Cloning And Expression Of Glucoamylase Genes From Aspergilus Niger CDNA Library In Yeast

Glucoamylase is an exo-glycosidase which also simplied as glucoamylase, containing mannose, glucose, galactose and uronic acid glycoprotein, molecular weight between50kD to112kD, usually accounted for3.2%to20%of the total carbohydrate. It can release of β-D-glucose from the non-reducing end of starch, dextrin or glycogen and other carbohydrates, the enzyme is widely distributed in bacteria, archaea and eukaryotes. The starch could be decomposed into glucose by Glucoamylase. The glucoamylase was widely used in pharmaceutical, wine and food fermentation industrial, which is one of the most important industrial enzymes. At present, the annual output of Glucoamylase about70000t. It was the largest yield of the enzyme species in China.An expression cDNA library was constructed from high-yielding glucoamylase strains of A.niger BU11-36and the glucoamylase gene was isolated, then to study the expression of the gene in Pichia pastoris. The cDNA sequence of glucoamylase from A.niger was obtained by RT-PCR. The cDNA fragment was cloned into the expression vector pPIC9K and the linearized recombinant vector was transformed to Pichia pastrois GS115by electroporation. The positive clones were analyzed subsequently. The recombined Pichia pastrois were cultured in the MM medium, using1%methanol to induce the expression of recombinant gene. The results showed that the maximum activity of glucoamylase was15.6U/mL when it gotten at72h. Sequence analysis revealed that glucoamylase had1908bp, which encodes a putative polypeptide of636amino acids. The expressed protein was purified from the fermented supernatant using column and determined by SDS-PAGE. The result of SDS-PAGE also showed that the molecular weights of the enzyme was80kD. Determination of recombinant glucoamylase enzymatic properties: enzyme optimum pH and temperature of50℃and5.0, respectively. Cu2+inhibition of recombinant glucoamylase was significantly stronger than that of Fe3+, Ca2+and Zn2+. The results show that anionic surfactant (SDS) was inhibition for he Enzyme activity of glucoamylase.However, The role of non-ionic surfactant could improve the Enzyme activity of glucoamylase.Using the same method of cloning a cDNA fragment of the glucoamylase gene of Aspergillus niger cDNA library with carrier pYES2connected to the recombinant vector was transformed wine Saccharomyces cerevisiae INVScl.The positive clones were selected and researched by iodine effects.The results showed that the maximum activity of glucoamylase was4.3u/mL when it gotten at60h. Determination of recombinant glucoamylase enzymatic properties: enzyme optimum pH and temperature of55℃and4.0. The starch hydrolysis force of the recombinant Saccharomyces cerevisiae was about 60%, and the alcoholic fermentation of recombinant Saccharomyces cerevisiae capacity was3.5%. The expressed protein was purified from the fermented supernatant using column and determined by SDS-PAGE. The result of SDS-PAGE also showed that the molecular weights of the enzyme was70kD. The result was the same as the predicted results.