An ecient procedure for the production of trans-4-hydroxy-L-proline using recombinantly expressed proline hydroxylase

Abstract. Due to the codon usage and high G+C content of the trans-4-proline-Lhydroxylase 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 modied plasmid pET-M-3C was found to yield the highest enzymatic activity. Additionally, after the primary fragment screening, the conversion eciency 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 various induction conditions with respect to the enzymatic activity of the engineered strain, the conversion eciency was more than 97% within 48 h.