Screening Of ?-glucosidase-producing Bacteria From Coral And Genes Expression For Preparation Gentiooligosaccharides

?-Glucosidase is a kind of cellulase,which is widely distributed in bacteria,fungi,plants and animals.It can be used to produce biofuels,isoflavones,enhance the flavor of fruit wine and synthesize gentiooligosaccharide.At present,?-glucosidase's industrial application was limited due to its low activity and poor glucose tolerance and other factors.Therefore,looking for ?-glucosidase with high enzyme activity and high glucose tolerance is of great significance in application.There are many kinds of symbiotic and epiphytic microorganisms in corals,which may contain new biological resources ?-glucosidase.In this study,the symbiotic and epiphytic microorganisms of coral were obtained by sucrose gradient centrifugation and in-situ simulation.The main results are as follows:(1)285 strains of bacteria were obtained sucrose density gradient centrifugation and in situ simulated culture.87 ?-glucosidase producing strain were obtained after functional screening.At the phylum level,they were Proteobacteria(67.82%),Firmicutes(25.29%),Actinobacteria(6.89%).And the dominant genera were Photobacterium sp(16.09%)and Paracoccus sp(13.79%).(2)The total DNA metagenomic library of ?-glucosidase-producing bacteria was constructed.High-volume sequencing and genomic annotation showed that total of 68 full-length ?-glucosidase hydrolyzing enzyme family 1(GH1)and 84full-length ?-glucosidase hydrolyzing enzyme family 3(GH3)were obtained.Three novel genes were screened from GH1 and GH3 families ?-glucosidase gene was expressed.The Km of mg 132,mg163 and mg9373 were 1.358 m M,0.319 m M and 15.113 m M,respectively,and the enzyme activities were 188.44 U/mg,341.41 U/mg and 372.23 U/mg,respectively.In 20% glucose solution,they still maintained more than 50%,30% and 25% of enzyme activity,respectively,showing good glucose tolerance.(3)The optimum conditions for the synthesis of gentiooligosaccharide by recombinant enzyme mg163 were as follows: 40 ?,p H 7.0,enzyme dosage 21.6U/g glucose,glucose:cellobiose=30%:15% for 48 h.The highest conversion of gentian oligosaccharide reached 15.63%.