Engineering amino acid racemase for DL-p-Hydroxyphenyglycine synthesis

• 1、School of Life Sciences and Health Engineering,Jiangnan University,Wuxi 214122
• 2、Key Laboratory of Industrail Biotechnology of Ministry of Education School of Biotechnology,Jiangnan University,Wuxi,214122

Abstract：D-Hydroxyphenylglycine (D-HPG) is an important raw material for the synthesis of broad-spectrum antibiotics (e.g., penicillin antibiotics, cephalosporin antibiotics) and is in great demand in the market. The preparation of D-HPG by using the racemic splitting of L-p-hydroxyphenylglycine (L-HPG) via a biocatalytic route has a promising application. However, the currently reported amino acid racemization enzymes generally have low or no racemization activity against bulky aromatic sEngineering amino acid racemase for DL-p-Hydroxyphenyglycine synthesisubstrates. To overcome this limitation, the amino acid racemase from Lactobacillus buchneri (LbAAR) was protein engineered in this study. Initial catalytic activity for HPG was obtained by eliminating the spatial site-blocking problem. The proton transfer efficiency was further enhanced by replacing the catalytic residues. The final screen yielded the double mutant L307S/D222H, whose activity was increased to 30.8 μmol/min/g, which was significantly better than that of the wild-type enzyme. The mutant efficiently completed the racemization of 15 g/L L-HPG in 2 h, which is the highest reported amino acid racemization efficiency for aromatic substrates. The present results provide new ideas and potential industrial applications for the efficient biosynthesis of D-amino acids.

Keywords： protein engineering DL-p-hydroxyphenylglycine amino acid racemase biocatalysis