Efficient synthesis of β-lactam antibiotics with in situ product removal by a newly isolated penicillin G acylase

Abstract

A penicillin G acylase (PGA) from Achromobacter xylosoxidans PX02 was newly isolated, and site-directed mutagenesis at three important positions αR141, αF142, βF24 was carried out for improving the enzymatic synthesis of β-lactam antibiotics. The efficient mutant βF24A was selected, and the (P_s/P_h)_ini (ratio between the initial rate of synthesis and hydrolysis of the activated acyl donor) dramatically increased from 1.42-1.50 to 23.8-24.1 by means of the optimization of reaction conditions. Interestingly, the efficient enzymatic synthesis of ampicillin (99.1% conversion) and amoxicillin (98.7% conversion) from a high concentration (600 mM) of substrate 6-APA in the low acyl donor/nucleus ratio (1.1:1) resulted in a large amount of products precipitation from aqueous reaction solution. Meanwhile, the by-product D-phenylglycine was hardly precipitated, and 93.5% yield of precipitated ampicillin (561 mM) and 94.6% yield of precipitated amoxicillin (568 mM) were achieved with high purity (99%), which significantly simplified the downstream purification. This was the first study to achieve efficient β-lactam antibiotics synthesis process with in situ product removal, with barely any by-product formation. The effect enzymatic synthesis of antibiotics in aqueous reaction solution with in situ product removal provides a promising model for the industrial semi-synthesis of β-lactam antibiotics.

Keywords: Enzymatic synthesis; In situ product removal; Penicillin G acylase; β-Lactam antibiotics.