Zhang Jian-Fen, Chen Wei-Qing, Chen Hong
College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou, 310015, China.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China.
World J Microbiol Biotechnol. 2017 Feb;33(2):21. doi: 10.1007/s11274-016-2187-0. Epub 2017 Jan 2.
In this study, we report the cloning and expression of a functional glucoside 3-dehydrogenase (G3DH) gene from Sphingobacterium faecium ZJF-D6. This gene is 1686 bp in length and encodes a peptide of 562 amino acids. The G3DH gene was successfully expressed in E. coli, and the recombinant enzyme could oxidize glucosides, galactosides and analogues at C-3 position. The sequence and multiple alignment analysis showed that the enzyme has highest identity with G3DHs from Paraglaciecola polaris LMG 21857, Aliiglaciecola lipolytica E3 and Halomonas sp. alpha-15. The recombinant G3DH was purified on Ni-NTA column and exhibited the highest activity at pH 7.6 and 30 °C. It was sensitive to acid and alkali, and showed well thermostability. The SfG3DH could oxidize a wild range of sugars. When recombinant E. coli BL21 cells were used as catalyst, a high rate of conversion to N-p-nitrophenyl-3-ketovalidamine was achieved, and no p-nitroaniline was detected. This process offers a promising approach to fulfill substrate of 3-ketovalidoxylamine A C-N lyase production.
在本研究中，我们报道了从屎鞘氨醇杆菌ZJF-D6克隆并表达功能性葡糖苷3-脱氢酶（G3DH）基因。该基因长度为1686 bp，编码一个由562个氨基酸组成的肽段。G3DH基因在大肠杆菌中成功表达，重组酶能够氧化C-3位的葡糖苷、半乳糖苷及其类似物。序列和多序列比对分析表明，该酶与极地副嗜冷杆菌LMG 21857、解脂嗜冷栖芽孢杆菌E3和嗜盐单胞菌属alpha-15的G3DH具有最高的同源性。重组G3DH在Ni-NTA柱上进行纯化，在pH 7.6和30 °C时表现出最高活性。它对酸碱敏感，但具有良好的热稳定性。屎鞘氨醇杆菌G3DH能够氧化多种糖类。当使用重组大肠杆菌BL21细胞作为催化剂时，可实现向N-对硝基苯基-3-酮缬氨霉素的高转化率，且未检测到对硝基苯胺。该过程为实现3-酮基缬氨氧基胺A C-N裂解酶生产的底物提供了一种有前景的方法。
