High Activity In The Directed Evolution Of The Alpha-acetolactate Decarboxylase And Low Temperature Conditions, The Mutant Enzyme Screening

a-acetolactate decarboxylase is critically important in beer industry. During beer or wine fermentation, the yeast produces a-acetolactate, and the a- acetolactate can be spontaneously decarboxylated into diacetyl. In beer or wine, very low concentration of diacetyl, i.e. 0.15ppm, will result in a very unpleasant taste. The a-ALDC can decarboxylate a-acetolactate directly to form acetoin without the formation of diacetyl. By adding a-ALDC to the fermenting wort, a-acetolactate will be removed as soon as it is formed, thereby which can avoid the formation of diacetyl and reduce or even skip the period for maturation. However, the normal temperature of fermentation is 10 C and the optimum temperature of a-ALDC is 30-40 C, which indicted that the activity of a-ALDC in the normal temperature of fermentation is 15-20% of that in the optimum temperature. Because the catalytic site of a-ALDC has not been certified and the catalytic mechanism has to be also further proved, it is difficult to enhance the enzyme activity in low temperature by the rational design. Therefore, we selected the method of directed evolution to modify the a-ALDC gene in order to obtain the mutants with high enzyme activity in low temperature.New and highly effective strategies for directed enzyme evolution in vitro have been developed in the protein engineering. It clearly allows us to engineer enzymes with novel functions and features by strategies, such as error-prone PCR, DNA shuffling and stagger extension process (StEP), when very little is known about spatial configuration of proteins. Therefore millions of years in nature can in principle be accomplished in the test within several years.According to the basic idea of directed evolution, we modified the a-ALDC gene by error-prone PCR. Approximately 7000 colonies were screened. One mutant was obtained, and its enzyme activity is 2.09-fold higher than that of wild-type a-ALDC in low temperature. The two nucleotide substitutions were occurred in the evolved a-ALDC gene. The mutant has been changed from DNA cordon AAG(Lys) to AGG(Arg) at 485bp. The wild-type and evolved a-ALDC were extracted and characterized. Both the pH stability and thermostabilization of evolved a-ALDC were higher than those of wild-type enzyme. This mutant is more suitable for the industrial production of beer. The replacement of Arg results in partial disruption of the a-helix. The primary structure is unchanged in the evolved a-ALDC, which implies that the catalytic mechanism of the evolved enzyme remained the same.