| ?-mannanase is a kind of hemicellulase,which can catalyze the random hydrolysis of ?-1,4-mannosidic linkages in the main chain of mannan by releasing short-and long-chain oligomannosides.?-mannanase can be useful in several process in the oil and gas wells development,paper bleaching,feed supplement industry,food additive field and pharmaceutical industry,demanding is gradually increasing.Therfore,the development and research of ?-mannanase is becoming hot issue of common concern.So it is vital significance to screen high yield strains of producing?-mannanase for the large-scale application and the engineering bacterium construction of ?-mannanase.The thesis mainly had been studying on three aspects as follows,namely screening and identification,gene cloning and expression,and enzymatic characteristics.1.The strain B19 with high yield of ?-mannanase(149.3 U/mg)was isolated from forest soil sample of Henan province,Xinyang city,using first screening(konjac powder as the sole carbon source and congo red staining method)and secondary screening(determination of ?-mannanase enzyme activity by DNS method).The strain was identified as Enterobacter aerogenes of Enterobacter sp.by electron microscopy,Gram-staining,physiological and biochemical identification,16 S rDNA sequence and phylogenetic tree analysis.The strain was named Enterobacter bugandensis B19,and the GenBank accession number was KU500561.2.By using bioinformatical methods,the amnio acid sequence of Enterobacter sp.produced ?-mannanase were aligned and analyzed.A set of primers were designed on basis of the conserved motifs.The ?-mannanase gene(1047bp)was cloned and encoded 348 amino acid residues showing the highest amino acid sequence identities of 98% with the putative glycosyl hydrolase(GH)family 26 ?-mannanase from Pantoea agglomerans strain A021(GenBank No.ACN30272)with a estimated molecular mass of 38.6 kDa.To efficiently express manB19 in E.coli BL21(DE3),manB19 gene was inserted into pET-28a(+)to yield a recombinant vector pET-28a(+)-manB19.Then the recombinant vector pET-28a(+)-manB19 was transformed andexpressed in E.coli BL21(DE3).After shaking overnight at 37?,200 r/min,IPTG was added at a final concentration of 1.0 mmol/L for 6h,the recombinant protein showed the highest expression with 497.9 U/mg and the protein concentration was0.203 mg/mL.The crude recombinant ?-mannanase was purified by a 6×His-Tag Protein Purification kit.The molecular mass of the purified ?-mannanase was estimated to be 38.6 kDa by SDS-PAGE analysis and the specific activity reached497.9 U/mg and protein concentration reached 0.203 mg/m L.3.The enzymatic characteristics analysis showed that the optimal temperature was around 55?,and it retained relatively stable at 55 ~ 60? for 60 min.Meanwhile,the optimal pH of the recombinant ?-mannanas was around 6.0 and was stable in the range from pH 4.0 ~ 10.0.In addition,Co2+ and Mn2+ markedly enhanced the recombinant ?-mannanase activity,while other metal ions had a different degree of negative effect on this enzyme.Km and Vmax of it using Locust bean gum as the substrate were 7.16 mg/mL and 117.64 ?mol/mL.min,respectively. |
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