College of Life Sciences, Hebei Normal University, No. 20, Road East of 2nd Ring South, Yuhua District, Shijiazhuang, 050024, China
Hebei Brant Pharmaceutical Co., LTD, Mayu Industrial Park, Jinzhou, 052260, China
Beijing Hongyisifang Radiation Technology Co., LTD, Beijing, 101113, China
Due to the codon usage and high G+C contentof thetrans-4-proline-L-hydroxylase gene from the Dactylosporangium sp.strain RH1, the whole gene was optimized and cloned into several vectors for expression. In biotransformations with resting cells, the activity of the enzyme was investigated. The in-house modified plasmid pet-M-3C was found to yield the highest enzymatic activity. Additionally, after the primary fragment screening, the conversion efficiency of fragment 1-257 aa was enhanced from 76.60% to 88.97% compared with the full-length proline 4-hydroxylase within 60 h; we also found that truncation of the gene improved the solubility of the encoded protein.After optimizing the various induction conditions with respect to the enzymatic activity of the engineered strain, including the concentration of the inducer IPTG, the cell density before the inducer was added, the induction temperature and the induction time, the conversion efficiency was more than 97%within 48 h.