Recombinant Expression,Purification And Characterization Of β-mannanase From Paenibacillus Polymyxa KF-1

Endo-1,4-β-mannosidase,EC 3.2.1.78 is a kind of hemicellulase,which catalyzes the breaking of β-1,4 glycosidic bond in mannan and is widely used in food,medicine and feed industries.A nonpathogenic bacterium Paenibacillus polymyxa KF-1 with hemicellulose and pectin degradation activity was screened in our laboratory.The extracellular fermentation broth was identified by LC-MS/MS,and it was found that endo-β-mannanase was contained in the fermentation broth.Through online analysis software analysis,it was found that endo-β-mannanase had signal peptide,belonging to glycoside hydrolase GH 26.In this paper,Using the genomic DNA of P.polymyxa KF-1 as template,the target gene of β-mannanase,named ppman26 a,was amplified by PCR.The full length of the gene was2001 bp,encoding 667 amino acid residues.The ppman26 a gene amplified by PCR was ligated to the expression vector p ET-28a(+)to construct the recombinant plasmid named PpMan26A-p ET-28a(+)and transformed into Escherichia coli BL21(DE3)competent cells.The final concentration of 0.5 m M IPTG was used to induce the recombinant bacteria,and the soluble expression of ppman26 a in recombinant E.coli was achieved.SDS-PAGE analysis showed that the size of the recombinant protein was about 70 k Da,which was consistent with the molecular weight of the protein expressed by ppman26 a gene fragment.The crude enzyme was purified by Ni NTA his tag purification agarose affinity column.The specific activity of the purified protease was 1849.7 U/m L using KG with final concentration of 5 mg/m L as substrate.The results showed that the optimum pH of the enzyme was 6.0,and more than 50% of the enzyme activity could be retained after 24 h incubation at pH 6.0-10.0;The optimum temperature was 45℃,which was stable in the range of 20-50℃.More than 60% of the enzyme activity remained after incubation at 50℃ for 2 h..The Km and Vmax of KG with final concentration of 5 mg/m L were 2.13 mg/m L and 357.14μmol/min/m L.Because of the strong adaptability of the recombinant enzyme to alkaline pH and high temperature,it has certain application potential.In addition,the hydrolytic ability of recombinant PpMan26 A to LBG、GG and KG was studied.The hydrolytic rates of three kinds of mannans were 32.8%,52.3% and 66.6%,respectively.The results of TLC analysis showed that the main products of the three substrates were mannan oligosaccharides and a small amount of mannose.The results of MALDI TOF/MS showed that the enzymatic hydrolysis product of KG was mannan oligosaccharide with different molecular weights from 3hex+Na to 12hex+Na.The in vitro probiotic activity of mannan oligosaccharide from KG hydrolyzed by recombinant enzyme was studied,It was found that the hydrolysate could promote the growth of Lactobacillus rhamnosus and Lactobacillus plantarum in vitro,and inhibit the growth of pathogenic bacteria,It suggested that the recombinant enzyme could be used in the enzymatic preparation of oligosaccharide prebiotics.Because the free enzyme is not easy to preserve and has poor reusability,the recombinant PpMan26 A was immobilized on the β-cyclodextrin modified sodium alginate matrix for immobilization experiment.Through single factor experiment and PB experiment,the three most significant factors affecting the activity of immobilized enzyme were selected,which were sodium alginate concentration,enzyme dosage and adsorption time.The interaction among the three variables was studied by the steepest ascent experiment and response surface analysis.The results of ANOVA showed that each factor had a significant effect on the immobilization effect(P<0.01).When the concentration of sodium alginate was3.6%,the amount of enzyme solution was 0.9 mg/g,and the adsorption time was 10 h,the best immobilization effect was achieved,and the immobilization rate was 82.3%.The results showed that the optimal pH of immobilized enzyme was 6.0 and the optimum temperature was 45℃,and the optimal pH value and the optimum temperature were not significantly changed compared with the free enzyme.However,the pH stability and thermal stability of the immobilized enzyme were significantly improved.The above studies are expected to lay a theoretical foundation for the application of β-mannanase.