Expression And Characterization Of The Novel GH32Hydrolases

Glycoside hydrolases family32include endoinulinases, exoinulinases, sucrases,endo β-2,6-levanases, etc. They hydrolyze β-2,6-fructofuranosidic linkages,β-2,1-fructofuranosidic linkages, or β-D-fructofuranosidic linkages to yield fructoseand FOS. The enzymes have some theoretical significance and various potentialapplications in feed, food, biofuels, washing, sugar, and medicine industries. Atpresent, the research of endolevanase from strain Sphingobacterium sp. has not beenreported. An exoinulinases with tolerance to ethanol, salt, protease, and surfactantwould have great value for basic research and versatile industrial applications but hasnever been reported. Furthermore, there are few reports on the study oflow-temperature active sucrase from microorganisms.In this study, three genes levAGN25, inuAJB13, and sucAHJ14were selectedfrom three bacterial strains Sphingobacterium sp., Sphingomonas sp., and Bacillus sp.,respectively. They encode endo β-2,6-levanase, exoinulinase, and sucrase,respectively. The three genes were inserted into pEASY-E1or pEASY-E2vector andexpressed successfully in Escherichia coli BL21(DE3). The recombinant enzymesLevAGN25, InuAJB13, and SucAHJ14were purified to electrophoretic homogeneityby Ni2+-NTA metal chelating affinity chromatography. Biochemical characterizationsof the three recombinant enzymes and their mutagenesis were detailed as follows:(1) Purified recombinant LevAGN25showed optimal activity at pH6.0and55°C, and retained13.5%of the maximum activity at10°C and32.6%of themaximum activity at20°C. The enzyme activity was stable at50°C and pH5.0-10.0for more than60min. LevAGN25was specific for levan and hydrolyzed the internalβ-2,6-fructofuranosidic linkages to yield FOS as the main products. This is the firstreport on the biochemical characterizations of a novel endo β-2,6-levanase from thestrain Sphingobacterium sp.. These properties suggest that LevAGN25is valuableboth for the basic research and various potential industrial applications, such as food,medical, and aquaculture industries.(2) Purified recombinant InuAJB13was apparently optimal at pH5.5and55°C.The enzyme activity was stable at the concentration of0.2-4.5M NaCl for60min,and the concentration of3.0-15.0%ethanol (v/v) for60min. Trypsin, proteinase K,surfactants, metal ions, and most commercially available liquid detergents had littleeffect on its activity. These properties suggest that InuAJB13is the first report of a novel exoinulinase with tolerance to ethanol, salt, protease, and surfactant. Thepurified enzyme has great value for basic research and versatile industrial applications,such as the mechanism of structural adaptation for high-salt activity, feed industry,washing, and the processing of sea material and saline food.(3) Purified recombinant SucAHJ14showed optimal activity at pH8.0, andremained stable at pH7.0-9.0. The recombinant enzyme displayed the typicalcharacteristics of low-temperatureactive enzyme (exhibiting optimum activity at35°C,26.6%at10°C, and64.0%at20°C; thermolability at≥30°C). SucAHJ14wascompletely inhibited by Hg2+, while activated by Mn2+and β-Mercaptoethanol.SucAHJ14had hydrolysis specificity towards sucrose. These properties suggest thatSucAHJ14is valuable for the basic research on the mechanism of structuraladaptation for low-temperature activity.(4) Based on amino acid sequence comparisons, homology modeling analysis,and site-directed mutagenesis: a. the three essential amino acid residues, includingD34in DP, D151in RDP, and E214in ECP, were involved in LevAGN25activity,among which D34and E214were the putative catalytic residues and D151might playan important role for substrate binding recognition; b. the essential amino acidresidues D40in WMNDPNG was involved in InuAJB13activity, and the mutation ofD40E had no effect on the alteration of acting mode of InuAJB13towards inulin.In summary, the three GH32enzymes were expressed and characterized,including an endo β-2,6-levanase with specific hydrolysis of levan fromSphingobacterium sp., an exoinulinase with tolerance to ethanol, salt, protease, andsurfactant from Sphingomonas sp., and a low-temperature active sucrase fromBacillus sp.. They showed great value for basic research and potential industrialapplications.