title: High-level expression of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas diminuta NK703 in Escherichia coli by combined optimization strategies.
abstract: In this work, a glutaryl-7-aminocephalosporanic acid acylase (GLA) coding gene was cloned from Pseudomonas diminuta NK703 which was screened from oilfield. The concerted effects of the expression system, inducing condition and culture medium on the expression of NK703 GLA in E. coli were firstly investigated. The best combination was the recombinant E. coli strain of pET-28a+GLA/BL21(DE3) with 2.0% (w/v) lactose inducing in YT medium at 25°C. Then, by optimizing the components of culture medium, a synthetic medium with dextrin and a feeding medium with glycerol as the carbon sources were developed to further enhance the GLA yield and improve the GLA solubility. In the end, the NK703 GLA activity increased about 50-fold, reached 14,470 ± 465 U/L, and the GLA productivity and the proportion of soluble GLA to the total soluble protein attained 206.0 ± 9.033 UL(-1)h(-1) and 60.13%, respectively. Scaling up the GLA production in 3.7 L fermenter under the optimized conditions identified in shake flask, the GLA activity also reached 12,406±521U/L, which was the highest report at fermenter level.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Ma
authors.forename: Erzheng
authors.initials: E
authors.lastname: Su
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Senwen
authors.initials: S
authors.lastname: Deng
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D002511:Cephalosporins / Q000096:biosynthesis*; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D007785:Lactose / Q000737:chemistry; D010399:Penicillin Amidase / Q000096:biosynthesis / Q000235:genetics*; D011549:Pseudomonas / Q000201:enzymology*
chemical_list: D002511:Cephalosporins; D003470:Culture Media; C081063:glutaryl-7-aminocephalosporanic acid; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase; D007785:Lactose
keywords: Glutaryl-7-aminocephalosporanic acid acylase (GLA); Lactose induction; Pseudomonas diminuta; Soluble expression