TY - JOUR
T1 - Combing multiple site-directed mutagenesis of penicillin G acylase from Achromobacter xylosoxidans PX02 with improved catalytic properties for cefamandole synthesis
AU - Li, Anni
AU - Cheng, Cheng
AU - Qi, Weimin
AU - Pan, Xin
AU - Xu, Xuanping
AU - Wang, Xinyu
AU - Wu, Chunying
AU - Chu, Jianlin
AU - He, Bingfang
N1 - Publisher Copyright:
© 2021
PY - 2021/4/1
Y1 - 2021/4/1
N2 - Penicillin G acylase (PGA) was an important biocatalyst for enzymatic production of second-generation cephalosporin. PGA from Achromobacter xylosoxidans PX02 (AxPGA) showed relatively lower identity to EcPGA (54.9% in α subunit and 51.7% in β subunit), which could synthesize cefamandole in the kinetically controlled N-acylation (kcNa). Semi-rational design of AxPGA and “small and smart” mutant libraries were developed with minimal screening to improve cefamandole production. A triple mutant αR141A/αF142I/βF24G by combining the mutational sites (βF24, αR141, and αF142) from different subunits of AxPGA showed better performance in cefamandole production, with 4.2-fold of improvement in the (kcat/Km)AD value for activated acyl donor (R)-Methyl mandelate. Meanwhile, the (kcat/Km)Ps value for cefamandole by mutant αR141A/αF142I/βF24G was sharply dropped by 25.5 times, indicating its highly synthetic activity and extremely low hydrolysis of cefamandole. Strikingly, the triple mutant αR141A/αF142I/βF24G could form cefamandole with a yield of 85% at an economical substrate ratio (acyl donor/nucleophile) of 1.3:1 (82% at 1.1:1), which advanced the greener and more sustainable process of cefamandole production than the wild type. Furtherly, the improved synthetic ability and lower hydrolysis of cefamandole by mutant were rationalized using molecular docking.
AB - Penicillin G acylase (PGA) was an important biocatalyst for enzymatic production of second-generation cephalosporin. PGA from Achromobacter xylosoxidans PX02 (AxPGA) showed relatively lower identity to EcPGA (54.9% in α subunit and 51.7% in β subunit), which could synthesize cefamandole in the kinetically controlled N-acylation (kcNa). Semi-rational design of AxPGA and “small and smart” mutant libraries were developed with minimal screening to improve cefamandole production. A triple mutant αR141A/αF142I/βF24G by combining the mutational sites (βF24, αR141, and αF142) from different subunits of AxPGA showed better performance in cefamandole production, with 4.2-fold of improvement in the (kcat/Km)AD value for activated acyl donor (R)-Methyl mandelate. Meanwhile, the (kcat/Km)Ps value for cefamandole by mutant αR141A/αF142I/βF24G was sharply dropped by 25.5 times, indicating its highly synthetic activity and extremely low hydrolysis of cefamandole. Strikingly, the triple mutant αR141A/αF142I/βF24G could form cefamandole with a yield of 85% at an economical substrate ratio (acyl donor/nucleophile) of 1.3:1 (82% at 1.1:1), which advanced the greener and more sustainable process of cefamandole production than the wild type. Furtherly, the improved synthetic ability and lower hydrolysis of cefamandole by mutant were rationalized using molecular docking.
KW - Cefamandole
KW - Penicillin G acylase
KW - Protein engineering
UR - http://www.scopus.com/inward/record.url?scp=85100625471&partnerID=8YFLogxK
U2 - 10.1016/j.ijbiomac.2021.01.194
DO - 10.1016/j.ijbiomac.2021.01.194
M3 - 文章
C2 - 33549660
AN - SCOPUS:85100625471
SN - 0141-8130
VL - 175
SP - 322
EP - 329
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
ER -