Heterologous expression of GH5 chitosanase in Pichia pastoris and antioxidant biological activity of its chitooligosacchride hydrolysate.

Chitosanase was widely used in the production of bioactive chitooligosacchride (CHOS) due to their safety, controllability, environmental protection, and biodegradability. Studies showed that the bioactivity of CHOS is closely related to its degree of polymerization. Therefore, the production of ideal polymerized CHOS becomes our primary goal. In this study, the glycosyl hydrolase (GH) family 5 chitosanase was successfully expressed heterologously in Pichia pastoris. After 96 h of high-density fermentation, the chitosanase activity reached 90.62 U·mL-1, the protein content reached 9.76 mg·mL-1. When 2% chitosan was hydrolyzed by crude enzyme (20 U/mL), the hydrolysis rate reached 91.2% after 8 h, producing a mixture of CHOS with 2-4 desirable degrees of polymerization (DP). Then, the antioxidant activity of CHOS mixture was investigated, and the results showed that the antioxidant effect was concentration-dependent and had great application potential in the field of nutrition.

Biocatalysis of heterogenously-expressed d-lactonohydrolases and its efficient preparation of desirable d-pantoic acid.

d-Pantoic acid (D-PA) is an essential intermediate for the production of d-pantolactone. Here, three d-lactonohydrolases (D-Lacs), namely, Fm-Lac from Fusarium moniliforme SW-902, Fp-Lac from Fusarium proliferatum Nirenberg ECU2002, and Fo-Lac from Fusarium oxysporum AKU3702 were heterogeneously expressed in Pichia pastoris. The constructed recombinant strains produced D-Lacs of 1263 U/mL, 1025 U/mL, and 948 U/mL in a 3-L fermenter, respectively. Simultaneously, these three D-Lacs were used to resolve racemic pantolactone (DL-PL), the hydrolysis rate by Fo-Lac over 40% and the enantiomeric excesses was 99% after 4 h reaction, which outperformed Fm-Lac and Fp-Lac. Under the 800 mL scale reaction, the hydrolysis rate of DL-PL reached 39.2% with a D-PA concentration of 144.6 g/L and space-time yield of 36.2 g/L/h correspondingly. This is the highest catalytic efficiency reported so far, which shows that D-Lac heterologously expressed by P. pastoris has excellent industrial application prospects.