Expression Of Xylanase Gene In Bacillus Subtilis Induced By Stresses

Xylan constitutes an important part of plant cell walls and is hard to be digested byanimals. Xylanases are glycosidaseswhich catalyze the endohydrolysis of1,4-β-D-xylosidiclinkages in xylan. β-1,4-D-xylanase can depolymerise xylan by the random hydrolysis ofxylan backbone, have promising applications in a wide range of industrial processes,including of animal feed, paper industry, food, textiles and ethanol production.Bacillus subtilis, regarded as a kind of food-grade expression host bacteia, has someimpotant advantages, including of clear genetic background, non-pathogenicity and secretionof target protein. Now it has been the second largest prokaryotic expression system afterEscherichia coli. Recently most Bacillus subtilis expression vectors are inducible, which canexpress target protein abundantly while adding certain inducer. However, most induciblepromoters used in these vectors exist some faults as follows:(i) low transcriptional intensity;(ii) inducers are very expensive;(iii) inducers have toxic effects on the host cell. Previousstudies have shown that expression levels of target genes, which were guided by Bacillussubtilis PgsiB promoter, have greatly improved than normal conditions under stressconditions.In this study, we constructed xylanase expression vector in Bacillus subtilis inducedby stresses, which contains xylanase gene (xynA) under control of promoter PgsiB. The genexynA was screened from Bacillus pumilus BYG5-20N, and we optimize conditions of stresstreatments. Subsequently, we subsituted signal peptide of xynA itself with other6signalpeptides from Bacillus subtilis and research effects of signal peptides on expreesion andsecretion of xylanase under the condition stresses.(1) Alkali resistance xylanase gene xynA was amplified from Bacillus pumilusstrain BYG5-20N genome by PCR; promter PgsiB fragment was amplified from prototrophicBacillus subtilis strain1A747. These two gene were connected to E. coli-Bacillus subtilisshuttle expression vector constructed formerly, leading to new expression vector pBXS. Thenwe transformed pBXS into B. Subtilis strain WB700and got the recombinant strain BXS-W.When strain BXS-W was grown in LB medium to the mid-logarithmic growth phase, we imposed five kinds of stress treatment including heat stress, ethanol stress, salt stress,oxidative stress and hypoxic stress. The results showed that these five stresses couldsignificantly improve the expression of xylanase gene (P<0.01) and ethanol stress was betterthan other four stresses.(2) In order to find a better stress treatment condition and increase xylanaseproduction, we chosed heat stress, ethanol stress and salt stress to design full-scale testing.The result showed that activity of xylanase can reach to the maximum,30.89U/ml, when weimposed strain BXS-W to2%ethanol+2%NaCl+45℃culture condition.(3) Signal peptides play a crucial role in the stability and maturation of proteinprecursors. In this experiment, we subsituted signal peptide of xynA itself with other6signalpeptides from Bacillus subtilis, got recombinant Bacillus subtilis strains. All of these strainswere imposed to2%ethanol+2%NaCl+45℃culture condition in order to induce xylanaseexpression. The result indicated that different signal peptides had some effects of xylanaseexpression. PhoB, PhrC and AbaA could increase production of xylanase significantly, SacCand DacB had no effect on xylanase expression and WapA reduced its production.