Surface Display And Characterization Of A Xylanase Gene From Bacillus Pumilus In E.Coli Expression

Xylan is a poly pentose,which is the major component of hemicellulose in plant cells.The structure of xylan is complicated and has highly heterogeneous branch,so its biodegradation obviously need a complex enzyme catalytic process.As one of the most abundant renewable resource in nature,the degradation products of xylan have considerable application value.Although xylanase has been a hot research area for a long time,and present progress also showed its application ability in some areas,but relatively in xylanase's widely potential application value,there is still a lack of breakthrough study.In the way of the production of xylanase,the in vitro secreted xylanase showed the advantage of application,but because of the cell wall barrier,it is difficult to study.In this study,we connected the gene of Bacillus pumilus NJ-M2 poly enzyme with the gene of green fluorescent protein.By comprehensive use of molecular biology,protein chemical purification and cell surface display technology,we successfully got a high expression of a novel fusion green fluorescent protein-wood xylanase fermentation strains,which showed the recombined xylanase displaying on the surface of Escherichia coli.We purified the fused green fluorescent protein-xylanase.Firstly,we synthesized the gene of xylanase from Bacillus pumilus NJ-M2,and cloned into pETsfgfp vector.The xylanase gene was connected with the 3'terminal of the ORF of the green fluorescent protein and a recombinant plasmid bearing coding sequence of the fused green fluorescent protein-xylanase was constructed.Then,the recombinant plasmid was transformed into Escherichia coli Rossetta Blue.With the shake-flask fermentation,the fused green fluorescent protein-xylanase was checked by fluorescent confocal microscopy,and the results showed the recombinant xylanase successfully displayed on the surface of the Escherichia coli cells.Then we further purified the recombinant xylanase by affinity chromatography,molecular sieve and other protein chemical methods.SDS-PAGE results showed the recombinant protein was 53KD in size,which is the same as theory.The results also showed expression system has a high production rate,the average per liter liquid can get more than 10mg of the purified protein.Finally,we detected enzymatic properties of this new fused green fluorescent protein-xylanase fermentation strains.The enzyme activity assay showed that the bacterial have the ability of hydrolysis xylanase substrate,which further confirmed that the recombiant xylanase was successfully displayed on the surface of Escherichia coli.Its specific activity was determined as 16U/mL.The optimum temperature was 50?,and the optimum pH was 7.5.We also detected the metal ion tolerance of this recombinant protein.The results showed that CO²⁺,Ca²⁺,Mg²⁺,Fe²⁺ could activate this enzyme.For further study,we purified fused green fluorescent protein-xylanase and the single xylanase.The specific activity of recombined xylanase was determined as 1538U/mL,which was higher than that of non-fused-green-fluorescent-protein xylanase.In summary,we constructed a novel fused green fluorescent protein-xylanase fermentation strain in this study.The enzymatic properties of the fused enzyme was also studied.Our experimental data provides an important theoretical basis for future industrialization of this enzyme.