Biotransformation Of Rare Sugar D-psicose And D-allose

Rare sugars are referred to a group of monosaccharides and their derivatives that exist in nature in very low amount and are generally of low caloriy.With the increasing of living standards,people pursuit more of health foods.Traditional high-energy sugars like sucrose will be gradually replaced by low-calorie,functional sugars.Therefore,rare sugars have become a research hotspot in recent years.There are many kinds of rare sugars,different types of rare sugars have their own unique physiological functions.Rare sugars have many applications in food,medicine,and cosmetics industries.D-psicose is a C-3 epimer compound of D-fructose,which is a rare sugar with a sweetness equivalent to sucrose and an energy value of 0.007 kcal/g.Known as a non-energy sweetener,it has important physiological functions such as decreasing blood sugar and blood fat,eliminating active oxygen radicals and being neuroprotection.D-allose is another important rare sugar synthesyzed from D-Psicose as a direct substrate It has wide range of physiological functions,especially excellent anti-cancer effects and assisting in cancer chemotherapy and radiotherapy,and has become a hot spot in the research.However,the current studies on D-psicose and D-allose are not comprehensive and systematic enough,resulting in the low production yield and high price,which limit their applications.Based on above analyses,this study aims to decrease the production cost and increase the production efficiency of D-psicose and D-allose by using multidisciplinary approachs such as microbiology,molecular biology,bioreaction engineering,and modeling and simulation etc.With the goal of achieving breakthroughs in technology and high integration of D-psicose production process,we have established advanced production and separation processes.On the other side,D-allose is produced by L-rhamnose isomerase as a catalyst using D-psicose as the substrate.Further,D-allose is produced using the mixed immobilized enzyme D-psicose 3-epimerase and L-rhamnose isomerase as the catalysts,and low-cost D-fructose as the substrate,which is much helpful for industrial production.The thesis includes the following sections1.Expression of D-psicose 3-epimerase in E.coli BL21Based on the dpe gene sequence of Ruminococcus sp.?5₁₃9BFAA?,the codons were optimized by the online codon optimization software http://gcua.schoedl.de/according to the online codon library http://www.kazusa.or.jp/codon/.The structures of mRNA of the genes before and after optimization were analyzed using the software DNASTAR?GeneQuest?.The free folding energy of the mRNA secondary structure after optimization is-131.71 kJ/mol,which is higher than-152.56 kJ/mol of before,and the mRNA structure after optimization is loose,and helps to improve the translation efficiency.Finally,the optimized gene sequence dpeY was obtained by chemical synthesis.In order to compare the effects of plasmids pET-22b,pET-28a-SUMO and pMAL-c2x on the expression of DPEase,the expression plasmids pET-22b-dpeY,pET-28a-SUMO-dpeY and pMAL-c2x-dpeY were successfully constructed.The plasmid pET-22b-dpeY was finally selected using E.coli as a host.In addition,the plasmid pET-22b-dpeY was transformed into Escherichia coli BL21-GroEL/GroES?DE3?containing the chaperone,and the effect of chaperone-GroEL/GroES on the soluble expression of DPE enzyme was further investigated Finally,we found that the chaperone molecule-GroEL/GroES could increase the soluble expression of DPEase.Furthermore,the cells of the recombinant strain BL21?GroEL/GroES-pET-22b-dpeY were more easily broken than the recombinant strain BL21-pET-22b-dpeY,in obtaining crude DPEase proteins.2.Immobilization of D-psicose 3-epimeraseImmobilization of DPEase was studied by adding 12-amino acid side chain to the C-terminus of DPEase.The addition of acidic amino acid side chains?-DDEDEDEDEDED?could change the isoelectric point?PI?of the DPEase.Compared with the original one,the side-chain C12-DPE has a larger value of |pI-pH| under the same pH condition and has stronger electrical force with ion exchange resin.The immobilized DPE was obtained using ion exchange resin D301 as the immobilization matrix,and the four important factors affecting the immobilization were optimized by single factor and orthogonal test:immobilized carrier?g?:enzyme activity U=1:150,immobilization pH of 8.0,immobilization time of 5 h,and immobilization temperature of 10?The immobilization yield of C12-DPE using the optimized immobilization protocol reaches 91%,and the activity of the immobilized enzyme reaches 60.8 ±2.1 U/g.Then,the enzymatic properties of immobilized and free enzymes were further compared.The optimum pH of 8.0 and the optimum temperature of 60? are the same for both immobilized and free enzymes.The immobilized enzyme has better thermal stability and broader pH and temperature ranges than that of the free enzyme.After reusing for 15 times,more than 70%of enzyme activity is still retained for the immobilized enzymeFinally,0.5 g of immobilized enzyme was added in every milliliter of the transformation solution,30%D-psicose could be obtained after 6 h transformation of 100 g/L D-fructose.3.Secretion expression of D-psicose 3-epimerase in B.pumilus SP3The gene dpe was inserted into the shuttle plasmid pNCMO2,and the recombinant plasmid pNCMO2-dpe was successfully constructed.The recombinant strain SP3-pNCMO2-dpe was constructed by electroporation transformation using SP3 as the host strain.Protein expression reached the highest level after 72 h of growth after optimizing the expression conditions.The secretory protein in the supernatant of the culture medium takes over about 60%analyzed by using Western-blot.Further,the immobilized enzyme was obtained by directly mixing the ion exchange resin D301 with the culture medium.The secretory expression of DPEase further simplifies the step of enzyme immobilization.Finally,recombinant cells with the non-secreted DPEase were immobilized using chitosan and sodium alginate.29.3%of D-psicose can be obtained from 100 g/L of D-fructose solution after 6 h reaction using the immobilized cells as the catalyst.4.Separation and purification of D-psicoseSeparation of D-psicose from D-fructose was achieved by using simulated moving bed?SMB?chromatography and bio-enzymatic conversion method.98.5%of D-psicose was obtained by using SMB and 91.2%of D-psicose was got by using bio-enzymatic conversion method.The purity of more than 99%of D-psicose can be obtained by crystallization from 80%of D-psicose.The principle of bio-enzymatic conversion method is to convert D-fructose into D-glucose by using the immobilized glucose isomerase?GI?at first,and then convert D-glucose into gluconic acid by using the immobilized glucose oxidase?GOD?,finally,gluconic acid is removed by adsorption using ion exchange resin.In the bio-enzymatic conversion method,the series connected continuous stirred tank reactor?CSTR-CSTR system?and the mathematical model for removal of D-fructose were constructed based on the enzyme kinetic parameters of the immobilized GI and GOD.5.Decolorization and crystallization of D-psicoseThe suitable decoloring method by using activated carbon was selected after comparing the three decoloring methods of resin,hydrogen peroxide and activated carbon.A good decolorization result of decolorization rate of 89%was obtained using the optimal decolorization conditions obtained by using single factor and orthogonal experimental design:activated carbon content of 3%,adsorption time of 60 min,and temperature of 60?.In addition,two crystallization methods of organic solvent addition and cooling were investigated,and the cooling method was used after comparation of the crystals formed by using the two methods.The specific step of it is:concentrate the sugar solution until its concentration is greater than 75%by using a rotary evaporator,and then add 0.1%D-psicose crystals of 400 meshes as the seeds,finally,decrease the temperature from 65? to 4?.After about 20 h,the crystallization was completed.The shape of the crystals obtained by cooling method is better,and the purity of the crystals is as high as 99.6%,which is equivalent to the purity of standard reagents sold on the market.6.Expression and immobilization of L-rhamnose isomerase and enzymatic transformation of D-alloseFirstly,the l-rhi gene from Bacillus subtilis 168 was cloned and the plasmid pET-22b-l-rhi was constructed.In order to compare the effects of host strain E.coli BL21?DE3?,E.coli BL21star?DE3?and E.coli Transetta?DE3?on the expression of L-RhI,the recombinant strains of BL21-pET-22b-l-rhi,BL21star-pET-22b-/-rhi and Transetta-pET-22b-l-rhi were constructed.The results showed that E.coli BL21star?DE3?with the mutant gene rne131 enhanced the expression of L-RhI possibilly by stabilizying the mRNA of the recombinant gene in the cells.Further,effects of different resins on the immobilization of L-rhamnose isomerase were compared,and amino resin cross-linked by using glutaraldehyde was finally chosen.The optimal protocal for enzyme immobilization was obtained based on the single factor and orthogonal experimental designs:the ratio of immobilized media?g?to enzyme activity?U?is 1:120,the concentration of crosslinking glutaraldehyde of 2%,the temperature of 25?,and the immobilization time of 10 h.About 89.5%of total L-RhI activity can be immobilized by using the optimized protocal and the activity of the immobilized L-Rhl is 48.5±1.1 U g-1.Then,the enzymatic properties of the immobilized enzyme L-RhI were studied.The optimum pH was 9.0,and optimum temperature was 70? for the immobilized L-Rhl.The immobilized L-RhI has a wider pH range and higher thermal stability than the free one.Also,the enzyme activity could maintain over 78%after reuses for 10 times.About 27.5%of D-allose could be obtained from 10 g/L D-psicose by using immobilized enzyme L-RhI as the catalyst.Finally,D-Allose can be efficiently produced by using one-step enzymatic process with the cheaper initial substrate of D-fructose than D-psicose.Using the low-cost D-fructose as substrate,the reusable immobilized enzymes of D-psicose 3-epimerase and L-rhamnose isomerase as the catalysts,the mass ratio of D-fructose,D-psicose and D-allose could reach at 6.0:2.4:1.6 after 7 h reaction performed at 65?.