Institute of Drug Technology, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil.
Institute of Drug Technology, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil.
Protein Expr Purif. 2023 Oct;210:106327. doi: 10.1016/j.pep.2023.106327. Epub 2023 Jun 20.
Penicillin G acylase (PGA) is a strategic enzyme in the production processes of beta-lactam antibiotics. High demand for β-lactam semisynthetic antibiotics explain the genetic and biochemical engineering strategies devoted towards novel ways for PGA production and application. This work presents a fermentation process for the heterologous production of PGA from Alcaligenes faecalis in Bacillus megaterium with optimization. The thermal stability from A. faecalis PGA is considerably higher than other described PGA and the recombinant enzyme is secreted to the culture medium by B. megaterium, which facilitates the separation and purification steps. Media optimization using fractional factorial design experiments was used to identify factors related to PGA activity detection in supernatant and cell lysates. The optimized medium resulted in almost 6-fold increased activity in the supernatant samples when compared with the basal medium. Maximum enzyme activity in optimized medium composition achieves values between 135 and 140 IU/ml. The results suggest a promising model for recombinant production of PGA in B. megaterium with possible extracellular expression of the active enzyme.
青霉素 G 酰化酶（PGA）是生产β-内酰胺类抗生素过程中的一种关键酶。β-内酰胺半合成抗生素的高需求解释了用于 PGA 生产和应用的新型方法的遗传和生化工程策略。本工作介绍了一种在巨大芽孢杆菌中异源生产粪产碱杆菌 PGA 的发酵工艺，并对其进行了优化。粪产碱杆菌 PGA 的热稳定性明显高于其他已描述的 PGA，并且重组酶通过巨大芽孢杆菌分泌到培养基中，这有利于分离和纯化步骤。使用部分因子设计实验对培养基进行了优化，以确定与上清液和细胞裂解物中 PGA 活性检测相关的因素。与基础培养基相比，优化后的培养基使上清液样品中的酶活增加了近 6 倍。在优化后的培养基组成中，最大酶活达到 135-140 IU/ml 之间。结果表明，在巨大芽孢杆菌中进行 PGA 的重组生产具有很大的发展潜力，可能实现活性酶的细胞外表达。
