Multiple Strategies for Recombinant Expression of Human Chymotrypsinogen C in <i>Pichia Pastoris</i>

ABSTRACT Chymotrypsin C (CTRC) is a multifunctional serine protease that have shown great application potential in the field of food processing. This study aims to efficiently express CTRC in Pichia pastoris and provide a safe and highly effective new enzyme formulation for the food industry. Here, we successfully expressed and secreted human chymotrypsinogen C (proCTRC) in P. pastoris through multiple strategies. First, we replaced the native AOX1 promoter with the stronger HpFMD promoter and used ribosomal DNA (rDNA) loci for gene integration to achieve significant expression and secretion of proCTRC. In addition, we designed a mutant pro2M that prevented host protease cleavage by replacing the dipeptide motif “KR” with “EK”. The enzyme activity of wild type and mutant pro2M in the fermentation supernatant reached 0.59 ± 0.003 U/mL and 3.94 ± 0.27 U/mL, respectively. Meanwhile, the mutant showed a specific activity of 54.84 U/mg, 3.3‐fold higher than the wild type's 16.6 U/mg. Further, the recombinant pro2M and proCTRC can exhibite good stability under neutral pH conditions. In summary, we have established P. pastoris as a powerful platform for high‐yield CTRC production and highlighted the importance of engineering protease‐resistant variants to optimize enzyme secretion and activity.