| Endo-β-1,4-xylanase is one of the main enzymes in xylanase system,and is widely used in the food industry.However,it is normally difficult to isolate high-yield strains with good enzymatic properties as well as to obtain purified protein due to the complexity of this enzyme system.Therefore,this study constructed a recombinant strain based on Bacillus subtilis to express neutral endo-β-1,4-xylanase from Bacillus pumilus,and compared its enzymatic properties with a commercial enzyme.In addition,the recombinant secretory expression was optimized based on the important role of signal peptide,signal peptidase and PrsA protein.The enzyme production level of recombinant bacteria was further improved by fermentation optimization.The full-length neutral endo-β-1,4-xylanase gene was amplified from B.pumilus genomic DNA and was then ligated to the downstream of the P43 promoter in the p WB980 vector.Thereafter,the recombinant vector was transformed into B.subtilis WB800 to construct the recombinant strain to realize extracellular expression.The enzyme activity in supernatant was5.33 U·m L-1 via shake flask fermentation in LB medium.Via the characterization of enzymatic properties,the optimum p H and temperature were 7.5 and 45 ℃,respectively.The enzyme could maintain a relative stable enzyme activity when the p H was between 6.5 and 8.5 within a range of temperature between 30 ℃ and 55 ℃.Compared with commercial enzyme,the studied enzyme showed higher activity in neutral and low temperature conditions.Although the adaptivity of the enzyme to temperature was slightly poorer,it was found to have a higher stability in alkaline condition.After the screen of 23 peptides originated from B.subtilis 168 genomic DNA,five signal peptides(Yla E,Yfh K,Egl S,Yqx I,Ypj P)were identified with effectivity.The corresponding enzyme activities were 7.15 U·m L-1,6.69 U·m L-1,6.36 U·m L-1,6.32 U·m L-1,and 6.18 U·m L-1,respectively.Subsequently,strain ZS and ZT were constructed respectively by the overexpression of two signal peptidases(Sip S and Sip T).The expression vectors which fused the above five signal peptides were then transformed into these two strains,respectively.It was found that overexpression of signal peptidase had a certain promotion on extracellular enzyme activity.Among these signal peptides,the secretion of endo-β-1,4-xylanase was promoted mostly by Sip S signal peptidase when fusion expression vector with Yfh K.The enzyme activity could be increased to 48.97 U·m L-1 via shake flask fermentation in TB medium.Meanwhile,it was found that there was significant difference in the effect of overexpression of Sip S and Sip T on the secretion of protein fused with different signal peptide.When fused with Yfh K,the secretion would be significantly promoted in strain ZS,while a better performance would be achieved when Sip S was fused in strain ZT.For the other three signal peptides,there was no obvious preference observed for these two signal peptidases,suggesting there was both preference and universality for the effect of signal peptidases on signal peptides.Based on the above result,strain ZP was further constructed by overexpressing the PrsA protein as well as transforming the expression vector fused with Yfh K signal peptide on the basis of strain ZS.Compared with strain ZS,the growth of strain ZP was abnormal,and the enzyme activity was lower(32.70 U·m L-1).In consideration of the possibility that PrsA protein could affect the cell membrane,the growth of the bacterial cell would be impacted when have these two membrane proteins(Sip S and PrsA)overexpressed simultaneously.Finally,the fermentation medium was optimized in shaking flasks.The optimum medium composition is glucose 31.25 g·L-1,cane molasses 5 m L·L-1,maltodextrin 53.73 g·L-1,(NH4)2HPO4 20 g·L-1 and urea5 g·L-1.Fermented with the above medium,the extracellular enzyme activity was achieved as 93.19 U·m L-1 after a fermentation in the shaking bed operated at 30 ℃ and 200 r·min-1 for 120 hours. |
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