Research On Expression And Enzymatic Characterization Of Fructosyltransferases AoFT From Aspergillus Oryzae

Fructosyltransferases are a group of enzymes(EC2.4.1.9)that catalyze the transfer of fructosyl units from one sucrose to another one.Sucralose is a new non-nutritive oligofructose,it is one of the most perfect and competitive sweetening agent by far.The sucrose-6-acetate is a key intermediate in the conventional synthesis pathway.Biological enzyme method to product sucrose-6-acetate is a hot topic recently,so fructosyltransferases is capable of synthesis sucrose-6-acetate from glucose-6-acetate and sucrose.The method has many advantages,such as specificity,efficient,less by-product,high percent conversion,so the method has a strong application potential.In our previous experiments,we screened a new fructosyltransferase(AoFT)from the fermentation broth of a strain of Aspergillus oryzae,and researches show that cell extract has a high activity in synthesis of sucrose-6-acetate.The purpose of this study was to clone and express the fructosyltransferase AoFT in the E.coli BL21 and characterize its enzymology properties.As the E.coli is intracellular expression system,product purification step is relatively complex,we will the fructosyltransferase AoFT gene express in the Pichia pastoris and research its properties,analysis the differences of the enzymology properties from the E.coli and pichia respectively,through the analysis of the glycosylation sites,explain the change of the enzymology properties.The specific research contents were as follows:(1)The target gene fructosyltransferase AoFT Aspergillus oryzae was cloned by the method of RACE-PCR and the recombinant plasmid pET22b-AoFT were constructed.Then the recombinant plasmid was transformed into E.coli BL21 and the recombinant enzymes were expressed induced by IPTG.Finally,the purified enzyme was obtained through ultrasonic crushing,centrifugal and nickel column affinity chromatography purification methods.The enzyme activity was determined.(2)The catalytic properties of the fructosyltransferase AoFT were researched including the optimum reaction temperature,the optimum pH,the thermal stability.The influence of some impact factors on the fructosyltransferase AoFT activity were futher studyed including metal ions,organic solvents,surfactants,reducing agent and protein inhibitors.(3)The target gene fructosyltransferase AoFT was obtained by double digests of the recombinant plasmid pET22b-AoFT,and the recombinant plasmid pPICZaA-AoFT were constructed.The plasmid after linearization by the Sac I enzyme electricity into pichia X33,X33 engineering bacterium expresse protein induced by methanol,the purified enzyme was obtained through collecting fermented liquid column and(NH4)SO4 precipitation and nickel affinity chromatography steps,and then to determine enzyme activity.(4)Analysis the differences of the enzymology properties from the E.coli and pichia respectively(temperature,pH,etc.),through the analysis of the glycosylation sites,explain the change of the enzymology properties.The results were as follows:(1)The recombinant enzyme fructosyltransferase AoFT was expressed stably in recombinant bacteria E.coli BL21.The target protein was purified and the molecular mass was about 68KDa showed by SDS-PAGE.The purification recovery rate of target protein was 33.6%and the specific activity was 65.8U/mg.(2)The optimal function temperature and the optimum pH of fructosyltransferase AoFT from BL21 were respectively 50? and 6.0,and more than 80%of its activity was recovered at 30-50?after treated for 1h.The fructosyltransferase AoFT also has high pH stability,and more than 80%of its activity was still kept after treated at pH 5.0-7.0 for 1 h.In addition,the enzyme has a good resistance to the majority of the metal ions,surfactant(Tween20,Triton-X100)and organic solvent(methanol,ethanol,acetone,n-butyl alcohol,formaldehyde,DMF,DMSO etc.).The enzyme activity was strongly inhibited by SDS and urea,but the enzyme activity is not affected by EDTA,which shows that the enzyme is not a metal enzyme.(3)The recombinant genetic engineering bacteria X33 expresse fructosyltransferase AoFT induced by methanol,The target protein was purified and the molecular mass was about 90KDa showed by SDS-PAGE.The purification recovery rate of target protein was 70.6%and the specific activity was 71.4U/mg.The optimal reaction temperature was 45 ?,and more than 50%of its activity was recovered at 20-60? after treated for 1h.The optimum pH was 5.5,and relative enzyme activity are kept at more than 60%in pH4.0-7.0 range..Most of the metal ions,common surfactants and EDTA has not obvious effects on the enzyme activity,and the enzymes in organic solvents has good resistance.In addition,reductant DTT has inhibitory effect to the enzyme activity,the more and more concentration of inhibition;and high concentration of inhibitor of the enzyme inhibitory effect is more apparent.(4)The fructosyltransferase AoFT from pichia expression had high enzyme activity,compared to the fructosyltransferase AoFT from E.coli expression,and they had similar enzymology properties,but the fructosyltransferase AoFT from pichia expression was closer to natural fructosyltransferase AoFT,this is because the pichia expression system had a high level of modification and processingof target protein after translated,some glycosylation modified results.