhodococcus sp. AN-22 grown on a medium without aniline, as well as on aniline medium. The bacterium synthesized CD in its cells grown on all the 21 non-aromatic substrates examined, including four natural media such as meat and yeast extracts, one sugar, six organic acids, and 10 amino acids as carbon, energy, and nitrogen sources. When the bacterium was incubated on a medium with D-glucose, L-malate, isoleucine, leucine, etc., it synthesized more CD than that in cells grown on aniline. Two CDs, which were prepared from cells grown on aniline and L-malate, were purified separately to homogeneity and characterized. The two enzymes were apparently identical in molecular and catalytic properties including molecular mass, optimal pH, stability to heating, and substrate specificity for catechol analogues. However, they differed in the substrate specificity and resistance to sulfhydryl and chelating agents from the inducible CDs produced by other aniline-assimilating bacteria reported previously.SOC BIOSCIENCE BIOENGINEERING JAPAN, Aug. 2004, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 98(2) (2), 71 - 76, English[Refereed]Scientific journal
- 2-Amino-5-carboxymuconic 6-semialdehyde is an unstable intermediate in the meta-cleavage pathway of 4-amino-3-hydroxybenzoic acid in Bordetella sp. strain 10d. In vitro, this compound is nonenzymatically converted to 2,5-pyridinedicarboxylic acid. Crude extracts of strain 10d grown on 4-amino-3-hydroxybenzoic acid converted 2-amino-5-carboxymuconic 6-semialdehyde formed from 4-amino-3-hydroxybenzoic acid by the first enzyme in the pathway, 4-amino-3-hydroxybenzoate 2,3-dioxygenase, to a yellow compound (epsilon(max) = 375 nm). The enzyme in the crude extract carrying out the next step was purified to homogeneity. The yellow compound formed from 4-amino-3-hydroxybenzoic acid by this purified enzyme and purified 4-amino-3-hydroxybenzoate 2,3-dioxygenase in a coupled assay was identified as 2-hydroxymuconic 6-semialdehyde by GC-MS analysis. A mechanism for the formation of 2-hydroxymuconic 6-semialdehyde via enzymatic deamination and nonenzymatic decarboxylation is proposed based on results of spectrophotometric analyses. The purified enzyme, designated 2-amino-5-carboxymuconic 6-semialdehyde deaminase, is a new type of deaminase that differs from the 2-aminomuconate deaminases reported previously in that it primarily and specifically attacks 2-amino-5-carboxymuconic 6-semialdehyde. The deamination step in the proposed pathway differs from that in the pathways for 2-aminophenol and its derivatives.BLACKWELL PUBLISHING LTD, Aug. 2004, EUROPEAN JOURNAL OF BIOCHEMISTRY, 271(15) (15), 3248 - 3254, English[Refereed]Scientific journal
- 2-Amino-5-carboxymuconic 6-semialdehyde is an unstable intermediate in the meta-cleavage pathway of 4-amino-3-hydroxybenzoic acid in Bordetella sp. strain 10d. In vitro, this compound is nonenzymatically converted to 2,5-pyridinedicarboxylic acid. Crude extracts of strain 10d grown on 4-amino-3-hydroxybenzoic acid converted 2-amino-5-carboxymuconic 6-semialdehyde formed from 4-amino-3-hydroxybenzoic acid by the first enzyme in the pathway, 4-amino-3-hydroxybenzoate 2,3-dioxygenase, to a yellow compound (epsilon(max) = 375 nm). The enzyme in the crude extract carrying out the next step was purified to homogeneity. The yellow compound formed from 4-amino-3-hydroxybenzoic acid by this purified enzyme and purified 4-amino-3-hydroxybenzoate 2,3-dioxygenase in a coupled assay was identified as 2-hydroxymuconic 6-semialdehyde by GC-MS analysis. A mechanism for the formation of 2-hydroxymuconic 6-semialdehyde via enzymatic deamination and nonenzymatic decarboxylation is proposed based on results of spectrophotometric analyses. The purified enzyme, designated 2-amino-5-carboxymuconic 6-semialdehyde deaminase, is a new type of deaminase that differs from the 2-aminomuconate deaminases reported previously in that it primarily and specifically attacks 2-amino-5-carboxymuconic 6-semialdehyde. The deamination step in the proposed pathway differs from that in the pathways for 2-aminophenol and its derivatives.BLACKWELL PUBLISHING LTD, Aug. 2004, EUROPEAN JOURNAL OF BIOCHEMISTRY, 271(15) (15), 3248 - 3254, English[Refereed]Scientific journal
- cis,cis-Muconate cycloisomerase (MC) was purified to homogeneity from benzamide-assimilating Arthrobacter sp. BA-5-17. The purified enzyme showed high activities for cis,cis-muconate and 3-methyl-cis,cis-muconate, and preferred the 3-substituted derivatives over the derivatives with the same substituent at the 2 position as a substrate. A gene encoding MC of strain BA-5-17 was cloned and named catB. The catB gene was clustered with catR encoding a putative LysR-type regulator, catC encoding a putative muconolactone isomerase, and catA-II encoding the catechol 1,2-dioxygenase isozymes CD-III-1 and III-2. These genes showed the same orientation in transcriptional direction and the organization of cloned genes was catRBCA-II. In the phylogenetic analysis of MCs and chloro-MCs, the BA-5-17 and Streptomyces setonii MCs formed a subfamily, clearly distinguished from those of other MCs. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.ELSEVIER SCIENCE BV, Feb. 2004, FEMS MICROBIOLOGY LETTERS, 231(1) (1), 119 - 124, English[Refereed]Scientific journal
- Bordetella sp. 10d produces a novel dioxygenase catalyzing the meta-cleavage of 4-amino-3-hydroxybenzoic acid, 4-amino-3-hydroxybenzoate 2,3-dioxygenase (4A3HBA23D). A gene encoding 4A3HBA23D was cloned and named ahdA. The deduced amino acid sequence of ahdA showed 29.2-24.2% identities to those of prokaryotic and eukaryotic 3-hydoxybenzoate 3,4-dioxygenases in reported meta-cleavage dioxygenases. However, no identities were observed in the amino-terminal sequences of the first 29 amino acid residues. An ORF was found downstream of ahdA. The deduced amino acid sequence of the ORF showed identities to those of LysR family regulators involved in protocatechuate metabolism and contained motifs conserved in the regulators. On the basis of these results, the ORF was named ahdR encoding a putative LysR family regulator. The transcription start point of ahdA was localized 414-bp upstream of the start codon of ahdA. Two DNA-binding motifs of LysR family regulators were found upstream of the transcription start point. These observations suggest that a LysR family regulator encoded by ahdR regulates the expression of ahdA. (C) 2003 Elsevier Inc. All rights reserved.ACADEMIC PRESS INC ELSEVIER SCIENCE, Feb. 2004, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 314(2) (2), 489 - 494, English[Refereed]Scientific journal
- Genes encoding an aniline dioxygenase of Frateuria sp. ANA-18, which metabolizes aniline via the ortho-cleavage pathway of catechol, were cloned and named tdn genes. The tdn genes were located on the chromosomal DNA of this bacterium and weren't clustered with catechol-degrading gene clusters. These results show that the ANA-18 aniline-degrading gene cluster is constructionally different from Pseudomonas tdn and Acinetobacter atd gene clusters, which degrade aniline via the meta-cleavage pathway of catechol and organize catechol-metabolic genes in the gene clusters. When cloned tdnQTA1A2B genes were expressed in Eschherichia coli, aniline dioxygenase activity was observed. Southern blot analysis revealed that homologues of the tdnA1A2B genes didn't exist in strain ANA-18. Disruption of the tdnA1A2 genes gave the parent strain ANA-18 a defect in aniline metabolism. On the basis of these results, we concluded that only the cloned tdn genes function as genes encoding aniline dioxygenase in strain ANA-18 although this bacterium had two catechol-degrading gene clusters.TAYLOR & FRANCIS LTD, Nov. 2003, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 67(11) (11), 2351 - 2358, English[Refereed]Scientific journal
- Burkholderia sp. strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source. A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis. Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol. 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid. The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with K-m and V-max values of 9.6 muM and 6.8 mumol min(-1) mg of protein(-1), respectively.AMER SOC MICROBIOLOGY, Sep. 2003, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 69(9) (9), 5410 - 5413, English[Refereed]Scientific journal
- A bacterial strain that grew on 4-amino-3-hydroxybenzoic acid was isolated from farm soil. The isolate, strain 10d, was identified as a species of Bordetella. Cell extracts of Bordetella sp. strain 10d grown on 4-amino-3-hydroxybenzoic acid contained a enzyme that cleaved this substrate. The enzyme was purified to homogeneity with a 110-fold increase in specific activity. The purified enzyme was characterized as a meta-cleavage dioxygenase that catalyzed the ring fission between C2 and C3 of 4-amino-3-hydroxybenzoic acid, with the consumption of 1 mol of O-2 per mol of substrate. The enzyme was therefore designated as 4-amino-3-hydroxybenzoate 2,3-dioxygenase. The molecular mass of the native enzyme was 40 kDa based on gel filtration; the enzyme is composed of two identical 21-kDa subunits according to SDS/PAGE. The enzyme showed a high dioxygenase activity only for 4-amino-3-hydroxybenzoic acid. The K-m and V-max values for this substrate were 35 muM and 12 mumol.min(-1).(mg protein)(-1), respectively. Of the 2-aminophenols tested, only 4-aminoresorcinol and 6-amino-m-cresol inhibited the enzyme. The enzyme reported here differs from previously reported extradiol dioxygenases, including 2-aminophenol 1,6-dioxygenase, in molecular mass, subunit structure and catalytic properties.BLACKWELL PUBLISHING LTD, Dec. 2002, EUROPEAN JOURNAL OF BIOCHEMISTRY, 269(23) (23), 5871 - 5877, English[Refereed]Scientific journal
- The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.SOC BIOSCIENCE BIOENGINEERING JAPAN, Jun. 2002, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 93(6) (6), 584 - 588, English[Refereed]Scientific journal
- A bacterial strain F-5-2, isolated from soil and identified as Klebsiella pneumoniae, removed NH4+ completely in 24 h of aerobic cultivation in a medium containing 1 mg/ml of NH4NO3. However, 70% of the NO3 originally provided remained. When 100 muM Fe2+ was added to the medium, both NH4+ and NO3- were removed simultaneously and completely from the culture within 6 It of incubation. In addition, the amount of MoO4- in the medium markedly affected the bacterial cell growth and utilization of NH4+ and NO3-. The bacterium could remove 4 mg/ml of NH4NO3 completely in 48 h of aerobic cultivation in a medium containing 100 muM Fe2+ and 0.8 pm MoO42-. The total nitrogen in the culture containing its cells was decreased to 14% of that in the NH4NO3 originally provided. GC-MS analysis demonstrated that N-2 was generated from the nitrogen atoms of both NH4+ and NO3-.TAYLOR & FRANCIS LTD, May 2002, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66(5) (5), 996 - 1001, English[Refereed]Scientific journal
- We isolated the two LysR-type regulatory proteins CatR(1) and CatR(2), which regulate the expression of cat(1) and cat(2) gene clusters, respectively, required for catechol degradation in the bacterium Frateuria sp. ANA-18. In a gel mobility shift assay using CatR(1) and the DNA fragment containing the catB(1) promoter region, the formation of two complexes, complex 1-1 (C1-1) and complex 1-2 (C1-2), was observed in the presence of cis,cis-muconate. On the other hand, CatR(2) and the DNA fragment containing the catB(2) promoter region formed only complex 2-2 (C2-2) at a lower concentration of cis,cis-muconate than that at which C1-1 and C1-2 were formed. As the concentration of cis, cis-muconate decreased, the production of the muconate cycloisomerase isozyme MC II encoded by catB(2) decreased as well as that of MC I encoded by catB(1). However, the amount of MC II synthesized was larger than that of MC I at low concentrations. On the basis of these results, we concluded that the catB(2) promoter was activated at low concentrations of cis,cis-muconate.TAYLOR & FRANCIS LTD, Oct. 2001, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 65(10) (10), 2146 - 2153, English[Refereed]Scientific journal
- Ralstonia sp. Ba-0323, a wild strain isolated from soil, produced catechol from benzoate and accumulated it outside the cells. The bacterium produced a maximal amount of catechol (1.6 mg/ml) from 3 mg/ml of sodium benzoate in a 20-h growing culture. The conversion rate of benzoate to catechol was 70% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 3 mg/ml of sodium benzoate reached 1.9 mg/ml at the conversion rate of 83% after 8 h of incubation. Catechol 1,2-dioxygenase, which catalyzed the ring cleavage of catechol, was purified to homogeneity from a cell extract of Ralstonia sp. Ba-0323 growing on benzoate and characterized. The specific activity of the purified enzyme was much lower than those of the dioxygenases from other microorganisms reported. The K-m for catechol of the purified enzyme was much higher than those of other dioxygenases. In addition, the NH2-terminal amino acid sequence of the enzyme was less similar to the other catechol 1,2-dioxygenases than they are to each other.TAYLOR & FRANCIS LTD, Sep. 2001, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 65(9) (9), 1957 - 1964, English[Refereed]Scientific journal
- Isolation of a benzoate-utilizing Pseudomonas strain from soil and production of catechol from benzoate by transpositional mutantsPseudomonas sp. Ba-0511 was isolated from soil by enrichment cultivation on a medium containing 6 mg/ml of sodium benzoate. The bacterium could grow on a medium containing 20 mg/ml of sodium benzoate by a successive enrichment culture. One hundred and twelve transpositional mutants of the bacterium produced catechol from benzoate and accumulated it outside of the cells. Among the mutants, strain BA(+)63 produced a maximal amount of catechol (2.3 mg/ml) from 6 mg/ml of sodium benzoate after growing for 10.5 h. The conversion rate of benzoate to catechol was 50% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 6 mg/ml of sodium benzoate reached 3.3 mg/ml at the conversion rate of 72% after 5 h of incubation. The resting cells converted m-methylbenzoic acid to 3- and 4-methylcatechol and m-chlorobenzoic acid to 3- and 4-chlorocatechol.URBAN & FISCHER VERLAG, 2001, MICROBIOLOGICAL RESEARCH, 156(2) (2), 151 - 158, English[Refereed]Scientific journal
- A 13.9-kb legion, which contained the 2-aminophenol 1,6-dioxygenase genes (amnBA) reported before, was cloned from the 3-aminophenol-assimilating bacterium Pseudomonas sp. AP-3. The complete nucleotide sequence of this region was determined and six genes were found downstream of amnBA. The eight genes together were designated amnBACFEDHG. Each gene was similar to the corresponding gene operating in the meta-cleavage pathway, except fur amnB, amnA, and amnD. The four 2-aminophenol-metabolizing enzymes, 2-aminomuconic 6-semialdehyde dehydrogenase, 2-aminomuconate deaminase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase, were purified and characterized. NH2-terminal amino acid sequences of each purified enzyme agreed with those deduced from amnC, amnF, amnE, and amnD, respectively. These genes were therefore assigned as the genes encoding these respective proteins. The tight clustering of the amn genes, which were all transcribed in the same direction, raised the possibility that these genes formed a single operon. The organization of the amn genes was entirely different from that of the atd, dmp, and xyl genes reported in the meta-cleavage pathway, although these latter genes clustered similarly.SPRINGER-VERLAG, Oct. 2000, ARCHIVES OF MICROBIOLOGY, 174(4) (4), 265 - 272, English[Refereed]Scientific journal
- Two Escherichia coli transformants with catechol 1,2-dioxygenase activity were selected from a gene library of the benzamide-assimilating bacterium Arthrobacter species strain BA-5-17, which produces four catechol 1,2-dioxygenase isozymes, A DNA fragment isolated from one transformant contained a complete open reading frame (ORF). The deduced amino acid sequence of the ORF shared high identity with hydroxyquinol 1,2-dioxygenase. An enzyme expressed by the ORF was purified to homogeneity and characterized. When hydroxyquinol was used as a substrate, the purified enzyme showed 6.8-fold activity of that for catechol. On the basis of the sequence identity and substrate specificity of the enzyme, we concluded that the ORF encoded hydroxyquinol 1,2-dioxygenase. When catechol was used as a substrate, cis,cis-muconic acid and 2-hydroxymuconic 6-semialdehyde, which were products by the intradiol and extradiol ring cleavage activities, respectively, were produced. These results showed that the hydroxyquinol 1,2-dioxygenase reported here was a novel dioxygenase that catalyzed both the intradiol and extradiol cleavage of catechol.TAYLOR & FRANCIS LTD, May 1999, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 63(5) (5), 859 - 865, English[Refereed]Scientific journal
- The aniline-assimilating bacterium Frateuria species ANA-18 produced two catechol 1,2-dioxygenases, CD I and CD II, and two muconate cycloisomerases, MC I and MC II. The catA genes catA(1) and catA(2) encoding CD I and CD II, respectively, were cloned from a gene library of this bacterium. The catA(1) gene was clustered with catB(1) encoding MC I, catC(1) encoding muconolactone isomerase (MI), catD encoding beta-ketoadipate enol-lactone hydrolase (ELH), and ORFR1 encoding a putative LysR-type regulator. The organization of these genes was ORF(R1)catB(1)C(1)D. The catA(2) gene also constructed a gene cluster involving catB(2) encoding MC II, catC(2) encoding MI, and ORFR2 encoding a putative LysR-type regulator with the alignment of ORF(R2)catB(2)A(2)C(2). The intergenic regions of ORFR1-catB(1) and ORFR2-catB(2) contained homologous sequences withe catR-catB intergenic region containing a repression binding site and activation binding site of CatR in Pseudomonas putida. These findings suggest that the two cat clusters were regulated independently in their expression. When a product of cloned catD was added to a reaction mixture containing beta-ketoadipate enol-lactone, beta-ketoadipate was produced. This observation showed that the cloned catD encoded ELH and was expressed in Escherichia coli. We found that Frateuria sp. ANA-18 had a large plasmid with a molecular size more than 100 kb. Polymerase chain reaction amplifying partial catA genes and Southern hybridization analyses with probes containing catA genes were conducted, to examine the localization of the two catA genes. We concluded that the catA(1) and catA(2) genes were located on the chromosomal and large plasmid DNAs, respectively, in Frateuria sp. ANA-18. (C) 1999 Elsevier Science B.V. All rights reserved.ELSEVIER SCIENCE BV, Jan. 1999, GENE, 226(2) (2), 189 - 198, English[Refereed]Scientific journal
- A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2,4-pentadienoic acid is the last metabolite containing an amino group. We, therefore, propose a modified meta-cleavage pathway for the 2-aminophenol metabolism.SPRINGER VERLAG, Aug. 1998, ARCHIVES OF MICROBIOLOGY, 170(2) (2), 132 - 137, English[Refereed]Scientific journal
- Catechol 2,3-dioxygenase (C23D; EC 1.13.1.2) was purified to homogeneity from a cell extract of Pseudomonas sp. AW-2 grown on aniline, and the purified C23D was characterized. The molecular mass estimated by gel filtration was 110 kDa. The enzyme dissociated into four identical subunits each with the molecular mass of 33 kDa. The enzyme had high activity for 3-methylcatechol as well as catechol, and differed from the enzyme from Pseudomonas putida mt-2, which carries the TOL plasmid, in optimal on for catechol, extradiol cleavage activities for 3-methylcatechol and 4-methylcatechol, and immunochemical properties. The amino acid sequence deduced from a C23D gene, alnE, from Pseudomonas sp. AW-2 was 85.7% identical to that of 3-methylcatechol 2,3-dioxygenase from toluidine-assimilating Pseudomonas putida UCC22. AlnE was 44.1% identical to the C23D encoded by xylE in P. putida mt-2. Because XylE has low activity for 3-methylcatechol, these results suggest that the differences in substrate specificity for 3-methylcatechol among the C23Ds reflected their sequence similarity.TAYLOR & FRANCIS LTD, Apr. 1998, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 62(4) (4), 747 - 752, English[Refereed]Scientific journal
- Muconate cycloisomerase was purified from aniline-assimilating Rhodococcus erythropolis AN-13. The purified enzyme exhibited a novel property in that it catalyzed cycloisomerization most efficiently when the reaction mixture contained Co2+ ion as a cofactor, even though Mn2+ ion has been reported to be the best cofactor for the muconate cycloisomerase reaction.SOC FERMENTATION BIOENGINEERING, JAPAN, 1998, JOURNAL OF FERMENTATION AND BIOENGINEERING, 85(5) (5), 521 - 524, English[Refereed]Scientific journal
- 2-Aminophenol 1,6-dioxygenase was purified from the cell extracts of Pseudomonas sp. AP-3 grown on 2-aminophenol. The product from 2-aminophenol by catalysis of the purified enzyme was identified as 2-aminomuconic 6-semialdehyde by gas chromatographic and mass spectrometric analyses. The molecular mass of the native enzyme was 140 kDa based on gel filtration. It was dissociated into molecular mass subunits of 32 (alpha-subunit) and 40 kDa (beta subunit) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase was a heterotetramer of alpha(2) beta(2). The genes coding for the alpha- and beta-subunits of the enzyme were cloned and sequenced. Open reading frames of the genes (amnA and amnB) were 816 and 918 base pairs in length, respectively. The amino acid sequences predicted from the open reading frames of amnA and amnB corresponded to the NH2-terminal amino acid sequences of the alpha-subunit (AmnA) and beta-subunit (AmnB), respectively. The deduced amino acid sequences of AmnB showed identities to some extent with HpaD (25.4%) and HpcB (24.4%) that are homoprotocatechuate 2,8-dioxygenases from Escherichia coli W and C, respectively, belonging to class III in the extradiol dioxygenases. On the other hand, AmnA had identity (23.3%) with only AmnB among the enzymes examined.AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, Jun. 1997, JOURNAL OF BIOLOGICAL CHEMISTRY, 272(23) (23), 14727 - 14732, English[Refereed]Scientific journal
- Partial purification and characterization of a bacterial dioxygenase that catalyzes the ring fission of 2-aminophenolA bacterial strain AP-3, grown on 2-aminophenol as a sole carbon, nitrogen and energy source and isolated from soil, was identified as a Pseudomonas species. When Pseudomonas sp. AP-3 grew with this substrate, it synthesized an enzyme acting on 2-aminophenol. This enzyme was purified with an 103-fold increase of the specific activity from its cell-free extracts. We proved that the purified enzyme was a dioxygenase catalyzing the ring fission between 1- and 6-positions of 2-aminophenol with the consumption of 1 mol of O-2 per mol of substrate. The enzyme showed the maximal activity at pH 7.5. It was stable between pH 7.5 and 9 and up to 35 degrees C. The molecular mass of this enzyme was 140 kDa by gel filtration.GUSTAV FISCHER VERLAG, Feb. 1997, MICROBIOLOGICAL RESEARCH, 152(1) (1), 33 - 38, English[Refereed]Scientific journal
- Resting cells of the transpositional mutant strain B-9, which was obtained from the aniline-assimilating Pseudomonas sp. AW-2 and produced catechol from aniline, exhibited the ability to oxidize 16 aromatic amines among 26 tested. Thirteen products from the 16 amines mere purified and crystallized, Analytical data including mass and nuclear magnetic resonance spectra of the products showed that methyl, dimethy, ethyl, chloro-, fluoro-, and hydroxyl derivatives of aniline were converted to the corresponding catechols, In particular, catechols were synthesized from o-, m-, and p-toluidine, p-fluoroaniline, and m-aminophenol at a conversion rate of 100%. It was revealed that strain B-9 cells exhibited extensive substrate specificity for aromatic amines, regardless of the position of substituent groups in their isomers.SOC FERMENTATION BIOENGINEERING, JAPAN, 1997, JOURNAL OF FERMENTATION AND BIOENGINEERING, 84(3) (3), 232 - 235, English[Refereed]Scientific journal
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- 日本生物工学会, 2010, 日本生物工学会大会講演要旨集, 22, 76 - 76, Japanese3P-1076 Characterization of N-acetyltransferase from Microbacterium paraoxydans FK-2-1
- Kobe University, 2003, The science reports of Faculty of Agriculture, Kobe University, 27, 60 - 64, JapaneseProduction of useful compounds from L-lysine by the microbial enzyme
- 社団法人日本農芸化学会, 05 Mar. 1996, 日本農藝化學會誌, 70, 281 - 281, Japaneseアニリン誘導体の微生物代謝(第5報) : Pseudomonas sp. AP-3における2-アミノフエノールの代謝経路 : 微生物
- 第73回日本生物工学会 沖縄大会（2021）, Oct. 2021Aspergillus chevalieri 由来カルボキシエステラーゼ/リパー ゼの特性解析Oral presentation
- 第73回日本生物工学会 沖縄大会（2021）, Oct. 2021Aspergillus sydowii の生産する耐塩性γ-グルタミルトランス ペプチダーゼの特性解析
- 第73回日本生物工学会 沖縄大会（2021）, Oct. 2021好乾性糸状菌が生産する耐塩性セルラーゼの探索と諸性質 の検討Oral presentation
- 日本農芸化学会（2020・2021年度）, Mar. 2021, Japanese鰹節カビAspergillus 属糸状菌が生産する脂質分解酵素 の特性解析Oral presentation
- 第71回 日本生物工学会大会, Sep. 2019, Japanese, 岡山, International conference卵殻膜分解活性を示す微生物由来エラスターゼの特性解析と加水分解物の生理活性評価Oral presentation
- 第71回 日本生物工学会大会, Sep. 2019, Japanese, 岡山, International conference鰹節のかび付け工程で働く好乾性糸状菌の菌叢解析Oral presentation
- 第71回 日本生物工学会大会, Sep. 2019, Japanese, 岡山, International conference鰹節カビ Aspergillus 属糸状菌が生産する加水分解酵素の解析Oral presentation
- The final joint seminar of core to core program advanced researc networks, Dec. 2018, English, International conferenceCharacterization and mutation analysis of a halotolerant serine protease from Bacillus subtilis isolated from Thai traditional fermented shrimp paseteOral presentation
- The 56th Annual Meeting of the BSJ, Sep. 2018, English, Okayama, Domestic conferenceOrigin of the anomalous uphill energy gap in the light-harvesting reaction center from purple photosynthetic bacterium strain 970Oral presentation
- The 56th Annual Meeting of the BSJ, Sep. 2018, English, Okayama, Domestic conferenceLight-induced FTIR spectroscopic studies on quinone exchange mechanism of the LH1-RC complexes from native and chimeric purple bacteriaPoster presentation
- 第70回 日本生物工学会大会, Sep. 2018, Japanese, 関西大学, Domestic conferenceAspergillus glaucus MA0196由来ハイマンノース型アスパルティックプロテアーゼの遺伝子クローニングと異種発現Oral presentation
- 第26回光合成セミナー：反応中心と色素系の多様性, Jul. 2018, Japanese, 神戸, Domestic conference紅色光合成細菌Thiorhodovibrio Strain 970が示す異常なLH1 Qyレッドシフトの起源Oral presentation
- 第26回光合成セミナー：反応中心と色素系の多様性, Jul. 2018, Japanese, 神戸, Domestic conference好熱性および好塩性を示すHalorhodospira halochloris由来光捕集反応中心複合体の特性解析Poster presentation
- 第26回光合成セミナー：反応中心と色素系の多様性, Jul. 2018, Japanese, 神戸, Domestic conference光誘起FTIR分光法を用いた紅色細菌由来LH1-RC複合体におけるキノン分子の追跡Poster presentation
- 第65回 日本生化学会 近畿支部例会, May 2018, Japanese, 兵庫医科大学, Domestic conferenceAspergillus glaucus MA0196におけるアスパルティックプロテアーゼの生産特性と酵素の性質Oral presentation
- 日本農芸化学会2018年度大会, Mar. 2018, Japanese, Nagoya, Domestic conference卵殻膜分解活性を示す微生物由来エラスターゼの検索と特性解析Oral presentation
- 2018年度 日本農芸化学会本大会, Mar. 2018, Japanese, Domestic conference卵殻膜分解活性を示す微生物由来エラスターゼの検索と特性解析Oral presentation
- 2017年度生命科学系学会合同年次大会, Dec. 2017, Japanese, Domestic conference微生物由来エラスターゼを用いた卵殻膜の可溶化と生理活性ペプチドの調製Others
- 2017年度生命科学系学会合同年次大会, Dec. 2017, Japanese, Domestic conferenceタイ産家蚕由来の廃棄繭のセリシン成分の分離と利活用：Bacillus halodurans SE5由来アルカリセリンプロテアーゼの特性解析Poster presentation
- 2017年度生命科学系学会合同年次大会, Dec. 2017, Japanese, Domestic conferenceAspergillus glaucus MA0196由来ハイマンノース型アスパルティックプロテアーゼの分泌生産と酵素特性の解析Poster presentation
- 第69回 日本生物工学会大会, Sep. 2017, Japanese, Domestic conferenceタイの塩蔵発酵食品から分離した Bacillus 属細菌の生産する耐塩性プロテアーゼの特性解析Poster presentation
- 日本農芸化学会関西・中四国・西日本支部2017年度合同大阪大会, Sep. 2017, Japanese, International conferenceタイの塩蔵発酵食品から分離したBacillus 属細菌の生産する耐塩性プロテアーゼの遺伝子クローニングと特性解析Oral presentation
- 農芸化学会関西・中四国・西日本支部2017年度合同大阪大会, Sep. 2017, Japanese, Domestic conferenceタイの塩蔵発酵食品から分離したBacillus属細菌の生産する耐塩性プロテアーゼの遺伝子クローニングと特性解析Oral presentation
- 2017年度日本農芸化学会大会, Mar. 2017, Japanese, Domestic conferenceChryseobacterium sp. 5-3B由来N-アセチルトランスフェラーゼの基質認識部位の探索Oral presentation
- The 2nd Joint seminar Core to Core Program A., Nov. 2016, English, International conferenceCharacterization of Lipase from Thermotolerant Streptomyces thermoviolaceus Strain TCW.Poster presentation
- The 2nd Joint seminar Core to Core Program A. Advanced Research Networks on “Establishment of an international research core for new bio-research fields with microbes from tropical areas”,, Nov. 2016, English, Bangsaen Heritage Hotel, Chonburi, Thailand, International conferenceCharacterization of Lipase from Thermotolerant Streptomyces thermoviolaceus Strain TCWPoster presentation
- 日本農芸化学会 2016年度大会, Mar. 2016, Japanese, 北海道 札幌, Domestic conference卵殻膜分解酵素の異種発現と同酵素を用いた卵殻膜からの生理活性ペプチドの取得Poster presentation
- 日本農芸化学会 2016年度大会, Mar. 2016, Japanese, Domestic conferenceタイ土壌から分離した耐熱性Streptomyces属放線菌の生産するリパーゼの特性解析Poster presentation
- 生物工学会 2015年度 本大会, Oct. 2015, Japanese, Domestic conference細菌由来ジアミントランスアミナーゼ遺伝子のクローニングと発現酵素の特性解析Poster presentation
- 生物工学会 2015年度 本大会, Oct. 2015, Japanese, Domestic conferenceかつお節のかび付けに使用される Aspergillus 属糸状菌由来アスパルティックプロテアーゼの特性 解析Poster presentation
- 生物工学会 2015年度 本大会, Oct. 2015, Japanese, 鹿児島, Domestic conferenceChryseobacterium sp. 5-3B 株由来 N-アセチルトランスフェラーゼの部位特異的アミノ酸置換と機能 解析Poster presentation
- IC-STAR 2015 (The 1st International Conference on Science, Technology and Interdisciplinary Research ), Sep. 2015, English, Bandar Lampung, Lampung, Indonesia, International conferenceBacterial Enzymes with Special Characteristics for Biotechnological ApplicationsKeynote oral presentation
- The 6th international conference on fermentation technology for value added agricultural products, Jul. 2015, English, centara hotel & convention centre, Khon Kaen, Thailand, International conferenceCharacterization of the native form and the carboxy-terminally trauncated halotolerant form of alpha-amylases from Bacillus subtilis FP-133Oral presentation
- 日本食品科学工学会, 2015, Japanese, 京都, Domestic conference卵殻膜由来ペプチドによるによるグルコース取り込み促進効果についてPoster presentation
- 生物工学会, Sep. 2014, Japanese, Domestic conferenceハイマンノース型糖鎖を有するAspergillus glaucus MA0196由来アスパルティックプロテアーゼの特性解析Oral presentation
- 生物工学会, Sep. 2014, English, Domestic conferenceChryseobacterium sp. 5-3B 由来N- アセチルトランスフェラーゼの特性解析Oral presentation
- 日本農芸化学会, Mar. 2014, Japanese, Domestic conferenceBacillus subtilisFP-133由来耐塩性アミラーゼの特性解析Oral presentation
- The 19th annual meeting of Japan Society of Food Factors, 2014, Japanese, Domestic conference卵殻膜由来ペプチドは筋肉細胞のグルコース取り込みを上昇させるPoster presentation
- 神戸大学研究基盤センター 若手フロンティア研究会2014, 2014, Japanese, Domestic conference卵殻膜由来ペプチドによる血糖値上昇抑制効果Poster presentation
- BioMicroWorld 2013, Oct. 2013, English, Mardid, Spain, International conferencePoly-β-hydroxybutyrate (PHB) accumulation in Bradyrhizobium japonicum depends on proteins referred to as phasins.Oral presentation
- BioMicroWorld 2013, Oct. 2013, English, Mardid, Spain, International conferenceA Bacillus subtilis cell factory to produce syllo-inositol, a disease-modifying therapeutic agent for Alzheimer's disease.Oral presentation
- 第65回日本生物工学会大会, Sep. 2013, Japanese, 広島, Domestic conference枯草菌フィターゼ分泌の高度効率化Oral presentation
- 第65回日本生物工学会大会, Sep. 2013, Japanese, 広島, Domestic conference枯草菌によるシローイノシトール生産の効率化Oral presentation
- 2013年度グラム陽性菌ゲノム機能会議, Sep. 2013, Japanese, 筑波, Domestic conferenceシローイノシトールの高効率バイオコンバージョン法の確立Oral presentation
- 日本農芸化学会関西・中四国・西日本支部 日本ビタミン学会近畿・中国四国・九州沖縄地区 2013年度 合同広島大会, Sep. 2013, Japanese, 広島, Domestic conferenceOptimizing secretion of heterologous thermostable cellulases in Bacillus subtilis.Oral presentation
- 2013年度グラム陽性菌ゲノム機能会議, Sep. 2013, Japanese, 筑波, Domestic conferenceBacillus subtilis FP-133由来C末端領域欠損アミラーゼの特性解析Poster presentation
- 7th International Conference on Gram-positive Microorganisms, Jun. 2013, English, Dr. Marta Perego, Montecatini Terme, Tuscany, Italy, International conferenceEfficient bioconversion of myo-inositol to scyllo-inoshitol by genetically modified Bacillus suntilis requires global metabolic change to improve NADPH regeneration.Poster presentation
- 7th International Conference on Gram-positive Microorganisms, Jun. 2013, English, Dr. Marta Perego, Montecatini Terme, Tuscany, Italy, International conferenceBacillus subtilis iolH encodes an additional triosephosphate isomerase.Poster presentation
- 第7回日本ゲノム微生物学会年会, Mar. 2013, Japanese, 長浜, Domestic conferenceBradyrhizobium japonicumのPHB蓄積に関わるパラログ遺伝子の機能解析Oral presentation
- 日本農芸化学会関西・中四国・西日本支部および日本ビタミン学会近畿・中国四国・九州沖縄地区合同大会, 2013, Japanese, 広島, Domestic conferencePseudomonas aeruginosa ME-4由来エラスターゼを用いた卵殻膜の可溶化と生理活性ペプチドの探索Oral presentation
- 日本農芸化学会関西支部442回講演会 講演要旨集p.2, Dec. 2005, Japanese, 日本農芸化学会関西支部, 神戸大学, Domestic conference芳香族化合物の代謝に関与するcat 遺伝子の構成的発現機構の解析Oral presentation
- 日本農芸化学会関西支部442回講演会 講演要旨集p.4, Dec. 2005, Japanese, 日本農芸化学会関西支部, 神戸大学, Domestic conferenceアニリン誘導体の微生物代謝（第32報）2-ヒドロキシ-1,4-ベンゾキノンレダクターゼアイソザイムの精製と特性解析Oral presentation
- 442th Conference of Kansai Branch, Japan Society of Bioscience, Biotechnology, and Agrochemistry. Abs.p.3, Dec. 2005, English, 日本農芸化学会関西支部, 神戸大学, Domestic conferenceComparison of three halotolerant proteases from Bacillus subtilis strain FP-133Oral presentation
- JSPS-NRCT Microbial Resources Symposium of BioThailand abs.p.292, Nov. 2005, English, 日本学術振興会, タイ学術研究会議, 山口大学, カセサート大学, Bangkok, International conferenceThe mechanism of nitrogen Utilization by Mesophilic and Thermotolerant Bacteria that can utilize ammonium and nitrate salts simultaneouslyOral presentation
- 日本農芸化学会2005年度大会講演要旨集p.166, Mar. 2005, Japanese, 日本農芸化学会, 札幌, Domestic conference第四級アンモニウム塩の微生物分解（第2 報） Pseudomonas fluorescens 7-6 における n-dodecyltrimethylammonium chloride の分解特性と代謝経路Oral presentation
- 日本農芸化学会2005年度大会講演要旨集p.330, Mar. 2005, Japanese, 日本農芸化学会, 札幌, Domestic conference硝酸アンモニウム排水の微生物処理（第9報）耐熱性微生物におけるNO3-代謝に関与する酵素系の解析およびNO3-の好気的除去に与える環境因子の影響Oral presentation
- 日本農芸化学会2005年度大会講演要旨集p.85, Mar. 2005, Japanese, 日本農芸化学会, 札幌, Domestic conferenceアニリン誘導体の微生物代謝（第31報）2-ヒドロキシムコン酸 6-セミアルデヒド分解酵素系の精製と特性解析Oral presentation
- 日本農芸化学会2005年度大会講演要旨集p.226, Mar. 2005, Japanese, 日本農芸化学会, 札幌, Domestic conferenceRhodococcus sp. AN-22のコールドショックタンパク質をコードするcspB2 遺伝子のクローニングとプロモーター領域の解析Oral presentation
- Annual meeting of Japan Society of Bioscience, Biotechnology, and Agrochemistry abs.p.84, Mar. 2005, English, 未記入, 札幌, Domestic conferenceMicrobial metabolism of aniline derivatives XXX: Metabolism of 4-phenylenediamine by Bacillus cereus 10-L-2.Oral presentation
- Annual Meeting of Japan Society of Bioscience, Biotechnology, and Agrochemistry abs.p.37, Mar. 2005, English, 未記入, 札幌, Domestic conferenceHalotolerant intracellular potease from non-halophilic Bacillus subtilis FP-133Oral presentation
- 日本農芸化学会関西支部436回講演会 講演要旨集p1, Feb. 2005, Japanese, 日本農芸化学会関西支部, 京都大学, Domestic conferenceBacillus halodurans 38C-2-1のアルカリ性耐熱性アミラーゼ遺伝子のクローニングと発現Oral presentation
- 日本農芸化学会2005年度大会講演要旨集p.329, 2005, Japanese, 日本農芸化学会, 札幌, Domestic conference硝酸アンモニウム排水の微生物処理（第8報）中温菌を用いたNH4+およびNO3-の同時除去に与える金属イオンの影響Oral presentation
- Joint Conference 2005 of Kansai, Tyushikoku, and Nishinihon branches, Japan Society of Bioscience, Biotechnology, and Agrochemistry abs.p.42, 2005, English, 未記入, 未記入, Domestic conferencePurification and characterization of two novel halotolerant extracellular proteases from Bacillus subtilis strain FP-133.Oral presentation
- 日本農芸化学会2004年度大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conference硝酸アンモニウム排水の微生物処理（第7報）NH4+およびNO3-を同時に除去する耐熱性微生物を用いた両イオン除去に与える金属イオンの影響Oral presentation
- 日本農芸化学会2004年度大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conferenceメラニンの微生物分解（第３報）、毛髪メラニン分解酵素の精製と特性解析Oral presentation
- 日本農芸化学会関西支部437回講演会, 2004, Japanese, 日本農芸化学会関西支部, 神戸大学, Domestic conferenceアニリン誘導体の微生物代謝（第29報）Bacillus cereus PDa-1における2-フェニレンジアミン代謝酵素系の解析Oral presentation
- 日本農芸化学会2004年度大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conferenceアニリン誘導体の微生物代謝（第28報）2-アミノ-5-カルボキシムコン酸 6-セミアルデヒドデアミナーゼの反応機構の解析Oral presentation
- 日本農芸化学会2004年度大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conferenceアニリン誘導体の微生物代謝（第27報）2-ヒドロキシー1,4-ベンゾキノンレダクターゼの特性解析と活性発現の特性Oral presentation
- 日本農芸化学会2004年度大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conferenceアニリン誘導体の微生物代謝（第26報）Bacillus sereus PDa-1における2-フェニレンジアミン代謝経路の解析Oral presentation
- 日本農芸化学会2004年度大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conferenceアニリン資化性菌Rhodococcus sp. AN-22の構成的に発現するカテコール代謝系の解析：構成的に生合成されるムコノラクトンイソメラーゼの精製および特性解析Oral presentation
- 日本農芸化学会2004年度大会, 2004, Japanese, 日本農芸化学会, 広島, Domestic conferenceアニリン資化性菌Rhodococcus sp. AN-22のコールドショックタンパク質をコードする遺伝子のクローニングOral presentation
- the 4th JSPS-NRCT joint seminar on development of thermotelerant microbial resources and their applications, 2004, English, 未記入, 未記入, International conferenceMetabolism of azo dyes by Lactobacillus casei TISTR 1500 and effects of various factors on decolorization.Oral presentation
- 10th International symposium on microbial ecology ISME-10 microbial planet: sub-surface to space., 2004, English, 未記入, 未記入, International conferenceDistribution and ecological significance of novel bacteria that can utilize ammonium and nitrate salts sumultaneously with heterotrophic nitrification and aerobic denitrification.Oral presentation
- the 4th JSPS-NRCT joint seminar on development of thermotelerant microbial resources and their applications, 2004, English, 未記入, 未記入, International conferenceCulture conditions of mesophilic Klebsiella pneumoniae and thermotolerant Bacillus licheniformis that can utiliza ammonium and nitrate salts simultaneously.Oral presentation
- the 4th JSPS-NRCT joint seminar on development of thermotelerant microbial resources and their applications, 2004, English, 未記入, 未記入, International conferenceCloning and sequencing analysis of a gene encoding alkaline and thermotolerant amylase from Bacillus halodurans 38C-2-1.Oral presentation
- 日本農芸化学会2003年度大会講演要旨集, Apr. 2003, Japanese, 日本農芸化学会, 日本大学湘南キャンパス, Domestic conference有用オリゴ糖の微生物生産（第2 報）キシロオリゴ糖生産のための培養条件の検討とキシロオリゴ糖高生産性変異株の取得Oral presentation
- 日本農芸化学会2003年度大会講演要旨集, Apr. 2003, Japanese, 日本農芸化学会, 日本大学湘南キャンパス, Domestic conference硝酸アンモニウム排水の微生物処理（第5報）NH4+およびNO3− を同時に除去する耐熱性微生物の分離と既報株を用いた両イオン除去に与える炭素源の影響Oral presentation
- 日本農芸化学会2003年度大会講演要旨集, Apr. 2003, Japanese, 日本農芸化学会, 日本大学湘南キャンパス, Domestic conferenceベンズアミド資化性菌Arthrobacter sp. BA-5-17 のムコン酸サイクロイソメラーゼ遺伝子のクローニングOral presentation
- 日本農芸化学会2003年度大会講演要旨集, Apr. 2003, Japanese, 日本農芸化学会, 日本大学湘南キャンパス, Domestic conferenceアニリン誘導体の微生物代謝（第24報）4-アミノフェノール資化性菌の分離と代謝関連酵素系の解析Oral presentation
- 日本農芸化学会2003年度大会講演要旨集, Apr. 2003, Japanese, 日本農芸化学会, 日本大学湘南キャンパス, Domestic conferenceアニリン誘導体の微生物代謝 （第23 報） フェニレンジアミン分解菌の分離Oral presentation
- 日本農芸化学会2003年度大会講演要旨集, Apr. 2003, Japanese, 日本農芸化学会, 日本大学湘南キャンパス, Domestic conferenceアニリン誘導体の微生物代謝（第22報）2-アミノ-5-カルボキシムコン酸 6-セミアルデヒド分解酵素の特性解析Oral presentation
- 日本農芸化学会2003年度大会講演要旨集, Apr. 2003, Japanese, 日本農芸化学会, 日本大学湘南キャンパス, Domestic conferenceアニリン誘導体の微生物代謝（第21報）Bordetella sp. 10d の4-アミノ-3-ヒドロキシ安息香酸2,3-ジオキシゲナーゼをコードする遺伝子のクローニングOral presentation
- 日本農芸化学会2003年度大会講演要旨集, Apr. 2003, Japanese, 日本農芸化学会, 日本大学湘南キャンパス, Domestic conferenceRhodococcus sp. AN-22 におけるアニリンジオキシゲナーゼ遺伝子の発現特性の解析Oral presentation
- 日本農芸化学会関西支部432回講演会、講演要旨集, 2003, Japanese, 日本農芸化学会関西支部, 未記入, Domestic conference硝酸アンモニウム排水の微生物処理（第6報）NH4+およびNO3− を同時に除去する微生物の検索Oral presentation
- 日本農芸化学会関西支部428回講演会、講演要旨集, 2003, Japanese, 日本農芸化学会関西支部, 未記入, Domestic conferenceアニリン誘導体の微生物代謝（第25 報）4-アミノフェノールの代謝に関与する酵素の精製と特性解析Oral presentation
■ Research Themes
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 神戸大学, 01 Apr. 2023 - 31 Mar. 2026好乾性糸状菌由来ペプチダーゼによる鰹節構成タンパク質の分解メカニズムの解明
- 公益財団法人 高橋産業経済研究財団, Apr. 2025 - Mar. 2026, Principal investigator麹菌による鰹節だしがらの新しい調味料の開発：発酵プロセスの最適化と製品評価
- 公益財団法人 伊藤記念財団, Apr. 2024 - Mar. 2025だしがら発酵物由来酵素カクテルを利用した食肉加工技術の開発
- 一般財団法人 東洋水産財団, Apr. 2022 - Mar. 2023鰹節の発酵熟成に関わるタンパク質分解酵素・遺伝子群の同定と特性解析
- 神戸大学, 神戸大学イノベーションファンドプログラム, 神戸大学, Apr. 2022 - Mar. 2023好乾性糸状菌による麦芽発酵エキスの調製と機能性評価
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, Apr. 2020 - Mar. 2023Characterization of extracellular lipases produced by xerophilic molds鰹節のかび付け発酵工程において、脂質分解酵素は、節に含まれる脂質の油焼けの防止（保存性向上）だけでなく、削り節から澄んだだしの素を得ることにも寄与する鍵酵素である。先行研究によって、鰹節カビが種々の脂質分解酵素を分泌生産することが明らかとなっているが、同定されている酵素群の触媒化学的諸性質や低水分活性下での活性発現や安定性についてはいまだ不明である。Aspergillus chevalieriおよびA. pseudoglaucus由来8種類のリパーゼ/カルボキシエステラーゼについて低水分活性環境下での脂質分解特性を明らかにするために、同脂質分解酵素の異種発現と特性解析を目的とした。 親株2種から8種の遺伝子をクローニングし、酵母および大腸菌の発現系にて組換え酵素の取得を試みた。既報の方法を参考に、形質転換株の培養および発現誘導を行い、得られた組換え酵素 (粗酵素) の加水分解活性を調べた。現段階では、rcLip 3以外の5種の組換え酵素の異種発現が完了した。これらの組換え酵素を精製し、p-nitrophenyl (pNP) エステルに対する加水分解特性を調べた結果、pNP -C2や-C4といった短鎖基質を好んで高い加水分解活性を示すグループ（3種）とpNP-C6～-C12等の中長鎖基質に対して幅広く高い加水分解活性を示すグループ（2種）に大別できた。つづいて、酵素反応系の水分活性を変化させた条件で活性に与える水分活性値の影響を調べた結果、2種の組換え酵素は、水分活性が低下するとともに加水分解活性は増大した。鰹肉由来脂質の分解特性を今後調べる必要があるが、見出した脂質分解酵素の中から低水分活性環境下で脂質分解に寄与するリパーゼ/カルボキシエステラーゼを見出すことができた。
- 公益財団法人 伊藤記念財団, Apr. 2021 - Mar. 2022, Principal investigator微生物由来アスパルティックプロテアーゼを利用した食肉加工技術の開発（III）
- 公益財団法人 伊藤記念財団, 一般研究助成, Apr. 2020 - Mar. 2021, Principal investigator微生物由来アスパルティックプロテアーゼを利用した食肉加工技術の開発（II）
- KIEIKAI RESEARCH FOUNDATION, 特別助成研究, Apr. 2020 - Mar. 2021, Principal investigatorHydrolysis of eggshell membrane by microbial elastase and characterization of produced bioactive peptides
- 公益財団法人伊藤記念, 研究助成, Apr. 2019 - Mar. 2020, Principal investigator微生物由来アスパルティックプロテアーゼを利用した食肉加工技術の開発Competitive research funding
- 一般財団法人 東和食品研究振興会, 学術奨励金, Apr. 2019 - Mar. 2020, Principal investigator鰹節の発酵熟成に関わる菌体外加水分解酵素の発現特性の解明Competitive research funding
- 公益財団法人 発酵研究所, 一般研究助成, Apr. 2018 - Mar. 2020, Principal investigatorかつお節かび付け工程で働く好乾性糸状菌の分類と菌叢解析に関する研究Competitive research funding
- 公益財団法人 ひょうご科学技術協会, 学術研究助成, Apr. 2018 - Mar. 2019, Principal investigator脂肪族アミン N- アセチルトランスフェラーゼの構造解析 に基づいた機能改変Competitive research funding
- 科学研究費補助金／基盤研究(B), Apr. 2015 - Mar. 2018Competitive research funding
- 学術研究助成基金助成金／基盤研究(C), Apr. 2014 - Mar. 2017, Principal investigatorCompetitive research funding
- 国立研究開発法人科学技術振興機構, 日本・アジア青少年サイエンス交流事業（さくらサイエンスプラン）, 2017, Principal investigatorタイの土壌より単離した耐熱性微生物とその酵素系の特性解析Competitive research funding
- 一般財団法人 旗影会, 研究助成＜特別助成＞, Apr. 2015 - Mar. 2016, Principal investigator卵殻膜由来生理活性ペプチドの効率的生産を目指した 卵殻膜分解酵素の大量発現Competitive research funding
- 一般財団法人 旗影会, 研究助成＜特別助成＞, Apr. 2012 - Mar. 2013, Principal investigator微生物由来加水分解酵素を用いた卵殻膜分解と 分解物中の生理活性ペプチドの検索Competitive research funding
- 科学研究費補助金／基盤研究(C), 2010, Principal investigatorCompetitive research funding
- 科学研究費補助金／基盤研究(C), 2007Competitive research funding
- 科学研究費補助金／若手研究(B), 2006, Principal investigatorCompetitive research funding
- 科学研究費補助金／若手研究(B), 2005, Principal investigatorCompetitive research funding
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- 脱色用プロテアーゼを用いる着色成分の脱色方法特願2009-032067, 13 Feb. 2009, 大学長, 特開2009-219483, 01 Oct. 2009, 特許5515017, 11 Apr. 2014Patent right
- ジオトリカム属菌を用いたキチン・キトサンを含む多糖体含有物の製造方法特願2006-308664, 15 Nov. 2006, 大学長, 特開2007-159569, 28 Jun. 2007, 特許5131676, 16 Nov. 2012Patent right
- 微生物及び微生物由来酵素によるアニリン誘導体のアセチル化特願2006-511293, 23 Mar. 2005, ＴＬＯ, 特許4812619, 02 Sep. 2011Patent right
- 微生物を利用してNH4+とNO3−とを同時除去する硝化・脱窒方法特願2003-275878, 17 Jul. 2003, 財団法人新産業創造研究機構, 特開2005-034783, 10 Feb. 2005, 特許第4631082号, 26 Nov. 2010Patent right
- ジオトリカム属菌を用いた発酵おから等の製造方法特願2006-013835, 23 Jan. 2006, 大学長, 特開2007-190001, 02 Aug. 2007, 特許4539867, 02 Jul. 2010Patent right
- 微生物及び微生物由来酵素によるアニリン誘導体のアセチル化JP2005005195, 23 Mar. 2005, 財団法人新産業創造研究機構, WO2005-090588, 29 Sep. 2005Patent right