Prokaryotic Expression And Characterization Of GH8 Endo-?-1,4-glucanase From Burkholderia Pyrrolica.

With the development of biotechnology,enzymatic treatment of agricultural waste has become one of the research hotspots.Endo-?-1,4-glucanases(EC 3.2.1.4)belong to cellulose glycosyl hydrolases,which can act on ?-1,4-glucoside bonds and play an important role in cellulose degradation.At present,many endoglucanases have been successfully expressed,but there are still some problems,such as low enzyme activity,enzyme properties can not meet the industrial needs.GH8 endoglucanase has a wide range of substrate specificity,which is very attractive in various industrial applications.In this study,GH8 EG,which comes from Burkholderia pyrrocinia,was used as the research object.The soluble expression of GH8 EG was carried out in prokaryotic system,and its enzymatic properties were systematically studied.The main conclusions are as follows:(1)The gene Bp EG01790 encoding EG was amplified from the genome of Burkholderia pyrrocinia.The soluble expression of the gene was realized on p Cold TF vector,and the enzyme level was higher after signal peptide was removed.The open reading frame(ORF)of the gene fragment is 1218 bp(the content of G + C is 50%),encoding 406 amino acid(AA)residues.The predicted molecular weight and p I are 43.0 k Da and 9.50,respectively.Bp EG01790 has more than 90% similarity with GH8,and the highest homology is 98.28% with GH8 cellulase from B.stabilis.At the same time,multiple sequence alignment showed that the amino acid residues E84 and D145 are the catalytic centers,which are consistent with the highly conserved catalytic active sites of GH8.(2)The gene was cloned into p ET-28(+)expression vector,but the results showed that the gene was not expressed.The soluble expression of EG was achieved by replacing p Cold TF vector,and the enzyme production level was higher after signal peptide was removed.(3)Plackett-Burman design and RSM response surface methodology were used to optimize the enzyme production conditions.The enzyme production level was increased from 3.1 U/m L to 39.9 U/m L.The results showed that the initial p H was 5.9,the post-induction period was 10 h,the pre-induction period was 12 h,the concentration of IPTG was 0.12 m M,the shaking speed was 200 rpm,the induction temperature was 20 ?,and the inoculation amount was 1%.(4)The results of enzymatic properties showed that the optimal p H of EG was 6.0,and the residual enzyme activity remained above 90% in the range of p H6.0-8.0.The optimum temperature is 40-45 ?,which is relatively stable in the environment below 25 ?.At the same time,the enzyme showed 30% of the highest activity at 10 ?,when the temperature increased,the enzyme activity decreased sharply,it belongs to cold-adaptive enzyme.EG has strict substrate specificity and can degrade dextran into oligosaccharides,which has a good application prospect in the production of oligosaccharides.In the early stage of the laboratory,we have been committed to the study of microorganisms in liquor brewing environment.In this study,the soluble expression of GH8 endo-?-1,4-glucanase from Burkholderia pyrrocinia was realized in prokaryotic system,and the GH8 EG was further expanded.The enzymatic characteristics of GH8 were systematically investigated,which provided more choices for industrial enzymes.At the same time,the use of biological enzyme treatment of agricultural waste,efficient environmental protection and improve the added value and utilization rate,solve the environmental problems.