Abstract
β-Mannanase has been widely used in industries such as food and feed processing and thus has been a target enzyme for biotechnological development. In this study, we sought to improve the stability and protease resistance of a recombinant β-mannanase, MAN47 from Armillariella tabescens, through rationally designed N-glycosylation. Based on homology modeling, molecular docking, secondary structure analysis and glycosylation feasibility analysis, an enhanced aromatic sequon sequence was introduced into specific MAN47 loop regions to facilitate N-glycosylation. The mutant enzymes were expressed in Pichia pastoris SMD1168, and their thermal stability, pH stability, trypsin resistance and pepsin resistance were determined. Two mutant MAN47 enzymes, g-123 and g-347, were glycosylated as expected when expressed in yeast, and their thermal stability, pH stability, and protease resistance were significantly improved compared to the wild-type enzyme. An enzyme with multiple stability characterizations has broad prospects in practical applications, and the rational design N-glycosylation strategy may have applications in simultaneously improving several properties of other biotechnological targets.
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