Purification Of Xylanase From Cellulosimicrobium Sp.and Gene Expression In Pichia Pastoris

Xylanase is a kind of enzyme that degrades the ?-1,4-xyloside bond in xylan molecules,including endo-?-1,4-xylanase and ?-xylosidase.Endo-?-1,4-xylanase in the xylan main chain of the internal?-1,4-xyloside bond,the main hydrolyzate is xylo-oligosaccharides and a small amount of xylose.There are obvious differences in the enzymatic properties of xylanase from different sources,and the properties of the hydrolyzate are not exactly the same.It can be widely used in renewable energy,beer brewing,feed,food and paper industry.In this study,based on the strain of the cellulosimicrobium sp.XM-8,which was screened from the laboratory,the wild enzyme named Xyn-8 with xylanase activity was purified by stepwise separation and its enzymatic properties was analyzed.The results showed that the optimum temperature of the wild enzyme was 60?,the optimum pH was 5.0,which had good temperature stability and pH stability.The enzyme activity was more than 70%after 8 hour of treatment at 50? and below,the activity was more than 80%after 12 hour of treatment between pH 4-10.The enzyme has a strong substrate specificity,only the xylan substrate enzyme activity.The 5 mM Na⁺,Mg²⁺ and Ba²⁺ had a slight effect on the activity of the enzyme.Ag⁺,Hg²⁺ and chemical reagent had a strong inhibitory effect on the enzyme activity,so that the enzyme activity was almost completely lost.The K_m and V_max of the soluble substrate Beechwood xylan were 2.167±0.18 g/L and 740.6± 15.77 ?mol/min·mg^-1.Under the optimum conditions,used as the insoluble substrate,the K_m and V_max were 7.91±0.56 g/L and 806.8±21.34 ?mol/min·mg^-1.The xylanase complete gene was obtained by cloning the design of degenerate primers by sequence comparison.The gene has a 10-family catalytic function with two carbohydrate-binding domains that were expressed by the use of the E.coli system and found that a large number of target proteins are present in the form of insoluble cell bodies.In order to obtain a large number of soluble expression and explored the effects of carbohydrate domain on thermal stability and other enzymatic properties,the recombinant gene Xyn-8a and the gene Xyn-8c,which removed the carbohydrate domain,were ligated into pPIC9K vector.The expression of high density of P.pastoris GS115 cells was carried out.The results showed that Xyn-8a and Xyn-8c obtained good soluble expression in P.pastoris expression system.The expression of Xyn-8c in P.pastoris GS115 was higher than that in E.coli,and the thermal stability was improved greatly.The optimum temperature was increased by 10?,and the temperature stability was decreased from 45? for 0.5 h at 50%increase to 55? for 8h there are still nearly 40%of the enzyme activity.The results showed that the affinity of recombinase and substrate decreased,while the optimum temperature and thermal stability of carbohydrate recombination enzyme were greatly improved,suggesting that the codon affinity and the N-glycosylation structure was different from the E.coli expression system and therefore had an effect on the enzymatic properties.