Cloning, Expression And Biological Characterization Of Chitinase From Saccharomyces Cerevisiae In Pichia Pastoris

The Chitinase of Saccharomyces cerevisiae(CTS1-2) is an Endochitinase which is endogenous in Saccharomyces cerevisiae. Its recognition and slipt site lies in the β -1,4 glucosidic linkage of homopolymer. Included in the amino acid sequence from N-terminal to C-terminal of the Saccharomyces cerevisiae chitinase are signal peptide sequence, catalytic domain, binding domain and a C-terminal shorten region. Some reports indicated that the chitinase can degrade chitin and have some extent efficiency of inhibiting fungi. Extensively, chitin exists in crustas, molluses, insects, fungi, higher plants and marine algae, animal arthrosis, hoof, feet and so forth. Natural environment yields is huge and insurmountable, the yield of every year is more than ten billion tons of chitin which occupy the most quantities of organized compounds on the earth with the exception of cellulose. These current problems to be resolved involve those infection of fungi on animals, crops and aquaculture. Currently, many internal or foreign research institutes engaged in researches on chitinase, chiefly concentrating in two aspects mentioned above. To some degree, it abtain a few achievements but not develop industrialization fashion.This study, using pichia pastoris as expression system, accomplished the high level of expression of chitinase derived from Saccharomyces cerevisiae genome in the fermentation supernatant via constructing the relevant expression vectors and recombinant strains and identification of some relevant bioactivities of chitinase, and, at the same time, explored the purification conditions of chitinase and thus provided the basis for next researches. After amplifying the chitinase gene from Saccharomyces cerevisiae genome, we constructed successively some vectors: pPIC9-CTS1-2 pPIC9K-CTS1-2 pPICZα-CTS1-2 (multiple cloning) and modified signal sequence. After the transformatin and screening, we got the high expression GS115 engineered strains, which can highly express the chitinase in secretory form, and the yield of the expression concentration can attain 297.4mg/L in shake flask. We carried out some study on bioactivities of chitinase including degradation of chitin and fungi-repressing test. Identification of chitinase on the level of nucleotide sequence and amino acid sequence proved that the expressed protein is correct and the protein of interest. We have...