Expression and biochemical characterization of wild-type and mutant β-amylases from Priestia flexa in Komagataella phaffii

Abstract

The β-amylases AmyPf1 and AmyPf2 from two distinct Priestia flexa strains were successfully expressed for the first time in Komagataella phaffii. The main biochemical characteristics of the recombinant enzymes were determined. β-Amylases exhibited similar optimal temperature (55 °C) and pH (pH 8.0), as well as high stability at 55 °C and across a broad pH range (4.0–10.0). The thermal inactivation half-lives at 55 °C for both recombinant enzymes were approximately 4 h. The AmyPf2 demonstrated superior specific activity (3836 U•mg⁻¹) and catalytic constant (6367 s⁻¹) compared to AmyPf1 (3411 U•mg⁻¹ and 4224 s⁻¹, respectively). Comparison of the amino acid sequences of the two β-amylases revealed four residue differences. Site-directed mutagenesis introducing AmyPf2-specific substitutions into the AmyPf1 sequence showed that the V180A mutation significantly enhanced specific activity (4009 U•mg⁻¹) and catalytic constant (6656 s⁻¹) of the resulting r-V180A variant. In fed-batch fermentation, the activity of r-V180A reached 5130 U•mL⁻¹. Hydrolysis of corn starch using r-V180A in combination with pullulanase (a starch-debranching enzyme) yielded 87.56 % maltose, indicating that a hydrolysate with high maltose content was obtained. These results highlight the potential of the recombinant r-V180A enzyme for industrial maltose production and underscore the utility of the K. phaffii expression system for efficient production of P. flexa β-amylases.