Cloning And Expressing Glucoamylase Gene Of Saccharomyces Diastaticus In Saccharomyces Cerevisiae

Ethanol has been promoted as a solution for a variety of complex problems related to energy and the environment. Compared to fossil fuels, ethanol has the advantages of being renewable, providing cleaner burning and producing no green-house gases. Methods for the fermentative production of ethanol from starch include using a genetically engineered microorganism that call directly ferment starch into ethanol and hence lead to an increase in the productivity of ethanol.Glucoamylase is an industrially extremely important enzyme, used in the enzymatic conversion of starch into high glucose and fructose syrups. The purpose of this study is to construct a glucoamylase producing recombinant yeast strains for the direct fermentation of soluble starch.In this study, two primers were designed according to the Genebank of NCBI. The glucoamylase DNA of STA1 was amplified by the method of PCR from the total DNA of Saccharomyces diastaticus. Then, the PCR segment was cloned into pUM-T vector to construct the middle vector pUM-T-stal that was then transformed into E coli to amplify and sequence. The STA1 segment was acquired by digesting pUM-T-stal using BspDI and Acc65I and ligated with Saccharomyces cerevisiae integrative expression vectors pRS416, named pRS416-stal. The recombinant glucoamylase was expressed in Saccharomyces cerevisiae.By inducing expression, the glucoamylase levels of Saccharomyces cerevisiae activity was 120U/mL. The result of determination of enzymatic properties of glucoamylase showed that the optimum temperature of glucoamylase was 60℃ with high stability in the condition of 40℃～70℃; the optimum pH of glucoamylase was 5.0 with high stability in the condition of pH4.0～6.0. After 2h of reaction of enzyme activity remained stable, the remaining activity can reach more than 90%.