High‐cell‐density fermentation of Escherichia coli for expression of a recombinant phenylalanine dehydrogenase mutant and its purification

Abstract BACKGROUND Phenylalanine dehydrogenase (PheDH; l ‐phenylalanine: NAD + oxidoreductase, deaminating (EC 1.4.1.20)) is widely used in the pharmaceutical industry. It is the main biocatalyst in the enantioselective synthesis of l ‐phenylalanine, related l ‐amino acids as well as some non‐natural amino acids which are important pharmaceutical intermediates. However, growing demands for PheDH and its limited production result in the shortage of the enzyme's supply so that it is necessary to explore and establish an industrial production process for PheDH. RESULTS In this study, the high‐cell‐density fermentation of recombinant Escherichia coli for expression of an engineered mutant R272M/E331Q/E196N of PheDH was investigated, which is a key catalyst with improved catalytic capability during the important intermediate production of saxagliptin. Cultivation media and induction conditions of recombinant E. coli were tested in shake flask experiments. A fed‐batch fermentation strategy using MR2 as cultivation medium and lactose as inducer was developed in a 30 L fermenter. After 24 h of cultivation, the dry cell weight reached 52.32 g L –1 (based on OD 600 ), and the specific activity of crude extracts was 0.49 U mg –1 . By applying the aqueous two‐phase system strategy, a purified PheDH productivity of 191.96 mg·L −1 ·h −1 was obtained. The specific activity, recovery, yield and purification fold of PheDH were 3.21 U mg –1 , 104.19%, 84.53% and 6.55, respectively. CONCLUSIONS This study shows that the application of high‐cell‐density fermentation in conjunction with aqueous two‐phase system strategy provides vital support for large‐scale production of recombinant PheDH. © 2020 Society of Chemical Industry (SCI)