宏蛋白质组学方法对清香大曲蛋白分析

Identification of crude proteins from Daqu(yeast for making hand liquor) for light-fragrant Chinese liquor by proteomics method

目的:分离鉴定清香大曲的蛋白质组分,为大曲功能菌的确认及深入理解大曲功能提供科学的理论依据。方法:挑选具有典型感官特征的清香大曲,用乙酸-乙酸钠提取粗酶液,粗酶液经SDS-PAGE后,将电泳条带分段切胶回收处理后用LC-MS/MS进行蛋白鉴定。结果:共鉴定蛋白122种。从蛋白功能分,这些蛋白质有酶类69种(56.6%),其中EMP途径的酶有17种,糖类水解酶10种,蛋白酶13种,氧化还原酶类5种,乙醛脱氢酶5种,其他代谢途径酶19种。热击蛋白15种(占12.3%),蛋白表达及核酸复制相关蛋白16种(占13.1%),其他的蛋白22种(占18.0%)。结论:糖化酶是大曲的主要生化指标,通过对清香大曲蛋白质成分分析发现,产生糖化酶的微生物只有米根霉,由此确认米根霉为清香白酒的功能菌。实验只检测到酿酒酵母合成的乙醇-O-酰基转移酶,确认其为清香白酒酯化功能菌。大曲还含有大量的EMP途径酶类,说明大曲还有助于葡萄糖到丙酮酸的代谢。

Objective： In order to understand the function of Daqu and their functional strains, proteins from Daqu for light-fragrant liquor making were isolated and identified. Methods： Daqu samples with typical sensory characteristics were selected as raw materials. Crude enzymes were extracted from raw materials by acetic acid-acetic acid sodium buffer. Then SDS-PAGE was implemented using the crude enzymes. The resulting gel was cut in sections, the proteins from different sections were reclaimed and identified by LC-MS/MS. Results： A total of 122 proteins were identified. The minimum pl is 4.40 （endo-l,4-β-xylanase F1）, the maximum pl is 10.9 （60S ribosomal protein L32）. Overall, 91 acidic proteins （pl〈7.0） accounted for 74.6%, 31 basic proteins （pl〉7.0） accounted for 25.4%. According to their functions, there were 69 kinds of enzymes （56.6%）, including 17 EMP pathway enzymes, 10 carbohydrate hydrolyzing enzymes, 13 protease, 5 oxidoreductases, 5 aldehyde dehydrogenase and 19 other pathway enzymes. Beside, there were 15 heat shock proteins （accounting for 12.3%）, 16 protein expression and DNA replication associated proteins （accounting for 13.1%） and 22 other proteins （accounting for 18.0%）. Conclusion： The activity of glucoamylase is one of the major characteristics of Daqu. It was found that only Rhizopus oryzae could produce glucoamylase by proteomics. So Rhizopus oryzae was confirmed to be the functional strain in lightfragrant liquor making. At a guess, estedfication could completed by endocellular acyltransferase from yeast in addition to extracelllular esterification enzyme during light-fragrant liquor making. In this experiment, only ethanoI-O-acyltransferase from Saccharomyces cerevisiae was detected, so it was proved to be the functional strain for esterification. A lot of enzymes of EMP pathway and acetaldehyde dehydrogenase were detected from Daqu, indicating that Daqu contributed to metabolize from glucose to pyruvate, ever to acetic acid.