College of Chemistry and Materials Science, Key Laboratory of Chemical Biology of Hebei Province, Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, Institute of Life Science and Green Development, Hebei University, Baoding, 071002, China.
College of Life Science, Microbial Technology Innovation Center for Feed of Hebei Province, Hebei Agricultural University, Baoding, 071001, China.
Appl Biochem Biotechnol. 2023 Dec;195(12):7553-7567. doi: 10.1007/s12010-023-04432-x. Epub 2023 Apr 4.
An efficient cofactor regeneration system has been developed to provide a hydride source for the preparation of optically pure alcohols by carbonyl reductase-catalyzed asymmetric reduction. This system employed a novel glucose dehydrogenase (BcGDH90) from Bacillus cereus HBL-AI. The gene encoding BcGDH90 was found through the genome-wide functional annotation. Homology-built model study revealed that BcGDH90 was a homo-tetramer, and each subunit was composed of βD-αE-αF-αG-βG motif, which was responsible for substrate binding and tetramer formation. The gene of BcGDH90 was cloned and expressed in Escherichia coli. The recombinant BcGDH90 exhibited maximum activity of 45.3 U/mg at pH 9.0 and 40 °C. BcGDH90 showed high stability in a wide pH range of 4.0-10.0 and was stable after the incubation at 55 °C for 5 h. BcGDH90 was not a metal ion-dependent enzyme, but Zn could seriously inhibit its activity. BcGDH90 displayed excellent tolerance to 90% of acetone, methanol, ethanol, n-propanol, and isopropanol. Furthermore, BcGDH90 was applied to regenerate NADPH for the asymmetric biosynthesis of (S)-(+)-1-phenyl-1,2-ethanediol ((S)-PED) from hydroxyacetophenone (2-HAP) with high concentration, which increased the final efficiency by 59.4%. These results suggest that BcGDH90 is potentially useful for coenzyme regeneration in the biological reduction.
开发了一种有效的辅因子再生系统，为通过羰基还原酶催化的不对称还原制备光学纯醇提供氢供体。该系统采用了来自蜡状芽孢杆菌 HBL-AI 的新型葡萄糖脱氢酶（BcGDH90）。通过全基因组功能注释发现了编码 BcGDH90 的基因。同源建模研究表明，BcGDH90 是一个同四聚体，每个亚基由βD-αE-αF-αG-βG 基序组成，该基序负责底物结合和四聚体形成。BcGDH90 的基因在大肠杆菌中克隆和表达。重组 BcGDH90 在 pH 9.0 和 40°C 时表现出最大活性 45.3 U/mg。BcGDH90 在 pH 4.0-10.0 的较宽范围内具有高稳定性，并且在 55°C 孵育 5 小时后仍然稳定。BcGDH90 不是金属离子依赖性酶，但 Zn 可以严重抑制其活性。BcGDH90 对 90%的丙酮、甲醇、乙醇、正丙醇和异丙醇具有出色的耐受性。此外，BcGDH90 被应用于再生 NADPH，用于从高浓度的对羟基苯乙酮（2-HAP）不对称生物合成（S）-（+）-1-苯基-1,2-乙二醇（（S）-PED），最终效率提高了 59.4%。这些结果表明，BcGDH90 在生物还原中辅酶再生方面具有潜在的应用价值。
