期刊
ENGINEERING IN LIFE SCIENCES
卷 17, 期 9, 页码 989-996出版社
WILEY
DOI: 10.1002/elsc.201600232
关键词
Bi-functional enzyme; Formate dehydrogenase; L-tert leucine; Leucine dehydrogenase; Peptide linker
资金
- National Natural Science Foundation of China [41176111, 41306124, 21336009]
- Foundation of South Oceanographic Research Center of China in Xiamen [14GYY011NF11]
- Fundamental Research Funds for the Central Universities [20720170033]
- Public science and technology research funds projects of ocean [201505032-6]
This study reports the application of peptide linker in the construction of bifunctional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L-tert leucine (L-tle) biotransformation. Seven FDH-LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzymeactivities. The incorporation order of FDH and LeuDH was investigated by predicting three-dimensional structures of LeuDH-FDH and FDH-LeuDH models using the I-TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N-, C-terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling.
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