Gene Cloning And Characterization Of GH23 Family Glycosidase

GH23 family glycoside hydrolase mainly includes lysozyme type G,chitinase and peptidoglycan lyase.Chitinase mainly hydrolyzes?-1,4-glucoside bond in N-acetyl glucosamine(NAG)in chitin,while the other two enzymes mainly hydrolyze the glycoside bond in N-acetyl-D-muramic acid(NAM)and NAG in peptidoglycan.Although these substrates are similar,there are differences in the cleavage mode and product type of the three enzymes to the substrate.The research on glycoside hydrolase of GH23 family is of great significance to the mechanism of action of the enzyme.So far,only 31 GH23 family glycoside hydrolases have been preliminarily characterized.The enzymes derived from bacteria mainly belong to chitinase and peptidoglyase,while most of the G-type lysozymes come from eukaryotes and only two of them from Bacillus subtilis.Therefore,it is necessary to study the new enzyme source mining,substrate selectivity and catalytic mechanism of GH23 family glycoside hydrolases,so as to clarify the foundation for the molecular mechanism of Glucohydrolase family catalysis.Acidothermus cellulolyticus 11B can grow at high temperature and high acidity,and produce rich glycoside hydrolase when using complex biomass such as cellulose as carbon source.According to the results of genome sequencing,it was predicted that the genome of the strain contained four kinds of GH23 family glycoside hydrolases.Therefore,in this paper,the related gene cloning,construction of genetic engineering bacteria,expression and characterization of recombinant proteins were carried out.The results of this paper will lay a foundation for further study of the catalytic mechanism of GH23.In this paper,the phylogenetic tree of characterized GH23 family was analyzed.The results showed that the protein ABK52619 sequence in A.cellulolyticus 11B had a close evolutionary relationship with CAB14053 and AIY92440 from Bacillus subtilis lysozyme.Sequence alignment showed that the sequence similarity of ABK52619 with CAB14053 and AIY92440 was about 40%.In this paper,the ABK52619 gene was cloned from the genome of A.cellulolyticus 11B and inserted into pET-28a plasmid.The recombinant plasmid was transferred into E.coli BL21,and the optimum induction conditions were obtained as follows:culture at 27 ? and IPTG concentration of 0.5 mM.The recombinant protein was expressed in soluble form.The recombinant protein was purified by Ni affinity chromatography.The substrate specificity of the recombinant protein was preliminarily tested.The results showed that the recombinant protein did not have lysozyme and chitinase activities,but showed a catalytic activity for 4-nitrophenyl?-D-galactose glucopyranoside.The results of TLC showed that the recombinant protein could hydrolyze lactose into galactose,but no glucose was produced.The optimum temperature and pH of the enzyme were 50 ? and 7.0,respectively,more than 50%of the enzyme activity could be retained after being stored at 60 ? for 6 hours.In this paper,a glycosidase from A.cellulolyticus 11B was cloned,expressed and purified.Preliminary analysis showed that the recombinant enzyme could hydrolyze lactose to produce galactose and showed high thermal stability.At present,the function of lactose hydrolyzing by enzymes in this family has not been reported.