In the present research, gene csn which consists of 1362 nucleotides and encodes chitosanase (CSN) of Bacillus cereus HN90 was amplified by PCR, ligated in vector pJET1.2 and transformed into Escherichia coli XL1-blue. After treatment of extracted plasmid from E. coli with both restriction enzymes EcoRI va XbaI, gene csn was inserted into plasmid pPICZaA to generate expression vector pPICZaA::csn. The resulting vector was then linearized at the AOX1 locus by Sad, transformed into Pichia pastoris X33 strain by electroporation and resulting transformants were screened on agar plate YPDS supplemented with antibiotic zeocine at various concentrations. The examination of yeast chromosomal DNA by PCR using specific primers 5' AOX1 va 3' AOX1 indicated that the gene csn was integrated into genome of P. pastoris X33. Among over 100 transformants tested, eight recombinant strains showed the both capabilities to grow on YPDS medium containing 1000-mg/ml zeocine and synthesize extracellular CSN. Based on the enzymatic assays, the recombinant strain P. pastoris L6 revealed the highest chitosanase activity, reaching up 322.4 U.L-1 after 72hour cultivation in BMMY medium induced by 1.5 percent (v/v) methanol.
Lọc theo danh mục
Lượt truy cập :
22,395,292
Xem toàn văn