Identification And Analysis Of A Novel ?-Glucosidase By Functional Screening Of Metagenomic Library

Due to the shortage of global energy and damage to the environment,there is an urgent need to utilize new renewable bio-energy.Cellulose,as a natural renewable resource,is widely distributed and easily available in nature.However,cellulose,a polysaccharide,needs to be degraded into glucose so as to be better utilized.?-glucosidase is a key enzyme which is involved in the final step of cellulose degradation,and catalyzes cellobiose into glucose.In addition,?-glucosidase also has some applications in food,medical treatment,agriculture and other fields,so it is very important to excavate novel ?-glucosidases with high enzyme activity,good stability,and special enzymatic properties.The proposal and development of metagenomics expands the scope of mining unknown functional genes.Metagenomics is a science that researchers extract all microbial DNA from a specific environment to construct metagenomic libraries and discover novel genes through function-based and sequence-based screening.In sequence-based metagenomic approach,novel genes can be identified through the analysis of the homologies between the metagenomic data and known genes.Alternatively,conserved sequences of known genes are used as probe to obtain homologous genes.In function-based metagenomic approach,clones harboring entire functional genes with low homologies to known genes can be discovered through some obvious characterizations such as color change,hydrolysis circle and inhibition zone.In this thesis,the metagenomic library is derived from Everest soil.Due to its complex environment,varied climates,and rich microbial resources,the cosmid library derived from Everest soil contains abundant genes.Because ?-glucosidase can hydrolysis esculin into esculetin which can react with Fe3+to product black substance,clones from the library are screened on LB medium containing the esculin and Fe3+.After screening about 320,000 clones,a novel ?-glucosidase gene zfbg3 is obtained,which belongs to the third family of glycoside hydrolase by blast search.ZFBG3 contains highly conserved amino acid residues Asp(D)and Glu(E)of the GH3 family.These two amino acid residues are related to the catalytic hydrolysis mechanism of the GH3 family.The zfbg3 is cloned and expressed in E.coli BL21(DE3).The size of the target protein after purification is consistent with the predicted molecular weight of 83 KDa.The enzymatic properties of ZFBG3 are studied using pNPG method.The optimum temperature and optimum pH of the enzyme is 55?,5.8,respectively.ZFBG3 could hold activity among wide pH range,and is tolerent to high temperature.Morever,the enzyme has good tolerance to CO(NH2)2.From a comprehensive perspective,ZFBG3 provides technical parameters for the further development of cellulose degradation.