Method for producing xylitol through two-step enzyme catalysis of hemicellulose hydrolysate

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for producing xylitol through two-step enzyme catalysis of hemicellulose hydrolysate, and the method comprises the following steps: taking hemicellulose hydrolysate as a raw material, adding arabinose dehydrogenase, NADH oxidase and coenzyme NADH, and carrying out first-step fermentation until no L-arabinose residue exists in fermentation liquor to obtain fermentation reaction liquor I; and adding xylose reductase and glucose dehydrogenase into the fermentation reaction liquid I, and carrying out second-step fermentation to obtain a fermentation product containing xylitol. According to the two-step enzyme catalysis method provided by the invention, xylitol is prepared, and high-purity xylose does not need to be used as a raw material; according to the present invention, the cheap hemicellulose hydrolysate can be directly adopted as the raw material, the xylose conversion rate and the xylitol yield can exceed 99%, the impurity arabitol content in the product is low, the other impurity saccharic acid is easy to separate, and the problems of complex process, large separation difficulty and high cost in the xylitol refining process are successfully solved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for producing xylitol from hemicellulose hydrolyzate by two-step enzyme catalysis. Background Art

[0002] Xylitol is an organic compound with the chemical formula C5H 12 O5, native to Finland, is a natural sweetener extracted from plant materials such as birch, oak, corn cobs, and sugarcane bagasse. Xylitol is widely distributed in nature, found in various fruits, vegetables, and grains, but at very low concentrations. Xylitol is not an imported product; it is an intermediate in normal carbohydrate metabolism. According to my country's "Hygienic Standard for the Use of Food Additives" (GB2760-1996), xylitol can replace sugar in candies, pastries, and beverages according to normal production needs. Labels should indicate that it is suitable for diabetics. In actual production, xylitol can be used as a sweetener and humectant. Reference dosages for food are: 43% for chocolate; 64% for chewing gum; 40% for jams and jellies; and 50% for ketchup. Xylitol can also be used in condensed milk, toffee, and soft candy. It does not cause browning when used in pastries.

[0003] Traditional xylitol production utilizes chemical hydrogenation, a process that requires a relatively high-purity xylose solution (xylose purity exceeding 98%). This is primarily because impurities in the xylose solution can easily poison the nickel catalyst used in the hydrogenation process and quickly deactivate it. Furthermore, chemical reduction has poor selectivity, and hemicellulose hydrolysate contains a variety of miscellaneous sugars, which are also reduced to their corresponding polyols during chemical catalytic hydrogenation. Because polyols are much more soluble than their corresponding sugars, separating xylitol from a polyol mixture is far more difficult than separating xylose from a mixed sugar mixture, requiring the use of complex separation techniques such as simulated moving beds, which reduces xylitol yield. Chemical hydrogenation requires multiple purification steps for the xylose hydrolysate prior to hydrogenation, significantly increasing the production cost of xylitol. The hydrogenation step also requires high temperatures (80-140°C) and high pressures (50 atm), and the nickel catalyst can pollute the environment.

[0004] Due to the above problems with traditional chemical hydrogenation, the development of bioconversion methods that can directly convert xylose in hemicellulose hydrolysates without using pure xylose as a substrate and under mild conditions, using synthetic biology as the underlying technology, has attracted increasing attention from researchers at home and abroad in recent years. There are two ways to produce xylitol by biological methods. One method mainly uses microorganisms such as bacteria, fungi, yeast, or recombinant genetically engineered bacteria to ferment and produce xylitol from different biomass raw materials. For example, Chinese invention patent application publication number CN104357339A discloses a tropical yeast that produces xylitol using high-concentration xylose as the sole carbon source. The xylitol concentration can reach 20-65g / L, and the residual sugar concentration is less than 5%. Chinese invention patent publication number CN110982850B discloses a genetically engineered Aspergillus oryzae that produces xylitol at a concentration of 13.5g / L using 50g / L xylan as the sole carbon source. The yield and productivity are 0.27g / g and 0.16g / L / h, respectively. Chinese invention patent publication number CN106661540B discloses a recombinant Pichia olmer yeast that produces 120 g / L of xylitol and 5 g / L of ribitol from 250 g / L of glucose monohydrate, with a xylitol yield and productivity of 0.48 g / g and 1.81 g / L / h, respectively. While xylitol production by fermentation avoids the harsh reaction conditions of chemical hydrogenation, the yield of the product relative to the raw material is generally low. Another biological method for producing xylitol is enzyme catalysis, including methods using resting cells (or immobilized cells) and free enzyme proteins (or immobilized enzymes) as catalysts. Chinese patent CN110628835 uses xylose reductase, glucose dehydrogenase, formate dehydrogenase, an electron carrier, and water to form a biocatalytic system to catalyze the reaction of xylose to produce xylitol. Within 24 hours, 278.4 g / L of xylitol was obtained from 2 M xylose, with a productivity of 11.6 g / L / h. Chinese patent CN108977432 uses immobilized recombinant E. coli cells co-expressing xylose reductase and glucose dehydrogenase as catalysts to produce xylitol from a xylose mother liquor. The recombinant cells catalyze 200 g / L of xylose for 30 hours, achieving a xylitol yield of 100%. Chinese patent CN108949852 uses recombinant E. coli containing the xylose reductase gene XR and the glucose dehydrogenase gene GDH as catalysts, forming a biocatalytic system with an aqueous solution containing xylose and glucose and CaCO3 to catalyze the reduction of xylose to produce xylitol. The product has a xylitol concentration of 145.81 g / L and a yield of 0.97 (g / g).

[0005] The advantage of bioenzymatic methods for producing xylitol is high reaction efficiency, with product yields generally exceeding 90%. However, current bioenzymatic methods can only use high-purity xylose as a raw material and cannot directly catalyze hemicellulose hydrolysates containing miscellaneous sugars to produce qualified xylitol products. This is mainly because hemicellulose hydrolysates contain a large amount of arabinose, which can be catalyzed by natural xylose reductase to produce arabinose (Chinese Patent CN118620969). The inclusion of arabinose in the product can result in substandard xylitol. Therefore, there is a need to develop new bioenzymatic methods that can directly use hemicellulose hydrolysates to produce xylitol. Summary of the Invention

[0006] In response to the demand for a production process for directly producing xylitol from hemicellulose hydrolysate in the prior art, the present application provides a two-step enzymatic method for producing xylitol from hemicellulose hydrolysate. The specific technical solution is as follows:

[0007] The present invention provides a two-step enzymatic method for producing xylitol from hemicellulose hydrolyzate, comprising:

[0008] (1) Using hemicellulose hydrolyzate as a raw material, adding arabinose dehydrogenase, NADH oxidase, and coenzyme NADH, and performing the first step of fermentation until no L-arabinose remains in the fermentation liquid, thereby obtaining a fermentation reaction liquid I;

[0009] (2) After adding xylose reductase and glucose dehydrogenase to the fermentation reaction liquid I, a second fermentation step is performed to obtain a fermentation product containing xylitol.

[0010] This application uses hemicellulose hydrolyzate as raw material to produce xylitol through a two-step enzyme-catalyzed reaction. In the first step, arabinose and galactose are converted into corresponding sugar acids using arabinose dehydrogenase, NADH oxidase, and coenzyme NADH. The arabinose dehydrogenase catalysis process consumes NAD + , and generate NADH, NADH oxidase can oxidize the generated NADH into NAD + , so that the coenzyme NADH can self-circulate; after the reaction of arabinose and galactose is complete, xylose reductase and glucose dehydrogenase are added to convert xylose into xylitol and glucose into gluconic acid. In the process of xylose reductase catalyzing the reduction of xylose in the hydrolyzate into xylitol, NADPH is consumed and NADP is generated. + Glucose dehydrogenase catalyzes the dehydrogenation of glucose to produce gluconic acid and consumes NADP + The enzyme also generates NADPH, allowing the coenzyme NADPH to self-circulate. After two steps of enzyme-catalyzed reactions, the xylose in the hemicellulose hydrolysate is completely converted into xylitol, while other miscellaneous sugars (glucose, galactose, and arabinose) are converted into their corresponding sugar acids. No arabitol is produced, facilitating subsequent separation.

[0011] Furthermore, the hemicellulose hydrolyzate is a hemicellulose hydrolyzate stock solution or a concentrated solution obtained by concentrating the hemicellulose hydrolyzate stock solution, and the hemicellulose hydrolyzate stock solution contains D-glucose, D-xylose, D-galactose and L-arabinose.

[0012] Furthermore, in the hemicellulose hydrolyzate stock solution, the concentrations of D-glucose, D-xylose, D-galactose, and L-arabinose are 5-7 g / L, 50-53 g / L, 3-4 g / L, and 7-8 g / L, respectively.

[0013] Furthermore, the arabinose dehydrogenase is obtained by crushing and extracting an engineered bacterium containing an arabinose dehydrogenase gene; and the NADH oxidase is obtained by crushing and extracting an engineered bacterium containing an NADH oxidase gene.

[0014] Furthermore, the xylose reductase is obtained by crushing and extracting an engineered bacterium containing a xylose reductase gene; and the glucose dehydrogenase is obtained by crushing and extracting an engineered bacterium containing a glucose dehydrogenase gene.

[0015] Furthermore, the accession number of the arabinose dehydrogenase gene in the NCBI library is WP_004041122.1, NC_003062.2 or KJ716853.1.

[0016] Furthermore, the accession number of the NADH oxidase gene in the NCBI library is AB035801.1, BBC74576.1, UPS09830.1, AF536177.1, CAG39343.1, X67220.1, BAL17746.1 or CUU11616.1.

[0017] Furthermore, the accession number of the xylose reductase gene in the NCBI library is AF451326.3, KF752418.1, AF074484.1, AB002106.1, ALO17776.1, EAA34695.1, AAA99507.1, NC_009045.1, SCU85332.1, AAO91803.1 or Q9P8R5.1.

[0018] Furthermore, the accession numbers of the glucose dehydrogenase gene in the NCBI library are WP_013055546.1, WP_012369122.1, and WP_274796929.1.

[0019] Furthermore, the host cell of the engineered bacteria is Escherichia coli.

[0020] Furthermore, the Escherichia coli is E. coli BL21.

[0021] Furthermore, in the first fermentation step, the pH is 7.5-9.5 and the temperature is 15-75°C; based on the wet cell mass, the added amount of the engineered bacteria containing the arabinose dehydrogenase gene and the engineered bacteria containing the NADH oxidase gene are both 1-50 g / L; the added concentration of the coenzyme NADH is 0.05 mM-1 mM.

[0022] Furthermore, in the first fermentation step, the pH value is 8.0-9.0, the amount of engineered bacteria added is 5-20 g / L, and the concentration of coenzyme NADH added is 0.1 mM-0.5 mM.

[0023] Furthermore, in the second fermentation step, the pH is 5.5-8.5, the temperature is 15-75°C, and the added amount of the engineered bacteria containing the xylose reductase gene and the engineered bacteria containing the glucose dehydrogenase gene is 1-50 g / L based on the added wet cell mass; the added concentration of the coenzyme NADPH is 0.05 mM-1 mM.

[0024] Furthermore, in the second fermentation step, the pH value is 6.0-8.0, the amount of engineered bacteria added is 5-20 g / L, and the concentration of coenzyme NADH added is 0.1 mM-0.5 mM.

[0025] Furthermore, glucose is added in the second fermentation step, and the mass of the added glucose is 80% to 100% of the mass of xylose in the hemicellulose hydrolyzate.

[0026] In the second step, since the glucose content in the hemicellulose hydrolysate is lower than the xylose content, a certain amount of glucose is added during the reaction to ensure that there is enough NADPH to participate in the reaction of xylose reductase to convert xylose, so that xylose can be completely converted into xylitol.

[0027] Furthermore, the method of adding glucose is to add glucose to the fermentation reaction liquid, or to flow a glucose aqueous solution into the fermentation reaction liquid.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The two-step enzymatic catalysis method provided by the present invention for preparing xylitol does not require the use of high-purity xylose as a raw material; inexpensive hemicellulose hydrolysate can be directly used as a raw material, the xylose conversion rate and the xylitol yield can both exceed 99%, the impurity arabitol content in the product is low, and other impurities, sugars and acids, are easily separated, successfully solving the problems of complex process, difficult separation and high cost in the xylitol refining process. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1This is the first step reaction equation for producing xylitol from hemicellulose hydrolyzate using two-step enzyme catalysis.

[0031] Figure 2 This is the reaction equation for the second step of producing xylitol from hemicellulose hydrolyzate by two-step enzyme catalysis of the present invention.

[0032] Figure 3 This is the HPLC detection spectrum of hemicellulose hydrolyzate from corn cob; among them, the peak at 16.35 min is D-glucose, the peak at 17.25 min is D-xylose, the peak at 19.12 min is D-galactose, and the peak at 20.38 min is L-arabinose. DETAILED DESCRIPTION

[0033] In order to make those skilled in the art better understand the present invention, the technical solution of the present invention is 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 embodiments.

[0034] 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.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled 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 detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0036] The experimental methods in the present invention are all conventional methods unless otherwise specified. For details on gene cloning operations, please refer to the "Molecular Cloning Experiment Guide" edited by J. Sambrook et al. The kits for gene manipulation were purchased from TAKARA for characterization. Reagents used in upstream genetic engineering: the restriction endonucleases and DNA ligases used in the examples of the present invention were purchased from TaKaRa and Takara Bioengineering (Dalian) Co., Ltd.; the genome extraction kit, plasmid extraction kit, and DNA recovery and purification kit 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, and agarose electrophoresis reagent were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the use of the above reagents can be referred to the product instructions. Other chemical reagents: xylose, arabinose, glucose, galactose, xylitol, arabitol, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0037] Detection of sugar alcohols: high performance liquid chromatography (HPLC) with ultrapure water as the mobile phase, a Carbomix Pb-NP10:5% column (7.8 mm x 300 mm), a column temperature of 75° C., and a flow rate of 0.5 mL / min.

[0038] For shake flask fermentation of genetically engineered bacteria, use LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in deionized water, adjusted to volume, sterilized at 121°C for 20 minutes, and set aside). Inoculate E. coli BL21(DE3) containing the relevant gene into 5 mL of LB medium containing 50 μg / mL kanamycin and incubate at 37°C with shaking for 12 hours. Transfer the culture to 250 mL of fresh LB medium also containing 50 μg / mL kanamycin and incubate at 37°C with shaking until the OD600 reaches approximately 0.8. IPTG is then added to a concentration of 0.1 mM and induced at 25°C for 16 hours. After incubation, centrifuge the culture at 4000 rpm for 15 minutes, discard the supernatant, and collect the bacterial cells for storage in a -70°C freezer until ready to use.

[0039] Definition of enzyme activity: The 1961 International Conference on Enzymology 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 (35°C), or the amount of enzyme that converts 1 micromole of related groups in the substrate.

[0040] Example 1 Recombinant expression and enzyme activity determination of arabinose dehydrogenase

[0041] Ten wild-type AraDH genes from different sources were selected and codon-optimized and fully synthesized in E. coli by Nanjing GenScript Biotechnology Co., Ltd. The genes were then constructed into the plasmid pET-28a and introduced into E. coli BL21 for shake flask fermentation and enzyme production to obtain an arabinose dehydrogenase enzyme library. + and 0.1 mM NAD + As a coenzyme, the enzyme activity of arabinose dehydrogenase on substrate L-arabinose (product is L-arabinonic acid) and substrate D-galactose (product is galactonic acid) was measured. The results are shown in Table 1. As shown in Table 1, the arabinose dehydrogenase from Pseudomonas fluorescens uses NADP + As a coenzyme, it has high enzyme activity on substrates L-arabinose and D-galactose, but NAD +When tested as a coenzyme, the enzyme activity against the two substrates was extremely low. + or NAD + It is a coenzyme and has high enzyme activity towards both L-arabinose and D-galactose. Arabinose dehydrogenases from other sources have basically no catalytic activity towards L-arabinose and D-galactose.

[0042] Table 1 Source of arabinose dehydrogenase and enzyme activity determination results

[0043]

[0044]

[0045] Note: The unit of enzyme activity is U / mL.

[0046] Example 2 Recombinant expression and enzyme activity determination of NADH oxidase

[0047] Eight wild-type NOX genes from different sources were codon-optimized and fully synthesized in E. coli by Nanjing GenScript Biotechnology Co., Ltd., then constructed into the pET-28a plasmid and introduced into E. coli BL21 for shake flask fermentation and enzyme production to generate an NADH oxidase library. The NADH oxidase activities against the two reduced coenzymes were measured using 1.0 mM NADPH and 1.0 mM NADH as coenzymes, respectively. The results are shown in Table 2. As shown in Table 2, all eight selected NADH oxidases exhibited good activity against NADH but showed little catalytic activity against NADPH.

[0048] Table 2. Source of NADH oxidase and enzyme activity determination results

[0049]

[0050] Example 3 Recombinant expression and enzyme activity determination of xylose reductase

[0051] Eleven wild-type XR genes from different sources were selected and codon-optimized and fully synthesized in E. coli by Nanjing GenScript Biotechnology Co., Ltd. The genes were then constructed onto the plasmid pET-28a and introduced into E. coli BL21 (DE3) for shake flask fermentation and enzyme production to obtain a xylose reductase enzyme library. The enzymatic activities of the xylose reductase on the substrate L-arabinose (product L-arabinitol) and the substrate D-xylose (product xylitol) were determined using 0.1 mM NADPH and 0.1 mM NADH as coenzymes, respectively. The results are shown in Table 3. For other procedures related to enzyme activity determination, refer to the literature Evolution in reverse: engineering a D-xylose-specific xylose reductase. Chembiochem, 2008, 9(8): 1213-5. The results in Table 3 show that, except for the xylose reductases from Candida boidinii and Candida parapsilosis, which showed little preference for coenzyme type and exhibited high enzymatic activity with both NADPH and NADH as coenzymes, the remaining xylose reductases all preferred NADPH as the coenzyme, exhibiting high enzymatic activity with NADPH as the coenzyme and extremely low activity with NADH as the coenzyme.

[0052] Table 3. Sources of xylose reductase and enzyme activity assay results on different substrates

[0053]

[0054] Note: The unit of enzyme activity is U / mL.

[0055] Example 4 Recombinant expression and enzyme activity determination of glucose dehydrogenase

[0056] Three wild-type glucose dehydrogenase genes from different sources were selected and codon optimized and fully synthesized in E. coli by Nanjing GenScript Biotechnology Co., Ltd. The genes were constructed into plasmid pET-28a and introduced into E. coli BL21 for shake flask fermentation and enzyme production to obtain a glucose dehydrogenase enzyme library. + The enzymatic activities of the arabinose dehydrogenases were measured using D-xylose, L-arabinose, D-galactose, and D-glucose as coenzymes, respectively. The results are shown in Table 3. As shown in Table 3, the three glucose dehydrogenases all exhibited significant catalytic activity towards the substrate glucose, but had no catalytic activity towards other substrates.

[0057] Table 4. Source of glucose dehydrogenase and enzyme activity determination results

[0058]

[0059] Example 5 Cascade catalytic reaction of arabinose dehydrogenase and NADH oxidase

[0060] A hemicellulose hydrolyzate from corn cobs was selected and the composition of various sugars was determined by HPLC, such as Figure 3 As shown in the figure, the hemicellulose hydrolysate mainly contains D-glucose, D-xylose, D-galactose and L-arabinose, with concentrations of 5.8 g / L, 51.7 g / L, 3.5 g / L and 7.2 g / L, respectively. The contents of other miscellaneous sugars are below the detection limit.

[0061] The enzyme-producing strains of arabinose dehydrogenase and NADH oxidase obtained in Example 1-2 were fermented in shake flasks to produce enzymes. 1 g of the wet cells containing the enzymes were taken, resuspended with 4 mL of 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH = 7.0), and then ultrasonically disrupted to obtain the corresponding crude enzyme solution. 4.0 mL of the above hemicellulose hydrolyzate was taken and the pH was adjusted to 7.0 with a 5 M aqueous sodium hydroxide solution. 0.5 mL of each of the above two enzymes was added, and then 50 μL of NADPH was added. + Aqueous solution (concentration 10 mM) was reacted in a 40°C water bath with magnetic stirring. After 12 hours of reaction, various sugars and sugar alcohols were detected, and the results are shown in Table 5. As shown in Table 5, D-galactose and L-arabinose were completely converted through the cascade catalysis of arabinose dehydrogenase and NADH oxidase, while D-glucose and D-xylose were largely unaffected by the reaction, and neither arabitol nor xylitol was produced.

[0062] Table 5. Detection results of arabinose dehydrogenase and NADH oxidase cascade catalytic hydrolysis solution

[0063]

[0064] Example 6 Cascade catalytic reaction of xylose reductase and glucose dehydrogenase

[0065] The xylose reductase and glucose dehydrogenase enzyme-producing strains obtained in Example 3-4 were fermented in shake flasks to produce enzymes. 1 g of the wet cells containing the enzymes were taken, resuspended with 4 mL of 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH = 7.0), and then ultrasonically disrupted to obtain the corresponding crude enzyme solution. 4.0 mL of corn cob hemicellulose hydrolyzate was taken and the pH was adjusted to 7.0 with a 5 M aqueous sodium hydroxide solution. 0.5 mL of each crude enzyme solution of xylose reductase and glucose dehydrogenase was added, and then 50 μL of NADP was added. +Aqueous solution (10 mM concentration) was reacted with magnetic stirring in a 35°C water bath. After 12 hours of reaction, various sugars and sugar alcohols were tested, and the results are shown in Table 6. The results in Table 6 show that D-glucose in the hydrolyzate was used by glucose dehydrogenase to regenerate NADPH; in the presence of NADPH, xylose reductase catalyzed the reduction of D-xylose to xylitol and L-arabinose to arabitol; D-galactose in the hydrolyzate was largely absent from the reaction. Due to the limited D-glucose content in the hydrolyzate, the reaction stagnated after complete conversion of the D-glucose.

[0066] Table 6. Detection results of xylose reductase and glucose dehydrogenase cascade catalytic hydrolysis solution

[0067]

[0068] Example 7 Two-step enzyme-catalyzed reaction

[0069] The AraDH2, NOX1, XR1, and GDH1 strains obtained in Examples 1-4 were fermented in shake flasks to produce enzymes. 1g of the resulting enzyme-containing wet cells was resuspended in 4mL of deionized water and ultrasonically disrupted to obtain the corresponding crude enzyme solution. 160mL of the hemicellulose hydrolyzate was added to a 250mL three-necked flask, magnetically stirred in a 35°C water bath, and the pH of the hydrolyzate was adjusted to 7.0 with 5M sodium hydroxide solution.

[0070] Step 1: First add the above 1g wet cell AraDH2 and NOX1 crude enzyme solution, then add 1.0mL NAD + Aqueous solution (concentration 10mM), start the reaction, the reaction equation is as follows Figure 1 As shown. During the reaction, oxygen was introduced by bubbling. After 2.0 h of reaction, the HPLC detection of substrates D-galactose and L-arabinose was below the detection limit, and the introduction of oxygen was stopped.

[0071] Step 2: Add 1g of the above wet cells to the XR1 and GDH1 crude enzyme solutions, and add 1.0mL of NADP + Aqueous solution (concentration 10mM), continue the reaction, the reaction equation is as follows Figure 2 The reaction was controlled at pH 7.0 ± 0.1 using 1M sodium hydroxide solution. After 6.0 hours, the reaction was terminated when the substrate D-glucose level fell below the detection limit by HPLC. A total of 201 mL of the reaction solution was measured, revealing a concentration of 3.81 g / L of the product xylitol, no detectable arabinitol, and 37.09 g / L of the substrate D-xylose.

[0072] Example 8 Two-step enzyme-catalyzed reaction

[0073] The AraDH3, NOX2, XR5, and GDH2 strains obtained in Examples 1-4 were fermented in shake flasks to produce enzymes. 1g of the resulting enzyme-containing wet cells was resuspended in 4mL of deionized water and ultrasonically disrupted to obtain the corresponding crude enzyme solution. 160mL of the hemicellulose hydrolyzate was added to a 250mL three-necked flask, magnetically stirred in a 40°C water bath, and the pH of the hydrolyzate was adjusted to 7.5 with 5M sodium hydroxide solution.

[0074] Step 1: First add the above 1g wet cells of AraDH3 and NOX2 crude enzyme solution, then add 1.0mL NAD + The reaction was started by bubbling oxygen in an aqueous solution (concentration 10 mM). After 1.0 h of reaction, the HPLC detection of substrates D-galactose and L-arabinose was below the detection limit, and the oxygen was stopped.

[0075] Step 2: Add the above 1g wet cells of XR3 and GDH2 crude enzyme solution, and add 1.0mL NADP + Aqueous solution (concentration 10mM), continue the reaction. During the reaction, the pH of the reaction solution is controlled at 7.5±0.1 with a 1M sodium hydroxide aqueous solution. After the total reaction time is 3.0h, 7.5g of anhydrous glucose is weighed and added to the reaction solution. After continuing the reaction for 6h, HPLC detection of the substrate D-xylose is below the detection limit and the reaction is stopped. The volume of the reaction solution is measured to be 210mL. The concentration of the product xylitol in the reaction solution is 38.95g / L, the product arabitol is not detected, and the substrate D-glucose remains at 1.05g / L. The D-xylose in the raw material is completely converted, and the yield of xylitol to xylose is calculated to be 98.9%.

[0076] Example 9 Two-step enzyme-catalyzed reaction

[0077] The AraDH9, NOX5, XR6, and GDH3 strains obtained in Examples 1-4 were fermented in shake flasks to produce enzymes. 1 g of the resulting enzyme-containing wet cells was resuspended in 4 mL of deionized water and ultrasonically disrupted to obtain the corresponding crude enzyme solution. 750 mL of the hemicellulose hydrolyzate prepared in Example 5 was measured, vacuum concentrated to 150 mL, and added to a 250 mL three-necked flask. The mixture was stirred magnetically in a 30°C water bath, and the pH of the hydrolyzate was adjusted to 8.5 with 5 M aqueous sodium hydroxide solution.

[0078] Step 1: First add the above 1g wet cells of AraDH9 and NOX5 crude enzyme solution, then add 1.0mL NAD + The reaction was started by bubbling oxygen in an aqueous solution (concentration 10 mM). After 10.0 h of reaction, the HPLC detection of the substrates D-galactose and L-arabinose was below the detection limit, and the oxygen was stopped.

[0079] Step 2: Add 1g of the above wet cells to the XR6 and GDH3 crude enzyme solution, and add 1.0mL of NADP + A 10 mM aqueous solution was added to continue the reaction. During the reaction, the pH of the reaction solution was controlled at 6.5 ± 0.1 using a 1 M sodium hydroxide aqueous solution. After a total reaction time of 16.0 h, a 50% (w / w) glucose aqueous solution was added at a flow rate of 5.66 g / h. The reaction was continued, and the conversion of the substrate D-xylose was monitored by HPLC. The reaction was stopped when the concentration of D-xylose fell below the detection limit. The total reaction time was 28 h. Finally, 230 mL of the reaction solution was measured, and the concentration of the product xylitol in the reaction solution was 167.88 g / L; the product arabinitol was not detected, and the substrate D-glucose remained at 0.02 g / L. The D-xylose in the raw material was completely converted, and the yield of xylitol to xylose was calculated to be 99.6%. The reaction solution was filtered through an ultrafiltration membrane to remove protein, and then separated by ion exchange chromatography to remove sugar and acid molecules. The solution was concentrated, crystallized once, and dried to obtain 18.13 g of xylitol product, with a xylitol purity of 99.2%.

[0080] Comparative Example 1 Four-enzyme one-pot catalytic reaction

[0081] The enzyme-producing strains obtained in Examples 1-4 were selected for shake flask fermentation to produce enzymes. 1 g of the wet cells containing enzymes were taken, resuspended with 4 mL of 100 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH = 7.0), and then ultrasonically disrupted to obtain the corresponding crude enzyme solution. 8.0 mL of corn cob hemicellulose hydrolyzate was taken and the pH was adjusted to 7.0 with 5 M sodium hydroxide aqueous solution. 0.5 mL of each of the four crude enzyme solutions was added, and then 50 μL of NADP was added. + aqueous solution (concentration 10 mM), and 50 μL NAD + Aqueous solution (10 mM concentration) was reacted in a 35°C water bath with magnetic stirring. After 24 hours of reaction, various sugars and sugar alcohols were detected, and the results are shown in Table 7. The results in Table 7 show that L-arabinose, D-galactose, and D-glucose in the hydrolyzate were completely converted, with a relatively high concentration of D-xylose remaining in the reaction solution. A certain amount of xylitol and arabitol was also produced.

[0082] Table 7. Test results of the four-enzyme one-pot cascade catalytic hydrolysis solution

[0083]

Claims

1. A two-step enzymatic method for producing xylitol from hemicellulose hydrolyzate, characterized in that: include: (1) Using hemicellulose hydrolyzate as a raw material, adding arabinose dehydrogenase, NADH oxidase, and coenzyme NADH, and performing the first step of fermentation until no L-arabinose remains in the fermentation liquid, thereby obtaining a fermentation reaction liquid I; (2) After adding xylose reductase and glucose dehydrogenase to the fermentation reaction liquid I, a second fermentation step is performed to obtain a fermentation product containing xylitol.

2. The method according to claim 1, characterized in that The arabinose dehydrogenase is obtained by crushing and extracting an engineered bacterium containing an arabinose dehydrogenase gene; and the NADH oxidase is obtained by crushing and extracting an engineered bacterium containing an NADH oxidase gene.

3. The method according to claim 1, characterized in that The xylose reductase is obtained by crushing and extracting an engineering bacterium containing a xylose reductase gene; and the glucose dehydrogenase is obtained by crushing and extracting an engineering bacterium containing a glucose dehydrogenase gene.

4. The method according to claim 2, characterized in that The accession number of the arabinose dehydrogenase gene in the NCBI database is WP_004041122.1, NC_003062.2 or KJ716853.

1.

5. The method according to claim 2, characterized in that The accession number of the NADH oxidase gene in the NCBI library is AB035801.1, BBC74576.1, UPS09830.1, AF536177.1, CAG39343.1, X67220.1, BAL17746.1 or CUU11616.

1.

6. The method according to claim 3, characterized in that The accession number of the xylose reductase gene in the NCBI library is AF451326.3, KF752418.1, AF074484.1, AB002106.1, ALO17776.1, EAA34695.1, AAA99507.1, NC_009045.1, SCU85332.1, AAO91803.1 or Q9P8R5.

1.

7. The method according to claim 3, characterized in that The accession numbers of the glucose dehydrogenase gene in the NCBI library are WP_013055546.1, WP_012369122.1, and WP_274796929.

1.

8. The method according to any one of claims 2 or 3, characterized in that The host cell of the engineering bacteria is Escherichia coli.

9. The method according to claim 8, characterized in that The Escherichia coli is E. coli BL21.

10. The method according to claim 2, characterized in that In the first fermentation step, the pH is 7.5-9.5 and the temperature is 15-75°C. The added amount of the engineered bacteria containing the arabinose dehydrogenase gene and the engineered bacteria containing the NADH oxidase gene is 1-50 g / L based on the wet cell mass. The added concentration of the coenzyme NADH is 0.05 mM-1 mM.

11. The method according to claim 3, characterized in that In the second step of fermentation, the pH is 5.5-8.5, the temperature is 15-75°C, and the added amount of the engineered bacteria containing the xylose reductase gene and the engineered bacteria containing the glucose dehydrogenase gene is 1-50 g / L based on the added wet cell mass; the added concentration of the coenzyme NADPH is 0.05 mM-1 mM.

12. The method according to claim 1, characterized in that Glucose is added in the second fermentation step, and the mass of the added glucose is 80% to 100% of the mass of xylose in the hemicellulose hydrolyzate.

Images