A rational design for trypsin-resistant improvement of Armillariella tabescens β-mannanase MAN47 based on molecular structure evaluation

Protease resistance of enzymes is required for the feed industry because of the presence of secretary proteases in the digestive tract. In this study, we report a rational method for protease-resistance improvement of enzymes based on molecular structure evaluation. The trypsin-resistance of β-mannanase MAN47 from Armillariella tabescens was investigated. Twelve tryptic sites within it were ordered by their positions in three-dimensional space from external to internal. Except of R144, R192 and R261, which were either conserved or highly related to the catalytic activity, the top external residue K280 and the most internal residue K371 were selected. With conducting computational design via H-bond analysis and molecular dynamics simulations, optimal mutants of K280N and K371Q were predicted. Meanwhile, a triple mutant K280N/K107H/R102N was also predicted. Half-lives of mutants K280N, K280N/K107H/R102N, K371Q and wild-type enzymes which were all pre-treated by trypsin at 40 °C were determined 185 min, 285 min, 102 min and 100 min, respectively. In addition, the temperature–activity and pH–activity profiles revealed that the mutations we designed had no obvious influence on the catalytic activity of the enzyme. Our results proved that trypsin-resistance of an enzyme could be improved by molecular rational evolution of homology modeling and molecular dynamics simulations.

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► A rational method for protease-resistance improvement of enzymes was introduced.
► Homology modeling and molecular dynamics simulation were employed in the method.
► Mutating trypsin digestion site located on enzyme surface improved its resistance.
► The mutations we designed had no obvious influence on the catalytic activity.