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

β-1,3-Glucanase is considered as a useful enzymatic tool forβ-1,3-glucan degradation to produce（1→3）-linkedβ-glucan oligosaccharides with pharmacological activity properties.The oligosaccharides are that hydrolysis of polysaccharides from P.cocos have anti-tumor and immunomodulatory activities.β-1,3-glucanase can act onβ-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 withβ-1,3-glucanase activities and studying the enzymatic properties ofβ-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 usingβ-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 exhibitedβ-1,3-glucanase activity.The isolate SYBCQL belonging to the genus Kitasatospora withβ-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 encodingβ-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 threeβ-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.