Biotransformation Of Rare Sugar(Alcohol)D-psicose And Allitol

International Society of Rare Sugars(ISRS)defines rare sugars as monosaccharides and their derivatives that exist in nature but are only present in small quantities.Rare sugars,because of their low calorie and diverse physiological functions,have important application potential in the fields of diet,health care and medicine,and attract more and more attentions of the scientists.The D-psicose studied in this paper is a rare sugar that has attracted wide attention from researchers in recent years.D-psicose has important physiological activities and has great application potential.Another important rare sugar alcohol,allitol,can be obtained by bioreaction using D-psicose as the direct substrate of the catalytic reaction.Allitol is a low-calorie sweetener that can be used in the food industry and drug synthesis.In addition,allitol can also be used as a substrate to synthesize other rare sugars with important physiological activities.At present,there are still some problems in the research of D-psicose and allitol,such as technical difficulties and lack of systematic and comprehensive research,and most of the research remain in the laboratory level,which limit their production and application.This reaearch aims to build a biotransformation method of these rare sugars with high substrate concentration,high conversion rate and simple craft by integrating the microbiology,molecular biology,fermentation engineering,chromatography and biological reaction engineering technologies etc,makes full use of multi-enzymatic reaction and advanced separation equipment,builds up the production process from fermentation,biotransformation,separation and purification to crystallization,and finally realizes the efficient production of D-psicose and allitol.This study is expected to provide experimental and theoretical bases as well as technical support for the industrial production of D-psicose and allitol.This paper mainly includes the following aspects:1.Biotransformation,separation,purification and crystallization of D-psicoseRecombinant E.coli BL21(DE3)-pET22(b)-dpe preserved in our lab was used as the biocatalyst to catalyze D-fructose into D-psicose.The medium for cultivation of the recombinant E.coli strain and the conditions for transformation of D-fructose into Dpsicose were optimized in order to decrease the cost and increase the transformation rate of D-psicose.Finally,the economical medium component for cultivation of the catalytic cells was determined as follows:15 g/L glucose,13.3 g/L KH2PO4,4 g/L(NH4)2HPO4,1.2 g/L MgSO4·7 H2O,1.7 g/L citric acid,10 g/L corn syrup.The optimum conversion temperature and pH were 60℃ and 8.0,respectively.When the substrate concentrations of D-fructose were 100,300,500 and 700 g/L,the optimal cell mass(OD600)was 2.When the substrate concentration of D-fructose was 900 g/L,the optimal OD600 was 3.In order to obtain a large number of recombinant E.coli with high cell catalytic activity,the feeding rate,the time of adding the inducer IPTG and the induction duration were optimized by using 1 L quadruple parallel fermenter.And Dpsicose production was operated in fermentor,and 139.3 g/L D-psicose was received when 500 g/L D-fructose was used as substrate.To further reduce the substrate cost,immobilized glucose isomerase and the recombinant E.coli BL21(DE3)-pET22(b)-dpe were used as the mixed catalysts to catalyze D-glucose into D-psicose.And the dosage of immobilized glucose isomerase and the adding time of the recombinant E.coli BL21(DE3)-pET22(b)-dpe were optimized during the production process.The experiment was carried out in a 10 L fermentor with 550 g/L D-glucose as the substrate under the optimal conditions,and finally 90.7 g/L D-psicose was obtained.Next,D-psicose was separated from the reaction mixture containing D-glucose,Dfructose and D-psicose by using simulated mobile bed chromatography(SMBC)system.The separation parameters were as follows:feed flow rate was 1 mL/min,desorbent flow rate was 20 mL/min,extract flow rate was 10 mL/min,raffinate flow rate was 11 mL/min,valve switching time was 800 s and the circulating water bath temperature was 35℃.Finally,D-psicose crystals with purity up to 99.8%were crystallized by a method combining organic reagent and cooling.2.Biotransformation of allitol with D-psicose as substrateFirstly,two recombinant E.coli strains,one expressing ribitol dehydrogenase(RDH)and formate dehydrogenase(FDH)in fusion(fusion expression strain)and the other expressing the above two enzymes separately(co-expression strain)were respectively constructed.Then,the carbon source in the medium for cultivation of the recombinant E.coli was optimized.The results showed that the recombinant E.coli cultured in the medium supplemented with glucose had higher catalytic activity.Finally,the conditions for the two recombinant E.coli strains in catalyzing Dpsicose to allitol were optimized and the advantages of the two recombinant E.coli strains in the production of allitol were compared.The experimental results showed that the co-expression recombinant E.coli strain had a significant advantage in production of allitol.When the substrate concentration of D-psicose and sodium formate were 500 mM and 1000 mM respectively,the allitol productivity was up to 58.5 g/L/h which was higher than the reports.3.Biotransformation of allitol from D-fructose or D-glucose by multiple enzymatic reactions and crystallization and purification of the productAt first,two recombinant E.coli strains(E.coli BL21(DE3)-pETDuet-1MCSIdpe-MCSIfdh-MCSIIrdh and E.coli BL21(DE3)-pET28(a)-dpe/pETDuet-1MCSIfdh-MCSIIrdh)which simultaneously expressed D-psicose 3-epimerase,ribitol dehydrogenase and formate dehydrogenase in one plasmid or two plasmids were constructed,and the ability of the two recombinant strains in production of allitol were compared.The results showed that the yield of allitol could reach 50%,when the concentration of substrate D-fructose was 100-150 g/L.But,compared with doubleplasmid recombinant E.coli,the single-plasmid recombinant E.coli was more stable and required only one antibiotic,which can effectively reduce the production cost.Next,the single-plasmid recombinant E.coli was immobilized by using polyethylenimine-glutaraldehyde crosslinking method.It was found that the optimum pH and temperature whether the immobilized cells or free cells were 6 and 45℃respectively.In addition,the effects of storage pH and storage temperature on immobilized cells were lower than th ose on free cells,and the reuse stability of immobilized cells were better than that of free cells.Further,immobilized glucose isomerase and the double-plasmid recombinant E.coli were used as mixed catalysts to catalyze D-glucose into allitol.The reaction system containing 50 g/L glucose and 150 g/L immobilized glucose isomerase was reacted at 70℃ and 100 rpm for 40 min.Then sodium formate was added into the reaction system to the final concentration of 37.5 g/L,and the double-plasmid recombinant E.coli was added to OD600 value of 80.After further reaction for 4 h,and finally 12.7 g/L allitol was produced.In addition,in order to obtain a large number of recombinant E.coli cells with high catalytic activity,the recombinant E.coli was cultivated by using fed-batch culture using a four parallel 1 L tank fermentor,and the feeding rate of 600 g/L glucose feeding solution and the IPTG induction conditions were optimized.The optimal conditions for fed-batch culture of the double-plasmid recombinant E.coli strain were that the feeding started at 7 h with the feeding rate of 5 g/L/h and the inducer was added when OD600 reached 9 and the cultivation was continued for further 12 h.The highest total cell catalytic activity of 2894.32 U was obtained and the final OD600 was up to 28.77.The optimal fed-batch culture of the single-plasmid recombinant E.coli strain were that the feeding strated at 6 h with the feeding rate of 2.5 g/L/h and the inducer was added when OD600 reached 9 and then the cultivation was continued for further 24 h.The highest total cell catalytic activity of 3492.47 U was obtained and the final OD600 was up to 37.23.Although the glucose feeding rate was low in the fed-batch culture process of single-plasmid strain,it had higher total cell catalytic activity and cell growth.Therefore,the single-plasmid recombinant E.coli strain was then scaled up in a 10 L fermenter to obtain a large number of bacteria cells and higher catalytic activity.The cultivated single-plasmid recombinant E.coli cells were resuspended using substrate solution containing 100 g/L D-fructose and 75 g/L sodium formate with the final OD600 value of 60.The resuspended broth was poured into a 5 L fermenter,reacted at 45℃and 200 rpm for 3 h,and 63.44 g/L allitol was finally obtained which was the highest report in the world.Finally,allitol cooling crystallization was applied by using uncontrolled and controlled cooling rates,respectively,and the crystallization yield and purity of allitol were investigated.The total crystallization yield of 32%and the purity of 99.7%were obtained by cooling rate uncontrolled crystallization method.The total crystallization yield up to 76.5%and the purity of 99.9%were achieved at a cooling rate of-1℃/min by using cooling rate controlled crystallization method.The way of cooling rate controlled crystallization method was more favorable than the uncontrolled crystallization method.4.Biotransformation of allitol to D-psicose by using recombinant cells expressing NAD(P)-dependent alcohol dehydrogenaseBased on the amino acid sequence of NAD(P)-dependent alcohol dehydrogenase from Gluconobacter frateurii NBRC 3264,the gene sequence of NAD(P)-dependent alcohol dehydrogenase was optimized and synthesized.The recombinant E.coli BL21 star(DE3)-pETDuet-1-MCSIIadh expressing NAD(P)-dependent alcohol dehydrogenase was constructed by genetic engineering technology and was used to catalyze allitol into D-psicose.The product was separated by preparation chromatography and then was identified as D-psicose by HPLC,specific optical rotation and mass spectrometry.Next,the induction conditions for cultivation of the cells and the biotransformztion conditions of allitol to D-psicose were optimized.The optimal induction conditions were as follows:the final concentration of inducer IPTG was 0.2 mM,and further cultivation for 12 h at 20℃ and 100 rpm.The optimal biotransformation conditions were as follows:the optimal pH was 10.0,the optimal temperature was 50℃ and the optimal OD600 was 80.At the substrate concentrations of 10 g/L and 100 g/L allitol,the transformation rates of D-psicose reached 88%and 52%,respectively,which were higher than the transformation rate obtained by epimerase catalyzed reaction.This was the first report on the production of D-psicose from allitol by using recombinant E.coli expressing NAD(P)-dependent alcohol dehydrogenase of Gluconobacter frateurii NBRC 3264.5.Preliminary study on physicochemical properties of NAD(P)-dependent alcohol dehydrogenaseIn order to confirm definitively that the NAD(P)-dependent alcohol dehydrogenase can catalyze allitol into D-psicose,the recombinant E.coli BL21 star(DE3)-pET22(b)-adh expressing NAD(P)-dependent alcohol dehydrogenase was constructed by genetic engineering technology.The ADH was produced by fermentation and was purified by nickel column.And 200 mM imidazole as the eluent could realize the purification of ADH enzyme.Next,the pure ADH enzyme solution was used for the conversion experiment with allitol as substrate.It was confirmed that the purified ADH could convert allitol into Dpsicose.This was the first confirmation that NAD(P)-dependent alcohol dehydrogenase from Gluconobacter frateurii NBRC 3264 can convert allitol into D-psicose.Finally,the effects of temperature,pH,metal ions and substrate concentration on ADH enzyme activity were studied.In particular,Co2+ can significantly increased ADH enzyme activity.When Co2+ was present in the reaction system with final concentration of 1 mM,the yield of D-psicose was 1.6±0.1 times higher than that without Co2+.