文献类型：期刊文献

英文题名：Spatially programmed assembling of oxidoreductases with single-stranded DNA for cofactor-required reactions

作者：Wang, Tianwen David[1];Ma, Fei[1];Ma, Xingyuan[1];Wang, Ping[1,2]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, BioMed Nanotechnol Ctr, Sch Biotechnol,NanoBioEngn Lab, Shanghai 200237, Peoples R China;[2]Univ Minnesota, Inst Biotechnol, Dept Bioprod & Biosyst Engn, St Paul, MN 55108 USA

年份：2015

卷号：99

期号：8

起止页码：3469

外文期刊名：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

收录：;EI(收录号：20144500163618);WOS:【SCI-EXPANDED(收录号:WOS:000351743600013)】；

基金：This research was financially supported by grants of the Natural Science Foundation of China (31300660), the National Science Research Project "Creation of significantly important new drugs" in the Eleventh Five-year Plan (2009ZX09103-693), the project of Chinese Education Ministry (20120074120027), and by Shanghai Pujiang Program (13PJD012) and partially supported by Open Funding Project of the State Key Laboratory of Bioreactor Engineering. Special thanks go to research technician Li Zhou for her generous assistance in HPLC analysis.

语种：英文

外文关键词：Coupled oxidoreductive reaction; Cofactor recycling; Spatially assembled enzymes; Single-stranded DNA; Substrate channeling; Proximity effect

摘要：Cofactor is especially important for biotransformation catalyzed by oxidoreductases. Many attempts in enhancing performance of the reactions by improving cofactor utilization have been reported. In this study, efficiency of cofactor-requiring biocatalysis was enhanced by improving cofactor recycling via spatially programmed assembling glycerol dehydrogenase (GlyDH, Escherichia coli MG1655) and glutamate dehydrogenase (GluDH, Bacillus subtilis str168), with the aid of single-stranded DNA (ssDNA). The two enzymes were first independently expressed as molecules fused with a phage protein A* that could covalently link ssDNA with certain features. After an enzymatic cross-linking reaction taking place under mild conditions, the conjugate of fused enzyme and ssDNA was assembled into desired structures through base pairing enabled by the ssDNA. Results showed that, to some extent, the fusion with protein A* could improve the specific activity of the enzymes (GlyDH-A*/GlyDH = 116.0 %; GluDH-A*/GluDH = 105.2 %). Additionally, in the coupled reaction system with glycerol and alpha-ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency of cofactor utilization was significantly enhanced (by 23.8- to 41.9-folds), indicating the existence of a substrate-channeling mechanism for cofactor utilization in the assembled reaction system due to the proximity effects. The degree of substrate channeling was calculated as from 1.65 to 1.73. Furthermore, the efficiency of cofactor utilization was influenced in an architecture-dependent manner when complexes with different stoichiometry of GlyDH and GluDH were utilized in biotransformation. This study demonstrated a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions.