Isolation Of Bacteria Producing α-acetolactate Decarboxylase And Cloning And Expression Of The Genes

Diacetyl(DA) is one of the most common off-flavors in beer fermentation, affects the quality of beer. Since reduction of DA flavor is the most time-consuming step in maturation of beer, acceleration of this step is economically important to the brewing industry.α-acetolactate decarboxylase(ALDC) can convertα-acetolactate (the precursor of diacetyl) to acetoin which has no effect on beer flavor. The addition of ALDC to freshly fermented beer is the best method to reduce the concentration of DA.In order to utilize theα-acetolactate decarboxylase in brewing industry, theα-acetolactate decarboxylases from bateria were studied in this thesis. Firstly,α-acetolactate was repared through several organic reactions,it was used as the substrate determining the activity ofα-acetolactate decarboxylase(ALDC EC.4.1.1.5). Secondly, seven strains with higher activity of ALDC were isolated from the soil and water. A higher activity ofα-acetolactate decarboxylase strain was identified as Bacillus subtilis by physiological and biochemical character studying. A 0.77 kb fragment was amplified from the strain by PCR using a pair of primers specified theα-acetolactate decarboxylase gene, and it was confirmed asα-acetolactate decarboxylase gene by DNA sequencing. 98% nucleotide sequence and 99.6% deduced amina acid sequence are same between our research and the...