Fermentation And Enzymatic Properties Of S. Cerevisiae Lactate Dehydrogenase And Its Gene Sequence Analysis

In the fermentation process, LDH catalytic pyruvate into lactic acid.Higher alcohols are important component of beer favor , Most of which formed simultaneously with alcohol during fermentation of beer brewing. Theorically, Higher alcohols producs mainly by the Harris pathway and the relation of the formation of the higher alcohols and the lactic acid is the reverse. Therefore we can increasing the production of lactic acid to reduce higher alcohols.However in brewing the regulation mechanism of LDH to control the higher alcohols is not clear. In this paper ,we studied the fermentation performance of different yeast lactic dehydrogenase enzyme.Adding Mg²⁺ and Zn²⁺ ions in the fermentation process to control vitality of LDH and control the production of higher alcohols.Morever , we are also studied in this paper the separation and purification methods of LDH of the Saccharomyces cerevisiae ,which laid the base for researches on the LDH enzyme nature. and analized lactic dehydrogenase coding sequence of the five strains of yeasts at the molecular level.we clarified the difference of different yeast LDH dynamic, which for the conditioning control high alcohol content of beer and provide a theoretical basis.In the beer fermentation process, the fermentation performance of different yeast lactic dehydrogenase enzyme had been measured. The data revealed the interrelationship between LDH activity and higher alcohols content. With LDH activity increasing , the higher alcohols content decreased during the beer fermentation ,which suggested competitive and inhabit relationship between LDH activity and higher alcohols.The metal ions Mg²⁺ and Zn²⁺ were added to the medium, and the lactic dehydrogenase activity and production of higher alcohols were determined in the fermentation process, the results showed that adding a metal ions Mg²⁺, Zn²⁺ in the beer fermentation increased the lactic dehydrogenase activity of yeast (P<0.05) significantly, higher alcohols in control were lower than formented products significantly. Also,Mg²⁺ plays more significant promotion role than Zn²⁺.The separation and purification methods of LDH from Saccharomyces cerevisiae had been established and optimized.The optimized purification process includes the steps that removing cells by the centrifugation,35%saturation ammonium sulfate precipitation , dialysis,ion exchange chromatography with DE52 and gel filtration chromatography with Superdex-25.Sedium dodecyl-sulfate-polyacrylamide gel electrophovesis(SDS-PAGE)was used to examine the purification effect ,and the results indicated that the final product obitained was homogeneous and had a molocular weight around 32.2KD.the purification rate and activity recovery of LDH was 14.3 and 24.2% respectively.The optimal reaction temperature and pH of LDH was 45℃and 9.4. The stability temperature of LDH enzyme was 35℃. Metalions such as Ca²⁺,Mg²⁺ and Zn²⁺ can activate LDH and 1.0mmol/L Ca²⁺ can activate LDH signicantly . Influence of Fe²⁺ was not apparent .However,K+ was found to deactive LDH signicantly as well as Ni+.Three pairs of primers were synthesized according to the sequence of Saccharomyces cerevisiae LDH published on the GenBank to amplify LDH genes of five srains of Saccharomyces cerevisiae with genomic DNA as the template. Amplified fragments had been sequenced . In this study analysis of the gene sequence differences among the laboratory five strains and the published Saccharomyces cerevisiae strain FLH201473.01X . It show that the difference among YZU03 , ST01 and SH01 mainly existed in 609-725th base of the whole sequence. These sequences were different from published at the site of the 213th,216th,217th,228th,314th and 315th bases.