Isolation,Purification And Properties Of Extracellular Pectinase Produced By Valsa Mali

Apple valsa canker is an apple tree branches skin disease caused by Valsa mali Mayabe et Yamada,which is a major disease on apple trees.This disease has seriously hampered the development of our apple industry.Various traditional methods demonstrated helplessly in the management of apple valsa canker,the reason should be attributed to the unclear pathogenic virulence factors and its pathogenic mechanism.It has been proved extracellular pectinase play important roles in pathogen infection process,but there is no research on the isolation of Valsa mali extracellular pectinase.In this work,the relationship between pathogenicity of Valsa mali and extracellular pectinase was studied.Then the optimal carbon and nitrogen sources for enzyme production was screened.Furthermore,the isolation,purification and enzymatic properties of Valsa mali extracellular pectinase were explored.The results are as follows:1.Studying the 3 kinds of Valsa mali with different virulence 03-8,X907 and T1 cultured in the same condition,we found a positive correlation between the virulence and the extracellular pectinase activity in first 10 days.Therefore,it indicated that pectinase was one of the pathogenic factors for this fungi.2.It showed that pectinase activity was relatively strong in the first 10 days fermentation in culture medium MS with 0.50%starch and 0.25%peptone as carbon and nitrogen sources respectively.3.The Ammonium sulfate gradient precipitation of fermentation showed that,when reaching 70%saturation,the total active part will be completely precipitated.4.There was a pectinase 2-S-2 purified by Sephacryl S-100 chromatography and DEAE-Sepharose Fast Flow chromatography.It was an electrophoretically pure enzyme detected with gel electrophoresis(SDS-PAGE).5.According to Rf-LogMr chart of the known molecular weight standard proteins in SDS-PAGE,we obtained 2-S-2 subunit molecular weight of 58.83 kDa.After LC-ESI-MS/MS techniques,by comparison to the NCBI database,2-S-2 was identified as rhamnogalacturonase with pI of 6.03.6.The study of properties showed the optimal temperature and pH of 2-S-2 were about 40 ? and pH 3.5,this enzyme was stable at pH 2.2-5.5 and temperature below 30 ?.The activity of the enzyme was strongly activated by Ca2+ and partially inhibited by K+,Fe2+,Pb2+,Zn2+,Cu2+,Mn2+ and Ni+,especially after adding Ba2+ and Mg2+,the activity of the pectinase was not detected.Other metal ions such as Li+ and Co2+ had little effects on the enzyme activity.7.The apparent Km and Vmax.value of 2-S-2 were 3.600 mg·mL-1 and 0.1627 mg/(mL·min)for the pectine,respectively.