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.
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