Improving the activity and stability of GL-7-ACA acylase CA130 by site-directed mutagenesis.
In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151alphaF and Q50betaN, showed two- to threefold-increased catalytic efficiency (k(cat)/K(m)) compared to wild-type CA130. Their K(m) values were decreased by ca. 50%, and the k(cat) values increased to 14.4 and 16.9 s(-1), respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121betaA and K198betaA had a 30 to 58% longer half-life than wild-type CA130, and K198betaA and D286betaA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50betaN/K198betaA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.
Zhang
Wei
W
Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Rd., Shanghai 200032, People's Republic of China.
Liu
Yuan
Y
Zheng
Huabao
H
Yang
Sheng
S
Jiang
Weihong
W
D002511:Cephalosporins / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D002511:Cephalosporins; C081063:glutaryl-7-aminocephalosporanic acid; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase