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Efficient synthesis of β-lactam antibiotics with very low product hydrolysis by a mutant Providencia rettgeri penicillin G acylase

Author:
Pan, Xin, Wang, Li, Ye, Jiajie, Qin, Song, He, Bingfang
Source:
Applied microbiology and biotechnology 2018 v.102 no.4 pp. 1749-1758
ISSN:
0175-7598
Subject:
Providencia rettgeri, amoxicillin, cefadroxil, hydrolysis, industrial applications, kinetics, molecular models, mutants, penicillin amidase, site-directed mutagenesis
Abstract:
Penicillin G acylase (PGA) was isolated from Providencia rettgeri PX04 (PrPGApx04) and utilized for the kinetically controlled synthesis of β-lactam antibiotics. Site-directed mutagenesis was performed to increase the process efficiency. Molecular docking was carried out to speculate the key mutant positions corresponding with synthetic activity, which resulted in the achievement of an efficient mutant, βF24G. It yielded higher conversions than the wild-type enzyme in the synthesis of amoxicillin (95 versus 17.2%) and cefadroxil (95.4 versus 43.2%). The reaction time for achieving the maximum conversion decreased from 14 to 16 h to 2–2.5 h. Furthermore, the secondary hydrolysis of produced antibiotics was hardly observed. Kinetic analysis showed that the (kcₐₜ/Kₘ)AD value for the activated acyl donor D-hydroxyphenylglycine methyl ester (D-HPGME) increased up to 41 times. In contrast, the (kcₐₜ/Kₘ)Pₛ values for the products amoxicillin and cefadroxil decreased 6.5 and 21 times, respectively. Consequently, the α value (kcₐₜ/Kₘ)Pₛ/(kcₐₜ/Kₘ)AD, which reflected the relative hydrolytic specificity of PGA for produced antibiotics with respect to the activated acyl donor, were only 0.028 and 0.043, respectively. The extremely low hydrolytic activity for the products of the βF24G mutant enabled greater product accumulation to occur during synthesis, which made it a promising enzyme for industrial applications.
Agid:
5900419