Screening Of Optimal Signal Peptide For Heterologous Xylanase Secretion By Bacillus Subtilis

Xylannase is compound hydrolases of endonuclease and excision enzyme.It can degrade xylan into xylo-oligosaccharide or xylose, in whichβ-1,4-D-xylanase is responsible for hydrolysis of xylan backbone,and has abroad application potential on food ,papermaking,feed and other industrial fields.However, the low enzyme activity increasing cost of production has become a major problem in industrialization.Bacillius Subtilis is a new expression system emerging in recent year,it has simple heredity background,compared with traditional expression system E.Coli,it is no pathogenicity and can secrete extracell protein directly,so its application prospect is very extensive.But the secretion of heterologous proteins must depend on the guidance of Bacillus Subtilis signal peptide itself.However,about 170 signal peptides has been found in B.subtilis,and studies have shown that there is Suitability between the signal peptide and heterologous protein.Therefor screening of signal peptides is effective way for optimization of the export of heterologous protein in B.subtilis.In this study,We constructed a screening vector for signal peptides from B.subtilis . The Alkali resistance xylanase gene(xynA) from Bacillus pumilus(BYG2-20) was chosen as repo- rter gene and cloned into E. coli and B. subtilis shuttle vector pGJ148 which has maltose- inducible promoter PglvMand Spectinomycin resistant gene. 24 Sec-type signal peptides (SPs) were amplify from B.subtilis 1A747 and cloned into screening vector,So we got 24 Xylanase enzymes secreted expression vectors. The plasmid was transformed into Bacillus subtilis WB700 and obtain a series of recombinant strains. The xylanase activity of the culture supernatant were detected after 24h incubation.The screening of these signal peptides revealed , The recombinant strain containing YnfF signal peptide showed the highest xylanase acitivity(37.2U/mL ).In addition ,we fusion xylanase and green fluorescent protein and hope to find the bottleneck of protein secretion by fluorescent labeling in sub-cellular.analysis the reason of differences in xylanase activity of the culture supernatants.Using overlapping PCR fusion xylanase and green fluorescent protein.Choose to pre-screened high secretion signal peptide YnfF secretion and low secretion signal peptide AmyE Clone into the vector.The plasmid was transformed into Bacillus subtilis WB700 and the xylanase activity of the culture supernatant were detected after 24h incubation.Observed Bacteria on Fluorescence microscope.but we did not detected the activity of xylanase.