Improving D-carbamoylase thermostability through salt bridge engineering for efficient D-p-hydroxyphenylglycine production

D -p -hydroxyphenylglycine (D-HPG) is an important intermediate in the pharmaceutical industry, and it is commonly synthesized by cascading D-hydantoinase (DHase) and D-carbamoylase (DCase). In this study, the stability of DCase was identified as the main problem that limits its application. Therefore, the complexed structure of Ak DCase (DCase from the Agrobacterium sp . strain KNK712) with the substrate N-carbamoyl-D- p -hydroxyphenylglycine (C p HPG) (with 2.52 Å resolution) and catalytic mechanism were resolved. Based on the catalytic mechanism and electrostatic stabilization, salt bridge engineering was adopted to improve Ak DCase thermostability. The best variant, Ak DCase D30A , increased the T m by 2.91 °C and half-life (t 1/2 ) at 40 and 60 °C by 18.43 h and 23.21 min, respectively. After Ak DCase D30A was assembled with Gs DHase (DHase from Geobacillus stearothermophilus SD-1) in a single Escherichia coli cell, the recombinant strain could produce 29.53 g/L D-HPG within 12 h, with a 97% conversion and a 2.46 g/(L·h) space–time yield (STY). The titer of D-HPG increased by 40.55% compared to the E. coli cell harboring pETduet-1- Ak DCase- Gs DHase. The recombinant strain could be used for two cycles. Our research provides a basis for the industrial production of D-HPG.