Cloning, expression and activity identification of chitin deacetylase gene from Saccharomyces cerevisiae
2025
HU Shiqi
To construct a non-methanol induced expression vector and engineering strain for chitin deacetylase in Pichia pastoris, two chitin deacetylase genes ScCDA1 and ScCDA2 were amplified and cloned from Saccharomyces cerevisiae and subjected to bioinformatics analysis. By homologous recombination technology, ScCDA1 and ScCDA2 genes were respectively cloned into the vector pGAPK containing a constitutive strong promoter pGAP and expressed in P. pastoris GS115, and their deacetylase activity was investigated. The degree of deacetylation was determined by alkalimetry, and the deacetylation ability of recombinant strains to substrate chitin was studied. The results showed that the full length of ScCDA1 and ScCDA2 genes were 906 bp and 939 bp, respectively, without introns, and the molecular mass of their encoded proteins were 34.64 kDa and 35.69 kDa. Both proteins did not contain transmembrane regions and signal peptides, and the homology was 51%. The high-copy recombinant strains pGAPK- ScCDA1/GS115 and pGAPK-ScCDA2/GS115 were successfully constructed. After 72 h of shake-flask fermentation, the deacetylase activity reached the highest level, which were 18.69 U/ml and 18.14 U/ml, respectively, with corresponding chitin deacetylation degrees reaching 59.20% and 53.55%, which was significantly higher than 3.58% of the control group (P<0.01).
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