| 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. |
