Method for preparing xylitol through co-immobilized enzyme coupling ion exchange continuous catalysis

Abstract

The invention belongs to the field of biochemical engineering, and particularly relates to a method for preparing xylitol through continuous catalysis of immobilized enzyme coupled ion exchange, which specifically comprises the following steps: co-immobilizing xylose reductase, glucose dehydrogenase and NADP + coenzyme, producing xylitol through enzyme catalysis by taking xylose and glucose as raw materials, and coupling an ion exchange reaction after the enzyme catalysis reaction to obtain the xylitol. Due to the arrangement of the ion exchange reaction, the byproduct gluconate can be separated, and the pH of the reaction liquid can be regulated and controlled by using alkaline anions exchanged in the ion exchange process, so that the normal operation of the reaction is further ensured; the xylitol and the by-product gluconate obtained by the reaction can be separated without an additional separation process. The method provided by the invention successfully solves the problems of complex process, high separation difficulty and high cost in the process of refining xylitol by a biological method.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering, and in particular relates to a method for preparing xylitol by co-immobilized enzyme coupled ion exchange continuous catalysis. Background Art

[0002] Xylitol is an organic compound with the chemical formula C5H 12 O5, white crystal, easily soluble in water. It is a natural sweetener and the sweetest known sugar alcohol, with a sweetness equivalent to that of sucrose. In nature, xylitol is widely found in various fruits, vegetables, and cereals, but the content is very low. It is mostly used as a sugar-free food additive. It is also an intermediate in the normal carbohydrate metabolism of animal bodies.

[0003] There are several main ways to prepare xylitol: the first is direct extraction. As early as 1890, German scientist Hermann Emil Fischer isolated xylitol from beech tree bark for the first time. It exists naturally in plant materials such as strawberry, yellow plum, broccoli, etc., with a content of 300-935 mg / 100 g dry weight. Although it can be obtained through solvent extraction, it accounts for too low a proportion in plant materials. Even in materials with relatively higher content such as apricot, plum and green plum, for example, green plum xylitol only accounts for about 1% of the dry weight. Extraction requires not only special equipment, but also huge energy consumption and high cost. The second is chemical synthesis. In the 1970s, Finland took the lead in using chromatography to separate D-xylose in wood hemicellulose, and then converted D-xylose into xylitol under high temperature, high pressure and hydrogen catalysis, and developed it into an industrial production method. It can be chemically synthesized with pure D-xylose or synthesized with xylose-rich wood cellulose biomass. It is commonly used at home and abroad. The method uses natural wheat straw, corn stalks, corn cobs and other polypentose raw materials, which are pretreated by acid hydrolysis, and xylose is purified from the hemicellulose fraction and then hydrogenated under the action of a catalyst; the third is the biological fermentation method, which uses biotechnology to hydrolyze corn cobs, bagasse, olive residues and other agricultural waste containing polypentose with dilute acid to obtain xylose hydrolyzate, and then uses microorganisms to reduce xylose to xylitol. This method has mild reaction conditions, simple operation, low environmental pollution, and reliable product quality. It is a low-cost alternative path. For example, patent CN104357339A uses tropical yeast to ferment high-concentration xylose, and the xylitol concentration can reach 20-65 g / L, residual sugar is less than 5%. Patent CN110982850B uses genetically engineered Aspergillus oryzae to ferment 50g / L xylan to produce 13.5 g / L xylitol, with a yield and productivity of 0.27 g / g and 0.16 g / L / h, respectively. Patent CN106661540B uses recombinant Pichia oemmerich to ferment 250 g / L glucose monohydrate as raw material to produce 120 g / L xylitol and 5 g / L ribitol, with a xylitol yield and productivity of 0.48 g / g and 1.81 g / L / h, respectively. However, the yield of raw materials for products produced by biological fermentation methods is generally not high.

[0004] In recent years, another biological method for producing xylitol has been developed. This method uses relevant enzymes to catalyze xylose to produce xylitol. It includes methods using resting cells, immobilized cells or free enzyme proteins as catalysts. Chinese patent CN110628835 uses xylose reductase, glucose dehydrogenase, formate dehydrogenase, electron carriers and water to form a biocatalytic system to catalyze the reaction of xylose to produce xylitol. 278.4 g / L of xylitol was obtained from 2 M xylose within 24 hours, with a yield of up to 11.6 g / L / h. Chinese patent CN108977432 uses recombinant Escherichia coli immobilized cells that co-express xylose reductase and glucose dehydrogenase as catalysts to catalyze xylose mother liquor to prepare xylitol. The recombinant cells catalyze 200g / L xylose. After 30 hours of reaction, the xylitol yield is 100%. Chinese patent CN108949852 uses recombinant Escherichia coli containing the xylose reductase gene XR and the glucose dehydrogenase gene GDH as a catalyst to form a biocatalytic system with an aqueous solution containing xylose and glucose and CaCO3 to catalyze the reduction reaction of xylose to produce xylitol. The xylitol concentration in the product is 145.81 g / L and the yield is 0.97 (g / g).

[0005] The advantage of bio-enzyme catalysis in producing xylitol is its high reaction efficiency, with the product yield generally exceeding 90%. However, the bio-enzyme catalysis method requires glucose as a co-substrate, and the gluconate formed after glucose conversion requires an additional separation process for separation, resulting in the bio-enzyme catalysis method having little advantage over the traditional chemical hydrogenation method. Therefore, it is necessary to develop a new bio-enzyme catalysis method that can simplify the production process of xylitol. Summary of the invention

[0006] In view of the need for simplifying the production process of xylitol in the prior art, the present invention provides a method for preparing xylitol by co-immobilized enzyme coupled with ion exchange continuous catalysis. The specific technical scheme is as follows: A method for preparing xylitol by co-immobilized enzyme coupled ion exchange continuous catalysis, comprising: continuous circulation of enzyme catalysis reaction and ion exchange reaction, and finally obtaining the product xylitol; The enzyme catalyzed reaction uses xylose and glucose as raw materials, and contains xylose reductase, glucose dehydrogenase and NADP + The co-immobilized enzyme of the coenzyme is used as a catalyst to generate a reaction solution containing xylitol and gluconate; The ion exchange reaction uses the reaction solution as the initial solution, and after ion exchange, gluconate is adsorbed to obtain a separation solution containing alkaline anions; the separation solution is refluxed into the reaction solution to achieve continuous circulation.

[0007] In the present application, xylose reductase is used to convert xylitol into xylitol. In this catalytic reaction, the required coenzyme species is NADP.+ , considering that the coenzyme will be converted into NADPH, glucose dehydrogenase is used to convert NADPH back into NADP + For the conversion of xylose, in this process, glucose will be converted into gluconic acid, which reduces the pH of the enzyme catalytic reaction, which is not conducive to the progress of the xylose reductase catalytic reaction. Therefore, an ion exchange reaction is set up after the enzyme catalytic reaction to separate the gluconate ions and release alkaline anions for adjusting the pH of the reaction solution, and the enzyme catalytic reaction and the ion exchange reaction are further set as a continuous cycle process, so that the ion exchange reaction and the enzyme catalytic reaction are carried out simultaneously and continuously, and the gluconate ions are continuously separated and the pH of the enzyme catalytic reaction is adjusted, thereby ensuring the normal progress of the immobilized enzyme catalytic reaction. In addition, the by-products produced in the production process of xylitol are separated during the reaction process, without the need for an additional separation process, and the xylitol product and the gluconate by-product can be obtained by simple concentration and crystallization, successfully solving the problems of complex process, high separation difficulty and high cost in the biological xylitol refining process.

[0008] Furthermore, the enzyme catalyzed reaction occurs in a reactor.

[0009] Furthermore, an anion exchange column is used to perform ion exchange on the reaction solution to adsorb gluconate.

[0010] Furthermore, after the ion exchange, the anion exchange column is desorbed to separate the by-product gluconate.

[0011] Furthermore, in the reaction solution, the mass ratio of xylose to glucose is 1:1 to 1:1.8, preferably 1:1.2 to 1:1.5, and preferably 1:1.125.

[0012] Furthermore, the initial concentration of xylose is 10 g / L to 300 g / L, and the initial concentration of glucose is 10 g / L to 540 g / L.

[0013] Furthermore, the initial concentration of xylose is preferably 50 g / L to 150 g / L, and more preferably 50 g / L to 115 g / L; the initial concentration of glucose is preferably 60 g / L to 225 g / L, and more preferably 60 g / L to 140 g / L.

[0014] Furthermore, in the enzyme-catalyzed reaction, the reaction pH is 5-9, preferably 6-8.

[0015] Furthermore, in the enzyme-catalyzed reaction, the reaction temperature is 15 to 50°C, preferably 20 to 40°C, and more preferably 35°C.

[0016] Furthermore, the total reaction time of the continuous cycle is 10 to 20 h, preferably 12 to 18 h.

[0017] Furthermore, in the co-immobilized enzymes, xylose reductase, glucose dehydrogenase and coenzyme NADP + The two are co-fixed on a carrier; the carrier is a resin, and the resin is an epoxy resin or an amino resin.

[0018] Furthermore, the xylose reductase is NADP + Dependent type.

[0019] Furthermore, in the co-immobilized enzyme, polyethyleneimine is used to coat the resin.

[0020] Furthermore, the mass ratio of polyethyleneimine to resin is 1:5 to 1:100, preferably 1:10 to 1:30.

[0021] Furthermore, in the co-immobilized enzyme, NADP + The ratio to the carrier is 1 to 50 μmol: 1 g, preferably 5 to 20 μmol: 1 g.

[0022] Furthermore, calculated based on the activity of the enzyme, the ratio of the co-immobilized enzyme to the reaction solution is 1-10 U:1 mL, preferably 2-5 U:1 mL.

[0023] In the present application, it is found that the xylose reductase, glucose dehydrogenase and coenzyme NADP + Compared with the separate fixation of the three enzymes, the co-immobilization has a better catalytic effect and can completely convert the substrate into xylitol in a shorter time. In addition, in the ion exchange process, if the coenzyme is immobilized alone, the coenzyme will be adsorbed by the anion exchange column. In the continuous cycle, the subsequent enzyme-catalyzed reaction will lose the coenzyme, and the ability of xylose reductase to catalyze xylose will gradually decrease, resulting in the obstruction of xylitol production. Therefore, in combination with the continuous cycle method of the enzyme-catalyzed reaction and the ion exchange reaction used in the present application, it is necessary to immobilize xylose reductase, glucose dehydrogenase and the coenzyme NADP + Co-fixation was performed.

[0024] Furthermore, the amount of enzyme is calculated based on the weight of wet cells, and the mass ratio of xylose reductase to glucose dehydrogenase to carrier is 1:10-500, preferably 1:50-200, wherein the mass ratio of xylose reductase wet cells to glucose dehydrogenase wet cells is 1:1-10, preferably 1:4-6.

[0025] Furthermore, the accession number of the xylose reductase in the NCBI library is ALO17776.1, EAA34695.1 or Q9P8R5.1; the accession number of the glucose dehydrogenase in the NCBI library is WP_012369122.1.

[0026] In the present application, the types of xylose reductase were screened so that the xylose reductase and glucose dehydrogenase in the co-immobilized enzymes in the present application both had better enzyme activity, and the required amount of glucose dehydrogenase was obtained by calculating the coenzyme required for the reaction of xylose reductase catalyzing xylose to produce xylitol. On the basis of obtaining the usage amount and ratio of xylose reductase and glucose dehydrogenase, the inventors of the present application repeatedly tested the loading ratio of the enzyme to the co-immobilized carrier to ensure that the co-immobilized enzyme had the best catalytic effect, and finally obtained the better usage ratio of xylose reductase and glucose dehydrogenase to the carrier.

[0027] Furthermore, the anion exchange column was pretreated with a NaOH solution.

[0028] Furthermore, the gluconate is sodium gluconate.

[0029] Furthermore, the alkaline anion is a hydroxide ion.

[0030] Furthermore, the gluconate ions adsorbed in the exchange column are desorbed using an alkaline solution; Furthermore, the alkali solution is NaOH.

[0031] In the present application, an anion exchange column is used for ion exchange reaction. An anion exchange column that adsorbs gluconate ions and flows out glucose, xylose and xylitol can be selected. In the embodiment of the present application, a strongly alkaline anion exchange resin pretreated with NaOH is used. After adsorbing gluconate ions, hydroxide ions can be released. The gluconate ions combine with sodium ions to form gluconate. The initial liquid of the anion exchange column flows out of the anion exchange column after ion exchange and mixes with the reaction liquid in the reactor. After the hydroxide ions enter the reaction liquid, the pH of the reaction liquid is increased, which is more suitable for the enzyme catalytic reaction.

[0032] Furthermore, in the anion exchange column, the volume ratio of the resin loading to the reaction solution is 1:1-10, preferably 1:2-5.

[0033] Furthermore, during the continuous circulation process, the flow rate of the reaction solution is calculated based on the volume of the anion exchange column, and is 0.1 to 5 BV / h, preferably 0.2 to 2 BV / h, wherein BV represents the volume of the anion exchange column.

[0034] Furthermore, the alkaline anion is OH- .

[0035] In the present application, in the enzyme-catalyzed reaction, the generated gluconic acid will lower the pH of the reaction system, and the hydroxide ions exchanged by the ions will increase the pH of the reaction system. Considering that the suitable pH range of the enzyme-catalyzed reaction is 6 to 8, it is necessary to control the amount of the exchanged hydroxide ions to prevent the amount of hydroxide ions from being too much or too little, so as to ensure that the pH of the enzyme-catalyzed reaction is always within an appropriate range.

[0036] The amount of resin loaded in the anion exchange column and the speed at which the reaction solution flows through the anion exchange column can not only affect the adsorption effect of the anion exchange column on gluconate, but also affect the amount of hydroxide ions exchanged in the anion exchange column. The larger the amount of resin loaded, the better the adsorption effect on gluconate, but more hydroxide ions will be released. Therefore, after comprehensively considering the adsorption effect of the anion exchange column on gluconate and the amount of hydroxide ions exchanged in the anion exchange column, the applicant has finally obtained a better range of resin loading and reaction solution flow rate after repeated experiments.

[0037] Furthermore, after the continuous cycle is completed, the reaction solution is filtered to separate the immobilized enzyme.

[0038] In the present application, after the production of xylitol is completed, the co-immobilized enzyme can be separated and used in subsequent production after treatment, which greatly reduces the cost of preparing xylitol by biological method.

[0039] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing xylitol by continuous catalysis of immobilized enzyme coupled with ion exchange, wherein xylose reductase, glucose dehydrogenase and NADP + The coenzyme is co-immobilized, xylose and glucose are used as raw materials for enzymatic catalysis to produce xylitol, and an ion exchange reaction is coupled after the enzyme catalytic reaction, which can not only separate the byproduct gluconate, but also use the alkaline anions exchanged in the ion exchange process to control the pH of the reaction solution, further ensuring the normal progress of the reaction; and the xylitol and gluconate obtained by the reaction are separated without an additional separation process. The method provided by the present invention successfully solves the problems of complex process, great separation difficulty and high cost in the biological method of xylitol refining process. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The following is the reaction equation for producing xylitol by the enzyme-catalyzed reaction of the present invention.

[0041] Figure 2 This is a schematic diagram of the device for producing xylitol of the present invention; wherein: 1. a reactor, 2. a separator.

[0042] Figure 3 This is a graph showing the results of the determination of the initial enzyme activity and residual enzyme activity of xylose reductase.

[0043] Figure 4 This is a graph showing the change in glucose dehydrogenase activity with insulation time. DETAILED DESCRIPTION

[0044] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0045] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0047] The experimental methods in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to the Molecular Cloning Experiment Guide compiled by J. Sambrook et al. The kits for gene manipulation were purchased from TAKARA for characterization. Reagents used in upstream genetic engineering: restriction endonucleases and DNA ligases used in the examples of the present invention were purchased from TaKaRa, Takara Biotechnology (Dalian) Co., Ltd.; genome extraction kits, plasmid extraction kits, and DNA recovery and purification kits were purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21 (DE3), plasmid pET-28a (+), etc. were purchased from Novagen; DNA marker, FastPfu DNA polymerase, low molecular weight standard protein, agarose electrophoresis reagent were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the use of the above reagents refers to the product manual. Other chemical reagents: xylose, arabinose, glucose, galactose, xylitol, arabitol, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0048] Detection of sugar alcohols: HPLC determination, the mobile phase is ultrapure water, Agilent Hi-Plex Ca chromatographic column (7.7 mm×300 mm), column temperature is 85℃; flow rate is 0.6 mL / min.

[0049] Detection of gluconic acid: HPLC determination, the mobile phase was 10 mmol / L K2HPO4-10 mmol / L tetrabutylammonium hydrogen sulfate (pH 7.2): methanol = 95:5 (V / V), chromatographic column C18 (4.6 mm × 250 mm), column temperature 30 ° C; flow rate was 0.7 mL / min.

[0050] Shake flask fermentation of genetically engineered bacteria: Use LB liquid medium (10g / L peptone, 5g / L yeast powder, 10g / L NaCl, dissolve with deionized water and make up to volume, sterilize at 121℃ for 20min, and set aside). Inoculate the engineered bacteria E. coliBL21(DE3) containing the relevant gene into 5mL LB liquid medium containing 50µg / mL kanamycin, and shake culture at 37℃ for 12 hours. Transfer to 250mL fresh LB liquid medium containing 50 µg / mL Kan, shake culture at 37℃ until OD600 reaches about 0.8, add IPTG to its concentration of 0.1 mM, and induce culture at 25℃ for 16 hours. After the culture is completed, centrifuge the culture solution at 4000 rpm for 15min, discard the supernatant, collect the bacterial cells, and store them in a -70℃ ultra-low temperature refrigerator for use.

[0051] Preparation of crude enzyme solution: The collected wet cells were resuspended in 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH = 8.0) to a cell solution with a bacterial concentration of 100 g / L; the resuspended cell solution was crushed under high pressure and centrifuged at 12000 rpm for 30 min to obtain the supernatant, which is the crude enzyme solution.

[0052] Definition of enzyme activity: The International Conference on Enzymology in 1961 stipulated that one unit of enzyme activity refers to the amount of enzyme that can convert 1 micromole of substrate within 1 minute under specific conditions, or the amount of enzyme that converts 1 micromole of related groups in the substrate.

[0053] In the following examples, the reaction equation for the production of xylitol by enzyme catalysis is as follows: Figure 1 as shown; Figure 2 As shown, the enzyme catalyzed reaction is carried out in a reactor 1, which is specifically a 5 L glass reactor with a jacket, coupled with a separator 2, which is specifically an anion exchange column, and the ion exchange reaction is carried out in the anion exchange column.

[0054] Example 1 Recombinant expression and screening of xylose reductase Eleven wild-type XR genes from different sources were selected, codon optimized and fully synthesized by Nanjing GenScript Biotechnology Co., Ltd., and then constructed into plasmid pET-28a and introduced into E. coli. E. coliDuring the BL21 genetic engineering, shake flask fermentation and enzyme production were carried out to obtain a xylose reductase enzyme library. The enzymatic activity of xylose reductase on the substrate D-xylose (the product is xylitol) was measured at 30°C with 0.1 mM NADPH coenzyme. The results are shown in Table 1. Other operations for enzyme activity determination refer to Evolution in reverse: engineering a D-xylose-specific xylose reductase. Chembiochem, 2008, 9(8): 1213-5. From the results in Table 1, it can be seen that after the expression of the xylose reductase gene, it showed good catalytic activity for D-xylose, and most of them had a catalytic activity greater than 200 U / mL. In addition, the xylose reductase (crude enzyme solution) was placed in a 35°C water bath for 12 hours to measure the residual enzyme activity. The results are shown in Figure 3 As shown, the only samples with residual enzyme activity greater than 200 U / mL were from Rhodotorula mucilaginosa , Neurospora crassa and Aspergillus niger Therefore, XR5, XR6 and XR11 were selected for immobilization.

[0055] Table 1 Source of xylose reductase and enzyme activity determination results

[0056] Example 2 Recombinant expression and screening of glucose dehydrogenase Four wild-type glucose dehydrogenase genes from different sources were selected, codon optimized and fully synthesized by Nanjing GenScript Biotechnology Co., Ltd., and then constructed into plasmid pET-28a and introduced into Escherichia coli. E. coli In BL21 genetic engineering, shake flask fermentation and enzyme production were carried out to obtain a glucose dehydrogenase enzyme library with 0.1 mM coenzyme NADP + , 10 g / L glucose as substrate, and the enzyme activity of each glucose dehydrogenase was measured at 30°C. The results are shown in Table 2. From the results in Table 2, it can be seen that the four glucose dehydrogenases all showed significant catalytic activity on the substrate glucose. Glucose dehydrogenase (crude enzyme solution) was placed in a 30°C and 35°C water bath for 6 hours, and the enzyme activity changes were measured. The results are shown in Figure 4 As shown, from Exiguobacterium artemia Glucose dehydrogenase GDH2 always maintained the highest activity. Therefore, GDH2 was selected for immobilization.

[0057] Table 2. Sources of glucose dehydrogenase and enzyme activity assay results

[0058] Example 3 Preparation of immobilized enzyme Pretreatment of immobilized carrier: Epoxy resin (EP) was washed with phosphate buffer (50 mM, pH 8.0) for 3 times, 2 h each time, with regular stirring, and the sand core funnel was drained for later use. Amino resin (EA) needs to be further activated with glutaraldehyde. Treatment method: 10 g of resin was placed in 100 mL of phosphate buffer (50 mM, pH 8.0) containing 4% glutaraldehyde for activation, stirred at 30 ° C for 2 h, and the sand core funnel was drained for later use.

[0059] Coenzyme NADP + Fixation: Prepare 0.5% polyethyleneimine solution with 50 mM phosphate buffer at pH 8.0. Put 10 g of treated resin into 100 mL polyethyleneimine solution for encapsulation reaction, stir continuously at 30°C, and react for 2 hours. Then use a sand core funnel to drain and set aside. Prepare 100 μM NADP disodium salt solution with phosphate buffer. Take 10 g of polyethyleneimine-treated resin and put it into NADP disodium salt solution for immobilization. Stir continuously at 25°C, react for 2 hours, then use a sand core funnel to drain and set aside.

[0060] Enzyme co-immobilization: Take 2 mL and 8 mL of XR and GDH crude enzyme solutions respectively, add 90 mL of buffer, mix, and add 10 g of immobilized coenzyme NADP + The immobilized material was stirred at 25℃ for 6 h. Finally, the mixture was drained with a sand core funnel, washed with buffer for 2-3 times, and drained to obtain the corresponding co-immobilized enzyme. The enzyme activity of the co-immobilized enzyme refers to the enzyme activity of the co-immobilized enzyme as xylose reductase. The results are shown in Table 3. The enzyme activity of the co-immobilized enzyme with epoxy resin as the carrier is significantly higher than that of the co-immobilized enzyme with amino resin as the carrier.

[0061] Table 3. Results of immobilized enzyme activity determination

[0062] Example 4 Preparation of xylitol by reaction separation coupling and co-immobilized enzyme catalysis In a 5 L glass reactor (with jacket), add 3.5 kg of deionized water, 400 g of xylose and 490 g of glucose, wash and activate the anion exchange resin, and then install it into a column with a column volume of about 2.0 L. Circulating water is passed through the jacket, and the circulating water temperature is controlled at 35°C. 100.0 g of IE1@EP co-immobilized enzyme is weighed and added to the reactor, stirring is started, and the reaction is carried out; the enzyme-catalyzed reaction liquid is continuously pumped from the bottom of the reactor into the anion exchange column by a peristaltic pump, and the anion exchange liquid is returned to the reactor. The peristaltic pump speed is controlled at 4.0 L / h.

[0063] After the total reaction time was 18.0 h, the substrate D-xylose was below the detection limit by HPLC, and the reaction was stopped. The reaction solution was filtered through a sand core funnel to separate the co-immobilized enzyme (for the next batch of reactions). The filtrate volume was 4080 mL. The concentration of the product xylitol in the reaction solution was 98.2 g / L, and the yield of xylitol was 99.5%. The substrate D-glucose remained at 2.3 g / L. After the reaction, the anion exchange column was regenerated with 4 L of 4% NaOH aqueous solution, and the regeneration liquid with a gluconate concentration greater than 1.0 g / L was collected. After the resin regeneration was completed, the exchange column was rinsed with deionized water until the effluent was close to neutral and then used for standby (for the next batch of reactions). The collected resin regeneration liquid was concentrated by vacuum to remove water to obtain 511.6 g of sodium gluconate as a byproduct, and the yield of sodium gluconate was calculated to be 88.0%.

[0064] Example 5 Preparation of xylitol by reaction separation coupling and co-immobilized enzyme catalysis In a 5 L glass reactor (with jacket), add 3.5 kg of deionized water, 400 g of xylose and 490 g of glucose, wash and activate the anion exchange resin, and then install it into a column with a column volume of about 2.0 L. Circulating water is passed through the jacket, and the circulating water temperature is controlled at 35°C. 100.0 g of IE1@EA co-immobilized enzyme is weighed and added to the reactor, stirring is started, and the reaction is carried out; the enzyme-catalyzed reaction liquid is continuously pumped from the bottom of the reactor into the anion exchange column by a peristaltic pump, and the anion exchange liquid is returned to the reactor. The peristaltic pump speed is controlled at 4.0 L / h.

[0065] After the total reaction time was 10.0 h, the substrate D-xylose was below the detection limit by HPLC, and the reaction was stopped. The reaction solution was filtered through a sand core funnel to separate the co-immobilized enzyme (for the next batch of reactions). The filtrate volume was 4050 mL. The concentration of the product xylitol in the reaction solution was 99.3 g / L, and the yield of xylitol was 99.2%. The substrate D-glucose remained at 3.0 g / L. After the reaction, the anion exchange column was regenerated with 4 L of 4% NaOH aqueous solution, and the regeneration liquid with a gluconate concentration greater than 1.0 g / L was collected. After the resin regeneration was completed, the exchange column was rinsed with deionized water until the effluent was close to neutral and then used for standby (for the next batch of reactions). The collected resin regeneration liquid was concentrated by vacuum to remove water to obtain 509.3 g of sodium gluconate as a byproduct, and the yield of sodium gluconate was calculated to be 88.3%.

[0066] Example 6 Preparation of xylitol by reaction separation coupling and co-immobilized enzyme catalysis In a 5 L glass reactor (with a jacket), 3.5 kg of deionized water, 200 g of xylose and 245 g of glucose were added. The anion exchange resin was cleaned and activated, and then loaded into a column with a column volume of about 1.0 L. Circulating water was passed through the jacket, and the circulating water temperature was controlled at 35°C. 100.0 g of IE1@EA co-immobilized enzyme was weighed and added to the reactor. Stirring was started to react. The enzyme-catalyzed reaction liquid was continuously pumped from the bottom of the reactor into the anion exchange column by a peristaltic pump, and the anion exchange liquid was returned to the reactor. The peristaltic pump speed was controlled at 2.0 L / h.

[0067] After the total reaction time was 8.0 h, the substrate D-xylose was below the detection limit by HPLC, and the reaction was stopped. The reaction solution was filtered through a sand core funnel to separate the co-immobilized enzyme (for the next batch of reactions). The filtrate volume was 4050 mL. The concentration of the product xylitol in the reaction solution was 49.3 g / L, the yield of xylitol was 99.2%, and the substrate D-glucose remained at 0.3 g / L. The anion exchange column was regenerated with 2 L of 4% NaOH aqueous solution, and the regeneration liquid with a gluconate concentration greater than 1.0 g / L was collected. After the resin regeneration was completed, the exchange column was rinsed with deionized water until the effluent was close to neutral and then used for standby. The collected resin regeneration liquid was concentrated by vacuum to remove water to obtain 250.2 g of sodium gluconate as a byproduct, and the yield of sodium gluconate was calculated to be 86.1%.

[0068] Example 7 Preparation of xylitol by reaction separation coupling and co-immobilized enzyme catalysis In a 5 L glass reactor (with a jacket), 3.5 kg of deionized water, 200 g of xylose and 245 g of glucose were added. The anion exchange resin was cleaned and activated, and then loaded into a column with a column volume of about 1.0 L. Circulating water was passed through the jacket, and the circulating water temperature was controlled at 35°C. 100.0 g of IE2@EA co-immobilized enzyme was weighed and added to the reactor. Stirring was started to react. The enzyme-catalyzed reaction liquid was continuously pumped from the bottom of the reactor into the anion exchange column by a peristaltic pump, and the anion exchange liquid was returned to the reactor. The peristaltic pump speed was controlled at 2.0 L / h.

[0069] After the total reaction time was 4.0 h, the substrate D-xylose was below the detection limit by HPLC, and the reaction was stopped. The reaction solution was filtered through a sand core funnel to separate the co-immobilized enzyme (for the next batch of reactions). The filtrate volume was 4060 mL. The concentration of the product xylitol in the reaction solution was 49.5 g / L, the yield of xylitol was 99.2%, and the substrate D-glucose remained 0.5 g / L. The anion exchange column was regenerated with 2 L of 4% NaOH aqueous solution, and the regeneration liquid with a gluconate concentration greater than 1.0 g / L was collected. After the resin regeneration was completed, the exchange column was rinsed with deionized water until the effluent was close to neutral and then used for standby. The collected resin regeneration liquid was concentrated by vacuum to remove water to obtain 245.1 g of sodium gluconate as a byproduct, and the yield of sodium gluconate was calculated to be 84.9%.

[0070] Example 8 Preparation of xylitol by reaction separation coupling and co-immobilized enzyme catalysis In a 5 L glass reactor (with a jacket), 3.5 kg of deionized water, 200 g of xylose and 245 g of glucose were added. The anion exchange resin was cleaned and activated, and then loaded into a column with a column volume of about 1.0 L. As a separator, circulating water was passed into the jacket, and the circulating water temperature was controlled at 40°C. 50.0 g of IE3@EA co-immobilized enzyme was weighed and added to the reactor. Stirring was started to react. The enzyme-catalyzed reaction liquid was continuously pumped from the bottom of the reactor into the anion exchange column by a peristaltic pump, and the anion exchange liquid was returned to the reactor. The peristaltic pump speed was controlled at 2.0 L / h.

[0071] After the total reaction time was 12.0 h, the substrate D-xylose was below the detection limit by HPLC, and the reaction was stopped. The reaction solution was filtered through a sand core funnel to separate the co-immobilized enzyme (for the next batch of reactions). The filtrate volume was 4010 mL. The concentration of the product xylitol in the reaction solution was 50.1 g / L, and the yield of xylitol was 99.8%. The substrate D-glucose remained 0.5 g / L. After the reaction, the anion exchange column was regenerated with 2 L of 4% NaOH aqueous solution, and the regeneration liquid with a gluconate concentration greater than 1.0 g / L was collected. After the resin regeneration was completed, the exchange column was rinsed with deionized water until the effluent was close to neutral and then used for standby (for the next batch of reactions). The collected resin regeneration liquid was concentrated by vacuum to remove water to obtain 261.5 g of sodium gluconate as a byproduct, and the yield of sodium gluconate was calculated to be 89.9%.

[0072] Comparative Example 1 According to the operation of Example 3, xylose reductase XR5 and glucose dehydrogenase GDH2 were immobilized respectively to obtain the corresponding immobilized enzymes XR5-NADP@EP and GDH2-NADP@EP, and the enzyme activities were measured to be 295.4 U / g and 40.3 U / g, respectively.

[0073] In a 5 L glass reactor (with a jacket), 3.5 kg of deionized water, 200 g of xylose and 245 g of glucose were added. The anion exchange resin was cleaned and activated, and then loaded into the column. The column volume was about 1.0 L. Circulating water was passed into the jacket, and the circulating water temperature was controlled at 30°C. 5.0 g of XR5-NADP@EP immobilized enzyme and 45.0 g of GDH2-NADP@EP immobilized enzyme were weighed and added to the reactor respectively. Stirring was started to react. The enzyme-catalyzed reaction liquid was continuously pumped from the bottom of the reactor into the anion exchange column by a peristaltic pump, and the anion exchange liquid was returned to the reactor. The pumping speed of the peristaltic pump was controlled at 1 L / h.

[0074] After a total reaction time of 24.0 h, HPLC detected that the concentration of substrate D-xylose was 38.9 g / L and the concentration of glucose was 50.3 g / L. The reaction was almost stagnant. After the immobilized enzyme was separated, the volume of the filtrate was 3980 mL. The concentration of the product xylitol in the reaction solution was 10.3 g / L, and the calculated xylitol yield was only 20.4%.

[0075] Comparative Example 2 The pretreatment of the immobilized carrier is consistent with that in Example 3.

[0076] Coenzyme NADP + Fixation: Prepare 0.5% polyethyleneimine solution with 50 mM phosphate buffer at pH 8.0. Put 10 g of treated resin into 100 mL polyethyleneimine solution for encapsulation reaction, stir continuously at 30°C, and react for 2 hours. Then use a sand core funnel to drain and set aside. Prepare 100 μM NADP disodium salt solution with phosphate buffer. Take 10 g of polyethyleneimine-treated resin and put it into NADP disodium salt solution for immobilization. Stir continuously at 25°C, react for 2 hours, then use a sand core funnel to drain and obtain immobilized coenzyme NADP + ,spare.

[0077] Enzyme immobilization: The crude enzyme solution of XR5 or GDH2 was taken and immobilized with the pretreated resin to obtain the corresponding immobilized enzymes XR5@EP and GDH2@EP without coenzyme. The enzyme activities were measured to be 181.1 U / g and 29.6 U / g, respectively.

[0078] In a 5 L glass reactor (with a jacket), 3.5 kg of deionized water, 200 g of xylose and 245 g of glucose were added. After the anion exchange resin was cleaned and activated, it was loaded into the column. The column volume was about 1.0 L. Circulating water was passed into the jacket. The circulating water temperature was controlled at 30 °C. 10.0 g of XR5@EP immobilized enzyme, 60.0 g of GDH2@EP immobilized enzyme and immobilized coenzyme NADP were weighed. +10 g were added into the reactor respectively, stirring was started to react; the enzyme-catalyzed reaction liquid was continuously pumped into the anion exchange column from the bottom of the reactor by a peristaltic pump, and the anion exchange liquid was returned to the reactor, and the peristaltic pump speed was controlled at 1 L / h.

[0079] After a total reaction time of 12.0 h, the concentration of the product xylitol in the reaction solution was 1.5 g / L by HPLC, indicating that the reaction had hardly proceeded.

[0080] Comparative Example 3 In a 5 L glass reactor (with jacket), add 3.5 kg of deionized water, 400 g of xylose and 490 g of glucose, and use circulating water temperature to control the temperature in the reactor at 35°C. Weigh 100.0 g of IE1@EP co-immobilized enzyme, add it to the reactor, start stirring, and react. After 2.0 h of reaction, the concentration of xylitol in the reaction solution detected by HPLC was 34.1 g / L; after 4.0 h of reaction, the concentration of xylitol was 33.9 g / L, and the product concentration no longer increased. The pH of the reaction solution was determined to be 2.56. Under this condition, the immobilized enzyme was inactivated, resulting in the termination of the reaction.

Claims

1. A method for preparing xylitol by co-immobilized enzyme coupled ion exchange continuous catalysis, characterized in that: include: The continuous cycle of enzyme catalysis and ion exchange reaction finally yields the product xylitol; The enzyme catalyzed reaction uses xylose and glucose as raw materials, and contains xylose reductase, glucose dehydrogenase and NADP + The co-immobilized enzyme of the coenzyme is used as a catalyst to generate a reaction solution containing xylitol and gluconate; The ion exchange reaction uses the reaction solution as the initial solution, and after ion exchange, gluconate is adsorbed to obtain a separation solution containing alkaline anions; The separation liquid is refluxed into the reaction liquid to realize continuous circulation; Among the co-immobilized enzymes, xylose reductase, glucose dehydrogenase and coenzyme NADP + Co-fixed on a carrier; the carrier is an epoxy resin or an amino resin; In the co-immobilized enzyme, the resin is coated with polyethyleneimine; The accession number of the xylose reductase in the NCBI library is ALO17776.1, EAA34695.1 or Q9P8R5.1; the accession number of the glucose dehydrogenase in the NCBI library is WP_012369122.1; In enzyme-catalyzed reactions, the reaction pH is 5~9.

2. The method according to claim 1, characterized in that In the raw materials, the mass ratio of xylose to glucose is 1:1-1.

8.

3. The method according to claim 2, wherein the ratio of the co-immobilized enzyme to the raw material is 1-10 U: 1 mL, calculated based on the activity of xylose reductase.

4. The method according to claim 1, characterized in that Using an anion exchange column to carry out ion exchange on the reaction solution to adsorb gluconate; After ion exchange, the anion exchange column is desorbed to separate the by-product gluconate.

5. The method according to claim 4, characterized in that The alkaline anion is a hydroxide ion, and the gluconate is sodium gluconate.

6. The method according to any one of claims 4 or 5, characterized in that According to the volume of the anion exchange column, the flow rate of the reaction solution in the anion exchange column is 0.1-5 BV / h.

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