Screening Of High-Yielding Pectinase Strains,Optimization Of Fermentation And Study Of Enzymatic Properties

Pectinase is commonly used in fruit juice beverage processing,feed additives,paper industry and other fields.For the juice beverage processing industry,pectinase has the effect of increasing the juice yield,reducing the process flow and decreasing the production cost.At present,in industrial production the specific enzyme activity of pectinase is relatively low.This thesis aims to screen a wild-type strain with higher pectinase activity than enzyme activity from the natural environment,and conduct research from the screening and identification of wild-type strains,the optimization of fermentation technology,the separation and purification of enzymes,and the properties of enzymes,to provide a certain theoretical basis for the production and application of pectinase.The research content of this article is as following:(1)Fifteen wild-type pectinase-producing strains were selected from soil from different sources such as strawberry plantations,vineyards,peach orchards,pear orchards,wheat fields,rape fields,and bayberry trees.Strains were separated and purified by plating streaking,Congo red staining to produce clear circles to screen pectinase-producing strains,and liquid fermentation to screen the strains with the highest pectinase activity to determine the experimental target strain CM3.The target strain CM3 was identified by microscopy,18S r RNA,and its sequence homology analysis.It was noted that the target strain was 99%similar to the strain Aspergillus sp.ZH14,so the target strain CM3 was named Aspergillus sp.CM3.(2)The fermentation process of the target strain CM3 to produce pectinase was optimized through a single factor experiment,culture condition optimization experiment and orthogonal experiment.The optimal fermentation process conditions are:bran 10 g/L,ammonium chloride 0.2 g/L,calcium chloride 0.08 mol/L,initial p H value3.0,fermentation temperature 28°C,inoculum volume 4%,liquid volume 40 m L.Under these fermentation conditions,the enzyme activity of the strain CM3 to produce pectinase reached 3942.7 U/m L,which was86.4%higher than that before the optimization of the fermentation process.(3)Use ammonium sulfate precipitation,disfiguration to remove salt,anion chromatography column Q Beads 6FF chromatographic separation,separation and purification of pectinase produced by liquid fermentation of strain CM3,after polyacrylamide gel electrophoresis and non-denaturing gel Electrophoresis experiments proved that the pectinase produced by the target strain is a single pectinase,with a protein molecular mass of about 38.34 KDa,a purification factor of 3.26 times,and an increase in specific enzyme activity to 174319.6 U/mg.(4)Assess the enzymatic properties of the purified pectinase.The optimum reaction temperature of pectinase is 50°C,and the optimum p H is 4.0.After treatment at 30?50°C for 2 h,the enzyme activity is still it can be maintained above 85%.In the range of p H 3.0?5.0,the enzyme activity of pectinase can be maintained above 80%within 2hours.the research results show that Ca²⁺,Co²⁺,Mg²⁺promote pectinase,and Mn²⁺has a strong inhibitory effect on pectinase.Substrate specificity experiments show that the purified pectinase in this study is a single pure enzyme,its K_mvalue is 25.14 mg/m L,V_max is 2.46 mg/(min·m L).The results of the fruit juice clarification experiment show that the purified pectinase acts on the juice clarification of apples and oranges,and the juice yield of the two fruits is increased by about 20%,which indicates that the purified pectinase can be used for juice clarification...