| Diacetyl was one of the most important flavors compound in beer. Because of its low threshold, the content of diacetyl has become an important indicator of beer maturation, a-acetolactate was the middle-product of Valine biosynthesis in brewing yeast during the fermentation and the precursor of diacetyl formation, a-acetolactate decarboxylase (ALDC) catalyze directly α-acetolactate into actone without diacetyl path. α-ALDC gene doesn't exist in brewing yeast, so there is no activity of α-ALDC in brewing yeast. The research is aiming at the selection of engineered strains with ALDC gene. Meanwhile, in the present research, the engineered yeast strains with ALDC gene were screened and their fermenting performances were investigated. Further study showed that engineered strains could produce α-ALDC to decompose precursor of diacetyl- α-acetolactate, accelerate deoxidization of diacetyl and shorten their fermenting period.Ten strains with high activity of α-ALDC were selected out from the received 250 ALDC positive transformants with the parameters of cell configuration, size, yeast growth, death rate, death temperature by the first round of selection. Five strains, 1# 2# 4# 9# 14# with low diacetyl peak, fast deoxidation, similar fermentation and flavor to original Tsingtao yeast were selected again .Enzymatic performance of α-ALDC was studied, the results showed that the activity of ALDC was induced evidently by different concentration of CuSO4 The activity of α-ALDC reached the top as the concentration of CuSO4 was 20umol/L, its pH is 5.0~7.0,optimum pH is 6.0, its temperature is 30~60, optimum temperature is 40; a -ALDC from recombinant yeast was sensitive to heat. During recombinant yeast culture, yeast cell density was positive correlation with a-ALDC amount, this shows that a-ALDC was compound, its produce was synchronized with yeast growth and the increase of yeast amount, α-ALDC activity will be advanced.Compared to receiver strain, engineered strains have high resistance ability to copper which is stable through continuous generation. Fermentation experiments were tested by using 20th generation yeast, the result showed that the content of diacetyl from engineered strains was 20-30% lower than receiver yeast. The result also showed that fermentation performance of recombinant yeast remained stable through generation.The total DMA was extracted respectively from engineered strains 4# 1st and 20th, digested by restruction enzyme Hind III, electrophoresised by agrose- gel, then was transfered to nitric cellulose membrane. The α-ALDC gene was used as probe to conduct southern blotting with the above DNA samples. The results showed that receiver yeast had no hybridization signal, while engineered yeast strains 4# 1st and 20th hadevident hybridization signal, this illustrated that engineered yeast strains had a-ALDC gene, their genetic stability is good.Eight continuous fermenting EBC tests were conducted with engineered strains No. 1,2,4. The results showed that engineered strains remain unchangeable on parameters of growth, death rate, fermenting performance and flavor compounds compared with receiver yeast through continuous generation fermentation; Compared with receiver yeast, engineered yeast decreased the diacetyl peak with 8.8~51.7%, the average value is 38.6% . NO 4# decreased by 32.4-51.7%. The diacetyl concentration of engineered yeast from fermenting liquor decreased to 0.08mg/L or less after fermenting for five days, while that of receiver yeast is 0.10 mg/L. a-ALDC activity from fermenting liquor of engineered yeast was determined continuously, the result showed that a-ALDC gene was expressed successfully and stably in engineered yeast, and the value of diacetyl peak and reductive activity was not identified with the increase of use generation in the engineered strains.The pilot-trials of engineered strains 100L scale was also tested, the parameters of yeast growth, attenuation, diacetyl reduce curve were determined continuously during 7th generation fermentation, the activit...
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