Isolation Of ?-1,3-glucanase-producing Actinomycetes And The Enzymes Genes From Them Were Cloned And Expressed

?-1,3-Glucanase is considered as a useful enzymatic tool for?-1,3-glucan degradation to produce?1?3?-linked?-glucan oligosaccharides with pharmacological activity properties.The oligosaccharides are that hydrolysis of polysaccharides from P.cocos have anti-tumor and immunomodulatory activities.?-1,3-glucanase can act on?-1,3-glycosidic bonds of soluble or insoluble polysaccharides in P.cocos to form oligosaccharides and promote its biological activity.Thus,it is meaningful and potential that screening isolates with?-1,3-glucanase activities and studying the enzymatic properties of?-1,3-glucanase for application.The study was to:?1?analyze the diversity and structure of microbial communities in soil samples by using the method of high-throughput sequencing,?2?isolate and identify glucan-degrading isolates,?3?degrade pachyman by using?-1,3-glucanase.Our findings collectively showed as follow:?1?The result of high-throughput sequencing show that the genera Streptomyces?1.90%?and Arthrobacter?0.78%?belonging to the order Actinomycetales?8.64%?in the phylum Actinobacteria?18.64%?were observed in soil for P.cocos cultivation?FTL₁?.The abundance of Actinobacteria is second only to Proteobacteria.Thus,FTL₁ is a preferred source for screening glucan-degrading actinomycetes.?2?Actinomycetes were isolated from FLT₁ by using dedicated medium.Out of 58 isolates,only 11 exhibited?-1,3-glucanase activity.The isolate SYBCQL belonging to the genus Kitasatospora with?-1,3-glucanase activity was found and reported for the first time.The isolate SYBC17 displayed the highest yield 1.02 U·mg^-1 among the isolates.The time of optimal fermentation in SYBC17 was 96 h.At this time,polysaccharides from P.cocos could be degraded well by SYBC17.?3?Two genes 17-W and 17-Q encoding?-1,3-glucanase in the isolate SYBC17 and the gene QLK1 in the isolate SYBCQL were cloned and analyzed for verifying in molecular level.The activities of the recombinant enzymes QLK1,17-W and 17-Q were 65.82 U·mg^-1,132.90 U·mg^-1and 14.70 U·mg^-1,respectively.17-W is the most productive isolate in all of purified recombinant enzymes.?4?Bioinformatics analysis revealed that all of enzyme proteins belong to the glycoside hydrolase family16,which contain a catalytic domain and a carbohydrate domain.The carbohydrate domain of QLK1 and 17-W was CMB13,the carbohydrate domain of 17-Q was CMB6.Three proteins were conducted to homologously modeled and the laminaribiose molecule were successfully modelled into the active-site cleft of three?-1,3-glucanase respectively.?5?The optimum temperature of QLK1 was 60°C,the optimum temperature of 17-W and17-Q was 55°C.The optimum pH of QLK1 and 17-Q was 5.5,the optimum pH of 17-W was 6.0,Three enzymes have good thermal stability and pH stability.The order of affinity of the three recombinant for the substrates was 17-W,QLK1,and 17-Q.?6?Pachyman were degraded by recombinant enzymes 17-Q and 17-W.TLC analysis of the products showed that the products of 17-W were two types of oligosaccharides under optimal conditions,and the products from 17-Q were three types of oligosaccharides.