The Screen Of The Strain Producing Levansucrase, Purification And Characteirzation Of Levansucrase And Enzymatic Synthesis Of Levan

Levan is a kind of homopolysaccharide consisting of a sucrose molecule that iselongated by a chain of fructosyl units connected through β-（2â†'6） linkages. However, theability of microorganisms producing levan has a great deal of difference and only a few ofthem have a high yield of levan, which limits the large-scale industrialization and applicationof levan. Beseides, the complex process of purifying levan also limits its production andapplication. Once the limitations are solved, the market of levan will gradually increase inmany fields. Thus it will have great significance that a new strain producing levan with highyield is screened.In this study, a novel levan producing strain Bacillus methylotrophicus SK21.002wasisolated from the soil of beet gardens. The structure of the biosynthetic product was evaluatedby sugars analysis, relative molecular weight measurement, Fourier transform-infraredanalysis and NMR analysis. In this paper, the culture medium and culture conditions ofBacillus methylotrophicus SK21.002producing levansucrases were investigated. Bysingle-factor experiments and orthogonal test, the optimal culture medium was determined asfollows: sucrose80g/L, yeast extract10g/L, tryptone10g/L, K2HPO48g/L, MgSO40.6g/L,NaCl2g/L, while the optimal culture conditions were initial pH6.5, fermentationtemperature30°C, medium volume30%（v/v）, inoculation ratio2%（v/v）, and fermentationtime21h. Under these conditions, the yield of levansucrases reached7.28U/mL.Levansucrase from Bacillus methylotrophicus SK21.002was purified by ammoniumsulfate fractionation, DEAE ion-exchange and gel filtration chromatography. The enzyme waspurified to homogeneity level with11.78-fold purification and a recovery of9.28%, thespecific activititiy of purified levansucrases was135.40U/mg. Its molecular mass wasestimated to be60kDa by gel filtration chromatography and SDS-PAGE.The properties of levansucrases were investigated. The enzyme exhibited optimumtransfructosylation activity at40°C, whereas maximal enzymatic hydrolytic activity wasmeasured at45°C. The maximum transfructosylation and hydrolytic activities both wereobtained at pH6.5. Cu²⁺, Fe²⁺, Zn²⁺and Ni²⁺inhibited both transfructosylation activity andhydrolytic activity up to100%, while Mn²⁺only inhibited hydrolytic activity distinctly. Ca²⁺and Mg²⁺stimulated the transfructosylation activity and hydrolytic activity obviously. Whenthe fructose concentration was below0.2mol/L, higher fructose concentration could promotethe transfructosylation activity and inhibit the hydrolytic activity. Chemical modifiers NBSand PMSF showed strong inhibition of the hydrolytic activity and transfructosylation activityof levansucrase, which indicated the tryptophan residues and hydroxy groups of Thr or Serwere essential to enzyme activity. Kmand V_maxvalues of the purified levansucrase were114 mmol/L and8.25μmol/（mL·min）, respectively.On the basis of the enzyme characteristic research, the optimal conditions of producinglevan were studied. The optimal sucrose concentration, enzyme dosage, and pH of levanproduction were300g/L,6U/g sucrose, and6.5, respectively. Its yield increased15%in thepresence of50mmol/L Mg²⁺. The optimal temperature and time of levan biosynthesis were37°C and16h, respectively. Under these conditions, the maximum levan yield finallyachieved100g/L.