Model-driven high-level expression of β-mannanase in Pichia pastoris for degrading mannan in palm kernel expeller

Palm kernel expeller (PKE), a copious byproduct, is limited in its application to non-ruminant diets due to the presence of antinutritive factors, predominantly galactomannan, necessitating effective enzymatic hydrolysis by β-mannanase to enhance its nutritional value. Here, a cross-strategy combining the predicting mutations with enhanced protein expression (PMEPE) model with B-factor analysis was employed to rationally modify the gene encoding β-mannanase CsMan5A in Pichia pastoris. The expression level of mutant CsMan5A-Q7S improved 17 % compared to the wild type, reaching 16.38 g/L, which resulted in an increased enzyme activity titer of 147,597 U/mL and specific enzyme activity of 9010.81 U/mg, respectively increasing by 32.87 % and 13.81 % compared to the wild-type. Then, the expression of CsMan5A-Q7S was further elevated to 18.70 g/L through cell wall engineering. Computational simulations explained the mechanism of the superior thermostability and activity observed in the CsMan5A-Q7S mutant, which presented a more stable overall structure than the wild type. The residue left after hydrolysis by CsMan5A-Q7S had lower fiber content, as well as higher protein content, thereby effectively enhancing its feed quality. This study achieved exceptional phenotypic enhancement of β-mannanase production in Pichia pastoris by an effective strategy through computational modeling, protein engineering, and cell wall modification strategies.