酿酒酵母乳酸脱氢酶发酵和酶学性质及其基因序列分析

【作者】 孟庆阳；

【导师】 方维明；

【作者基本信息】 扬州大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 在酿酒酵母的正常发酵过程中,乳酸脱氢酶催化丙酮酸还原为乳酸。高级醇是啤酒发酵中最重要的风味物质之一,大多数高级醇和酒精发酵同步形成,主要是由合成途径产生的。理论上分析高级醇的生成与乳酸的生成是反向的关系,因此可以通过增加乳酸的生成量来降低啤酒发酵中高级醇的生成量。然而在啤酒酿造中乳酸脱氢酶对高级醇的调控机制尚不清楚。本文拟研究酿酒酵母乳酸脱氢酶的发酵性质,在发酵过程中通过添加镁锌离子,调控乳酸脱氢酶的活力控制高级醇的含量。建立酿酒酵母乳酸脱氢酶分离纯化的方法,并分析不同菌株乳酸脱氢酶编码基因的序列差异,阐明不同酵母乳酸脱氢酶活力差异,为调节控制啤酒高级醇含量提供理论依据。本研究测定5株不同酿酒酵母菌株乳酸脱氢酶的发酵性质,结果表明不同酵母菌株之间的乳酸脱氢酶酶活力存在差异。在啤酒发酵过程中随着LDH活性增加,啤酒中的高级醇含量则下降,显示了LDH活性与高级醇生成量呈负相关性。通过向发酵液中添加金属离子Mg²⁺（500mg/L）和Zn²⁺（0.17mg/L）,测定发酵过程中发酵液的乳酸脱氢酶活力和高级醇生成量,结果显示Mg²⁺和Zn²⁺的添加,显著提高了酵母的乳酸脱氢酶活力,生成产物中高级醇的含量比对照降低,并且Mg²⁺比Zn²⁺的促进作用更为显著。酵母乳酸脱氢酶的分离纯化条件进行优化。结果表明:发酵液经细胞破碎,透析, 40%的（NH₄）₂SO₄盐析, DEAE-Sepharos（eDE-52）离子交换层析,Sephadex-G25凝胶过滤等纯化步骤,分离的乳酸脱氢酶经SDS-聚丙烯酰胺凝胶电泳检测为单一蛋白条带,纯化倍数为14.3,回收率为24%。凝胶法测定该酶的分子量约为32.2kD。对酵母乳酸脱氢酶的性质测定结果表明:该酶的最适反应温度为45℃,在35℃温度下比较稳定,最适反应pH值为9.4。金属离子对该酶活性具有显著影响,在0.5 mmol/L和1.0 mmol/L的金属离子浓度下:Mg²⁺,Zn²⁺对酶活有明显的促进作用,其中1.0mmol/L Ca²⁺离子作用最显著,Ni⁺和K⁺对它有明显的抑制作用,Fe²⁺的作用不是太明显。以酿酒酵母的基因组DNA为模板,根据Genbank发表的全序列,设计3对引物,扩增LDH基因的不同片断,对扩增片断测序。对本研究中5株酵母菌株和酿酒酵母（FLH201473.01X）的LDH基因（GenBank,accession No.DQ332540）之间的序列差异进行比对分析。结果表明: YZU03与其中的ST01和SH01的差异在LDH基因的第609-725碱基之间。而它们和FLH201473.01X的LDH基因的差异在该基因的第213、216、217、228、314和315碱基。更多还原

【Abstract】 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.更多还原