Heterologous Expression and Characterization of Maltotetraose Amylase in Bacillus subtilis Based on Maltose-Inducible Promoter
Maltotetraose amylase can cut successively the fourth α-1,4 glycosidic bond from the non-reducing end of starch to produce maltotetraose,which is widely used to date in the food,healthcare,and paper industries.However,the urgent and realistic need to reduce production cost is to establish safe,high efficient expression system to strength-en the recombinant expression of maltotetraose amylase.In this study,a maltotetraose amylase gene nta from Pseudo-monas saccharophila(DSM 654)was expressed in Bacillus subtilis using a maltose-inducible promoter to realize its safe and high efficient expression,followed by isolation purification and feature characterization of the recombinant en-zyme.The results showed that recombinant plasmid carrying the maltose-inducible promoter Pglv with expression carrier was transformed into B.subtilis WB800N for the expression.After sucessful construction of the engineered strain's ex-pression was induced and successfully obtained pure enzyme Mta adopting metal ion chelate chromatography.The re-sult of enzymological study showed that its optimized reaction temperature was at 55 ℃ and pH at 7.5.And its kinetic constant Km was at(1.26±0.17)g/L,and its kcat/Km was at(2 275.07±32.83)L/(s·g).The purified enzyme could preserve 40％of its original activity after 14 days of storage at 4 ℃.To summarized,in this study a safe and high efficient expression system of maltotetraose amylase was successfully constructed and provided a new strategy for its large-scale preparation and application.
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