A method for continuously preparing xylitol by co-immobilized enzyme coupling ion exchange catalysis

Abstract

The present invention belongs to the field of biochemistry, and particularly relates to a method for continuously catalyzing the preparation of xylitol by immobilized enzyme coupling ion exchange. Specifically, xylose reductase, glucose dehydrogenase and NADP + coenzyme are co-immobilized, and xylose and glucose are used as raw materials for enzymatic production of xylitol. After the enzymatic reaction, an ion exchange reaction is coupled. The setting of the ion exchange reaction can not only separate the by-product gluconate, but also use the basic anions exchanged during the ion exchange process to regulate the pH of the reaction solution, further ensuring the normal progress of the reaction; the xylitol and the by-product gluconate obtained by the reaction are separated without additional separation procedures. The method provided by the present invention successfully solves the problems of complex process, difficult separation and high cost in the refining process of xylitol by biological methods.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and particularly relates to a method for continuously catalyzing the preparation of xylitol by co-immobilized enzyme coupling ion exchange. Background Art

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

[0003] The main preparation methods of xylitol are as follows: The first is the direct extraction method. As early as 1890, the German scientist Hermann Emil Fischer first isolated xylitol from the bark of beech trees. It naturally exists in plant raw materials such as strawberries, yellow plums, and broccoli, with a content in the range of 300 - 935 mg / 100 g dry weight. Although it can be obtained by solvent extraction, due to its extremely low proportion in plant raw materials, even in raw materials with relatively higher content such as apricot plums and greengage plums, for example, xylitol in greengage plums only accounts for about 1% of the dry weight. The extraction not only requires special equipment but also consumes a large amount of energy, resulting in high costs. The second is the chemical synthesis method. In the 1970s, Finland took the lead in using chromatography to separate D-xylose from lignocellulose. Subsequently, D-xylose was converted into xylitol under high temperature, high pressure, and hydrogen catalysis, and it has developed into an industrial production method. It can be chemically synthesized with pure D-xylose or with lignocellulosic biomass rich in xylose. Commonly used raw materials with pentosans at home and abroad include natural wheat straw, corn straw, corn cobs, etc. After acid hydrolysis pretreatment, xylose is purified from the hemicellulose fraction and then undergoes a hydrogenation reaction under the action of a catalyst. The third is the biological fermentation method. With the help of biotechnology, agricultural waste containing pentosans such as corn cobs, bagasse, and olive residues is hydrolyzed with dilute acid to obtain a xylose hydrolysate, and then microorganisms are used to reduce xylose to xylitol. This method has mild reaction conditions, simple operation, low environmental pollution, and reliable product quality, and is a low-cost alternative path. For example, in patent CN104357339A, Candida tropicalis is used to ferment high-concentration xylose, and the xylitol concentration can reach 20 - 65 g / L, with the residual sugar being less than 5%. In patent CN110982850B, an Aspergillus oryzae genetically engineered bacterium is used to ferment 50 g / L xylan, producing 13.5 g / L of xylitol, and the yields and productivities are 0.27 g / g and 0.16 g / L / h respectively. In patent CN106661540B, recombinant Pichia ohmeri is used with 250 g / L of glucose monohydrate as the raw material, producing 120 g / L of xylitol and 5 g / L of ribitol, and the xylitol yields and productivities are 0.48 g / g and 1.81 g / L / h respectively. However, the yields of products by the biological fermentation method from raw materials are generally not high.

[0004] In recent years, another biological method for producing xylitol has been developed, which uses related enzymes to catalyze the production of xylitol from xylose. This includes methods using resting cells, immobilized cells, or free enzyme proteins as catalysts. Chinese Patent CN110628835 uses xylose reductase, glucose dehydrogenase, formate dehydrogenase, an electron carrier, and water to form a biocatalytic system that catalyzes 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 as high as 11.6 g / L / h. Chinese Patent CN108977432 uses immobilized cells of recombinant Escherichia coli co-expressing xylose reductase and glucose dehydrogenase as a catalyst to catalyze the preparation of xylitol from xylose mother liquor. After the recombinant cells catalyzed 200 g / L of xylose for 30 h, the xylitol yield was 100%. Chinese Patent CN108949852 uses recombinant Escherichia coli containing the xylose reductase gene XR and the glucose dehydrogenase gene GDH as a catalyst, which forms 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 was 145.81 g / L, and the yield was 0.97 (g / g).

[0005] The advantage of producing xylitol by biocatalysis with enzymes is high reaction efficiency, and the product yield generally exceeds 90%. However, the production of xylitol by biocatalysis with enzymes requires a co-substrate glucose, and the gluconate formed after glucose conversion needs an additional separation process for separation. As a result, compared with the traditional chemical hydrogenation method, the biocatalysis with enzymes method has little advantage. Therefore, it is necessary to develop a new biocatalysis with enzymes method that can simplify the production process of xylitol. Summary of the Invention

[0006] In response to the need to simplify the production process of xylitol in the prior art, the present invention provides a method for continuously catalyzing the preparation of xylitol by co-immobilized enzyme coupling ion exchange, and the specific technical solution is as follows:

[0007] A method for continuously catalyzing the preparation of xylitol by co-immobilized enzyme coupling ion exchange includes: continuous circulation of an enzyme-catalyzed reaction and an ion exchange reaction to finally obtain the product xylitol;

[0008] The enzyme-catalyzed reaction uses xylose and glucose as raw materials, and a co-immobilized enzyme containing xylose reductase, glucose dehydrogenase, and NADP + coenzyme as a catalyst, and the reaction produces a reaction solution containing xylitol and gluconate ions;

[0009] The ion exchange reaction uses the reaction solution as the initial solution. After ion exchange, gluconate ions are adsorbed to obtain a separation solution containing alkaline anions; the separation solution is refluxed into the reaction solution to achieve continuous circulation.

[0010] In this application, xylose is converted into xylitol by xylose reductase. In this catalytic reaction, the required coenzyme type is NADP + , considering that the coenzyme will be converted into NADPH, glucose dehydrogenase is used to reconvert NADPH into NADP + for the conversion of xylose. In this process, glucose will be converted into gluconic acid, which reduces the pH of the enzymatic reaction and is not conducive to the progress of the xylose reductase-catalyzed reaction. Therefore, an ion exchange reaction is set up after the enzymatic reaction to separate gluconate ions and release basic anions to adjust the pH of the reaction solution. Further, the enzymatic reaction and the ion exchange reaction are set as a continuous cyclic process, so that the ion exchange reaction and the enzymatic reaction occur simultaneously and continuously, constantly separating gluconate ions and adjusting the pH of the enzymatic reaction, thereby ensuring the normal progress of the immobilized enzyme-catalyzed reaction. In addition, the by-products produced in the production process of xylitol are separated during the reaction process, and no additional separation process is required. Xylitol products and gluconate by-products can be obtained through simple concentration and crystallization, successfully solving the problems of complex process, difficult separation and high cost in the refining process of biotechnological xylitol.

[0011] Furthermore, the enzymatic reaction occurs in a reaction kettle.

[0012] Furthermore, an anion exchange column is used to perform ion exchange on the reaction solution to adsorb gluconate ions.

[0013] Furthermore, after ion exchange, the anion exchange column is desorbed to separate the by-product gluconate.

[0014] 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 more preferably 1:1.125.

[0015] Even further, 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.

[0016] Even further, 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.

[0017] Furthermore, in the enzymatic reaction, the reaction pH is 5 to 9, preferably 6 to 8.

[0018] Furthermore, in the enzymatic reaction, the reaction temperature is 15 to 50 °C, preferably 20 to 40 °C, and more preferably 35 °C.

[0019] Furthermore, the total reaction time of the continuous cycle is 10 to 20 h, preferably 12 to 18 h.

[0020] Furthermore, in the co-immobilized enzyme, xylose reductase, glucose dehydrogenase, and coenzyme NADP + are co-immobilized on a carrier; the carrier is a resin, and the resin is an epoxy resin or an amino resin.

[0021] Furthermore, the xylose reductase is NADP + -dependent.

[0022] Furthermore, in the co-immobilized enzyme, the resin is coated with polyethyleneimine.

[0023] Furthermore, the mass ratio of polyethyleneimine to the resin is 1:5 to 1:100, preferably 1:10 to 1:30.

[0024] Furthermore, in the co-immobilized enzyme, the ratio of NADP + to the carrier is 1 to 50 μmol:1 g, preferably 5 to 20 μmol:1 g.

[0025] Furthermore, calculated by the enzyme activity, the ratio of the co-immobilized enzyme to the reaction solution is 1 to 10 U:1 mL, preferably 2 to 5 U:1 mL.

[0026] In this application, it is found that co-immobilizing the xylose reductase, glucose dehydrogenase, and coenzyme NADP used + has a better catalytic effect compared to immobilizing these three enzymes separately, and the substrate can be completely converted into xylitol in a shorter time. And during the ion exchange process, if the coenzyme is immobilized separately, the coenzyme will be adsorbed by the anion exchange column. In the continuous cycle, in the subsequent enzyme-catalyzed reaction, due to the loss of the coenzyme, the ability of xylose reductase to catalyze xylose will gradually decline, resulting in the hindrance of xylitol production. Therefore, combined with the continuous cycle method of the enzyme-catalyzed reaction and the ion exchange reaction used in this application, it is necessary to co-immobilize xylose reductase, glucose dehydrogenase, and coenzyme NADP + for co-immobilization.

[0027] Furthermore, calculated by the weight of wet cells, the mass ratio of xylose reductase, glucose dehydrogenase to the carrier is 1:10 to 500, preferably 1:50 to 200, and the mass ratio of wet cells of xylose reductase to wet cells of glucose dehydrogenase is 1:1 to 10, preferably 1:4 to 6.

[0028] Further, the xylose reductase has an accession number of ALO17776.1, EAA34695.1 or Q9P8R5.1 in the NCBI database; the glucose dehydrogenase has an accession number of WP_012369122.1 in the NCBI database.

[0029] In this application, the types of xylose reductase were screened to ensure that both the xylose reductase and the glucose dehydrogenase in the co-immobilized enzyme in this application have relatively excellent enzyme activities. By calculating the coenzyme required in the reaction of xylose reductase catalyzing xylose to produce xylitol, the dosage of the required glucose dehydrogenase was obtained. On the basis of obtaining the usage amounts and ratios of the xylose reductase and the glucose dehydrogenase, the inventors of this application repeatedly tested the loading ratio of the enzyme to the co-immobilized carrier to ensure that the co-immobilized enzyme has the optimal catalytic effect, and finally obtained the optimal usage ratio of the xylose reductase, the glucose dehydrogenase and the carrier.

[0030] Furthermore, the anion exchange column is pretreated with a NaOH solution.

[0031] Furthermore, the gluconate is sodium gluconate.

[0032] Furthermore, the basic anion is a hydroxide ion.

[0033] Furthermore, the gluconate ions adsorbed in the exchange column are desorbed using an alkali solution;

[0034] Furthermore, the alkali solution is NaOH.

[0035] In this application, an anion exchange column is used for the ion exchange reaction. Any anion exchange column that can adsorb gluconate ions and flow out glucose, xylose and xylitol can be selected. In the examples of this application, a strongly basic anion exchange resin pretreated with NaOH is used. After adsorbing gluconate ions, it can release hydroxide ions, and 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 is mixed with the reaction liquid in the reaction kettle. After the hydroxide ions enter the reaction liquid, the pH of the reaction liquid increases, making it more suitable for the enzyme-catalyzed reaction to proceed.

[0036] Furthermore, in the anion exchange column, the volume ratio of the resin packing amount to the volume of the reaction liquid is 1:1 to 10, preferably 1:2 to 5.

[0037] Furthermore, in the continuous circulation process, calculated according to the volume of the anion exchange column, the flow rate of the reaction liquid is 0.1 to 5 BV / h, preferably 0.2 to 2 BV / h, where BV represents the volume of the anion exchange column.

[0038] Further, the alkaline anion is OH - .

[0039] In this application, in the enzymatic reaction, the gluconic acid generated will lower the pH of the reaction system, and the hydroxide ions exchanged out will raise the pH of the reaction system. Considering that the suitable pH range for the enzymatic reaction is 6 - 8, it is necessary to control the amount of hydroxide ions exchanged out to prevent the amount of hydroxide ions from being too much or too little, and ensure that the pH of the enzymatic reaction always remains within a suitable range.

[0040] The packing amount of the resin in the anion exchange column and the flow rate of the reaction solution through the anion exchange column can not only affect the adsorption effect of the anion exchange column on gluconate ions, but also affect the amount of hydroxide ions exchanged out in the anion exchange column. The larger the packing amount of the resin, the better the adsorption effect on gluconate ions, but more hydroxide ions will be released. Therefore, after comprehensively considering the adsorption effect of the anion exchange column on gluconate ions and the amount of hydroxide ions exchanged out in the anion exchange column, through repeated experiments by the applicant, the optimal resin packing amount range and reaction solution flow rate range are finally obtained.

[0041] Further, after the continuous cycle ends, the reaction solution is filtered to separate the immobilized enzyme.

[0042] In this 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 producing xylitol by the biological method.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention provides a method for continuously catalyzing the preparation of xylitol by coupling immobilized enzymes with ion exchange. Xylose reductase, glucose dehydrogenase, and NADP + coenzyme are co - immobilized, and xylose and glucose are used as raw materials for enzymatic production of xylitol. After the enzymatic reaction, an ion exchange reaction is coupled. It can not only separate the by - product gluconate, but also use the alkaline anions exchanged out during the ion exchange process to regulate the pH of the reaction solution, further ensuring the normal progress of the reaction; and the xylitol and gluconate obtained from the reaction are separated without additional separation processes. The method provided by the present invention successfully solves the problems of complex process, difficult separation, and high cost in the refining process of biological xylitol. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the reaction equation for the enzymatic production of xylitol in the present invention.

[0046] Figure 2Schematic diagram of the device for producing xylitol according to the present invention; wherein: 1. Reactor, 2. Separator.

[0047] Figure 3 Graph showing the determination results of the initial enzyme activity and residual enzyme activity of xylose reductase.

[0048] Figure 4 Graph showing the change of glucose dehydrogenase enzyme activity with the incubation time. Detailed implementation manners

[0049] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all of the embodiments.

[0050] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the scope of protection of the present invention.

[0051] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be obtained through commercial channels. The experimental methods without detailed conditions are carried out according to conventional experimental methods or according to the operation manuals recommended by the suppliers.

[0052] The experimental methods in the present invention are all conventional methods unless otherwise specified. For specific gene cloning operations, reference can be made to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al. The gene operation kits are purchased from TAKARA for characterization. Reagents used in upstream genetic engineering: The restriction endonucleases and DNA ligases used in the embodiments of the present invention are all purchased from TaKaRa, Takara Bio Inc. (Dalian); the genomic DNA extraction kit, plasmid extraction kit, and DNA recovery and purification kit are purchased from Axygen (Hangzhou) Co., Ltd. E. coli BL21(DE3), plasmid pET-28a(+), etc. are purchased from Novagen; DNA marker, FastPfu DNA polymerase, low molecular weight standard protein, and agarose electrophoresis reagents are purchased from Beijing TransGen Biotech Co., Ltd.; the usage methods of the above reagents refer to the product manuals. Other chemical reagents: xylose, arabinose, glucose, galactose, xylitol, arabinitol, etc. are purchased from Sinopharm Chemical Reagent Co., Ltd.

[0053] Detection of sugar alcohols: Determined by high performance liquid chromatography (HPLC). The mobile phase was ultrapure water, and the column used was an Agilent Hi-Plex Ca column (7.7 mm×300 mm). The column temperature was 85°C, and the flow rate was 0.6 mL / min.

[0054] Detection of gluconic acid: Determined by high performance liquid chromatography (HPLC). The mobile phase was 10 mmol / L K2HPO4 - 10 mmol / L tetrabutylammonium hydrogen sulfate (pH 7.2): methanol = 95:5 (V / V). The column used was a C18 column (4.6mm×250 mm). The column temperature was 30°C, and the flow rate was 0.7 mL / min.

[0055] Flask fermentation culture of genetically engineered bacteria: An LB liquid medium was used (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in deionized water and made up to volume, sterilized at 121°C for 20 min for later use). The engineered bacteria E. coli BL21(DE3) containing the relevant gene was inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured with shaking at 37°C for 12 h. Then it was transferred to 250 mL of fresh LB liquid medium containing 50 μg / mL Kan and cultured with shaking at 37°C until the OD600 reached about 0.8. IPTG was added to a concentration of 0.1 mM, and the culture was induced at 25°C for 16 h. After the culture was completed, the culture broth was centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the cells were collected and stored in a -70°C ultra-low temperature freezer for later use.

[0056] Preparation of crude enzyme solution: The collected wet cells were resuspended in a 50 mM sodium hydrogen phosphate - sodium dihydrogen phosphate buffer (pH = 8.0) to obtain a cell suspension with a cell concentration of 100 g / L. The resuspended cell suspension was subjected to high-pressure crushing and centrifuged at 12000 rpm for 30 min, and the supernatant was the crude enzyme solution.

[0057] Definition of enzyme activity: As stipulated by the International Enzyme Commission in 1961, one enzyme activity unit refers to the amount of enzyme that can convert 1 micromole of substrate per minute under specific conditions, or the amount of enzyme that can convert 1 micromole of the relevant group in the substrate.

[0058] In the following examples, the reaction equation for the production of xylitol by enzymatic catalysis is as Figure 1 shown; as Figure 2 shown, the enzymatic catalysis reaction was carried out in reactor 1, which was specifically a 5 L glass reaction kettle with a jacket, coupled with separator 2, which was specifically an anion exchange column, and the ion exchange reaction was carried out in the anion exchange column.

[0059] Example 1 Recombinant expression and screening of xylose reductase

[0060] Eleven wild-type XR genes from different sources were selected, codon-optimized and fully gene-synthesized in Escherichia coli by Nanjing Genscript Biotech Co., Ltd., constructed onto plasmid pET-28a, and introduced into Escherichia coli E.coli BL21. Flask fermentation and enzyme production were carried out in genetic engineering to obtain a xylose reductase enzyme library. With 0.1 mM NADPH coenzyme, the enzyme activity of xylose reductase towards the substrate D-xylose (the product is xylitol) was measured at 30 °C. The results are shown in Table 1. For other operations in enzyme activity measurement, refer to the literature 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 xylose reductase genes, they all showed good catalytic activity towards D-xylose, and the catalytic activity of most of them was greater than 200 U / mL. In addition, the residual enzyme activity of xylose reductase (crude enzyme solution) was measured after incubating in a 35 °C water bath for 12 hours. The results are as Figure 3 shown. Only the xylose reductases from Rhodotorula mucilaginosa , Neurospora crassa and Aspergillus niger had a residual enzyme activity greater than 200 U / mL. Therefore, XR5, XR6, and XR11 were selected for immobilization.

[0061] Table 1 Sources of xylose reductase and results of enzyme activity measurement

[0062]

[0063] Example 2 Recombinant expression and screening of glucose dehydrogenase

[0064] Four wild-type glucose dehydrogenase genes from different sources were selected, codon-optimized and fully gene-synthesized in Escherichia coli by Nanjing Genscript Biotech Co., Ltd., constructed onto plasmid pET-28a, and introduced into Escherichia coli E.coli BL21. Flask fermentation and enzyme production were carried out in genetic engineering to obtain a glucose dehydrogenase enzyme library. With 0.1 mM coenzyme NADP + , 10 g / L glucose as the substrate, 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 towards the substrate glucose. The glucose dehydrogenase (crude enzyme solution) was incubated in a 30 °C and 35 °C water bath for 6 hours, and the change in enzyme activity was measured. The results are as Figure 4 shown. The glucose dehydrogenase GDH2 from Exiguobacterium artemiae always maintained the highest activity. Therefore, GDH2 was selected for immobilization.

[0065] Table 2. Sources of Glucose Dehydrogenase and Results of Enzyme Activity Assay

[0066]

[0067] Example 3 Preparation of Immobilized Enzyme

[0068] Pretreatment of immobilization carrier: Epoxy resin (EP) was washed 3 times with phosphate buffer (50 mM, pH 8.0), 2 h each time, with regular stirring, and then dried by suction through a sintered glass funnel for standby. Amino resin (EA) needed to be further activated with glutaraldehyde. Treatment method: 10 g of resin was put into 100 mL of phosphate buffer (50 mM, pH 8.0) containing 4% glutaraldehyde for activation, stirred and reacted at 30 °C for 2 h, and then dried by suction through a sintered glass funnel for standby.

[0069] Immobilization of coenzyme NADP + : Prepare a 0.5% polyethyleneimine solution with 50 mM phosphate buffer at pH 8.0. Put 10 g of pretreated resin into 100 mL of polyethyleneimine solution for wrapping reaction, stir continuously at 30 °C for 2 h. Then dry by suction through a sintered glass funnel for standby. Prepare a 100 μM NADP disodium salt solution with phosphate buffer. Take 10 g of resin treated with polyethyleneimine and put it into the NADP disodium salt solution for immobilization. Stir continuously at 25 °C for 2 h, and then dry by suction through a sintered glass funnel for standby.

[0070] Co-immobilization of enzymes: Take 2 mL of XR crude enzyme solution and 8 mL of GDH crude enzyme solution respectively, add 90 mL of buffer, mix, and then add 10 g of immobilized material immobilizing coenzyme NADP + . Stir and react at 25 °C for 6 h. Finally, dry by suction through a sintered glass funnel, wash with buffer 2 - 3 times and then dry by suction to obtain the corresponding co-immobilized enzyme, and measure the enzyme activity of the co-immobilized enzyme. The enzyme activity of the co-immobilized enzyme refers to its enzyme activity 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.

[0071] Table 3. Results of Enzyme Activity Assay of Immobilized Enzyme

[0072]

[0073] Example 4 Catalytic Preparation of Xylitol by Reaction-Separation Coupled Co-immobilized Enzyme

[0074] Add 3.5 kg of deionized water into a 5 L glass reactor (with jacket), add 400 g of xylose and 490 g of glucose. After cleaning and activating the anion exchange resin, load it into a column with a volume of about 2.0 L. Pass circulating water through the jacket and control the circulating water temperature at 35°C. Weigh 100.0 g of IE1@EP co-immobilized enzyme and add it into the reactor. Start stirring to carry out the reaction. Continuously pump the enzyme-catalyzed reaction solution from the bottom of the reactor into the anion exchange column through a peristaltic pump, and the anion exchange solution returns to the reactor. Control the pump speed of the peristaltic pump at 4.0 L / h.

[0075] After the total reaction time of 18.0 h, HPLC detection shows that the substrate D-xylose is below the detection limit and the reaction stops. Filter the reaction solution through a sintered funnel to separate the co-immobilized enzyme (for the next batch of reactions). Measure the volume of the filtrate as 4080 mL. The concentration of the product xylitol in the reaction solution is 98.2 g / L, and the yield of xylitol is 99.5%. The remaining amount of the substrate D-glucose is 2.3 g / L. After the reaction, regenerate the anion exchange column with 4 L of 4% NaOH aqueous solution, and collect the regenerated solution with a gluconate concentration greater than 1.0 g / L. After the resin regeneration is completed, rinse the exchange column with deionized water until the effluent is close to neutral and reserve it for the next batch of reactions. Remove the water from the collected resin regenerated solution by vacuum concentration to obtain 511.6 g of by-product sodium gluconate, and calculate the yield of sodium gluconate as 88.0%.

[0076] Example 5: Preparation of xylitol by reaction-separation coupled co-immobilized enzyme catalysis

[0077] Add 3.5 kg of deionized water into a 5 L glass reactor (with jacket), add 400 g of xylose and 490 g of glucose. After cleaning and activating the anion exchange resin, load it into a column with a volume of about 2.0 L. Pass circulating water through the jacket and control the circulating water temperature at 35°C. Weigh 100.0 g of IE1@EA co-immobilized enzyme and add it into the reactor. Start stirring to carry out the reaction. Continuously pump the enzyme-catalyzed reaction solution from the bottom of the reactor into the anion exchange column through a peristaltic pump, and the anion exchange solution returns to the reactor. Control the pump speed of the peristaltic pump at 4.0 L / h.

[0078] After the total reaction time of 10.0 h, the substrate D-xylose was below the detection limit by HPLC, and the reaction stopped. The reaction solution was filtered by a sintered funnel to separate the co-immobilized enzyme (for the next batch of reactions). The volume of the filtrate was measured as 4050 mL. The concentration of xylitol in the reaction solution was 99.3 g / L, and the yield of xylitol was 99.2%. The remaining substrate D-glucose was 3.0 g / L. After the reaction, the anion exchange column was regenerated with 4 L of 4% NaOH aqueous solution, and the regenerated solution with 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 reserved for the next batch of reactions. The collected resin regenerated solution was concentrated under vacuum to remove water, obtaining 509.3 g of by-product sodium gluconate, and the yield of sodium gluconate was calculated as 88.3%.

[0079] Example 6 Preparation of Xylitol by Reaction-Separation Coupled with Co-Immobilized Enzyme Catalysis

[0080] Add 3.5 kg of deionized water into a 5 L glass reactor (with a jacket), add 200 g of xylose and 245 g of glucose. After washing and activating the anion exchange resin, load it into a column with a column volume of about 1.0 L. Pass circulating water through the jacket, and control the circulating water temperature at 35°C. Weigh 100.0 g of IE1@EA co-immobilized enzyme and add it into the reactor. Start stirring to carry out the reaction. Continuously pump the enzyme-catalyzed reaction solution from the bottom of the reactor into the anion exchange column through a peristaltic pump, and the anion exchange solution returns to the reactor again. The pump speed of the peristaltic pump is controlled at 2.0 L / h.

[0081] After the total reaction time of 8.0 h, the substrate D-xylose was below the detection limit by HPLC, and the reaction stopped. The reaction solution was filtered by a sintered funnel to separate the co-immobilized enzyme (for the next batch of reactions). The volume of the filtrate was measured as 4050 mL. The concentration of xylitol in the reaction solution was 49.3 g / L, and the yield of xylitol was 99.2%. The remaining substrate D-glucose was 0.3 g / L. The anion exchange column was regenerated with 2 L of 4% NaOH aqueous solution, and the regenerated solution with 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 reserved. The collected resin regenerated solution was concentrated under vacuum to remove water, obtaining 250.2 g of by-product sodium gluconate, and the yield of sodium gluconate was calculated as 86.1%.

[0082] Example 7 Preparation of Xylitol by Reaction-Separation Coupled with Co-Immobilized Enzyme Catalysis

[0083] Add 3.5 kg of deionized water into a 5 L glass reactor (with a jacket), add 200 g of xylose and 245 g of glucose. After cleaning and activating the anion exchange resin, pack it into a column with a volume of about 1.0 L. Pass circulating water through the jacket and control the circulating water temperature at 35°C. Weigh 100.0 g of the IE2@EA co-immobilized enzyme, add it to the reactor, turn on the stirrer, and carry out the reaction. Continuously pump the enzyme-catalyzed reaction solution from the bottom of the reactor into the anion exchange column through a peristaltic pump, and the anion exchange solution returns to the reactor. Control the pump speed of the peristaltic pump at 2.0 L / h.

[0084] After the total reaction time of 4.0 h, HPLC detection shows that the substrate D-xylose is below the detection limit, and the reaction stops. Filter the reaction solution through a sintered funnel to separate the co-immobilized enzyme (for the next batch of reactions). Measure the volume of the filtrate to be 4060 mL. The concentration of the product xylitol in the reaction solution is 49.5 g / L, the yield of xylitol is 99.2%, and the remaining substrate D-glucose is 0.5 g / L. Regenerate the anion exchange column with 2 L of 4% NaOH aqueous solution, and collect the regeneration solution with a gluconate concentration greater than 1.0 g / L. After the resin regeneration is completed, rinse the exchange column with deionized water until the effluent is close to neutral and then set it aside. Remove the water from the collected resin regeneration solution by vacuum concentration to obtain 245.1 g of the by-product sodium gluconate, and calculate the yield of sodium gluconate to be 84.9%.

[0085] Example 8: Preparation of xylitol by reaction-separation coupling with co-immobilized enzyme

[0086] Add 3.5 kg of deionized water into a 5 L glass reactor (with a jacket), add 200 g of xylose and 245 g of glucose. After cleaning and activating the anion exchange resin, pack it into a column with a volume of about 1.0 L as a separator. Pass circulating water through the jacket and control the circulating water temperature at 40°C. Weigh 50.0 g of the IE3@EA co-immobilized enzyme, add it to the reactor, turn on the stirrer, and carry out the reaction. Continuously pump the enzyme-catalyzed reaction solution from the bottom of the reactor into the anion exchange column through a peristaltic pump, and the anion exchange solution returns to the reactor. Control the pump speed of the peristaltic pump at 2.0 L / h.

[0087] After 12.0 h of the total reaction time, the substrate D-xylose was below the detection limit by HPLC, and the reaction stopped. The reaction solution was filtered by suction through a sintered glass funnel to separate the co-immobilized enzyme (for the next batch of reactions). The volume of the filtrate was measured to be 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 remaining amount of the substrate D-glucose was 0.5 g / L. After the reaction, the anion exchange column was regenerated with 2 L of 4% NaOH aqueous solution, and the regenerated solution with 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 reserved for the next batch of reactions. The collected resin regenerated solution was concentrated under vacuum to remove water, obtaining 261.5 g of by-product sodium gluconate, and the yield of sodium gluconate was calculated to be 89.9%.

[0088] Comparative Example 1

[0089] 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.

[0090] 3.5 kg of deionized water was added to a 5 L glass reaction kettle (with jacket), 200 g of xylose and 245 g of glucose were added. After the anion exchange resin was washed and activated, it was packed into a column with a column volume of about 1.0 L. The jacket was passed through circulating water, and the circulating water temperature was controlled at 30 °C. 5.0 g of the immobilized enzyme XR5-NADP @EP and 45.0 g of the immobilized enzyme GDH2-NADP @EP were weighed and added to the reaction kettle respectively. Stirring was started for the reaction; the enzyme-catalyzed reaction solution was continuously pumped from the bottom of the kettle into the anion exchange column through a peristaltic pump, and the anion exchange solution was then returned to the reaction kettle. The pump speed of the peristaltic pump was controlled at 1 L / h.

[0091] After 24.0 h of the total reaction time, the concentration of the substrate D-xylose was 38.9 g / L and the glucose concentration was 50.3 g / L by HPLC, and the reaction almost stalled. After separating the immobilized enzyme, the volume of the filtrate was measured to be 3980 mL. The concentration of the product xylitol in the reaction solution was 10.3 g / L, and the calculated yield of xylitol was only 20.4%.

[0092] Comparative Example 2

[0093] The pretreatment of the immobilization carrier was the same as that in Example 3.

[0094] Coenzyme NADP +Immobilization: Prepare a 0.5% polyethylenimine solution with 50 mM phosphate buffer at pH 8.0. Put 10 g of the treated resin into 100 mL of the polyethylenimine solution for encapsulation reaction, stir continuously at 30 °C for 2 h. Then use a sintered glass funnel to drain and set aside. Prepare a 100 μM solution of NADP disodium salt with phosphate buffer. Take 10 g of the resin treated with polyethylenimine and put it into the NADP disodium salt solution for immobilization. Stir continuously at 25 °C for 2 h, then use a sintered glass funnel to drain to obtain immobilized coenzyme NADP + , for standby.

[0095] Immobilization of enzymes: Take the crude enzyme solutions of XR5 or GDH2 respectively, and immobilize them with the pretreated resin to obtain the corresponding coenzyme-free immobilized enzymes XR5@EP and GDH2@EP. The measured enzyme activities are 181.1 U / g and 29.6 U / g respectively.

[0096] Add 3.5 kg of deionized water to a 5 L glass reactor (with jacket), add 200 g of xylose and 245 g of glucose. After washing and activating the anion exchange resin, pack it into a column with a column volume of about 1.0 L. Pass circulating water through the jacket and control the circulating water temperature at 30 °C. Weigh 10.0 g of the immobilized enzyme XR5@EP, 60.0 g of the immobilized enzyme GDH2@EP and the immobilized coenzyme NADP + 10 g, and add them into the reactor respectively. Start stirring for reaction; continuously pump the enzyme-catalyzed reaction solution from the bottom of the reactor into the anion exchange column through a peristaltic pump, and the anion exchange solution returns to the reactor. Control the pump speed of the peristaltic pump at 1 L / h.

[0097] After the total reaction time of 12.0 h, HPLC detects that the concentration of the product xylitol in the reaction solution is 1.5 g / L, and the reaction hardly proceeds.

[0098] Comparative Example 3

[0099] Add 3.5 kg of deionized water to a 5 L glass reactor (with jacket), add 400 g of xylose and 490 g of glucose, and control the temperature inside the reactor at 35 °C with circulating water temperature. Weigh 100.0 g of the co-immobilized enzyme IE1@EP and add it into the reactor. Start stirring for reaction. After 2.0 h of reaction, HPLC detects that the concentration of the product xylitol in the reaction solution is 34.1 g / L; after 4.0 h of reaction, detect the xylitol concentration to be 33.9 g / L, and the product concentration no longer increases. Measure the pH of the reaction solution to be 2.56. Under this condition, the immobilized enzyme is inactivated, resulting in the termination of the reaction.

Claims

1. A method for continuously catalyzing the preparation of xylitol by co-immobilized enzyme coupling ion exchange, characterized in that, Comprising: A continuous cycle of an enzymatic reaction and an ion exchange reaction, finally obtaining the product xylitol; The enzymatic reaction uses xylose and glucose as raw materials, and a co-immobilized enzyme containing xylose reductase, glucose dehydrogenase, and NADP+ coenzyme as a catalyst, and the reaction generates a reaction solution containing xylitol and gluconate; The ion exchange reaction uses the reaction solution as the initial solution, and after ion exchange, adsorbs gluconate to obtain a separation solution containing basic anions; The separation solution is refluxed into the reaction solution to achieve a continuous cycle; In the co-immobilized enzyme, xylose reductase, glucose dehydrogenase, and coenzyme NADP+ are co-immobilized on a carrier; the carrier is an epoxy resin or an amino resin; In the co-immobilized enzyme, polyethyleneimine is used to coat the resin; The accession number of the xylose reductase in the NCBI database is ALO17776.1, EAA34695.1, or Q9P8R5.1; the accession number of the glucose dehydrogenase in the NCBI database is WP_012369122.1; In the enzymatic reaction, the reaction pH is 5 to 9; The reaction solution is subjected to ion exchange using an anion exchange column to adsorb gluconate; In the anion exchange column, the ratio of the resin packing amount to the volume of the reaction solution is 1:2 to 5; Calculated according to the volume of the anion exchange column, the flow rate of the reaction solution in the anion exchange column is 0.2 to 2 BV / h.

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

8.

3. According to the method described in claim 2, calculated based on the activity of xylose reductase, the ratio of the co-immobilized enzyme to the raw materials is 1 to 10 U:1 mL.

4. The method according to claim 1, wherein After ion exchange, the anion exchange column is desorbed to separately obtain the by-product gluconate.

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

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