The Expression,purification And Its Fermentation Of D-psicose 3-epimerase

Rare sugar is a kind of sugar which exists naturally in nature,but has very little content,such as D-aloxone sugar,trehalose,fructose oligosaccharide and arabinose and so on.Among them,D-aloxone is an important rare sugar with low calorie and high sweetness(similar to sucrose).Some studies have also shown that D-aloxanose has the function of regulating blood sugar,lowering blood lipid and so on.Based on these advantages,D-aloxolose is expected to be a new low-calorie sweet additive.At present,the production of aloxanose mainly depends on the bioenzyme conversion method.Among them,D-aloxone 3-differential isomerase(DPEase)is a kind of important functional enzymes that can convert fructose to aloxone sugar.However,the natural source of DPEase content is very low,can not be expanded production.Therefore,the gene of the DPEase derived from Agrobacterium tumefaciens(At-DPEase)was cloned and expressed.And the expression conditions for At-DPEase in the recombinant ewngineered bacteria were optimized.Finally,the genetic engineering strain with higher At-DPEase enzyme yield was obtained.Firstly,the molecular weight,amino acid sequence,hydrophilicity/hydrophobicity,properties and tertiary structure of the At-DPEase protein are predicted and analyzed by using the Ex PASy protein analysis system website.This couldprovide theoretical basis for subsequent protein expression and purification.At-DPase protein mainly exists as tetramer in the cell.The molecular weight of monomer protein is 32 KDa,the isoelectric point p I=5.88,and the average coefficient of hydrophilicity is-0.195.According to the above prediction results,it is presumed that At-DPEase will be soluble expressed in genetically engineered bacteria E.coli,but if the molecular weight is too large,inclusion bodies will easily form in cells.Subsequently,the codons of At-DPEase protein were analyzed,and the codon preferences of E.coli and Agrobacterium tumefaciens(A.tumefaciens)were compared.The codons of 13 amino acids of At-DPEase protein(translation efficiency<40%in E.coli)were optimized and mutated into a more preferred codon sequence in the host E.coli.According to the optimized sequence,the whole gene of the protein is synthesized,6×Histidine tag sequences are introduced into the C-terminal of the protein,and the expression vector p ET22b-DPEase(O)is constructed by introducing the sequences into p ET 22b plasmid.The recombinant engineering bacterium BL21(DE3)/p ET 22b-At-DPEase(O)was successfully constructed by heat shock method.IPTG was initially used to induce the expression of the target protein.SDS-PAGE showed that the BL21(DE3)/p ET 22b-At-DPEase(O)could successfully express soluble At-DPEase protein.TLC,NMR and HPLC results show that cells expressing At-DPEase protein can successfully convert D-fructose into D-Psicose.On this basis,we optimized the induced expression conditions of BL21(DE3)/p ET 22b-At-DPEase(O)and successfully increased the expression of At-DPEase protein by recombinant genetically engineered bacteria.The optimized conditions include induction time,IPTG concentration,induction time,p H,temperature,inoculation amount,liquid loading amount and rotation speed.Comparing the whole cell transformation activity and the expression level of the target protein,the optimal fermentation conditions of BL21(DE3)/p ET 22b-At-DPEase(O)were determined.The optimal fermentation conditions were as follows:the overnight activated seed solution was inoculated into 50%Lysogeny broth containing 50 ug/m L Amp⁺with 1%inoculum(p H 8.5),cultured in 200 r/mim shaker for 4 h,IPTG was added to the solution to a final concentration of 0.3 mmol/L,and induced in 200r/mim shaker for 5 h.The conversion rate of At-DPEase reached 33.53%,which was 1.7 times higher than that before optimization(19.53%).In the experiment,high purity At-DTE protein was obtained by Ni²⁺-sepharose.Calculations show that the enzyme yield of recombinant genetically engineered bacteria is 3.4 g/L.