Cloning And Expression Of α-Acetolactate Decarboxylase

α-acetolactate decarboxylase( ALDC, EC4.1.1.5) can convert the precursor of diacetyl, α- acetolactate, to acetoin which has no effect on beer flavor. diacetyl(DA) is one of the most common off-flavors in beer fermentation. beacause reduction of deflavor is the most time-consuming step in maturation of beer, acceleration of this step is economically important to the brewing industry.This paper comprises the isolation and expression of α-acetolactate decarboxylase gene from Bacillus subtilis 168 and its expression in E. coli; E. coli transfer RNA(tRNA^Arg) of rare codon are utilized to enhance α-acetolactate decarboxylase gene expression and increase the activity level; Secreted expression α-acetolactate decarboxylase in Pichia Yeast GS115; Construct the E. coli-Yeast shuttle recombinant plasmid pYES2-ALDC and got the positive Saccharomyces cerevisiae transformants.Amplified the α-acetolactate decarboxylase gene with PCR from B. subtilis 168 and cloned in plasmid pUC18 and pQE60, construct the recombinant plasmid pUC18-ALDC and pQE60-ALDC. Sequencing result shown that recombinant plasmid pUC18-ALDC contain whole α-acetolactate decarboxylase gene. Under T5 promoter, DH5α/pQE60-ALDC expressed ALDC with high level induced with IPTG and assaied the activity of ALDC. Expressed product activity is determined to be 0.11U/mL. To increase ALDC activity, constrct two pOK12-argU and pOK12-ileX. pOK12-ileX contains ileX gene which transcripte tRNA of ileX. Transform these two plasmids and pQE60-ALDC in E.coli strain Top10 respectively, the results shown expressed ALDC and its enzyme activity is higher than control by activity assay and SDS-PAGE analysis.Cloned the ALDC gene in E. coli-Yeast shuttle vector pPIC9 and obtained pPIC9-ALDC, electrotransformed Pichia pastoris GS115 strain and α-acetolactate decarboxylase gene integrate in chromosome of P. pastoris and engineering Yeast is got. Inducement whith the methanol, the recombinant protein was proved to have biological activity of α-acetolactate decarboxylase, which is secreted into culture medium. It is shown that the recombinant, GS115/pPIC9-ALDC, secreted ALDC under induction with methanol. A band of about 62 kDa was detected under SDS-GAGE assay. The biological activity of the recombinant is determined to be 0.03 U/mL. The purification of expression product has a high level.