Of ¦Á-acetolactate Decarboxylase Gene Engineering Strain Build And Optimize The Conditions For Enzyme Production,

The diacetyl is an important substance in the beer, but its high content will effectthe quality of the beer. The taste standard is low which is between 0.1 and 0.15 mg/L,if exceeding this standard, the beer will have unhappy taste, so the traditionalproduction of beer needs long time storage at low temperature to reduce the content ofdiacetyl.Alpha-acetolactate decarboxylase(a-ALDC, EC.4.1.1.5) can transfer theα-acetolactic acid to the acetoin, thus the content of diacetyl is reduced fast to promote thebeer to mature. The fermentation period of beer production will be shortened, the costof production will be reduced.In this study, a recombinant yeast strain was constructed which can produce thediacetyl in small amounts.First,two primers were designed based on the gene sequenceof ALDC in the GenBank(L04470), ALDC gene was cloned using PCR by the templateof the Bacillus subttlis strain, 780bp fragment was obtained after PCR reaction and thisresult was 93.6% similar to the sequence of the GenBank,Which was the ALDC gene.then the gene was combined with the pYC6/CT and transferred into the Saccharomycescerevisiae HDY-01 using LiAc transfer method. Transformants was screened byenzyme protein activity, then some strains in which the ALDC gene can be expressedwere obtained. ALDC activity of these strains was determined and one strain named1-27 was selected whose ALDC activity was highest, the activity was 0.0081U/mL,after optimization the ALDC enzyme activity can be 0.0087U/mL in the condition of36h,30℃,10% inoculum size. The fermentation experiment at 3% inoculum size and13℃shows that the diacetyl content was reduced to 0.025ppm at the eighth day. Thedetermination of twelve substance includes acetaldehyde shows that the transformstrain was conform to the standard.The research is the basement of constrction low diacetyl yeast strain.