The Cloning And Expression Of CDNA Encoding α-Transglucosidase From Aspergillus Niger

Alpha-transglucosidase is the key enzyme for yielding iso-maltooligosaccharide.Iso-maltooligosaccharide,one of theoligosaccharide,has special physiological function and benefits to human body.Aspergillus niger M-1,a higher a-transglucosidase-producing strain,was screened from soil by Food & Fermentation Engineering Institute of Guangxi University.Complementary DNA(cDNA) encoding a-transglucosidase was cloned from Aspergillus niger M~l in E.coli . About 3.0kb DNA fragment was obtained by RT-PCR from total RNA of Aspergillus niger using a specific 5'- end and 3'- end primer.PCR product was cleaved with restriction enzyme EcoRI and BamHI and ligated into the EcoRI/BamHI-digested restriction site of vector pSE380.After transformation of the competent E.coli JM109,transformants were obtained. When the transformant DNA was digested by the same enzymes, 4.4kb and 3.0kb fragment were obtained respectively, which were similarto the length of PCR product and pSE380 vector.lt primarily proved that the transformant DNA was the complementary DNA of a-transglucosidase.After 12 hours of continuous IPTG induction, it was observed that the total secreted protein in culture migrated as the main bands of 110KD on SDS-PAGE. Alpha-transglucosidase activity was up to 226.8 U / ml when assayed using methyl- a -D-glucopyranoside as specific substrate.Successfully cloning and expression of this recombinant plasmid laid a solid basis for further study and built a set of successful methods for constructing other recombinant plasmid by using the vector pSE380.