title: Simultaneously improving stability and specificity of cell surface displayed glucose dehydrogenase mutants to construct whole-cell biocatalyst for glucose biosensor application.
abstract: The improved stability and substrate specificity of cell surface displayed glucose dehydrogenase (GDH) mutants by replacing four amino acids from Bacillus subtilis by using site-directed mutagenesis was systematically investigated. A series of mutated GDHs including E170R/Q252L, V149K/E170R/Q252L, E170R/Q252L/G259A and V149K/E170R/Q252L/G259A, were fused to the ice nucleation protein for displaying on cell surface of Eschericia coli. Q252L/E170R/V149K, Q252L/E170R/G259A and Q252L/E170R/V149K/G259A variants were found stable at a wide pH range and shown excellent thermostability. Especially, the Q252L/E170R/V149K/G259A mutant showed half-life of ~3.8days at 70 °C. Q252L/E170R/V149K/G259A variant exhibited the narrowest substrate specificity for d-glucose. The whole cell displayed GDH mutant could be cultured in a large scale with excellent enzyme activity and productivity. In addition, a sensitive and stable electrochemical glucose biosensor can be prepared using the GDH-mutant bacteria modified electrode. Thus, the whole cell biocatalysts are promising candidates for exploitation in a wide range of industrial applications.
authors.affiliation: Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Liang
authors.affiliation: Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors.forename: Qiaolin
authors.initials: Q
authors.lastname: Lang
authors.affiliation: Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors.forename: Xiangjiang
authors.initials: X
authors.lastname: Tang
authors.affiliation: Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China. Electronic address: liuah@qibebt.ac.cn.
authors.forename: Aihua
authors.initials: A
authors.lastname: Liu
mesh_terms: D055162:Biocatalysis; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000201:enzymology; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D042843:Glucose 1-Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D042843:Glucose 1-Dehydrogenase; D005947:Glucose
keywords: Enzyme stability; Glucose biosensor; Glucose dehydrogenase mutant; Substrate specificity; Surface display