【作者】 孙莹；

【导师】 吴丹；

【作者基本信息】 江南大学 ， 生物化学与分子生物学， 2022， 硕士

【摘要】 果胶酯酶（Pectinesterase,EC 3.1.1.11,PE）是果胶酶的一种,属于碳水化合物酯酶的第8类（CE8）,催化果胶同型半乳糖醛酸（HG）结构域中半乳糖醛酸（Gal A）残基在C-6处的甲酯键的水解,进行去酯化反应生成果胶酸,从而将高酯果胶转化为低酯果胶。PE在食品、纺织、医药等方面应用广泛,尤其是在食品工业方面,低酯果胶由于更符合人们少油低糖、健康饮食的需求而受到关注。目前国内的商品果胶酶制剂多是果胶酯酶、多聚半乳糖醛酸酶和果胶酸裂解酶的混合制剂。与其他工业酶一样,果胶酯酶在工业生产中也面临着耐热性低和低酶活的问题。本研究以毕赤酵母X33（Pichia pastoris X33）作为生产PE的宿主菌,研究了重组PE的酶学性质,优化了重组PE的脱酯条件,并通过构建突变体提高了PE的耐热性,主要内容如下:（1）将黑曲霉（Aspergillus niger）的PE基因,与载体pPICZαA连接后,在P.pastoris X33中进行异源表达。先进行摇瓶水平发酵,根据发酵结果筛选得到的一株较好的重组菌株进行3 L和50 L发酵罐的扩大发酵。在3 L发酵罐内甲醇诱导96 h后,粗酶液酶活为85.12U·mL^-1,50 L发酵罐甲醇诱导108 h后,粗酶液酶活为138.32 U·mL^-1。摇瓶发酵液上清经分离纯化后进行酶学性质的测定。结果显示该酶的最适温度和pH分别是55℃、5.0,pH耐受范围宽泛,在pH 3.5-6.5的环境中处理120 min,仍有60%以上的活性,但该酶的耐热性较差。（2）3 L罐发酵液上清分离纯化后进行PE的脱酯工艺的条件优化。得到最佳脱酯工艺条件:3%果胶浓度、酶量65.4 U·g^-1、脱酯温度50℃、脱酯初始pH 5.5、脱酯时间60 min。在最佳下,该PE将果胶的酯化度从70.8%降至13.6%,是目前酶法脱酯制备低酯果胶的研究中脱酯效率较高且获得的低酯果胶的酯化度最低的研究。（3）使用二硫键计算软件MODIP和BridgeD预测了潜在的二硫键位点,进行定点突变,构建突变菌株。酶学性质分析结果表明,突变位点并不影响PE的最适pH及温度,但突变体的温度稳定性得到明显提高,其中PE-AB的耐热性最好,在90℃保温10 min后仍剩余50%以上的酶活,而野生型PE在80℃保温10 min后即失去活性。最后,对6个突变体的脱酯效率与野生型PE进行比较分析,发现突变位点对PE的脱酯效果影响不大。（4）为了确定突变体热稳定性提高的分子机制,将三个组合突变体和野生型PE的氨基酸序列上传至SWISS-MODEL网站,以黑曲霉的PE晶体结构5c1e为模板,进行同源建模,用PyMOL软件对蛋白质结构进行分析比较,初步认为突变后引入的额外二硫键和氢键是热稳定性提高的主要原因。更多还原

【Abstract】 Pectinesterase（EC 3.1.1.11,PE）is one of the pectinase,belongs to the 8th group of carbohydrate esterase（CE8）,catalyzes the hydrolysis of methyl ester at C-6 of Gal A residue in the homogalacturonic acid（HG）domain of pectin,and the high methoxyl pectin is converted to low methoxyl pectin by de-esterification to produce pectin acid.Pectinesterase is widely applied in food,textile,medicine and other fields.Especially in the food industry,low methoxyl pectin has gained much attention because it is more in line with people’s need for healthy diet with less oil and sugar.At present,domestic commercial pectinase is mostly a mixture of pectinesterase,polygalacturonase and pectate lyase.Like other industrial enzymes,pectinesterase is also faced with problems of low heat resistance and low enzyme activity in industrial production.In this study,Pichia pastoris X33 was used as host bacteria to produce PE.The enzymatic properties of the recombinant PE were studied,and the de-esterification conditions were optimized,and the heat resistance of PE was improved by constructing mutants.（1）The pectinesterase gene from Aspergillus Niger was linked with vector pPICZαA and heterologous expression was carried out in P.Pastoris X33.Firstly,flask fermentation was carried out.According to the fermentation results,a better recombinant strain was screened for 3L and 50 L expand fermentation.The activity of crude enzyme solution was 85.12 U·mL^-1 after96 h of methanol induction in 3 L fermenter,and 138.32 U·mL^-1 after 108 h of methanol induction in 50 L fermenter.The enzymatic properties of the supernatant of flask fermentation broth were determined after separation and purification.The results indicate that the optimal temperature and pH of PE was 55℃and 5.0,and the pH tolerance range was wide.The PE still had more than 60%activity in the environment of pH 3.5-6.5 for 120 min,but its heat resistance was poor.（2）The conditions of PE de-esterification reaction were optimized.The optimal conditions were as follows:3%pectin concentration,65.4 U·g^-1 enzyme amount,50℃de-esterification temperature,initial pH 5.5 and 60 min de-esterification time.Under these conditions,the DE of pectin decreased from 70.8%to 13.6%.At present,it is the research related to enzymatic de-esterification,which has higher de-esterification efficiency and the lowest esterification degree of low methoxyl pectin.（3）The disulfide bond calculation software MODIP and BridgeD were used to predict the potential disulfide bond sites,and site-directed mutation was carried out to construct mutant strains.The results of enzymatic properties showed that the mutant sites did not affect the optimal pH and temperature of PE,but the thermostability of the mutants was obviously improved.Among them,PE-AB had the best heat resistance,and more than 50%enzyme activity remained after holding at 90℃for 10 min,while wild-type PE lost its activity at 80℃for 10 min.Finally,by comparing the de-esterification efficiency of six mutants with that of wild-type PE,it was found that the mutation sites had little effect on the de-esterification efficiency of PE.（4）In order to determine the molecular mechanism of increased the thermostability of mutants,the amino acid sequences of the three combined mutants and wild-type PE were uploaded to SWISS-MODEL website,and 5c1e crystal structure of PE of Aspergillus Niger was used as template for homology modeling.The structures of protein were analyzed and compared by PyMOL,and it was preliminarily concluded that the additional disulfide bonds and hydrogen bonds introduced after mutation might be the main reasons for the improvement of thermostability.更多还原