Pongtharangkul Thunyarat, Chuekitkumchorn Pattra, Suwanampa Nhuengtida, Payongsri Panwajee, Honda Kohsuke, Panbangred Watanalai
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.
Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan.
AMB Express. 2015 Dec;5(1):68. doi: 10.1186/s13568-015-0157-9. Epub 2015 Nov 4.
Glucose dehydrogenases (GluDH) from Bacillus species offer several advantages over other NAD(P)H regeneration systems including high stability, inexpensive substrate, thermodynamically favorable reaction and flexibility to regenerate both NADH and NADPH. In this research, characteristics of GluDH from Bacillus amyloliquefaciens SB5 (GluDH-BA) was reported for the first time. Despite a highly similar amino acid sequence when comparing with GluDH from Bacillus subtilis (GluDH-BS), GluDH-BA exhibited significantly higher specific activity (4.7-fold) and stability when pH was higher than 6. While an optimum activity of GluDH-BA was observed at a temperature of 50 °C, the enzyme was stable only up to 42 °C. GluDH-BA exhibited an extreme tolerance towards n-hexane and its respective alcohols. The productivity of GluDH obtained in this study (8.42 mg-GluDH/g-wet cells; 1035 U/g-wet cells) was among the highest productivity reported for recombinant E. coli. With its low K M-value towards glucose (5.5 mM) and NADP(+) (0.05 mM), GluDH-BA was highly suitable for in vivo applications. In this work, a recombinant solvent-tolerant B. subtilis BA overexpressing GluDH-BA was developed and evaluated by coupling with B. subtilis overexpressing an enzyme P450 BM3 F87V for a whole-cell hydroxylation of n-hexane. Significantly higher products obtained clearly proved that B. subtilis BA was an effective cofactor regenerator, a valuable asset for bioproduction of value-added chemicals.
与其他NAD(P)H再生系统相比，芽孢杆菌属的葡萄糖脱氢酶（GluDH）具有多种优势，包括高稳定性、廉价底物、热力学有利反应以及再生NADH和NADPH的灵活性。在本研究中，首次报道了解淀粉芽孢杆菌SB5的GluDH（GluDH-BA）的特性。尽管与枯草芽孢杆菌的GluDH（GluDH-BS）相比，其氨基酸序列高度相似，但当pH高于6时，GluDH-BA表现出显著更高的比活性（4.7倍）和稳定性。虽然GluDH-BA在50°C的温度下观察到最佳活性，但该酶仅在42°C以下稳定。GluDH-BA对正己烷及其相应的醇类表现出极强的耐受性。本研究中获得的GluDH的产量（8.42 mg-GluDH/g湿细胞；1035 U/g湿细胞）是重组大肠杆菌报道的最高产量之一。由于其对葡萄糖（5.5 mM）和NADP(+)（0.05 mM）的低K M值，GluDH-BA非常适合体内应用。在这项工作中，构建了一种过表达GluDH-BA的重组耐溶剂枯草芽孢杆菌BA，并通过与过表达酶P450 BM3 F87V的枯草芽孢杆菌偶联，用于正己烷的全细胞羟基化反应，对其进行了评估。获得的显著更高的产物清楚地证明了枯草芽孢杆菌BA是一种有效的辅因子再生剂，是生物生产增值化学品的宝贵资产。
