| Chitosanase is a kind of specific enzyme to hydrolyze linear chitosan. Chitooligosaccharides(CHOS), the product from the degradation of chitosan by chitosanase, has many biological functions and has an attractive prospect in food additives, medicine and health care, diagnostic reagents. In addition, it is high efficient and environmental friendly to produce CHOS with chitosanase in industry. Therefore, looking for chitosanase with high efficiency has become a hot point in research. There is few commercial chitosanase at present and can not meet the needs of industrial production of CHOS.In our laboratory, an inducible chitosanase-producing bacterium was screened in the soil samples from the lakeshore of Nan Hu, where shrimp and crab shells were degraded. And the bacterium was identified as Mitsuaria sp.141, which get a chitosanase activity of 4 U/mL in the culture after fermentation in appropriate condition in 500 mL shaking flask for 2 days. Based on this high activity, the main purpose of this study is to clone the chitosanase gene from Mitsuaria sp.141 and utilize genetic engineering technology to achieve high expression of chitosanase to meet the needs of industial production.After the extraction of total DNA from Mitsuaria sp.141, the gene encoding chitosanase choAl was isolated by PCR method. This gene had an open reading frame of 1176 bp, encoding a peptide of 391-amino acid residue, in which the 80-amino acids at N-terminal indicate a signal peptide. By BLAST, the chitosanase from Mitsuaria sp.141 belongs to glycoside hydrolases (GH) family 80.To achieve high expression of this chitosanase in Pichia pastor is, the sequence of the gene choA1 had been modified in several aspects:(1)removing the signal peptide; (2)codon optimization; (3)adjustment of GC content; (4)reducing the energy value of mRNA second structure. Then the unoptimized sequence ori-choA1 and the optimized sequence opt-choA1 were inserted into the pPIC9K vector of Pichia pastoris between the SnaBⅠand AvrⅡsites to construct the recombinant plasmids pPIC9K-ori-choA1 and pPIC9K-opt-choA1, respectively. These two plasmids were both digested by SacⅠor DraⅠand transformed into Pichia pastoris. As a result, four strains with different phenotypes and methanol consumption speed were obtained:ori-mut+、ori-muts、opt-nut+、opt-muts, all of which were screened on G418 plates and a strain opt-muts3# shows the highest chitosanase activity. This strain get an activity of 55.6 U/mL in the culture after fermentation in 500 mL shaking flask for 4 days and get an activity of 422 U/mL in the culture after high cell density fermentation in 50 L bioreactor for 140 h. Purified native chitosanase from Mitsuaria sp.141 was estimated to be 33.6 kDa on the gel figure by SDS-PAGE, while the purified recombinant chitosanase from Pichia pastoris showed smear bands ranging from 38 to 55 kDa but no band at 33.6 kDa. MALDI-TOF analysis indicated the protein in the smear bands were identical to the amino acid sequence of the cloned chitosanase, and the result of Western blotting also indicated proteins in the smear bands were his-tagged. Identification by glycopeptidase showed that the recombinant chitosanase is indeed glycosylated in Pichia pastoris and bring about smear bands in protein gel.The native and recombinant chitosanases showed the same optimal substrate colloidal chitosan and the same optimal pH for reaction at 5.5. The native-CHOA1 and recombinant-CHOA1 showed optimal temperature for reaction at 65℃and 55℃, respectively.This study provided the chitosanase gene from Mitsuaria sp.141. Moreover, a strain of recombinant Pichia pastoris with high efficiency of producing chitosanase was abtained, which is valuable in the industrial application. |
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