Cloning And Heteroexpression Of α-dextranase From Chaetomium Gracile

Dextranase could hydrolyze dextran by recognizing and cleaving the a-1,6-glucosidic linkages specifically, it shows important application potential in sugar industry and dental plaque prevention. A variety of dextranases have been isolated from bacteria, fungi and yeasts. Dextranase from Chaetomium gracile has been proved to have excellent enzymatic properities which could stand higher temperature and maintain better stablility with higher dextranase activity in a wide range of pH, thus it performs great application prospects in sugar industry. Nevertheless, the gene sequence of C. gracile dextranase has not been report yet.In this paper, C. gracile CGMCC3.3783was selected as the research target, the purification of C. gracile dextranases was conducted then two purified dextranases (named CGD-1and CGD-2) with approximate molecular weights of70kDa and67kDa were obtained, respectively. Analysis of enzymatic properities indicated that CGD-2showed a better enzymatic properities than CGD-1. Genomic DNA and total RNA of C. gracile were isolated, then a dextranase gene (cgdex) was obtained by Genome Walking and RACE. The open reading frame (ORF) of cgdex was determined to be1788bp long without introns. The deduced protein consisted of595amino acids with an estimated molecular mass of65.96kDa. Then cgdex gene was separately heterologous expressed in E. coli BL21(XDE3), Pichia pastoris and Yarrowia lipolytica. The recombinant dextranase presented the best expression result in Pichia pastoris GS115, with the highest dextranase activity of29.36U/ml and a protein concentration of0.61mg/ml, separately. The optimum temperature and pH of recombinant dextranase expressed by GS115were56℃and5.0, which was similar with the original protein CGD-2. By site-directed mutagenesis, we found the Asp residue401in the conserved region of C. gracile dextranase has an important impact on the dextranase activity and speculated the site may be located in the enzyme activity center.This study would have positive significance for the large-scale commercial production and application of dextranase.