Method for producing xylitol by catalyzing hemicellulose hydrolysate through biological enzyme

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for producing xylitol by catalyzing hemicellulose hydrolysate through a biological enzyme, which comprises two stages: in the first stage, adding hemicellulose hydrolysate, xylose reductase, arabinose dehydrogenase and glucose dehydrogenase to form a fermentation system, adjusting the pH value of the system to 8.0-9.5, and fermenting, adding L-arabinose until no L-arabinose residue exists in the fermentation liquid to obtain a fermentation reaction liquid I; in the second stage, the pH value of the fermentation reaction liquid I is adjusted to 6.5-7.5, fermentation continues to be conducted, and a fermented product containing xylitol is obtained. According to the method for preparing the xylitol through the biological enzyme catalysis method, cheap hemicellulose hydrolysate can be directly adopted as a raw material, and high-purity xylose does not need to be used as the raw material; the xylose reductase, the arabinose dehydrogenase and the glucose dehydrogenase used in the method are wide in gene source, the three-enzyme one-pot catalytic process is simple and efficient, the conversion rate of xylose and the yield of xylitol both can exceed 99%, the content of impurity arabitol in the product is low, other impurity saccharic acid is easy to separate, and the method is suitable for industrial production. The problems of complex process, high 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 catalyzed by biological enzymes. Background Art

[0002] Xylitol, also known as (2R,3R,4S)-pentapentol, is a type of sugar alcohol. It is a five-carbon sugar alcohol that can be used as a sucrose substitute. It is a product of xylose metabolism. Xylitol is primarily produced by the hydrogenation reduction of xylose. Xylitol is used in sugar foods and sugar substitutes, and its E code is E967. Xylitol is an edible food additive for humans. Its sweetness is comparable to sucrose, but its calorie content is only 60% of that of sucrose of the same weight, making it a suitable sucrose substitute.

[0003] Biological production of xylitol is a novel process for producing xylitol. Its mild reaction conditions and relatively low production costs make it a promising alternative to traditional chemical hydrogenation production processes and have garnered widespread attention both domestically and internationally in recent years. Two methods exist for producing xylitol through biological methods. One method primarily utilizes microorganisms such as bacteria, fungi, yeast, or recombinant genetically engineered bacteria to ferment various biomass feedstocks to produce xylitol. For example, Chinese invention patent application publication number CN104357339A discloses a Candida tropicalis yeast that produces xylitol using high-concentration xylose as the sole carbon source. The fermentation yield can reach 20-65 g / L, with a residual sugar concentration below 5%. Chinese invention patent publication number CN110982850B discloses a genetically engineered Aspergillus oryzae strain that produces xylitol at a concentration of 13.5 g / L using 50 g / L xylan as the sole carbon source. The yield and productivity are 0.27 g / g and 0.16 g / 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 biological fermentation for xylitol production 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, which involves using resting cells (or immobilized cells) and free enzyme protein (or immobilized enzyme) as catalysts. Chinese patent CN117143929 utilizes an immobilized enzyme column to catalyze the production of xylitol from a xylose solution with a xylose purity greater than 90%, increasing the total xylitol yield by four percentage points. 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 yield of up to 11.6 g / L / h. Chinese patent CN108977432 uses immobilized recombinant Escherichia coli cells co-expressing xylose reductase and glucose dehydrogenase as a catalyst to produce xylitol from a xylose mother liquor. The recombinant cells catalyzed the reaction of 200 g / L of xylose, achieving a xylitol yield of 100% after 30 hours. 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, catalyzing 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).

[0004] The advantage of the enzymatic method for producing xylitol is its high reaction efficiency, with product yields generally exceeding 90%. However, this method can only produce xylitol using high-purity xylose as a raw material and cannot directly catalyze the production of xylitol from hemicellulose hydrolysates containing miscellaneous sugars. This is primarily because hemicellulose hydrolysates contain a large amount of arabinose, and natural xylose reductase can catalyze the conversion of arabinose to arabitol (Chinese patent CN118620969). Arabitol is an isomer of xylitol and is difficult to separate. If it is introduced into the product, it will result in substandard xylitol. Although hemicellulose is the largest natural pentose biomass raw material, and producing xylitol from hemicellulose hydrolysates is the most economical option, new enzymatic methods are needed to directly produce xylitol from hemicellulose hydrolysates. Summary of the Invention

[0005] In response to the existing demand for a process for producing xylitol from hemicellulose hydrolysate, the present invention provides a method for producing xylitol from hemicellulose hydrolysate using bio-enzyme catalysis. The specific technical solution is as follows: A method for producing xylitol from hemicellulose hydrolyzate using enzyme catalysis, comprising: (1) adding hemicellulose hydrolyzate, xylose reductase, arabinose dehydrogenase, and glucose dehydrogenase to form a fermentation system, adjusting the pH of the system to 8.0-9.5, and fermenting until no L-arabinose remains in the fermentation liquid, thereby obtaining fermentation reaction liquid I; (2) The pH of the fermentation reaction liquid I is adjusted to 6.5-7.5, and the fermentation is continued to obtain a fermentation product containing xylitol.

[0006] The method of the present invention uses hemicellulose hydrolyzate as raw material and produces xylitol through the co-catalysis of xylose reductase, arabinose dehydrogenase, and glucose dehydrogenase. To reduce the production of arabinitol, the reaction process is controlled by controlling the pH. In the first stage of the reaction, the pH is controlled within the range of 8-9.5. This pH range is conducive to the catalysis of arabinose dehydrogenase, reducing the rate at which arabinose is converted to arabinitol by xylose reductase, thereby converting more arabinose into arabinonic acid. In the second stage of the reaction, the pH is controlled within the range of 6-8. This pH range is conducive to the catalysis of xylose reductase and glucose dehydrogenase, allowing the remaining xylose in the hydrolyzate to be quickly converted into xylitol.

[0007] Furthermore, the hemicellulose hydrolyzate is added to the fermentation system after being concentrated; the hemicellulose hydrolyzate raw material contains D-xylose, L-arabinose, D-galactose and D-glucose.

[0008] Furthermore, after concentration, the volume of the hemicellulose hydrolyzate is 1 to 1 / 4 of the original volume.

[0009] Furthermore, the accession number of the xylose reductase in the NCBI library is KF752418.1, EAA34695.1, SCU85332.1, or Q9P8R5.1.

[0010] Furthermore, the accession number of the arabinose dehydrogenase in the NCBI library is WP_004041122.1, NC_003062.2, KJ716853.1 or KJ716856.1.

[0011] Furthermore, the accession number of the glucose dehydrogenase in the NCBI library is WP_013055546.1, WP_012369122.1 or WP_274796929.1.

[0012] The method provided herein utilizes genetically engineered bacteria to express xylose reductase, arabinose dehydrogenase, and glucose dehydrogenase genes. The genes for these three enzymes can be derived from any source, as long as they can be functionally expressed in the engineered bacteria and exhibit the corresponding catalytic activity. The xylose reductase preferably exhibits greater selectivity for xylose than for arabinose.

[0013] Furthermore, in (1), the hemicellulose hydrolyzate is added to the fermentation system in a feeding manner.

[0014] Furthermore, the addition rate of the hemicellulose hydrolyzate is 0.04-0.25 g / (g·h), expressed as a ratio of L-arabinose contained in the hemicellulose hydrolyzate to the amount of arabinose dehydrogenase added, wherein the amount of arabinose dehydrogenase added is calculated based on the mass of the wet cells added.

[0015] In the present invention, in the first stage, the hemicellulose hydrolyzate is added to the reaction system in a continuous flow manner, so that the concentration of arabinose in the reaction solution is maintained at a low level, and almost all of the arabinose is converted into arabinonic acid, and the cumulative amount of arabitol in the reaction system is further reduced.

[0016] Furthermore, after step (1) is completed, glucose feeding is added to the fermentation reaction liquid I, and the mass ratio of the added glucose to the xylose in the hemicellulose hydrolyzate added in step (1) is 0.8~1:1.

[0017] During the second stage of catalysis, a certain amount of glucose is added, and under the catalysis of xylose reductase and glucose dehydrogenase, xylose is completely converted into xylitol.

[0018] Furthermore, the mass ratio of the glucose to the xylose added to the hemicellulose hydrolyzate in step (1) is 0.85-0.95:1.

[0019] Furthermore, glucose feed was added, and the pH of the reaction solution naturally decreased to 6.5-7.5, and then the pH of the reaction solution was controlled at 6.5-7.5.

[0020] Furthermore, the xylose reductase, arabinose dehydrogenase and glucose dehydrogenase are obtained by crushing and extracting engineering bacteria containing xylose reductase gene, arabinose dehydrogenase gene and glucose dehydrogenase gene, respectively.

[0021] Furthermore, the host cell of the engineered bacteria is Escherichia coli.

[0022] Furthermore, the host cell of the engineered bacteria is Escherichia coli E. coli BL21(DE3).

[0023] Furthermore, the xylose reductase, arabinose dehydrogenase, and glucose dehydrogenase are all added in an amount of 5 to 50 g / L based on the concentration added to wet cells.

[0024] Furthermore, the added amount of xylose reductase, arabinose dehydrogenase, and glucose dehydrogenase is 10-30 g / L.

[0025] Furthermore, in step (1), 0.05 mM to 1 mM coenzyme NADP is added + .

[0026] Furthermore, the coenzyme NADP + The amount of addition is 0.1 mM~0.5 mM.

[0027] Furthermore, the fermentation temperature is 15-75°C.

[0028] Furthermore, the fermentation temperature is 30-40°C.

[0029] Compared with the prior art, the present invention has the following beneficial effects: The bio-enzyme catalysis method provided by the present invention for preparing xylitol can directly use cheap hemicellulose hydrolyzate as a raw material, without the need to use high-purity xylose as a raw material; the xylose reductase, arabinose dehydrogenase and glucose dehydrogenase genes used in the present invention are from a wide range of sources, the three-enzyme one-pot catalysis process is simple and efficient, the xylose conversion rate and xylitol yield can both exceed 99%, the impurity arabinitol content in the product is low, and other impurities, sugars and acids, are easy to separate, successfully solving the problems of complex process, difficult separation and high cost in the xylitol refining process. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the reaction equation for producing xylitol from hemicellulose hydrolyzate catalyzed by the bio-enzyme of the present invention.

[0031] Figure 2This 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

[0032] 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.

[0033] 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.

[0034] 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.

[0035] Example 1 Recombinant expression of xylose reductase and substrate selectivity determination Eleven wild-type XR genes from different sources were selected and codon optimized and fully synthesized by Nanjing GenScript Biotechnology Co., Ltd. in E. coli. The genes were then constructed onto the plasmid pET-28a and introduced into E. coli. E. coli The xylose reductase enzyme library was obtained by shake flask fermentation and enzyme production in BL21(DE3) genetic engineering. The kinetic parameters of xylose reductase using L-arabinose as substrate (product L-arabitol) were determined using NADPH as coenzyme. k cat and K M Values, and kinetic parameters with D-xylose as substrate (product is xylitol) k cat and K M value, with k cat / K M（木糖） and k cat / K M（阿拉伯糖）The substrate selectivity of xylose reductase was characterized by the ratio of , and the results are shown in Table 1. The operation of enzyme kinetic parameter determination is referenced to Applied and environmental microbiology, Vol. 71, No. 3, p. 1642–1647. From the results in Table 1, it can be seen that the substrate selectivity of xylose reductase is Rhizopus oryzae 、 Neurospora crassa 、 Saccharmoyces cerivisiae 、 Aspergillus niger The selectivity of xylose reductase for the substrate D-xylose is significantly higher than that for L-arabinose.

[0036] Table 1 Source of xylose reductase and selectivity determination results

[0037] Example 2 Recombinant expression of arabinose dehydrogenase and enzyme activity determination Ten wild-type AraDH genes from different sources were selected and codon optimized and fully synthesized by Nanjing GenScript Biotechnology Co., Ltd. for E. coli. The genes were then constructed onto the plasmid pET-28a and introduced into E. coli. E. coli The BL21(DE3) gene engineering was carried out by shake flask fermentation and enzyme production to obtain an arabinose dehydrogenase enzyme library with NADP + The enzymatic activities of various arabinose dehydrogenases were measured using D-xylose, L-arabinose, D-galactose, and D-glucose as coenzymes, and the results are shown in Table 2. As can be seen from the results in Table 2, except for a few arabinose dehydrogenases with extremely low enzymatic activities on all substrates, the other arabinose dehydrogenases exhibited good catalytic activity towards L-arabinose and D-galactose. The arabinose dehydrogenases with high enzymatic activities had essentially no catalytic activity towards D-xylose and D-glucose.

[0038] Table 2 Source of arabinose dehydrogenase and enzyme activity determination results

[0039] Example 3 Recombinant expression of glucose dehydrogenase and enzyme activity determination Three wild-type glucose dehydrogenase genes from different sources were selected and codon optimized and fully synthesized by Nanjing GenScript Biotechnology Co., Ltd. for E. coli. The genes were then constructed onto the plasmid pET-28a and introduced into E. coli. E. coli In the BL21 genetic engineering, shake flask fermentation and enzyme production were carried out to obtain a glucose dehydrogenase enzyme library with NADP + 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.

[0040] Table 3 Sources of glucose dehydrogenase and enzyme activity determination results

[0041] Example 4 The hemicellulose hydrolysate from corn cobs was selected and the composition of various sugars was determined by HPLC. The results are as follows: Figure 2 As shown in Table 4, the hemicellulose hydrolysate mainly contains D-xylose, L-arabinose, D-galactose and D-glucose, and the contents of other miscellaneous sugars are below the detection limit.

[0042] Table 4 Determination results of hemicellulose hydrolyzate from corn cobs

[0043] The strains with enzyme activity obtained in Examples 1-3 were fermented in shake flasks 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 = 7.5), and then ultrasonically disrupted to obtain the corresponding crude enzyme solution. 30 mL of the above hemicellulose hydrolyzate was taken, 1.5 g of anhydrous glucose was weighed and added thereto, and the pH was adjusted to 7.5 with 5 M sodium hydroxide aqueous solution, and finally the volume was adjusted to 35 mL with deionized water. The crude enzyme solutions of the three enzymes were added, and then 500 μL of NADP was added. + Aqueous solution (concentration 10 mM) was reacted in a 35°C water bath with magnetic stirring. After 24 h of reaction, various sugars and sugar alcohols were detected. The results are shown in Table 5.

[0044] Table 5. Test results of enzyme-catalyzed hemicellulose hydrolysate

[0045] Example 5 The strains with enzyme activity obtained in Examples 1-3 were fermented in shake flasks to produce enzymes. 1 g of the wet cells containing enzymes were taken, resuspended with 4 mL of 100 mM sodium carbonate-sodium bicarbonate buffer (pH = 9.0), and then ultrasonically disrupted to obtain the corresponding crude enzyme solution. 30 mL of the hemicellulose hydrolyzate obtained in Example 4 was taken, 1.5 g of anhydrous glucose was weighed and added thereto, and the pH was adjusted to 9.0 with a 5 M aqueous sodium hydroxide solution. Finally, the volume was adjusted to 35 mL with deionized water. The crude enzyme solutions of the three enzymes were added, and then 500 μL of NADP was added. + Aqueous solution (concentration 10 mM) was reacted in a 35°C water bath with magnetic stirring. After 24 h of reaction, various sugars and sugar alcohols were detected. The results are shown in Table 6.

[0046] Table 6 Detection results of enzyme-catalyzed hemicellulose hydrolysis solution for 24 h

[0047] After 24 hours of reaction, due to incomplete xylose reaction, 2.5 mL of crude xylose reductase and glucose dehydrogenase enzyme solutions were added, and the reaction continued. After 48 hours of total reaction, various sugars and sugar alcohols were assayed. The results are shown in Table 7. The final pH of each reaction solution was measured and ranged from 7.4 to 8.2.

[0048] Table 7 Detection results of enzyme-catalyzed hemicellulose hydrolysis solution for 48 h

[0049] Example 6 The hemicellulose hydrolyzate from corn cobs was concentrated to about 1 / 2 of its original volume, 150 mL of concentrated corn cob hydrolyzate was measured and placed in a 250 mL three-necked flask, magnetically stirred in a 35°C water bath, and the pH of the hydrolyzate was adjusted to 9.0 with a 5 M aqueous sodium hydroxide solution. The XR2, AraDH3, and GDH2 strains obtained in Examples 1-3 were fermented in shake flasks to produce enzymes. 1 g of the wet cells containing enzymes were taken, resuspended with 4 mL of deionized water, and ultrasonically broken to obtain the corresponding crude enzyme solution. The crude enzyme solution was added to the three-necked flask, and 1 mL of NADP was added. + The reaction started with an aqueous solution (concentration 10 mM). Figure 1 As shown, the reaction solution initially maintained a pH of 9.0 ± 0.1 using 5 M sodium hydroxide solution. After 9.5 hours of reaction, L-arabinose was below the detection limit by HPLC. 6.5 g of glucose was added to the reaction solution and the reaction continued. After the pH naturally dropped to 7.0, the pH was then controlled to 7.0 ± 0.1 using 5 M sodium hydroxide solution, and the reaction progress was monitored. After 34 hours of reaction, the xylose concentration in the reaction solution fell below the detection limit, and the reaction was terminated. The reaction volume was approximately 181 mL. HPLC analysis of the concentrations of other components in the reaction solution revealed: D-galactose below the detection limit, glucose 1.38 g / L, arabitol 0.17 g / L, and xylitol 85.54 g / L. The calculated xylitol-to-xylose yield was greater than 99.3%.

[0050] Example 7 The hemicellulose hydrolyzate from corn cobs was concentrated to about 1 / 2 of its original volume, 150 mL of the concentrated corn cob hydrolyzate was measured and placed in a 250 mL three-necked flask, and magnetic stirring was started in a 35°C water bath. The pH of the hydrolyzate was adjusted to 9.0 with a 5 M aqueous sodium hydroxide solution. The XR6, AraDH2, and GDH1 strains obtained in Examples 1-3 were fermented in shake flasks to produce enzymes. 1 g of the wet cells containing enzymes were taken, resuspended with 4 mL of deionized water, and ultrasonically broken to obtain the corresponding crude enzyme solution. The crude enzyme solution was added to the three-necked flask, and 1 mL of NADP was added. + The reaction was initiated with a 10 mM aqueous solution. Initially, the pH of the reaction solution was maintained at 8.8 ± 0.1 using 5 M sodium hydroxide. After 11.4 h of reaction, L-arabinose was below the detection limit by HPLC. 6.5 g of glucose was added to the reaction solution and the reaction continued. After the pH naturally dropped to 6.5, the pH was maintained at 6.5 ± 0.1 using 5 M sodium hydroxide, and the reaction progress was monitored. After 27.5 h, the xylose concentration in the reaction solution fell below the detection limit, and the reaction was terminated. The reaction volume was approximately 184 mL. HPLC analysis of the concentrations of other components in the reaction solution revealed: D-galactose below the detection limit, glucose 0.95 g / L, arabinitol 0.29 g / L, and xylitol 83.98 g / L. The calculated yield of xylitol to xylose was greater than 98.9%.

[0051] Example 8 The hemicellulose hydrolyzate from corn cobs was concentrated to about 1 / 2 of its original volume, 150 mL of the concentrated corn cob hydrolyzate was measured and placed in a 250 mL three-necked flask, and magnetic stirring was started in a 35°C water bath. The pH of the hydrolyzate was adjusted to 9.0 with a 5 M aqueous sodium hydroxide solution. The XR8, AraDH10, and GDH1 strains obtained in Examples 1-3 were fermented in shake flasks to produce enzymes. 1 g of the wet cells containing enzymes were taken, resuspended with 4 mL of deionized water, and ultrasonically broken to obtain the corresponding crude enzyme solution. The crude enzyme solution was added to the three-necked flask, and 1 mL of NADP was added. +The reaction was initiated with a 10 mM aqueous solution. Initially, the pH of the reaction solution was controlled at 9.0 ± 0.1 using 5 M sodium hydroxide solution. After 12 h of reaction, L-arabinose was below the detection limit by HPLC. 6.5 g of glucose was added to the reaction solution and the reaction continued. After the pH naturally dropped to 7.0, the pH was controlled at 7.0 ± 0.1 using 5 M sodium hydroxide solution, and the reaction progress was monitored. After 36 h of reaction, the xylose concentration in the reaction solution fell below the detection limit, and the reaction was terminated. The reaction volume was approximately 187 mL. HPLC analysis of the concentrations of other components in the reaction solution revealed: D-galactose below the detection limit, glucose 1.65 g / L, arabinitol 0.57 g / L, and xylitol 85.27 g / L. The calculated yield of xylitol to xylose was greater than 99.7%.

[0052] Example 9 The hemicellulose hydrolyzate from corn cobs was concentrated to about 1 / 2 of its original volume, 150 mL of concentrated corn cob hydrolyzate was measured and placed in a 250 mL three-necked flask, magnetically stirred in a 35°C water bath, and the pH of the hydrolyzate was adjusted to 9.0 with a 5 M aqueous sodium hydroxide solution. The XR9, AraDH9, and GDH3 strains obtained in Examples 1-3 were fermented in shake flasks to produce enzymes. 1 g of the wet cells containing enzymes were taken, resuspended with 4 mL of deionized water, and ultrasonically broken to obtain the corresponding crude enzyme solution. The crude enzyme solution was added to the three-necked flask, and 1 mL of NADP was added. + The reaction was initiated with a 10 mM aqueous solution. Initially, the pH of the reaction solution was maintained at 9.4 ± 0.1 using 5 M sodium hydroxide. After 9.0 h of reaction, L-arabinose was below the detection limit by HPLC. 6.5 g of glucose was added to the reaction solution and the reaction continued. After the pH naturally dropped to 7.5, the pH was maintained at 7.5 ± 0.1 using 5 M sodium hydroxide, and the reaction progress was monitored. After 33.0 h, the xylose concentration in the reaction solution fell below the detection limit, and the reaction was terminated. The reaction volume was approximately 180 mL. HPLC analysis of the concentrations of other components in the reaction solution revealed: D-galactose below the detection limit, glucose 1.15 g / L, arabinitol 0.21 g / L, and xylitol 86.01 g / L. The calculated yield of xylitol to xylose was greater than 99.4%.

[0053] Example 10 The XR4, AraDH10, and GDH3 strains obtained in Examples 1-3 were fermented in shake flasks to produce enzymes. 5 g of wet cells containing enzymes were taken, resuspended in 20 mL of deionized water, and then ultrasonically disrupted to obtain the corresponding crude enzyme solution. The crude enzyme solution was added to a 250 mL three-necked flask, and 1 mL of NADP was added. +Aqueous solution (10 mM concentration). Corncob hemicellulose hydrolysate was concentrated to approximately one-third of its original volume. 100 mL of the concentrated corncob hydrolysate was continuously added to a three-necked flask via a peristaltic pump at a flow rate of 8.4 mL / h. The pH of the reaction solution was controlled at 9.0 ± 0.1 using 5 M sodium hydroxide solution. The hydrolysate addition was complete after 12 h, and L-arabinose was below the detection limit by HPLC at 15 h. 17 g of glucose was added to the reaction solution and the reaction continued. After the pH naturally decreased to 7.0, the pH was controlled at 7.0 ± 0.1 using 5 M sodium hydroxide solution, and the reaction progress was continuously monitored. After 35 h, the xylose concentration in the reaction solution fell below the detection limit, and the reaction was terminated. The reaction volume was approximately 205 mL. HPLC analysis of the concentrations of other components in the reaction solution revealed: D-galactose below the detection limit, glucose at 0.24 g / L, arabitol at 0.07 g / L, and xylitol at 101.79 g / L. The yield of xylitol to xylose was calculated to be greater than 99.5%. The reaction solution was filtered through an ultrafiltration membrane to remove protein and then subjected to ion exchange chromatography to remove sugar and acid molecules. The solution was concentrated, crystallized, and dried to yield 12.5 g of xylitol, with a purity of 99.4%.

[0054] Example 11 The XR7, AraDH9, and GDH1 strains obtained in Examples 1-3 were fermented in shake flasks to produce enzymes. 5 g of wet cells containing enzymes were taken, resuspended in 20 mL of deionized water, and then ultrasonically disrupted to obtain the corresponding crude enzyme solution. The crude enzyme solution was added to a 250 mL three-necked flask, and 1 mL of NADP was added. +Aqueous solution (10 mM concentration). Corncob hemicellulose hydrolysate was concentrated to approximately one-third of its original volume. 100 mL of the concentrated corncob hydrolysate was continuously added to a three-necked flask via a peristaltic pump at a flow rate of 8.5 mL / h. The pH of the reaction solution was controlled at 9.0 ± 0.1 using 5 M sodium hydroxide solution. The hydrolysate addition was complete after 12 h, and the L-arabinose concentration by HPLC was below the detection limit at 15 h. 17 g of glucose was added to the reaction solution and the reaction continued. After the pH naturally decreased to 7.0, the pH was controlled at 7.0 ± 0.1 using 5 M sodium hydroxide solution, and the reaction progress was continuously monitored. After 35 h, the xylose concentration in the reaction solution fell below the detection limit, and the reaction was terminated. The reaction volume was approximately 210 mL. HPLC analysis of the concentrations of other components in the reaction solution revealed: D-galactose below the detection limit, glucose at 0.35 g / L, arabitol at 0.10 g / L, and xylitol at 99.62 g / L. The calculated yield of xylitol to xylose was greater than 99.5%. The reaction solution was filtered through an ultrafiltration membrane to remove protein and then subjected to ion exchange chromatography to remove sugar and acid molecules. The solution was concentrated, crystallized once, and dried to yield 11.8 g of xylitol, with a purity of 99.5%.

[0055] Example 12 The XR1, AraDH3, and GDH2 strains obtained in Examples 1-3 were fermented in shake flasks to produce enzymes. 5 g of wet cells containing enzymes were taken, resuspended in 20 mL of deionized water, and then ultrasonically disrupted to obtain the corresponding crude enzyme solution. The crude enzyme solution was added to a 250 mL three-necked flask, and 1 mL of NADP was added. +Aqueous solution (10 mM concentration). Corncob hemicellulose hydrolysate was concentrated to approximately one-third of its original volume. 100 mL of the concentrated corncob hydrolysate was continuously added to a three-necked flask via a peristaltic pump at a flow rate of 8.4 mL / h. The pH of the reaction solution was controlled at 9.0 ± 0.1 using 5 M sodium hydroxide solution. The hydrolysate addition was complete after 12 h, and L-arabinose was below the detection limit by HPLC at 15 h. 17 g of glucose was added to the reaction solution and the reaction continued. After the pH naturally decreased to 7.0, the pH was controlled at 7.0 ± 0.1 using 5 M sodium hydroxide solution, and the reaction progress was continuously monitored. After 35 h, the xylose concentration in the reaction solution fell below the detection limit, and the reaction was terminated. The reaction volume was approximately 202 mL. HPLC analysis of the concentrations of other components in the reaction solution revealed: D-galactose below the detection limit, glucose at 0.19 g / L, arabitol at 0.05 g / L, and xylitol at 103.69 g / L. The calculated yield of xylitol to xylose was greater than 99.5%. The reaction solution was filtered through an ultrafiltration membrane to remove protein and then subjected to ion exchange chromatography to remove sugar and acid molecules. The solution was concentrated, crystallized, and dried to yield 13.0 g of xylitol, with a purity of 99.5%.

Claims

1. A method for producing xylitol from hemicellulose hydrolyzate catalyzed by biological enzymes, characterized in that: include: (1) adding hemicellulose hydrolyzate, xylose reductase, arabinose dehydrogenase, and glucose dehydrogenase to form a fermentation system, adjusting the pH of the system to 8.0-9.5, and fermenting until no L-arabinose remains in the fermentation liquid, thereby obtaining fermentation reaction liquid I; (2) The pH of the fermentation reaction liquid I is adjusted to 6.5-7.5, and the fermentation is continued to obtain a fermentation product containing xylitol.

2. The method according to claim 1, characterized in that The accession number of the xylose reductase in the NCBI database is KF752418.1, EAA34695.1, SCU85332.1, or Q9P8R5.

1.

3. The method according to claim 1, characterized in that The accession number of the arabinose dehydrogenase in the NCBI database is WP_004041122.1, NC_003062.2, KJ716853.1 or KJ716856.

1.

4. The method according to claim 1, wherein The accession number of the glucose dehydrogenase in the NCBI library is WP_013055546.1, WP_012369122.1 or WP_274796929.

1.

5. The method according to claim 1, wherein In (1), the hemicellulose hydrolyzate is added to the fermentation system in a feeding manner.

6. The method according to claim 1, wherein After step (1) is completed, glucose feeding is added to the fermentation reaction liquid I, and the mass ratio of the added glucose to the xylose in the hemicellulose hydrolyzate added in step (1) is 0.8~1:

1.

7. The method according to claim 5, characterized in that Glucose feed was added, and the pH of the reaction solution naturally dropped to 6.5-7.5, and then the pH of the reaction solution was controlled at 6.5-7.

5.

8. The method according to claim 1, characterized in that The xylose reductase, arabinose dehydrogenase and glucose dehydrogenase are obtained by crushing and extracting engineering bacteria containing xylose reductase gene, arabinose dehydrogenase gene and glucose dehydrogenase gene respectively.

9. The method according to claim 8, wherein The xylose reductase, arabinose dehydrogenase and glucose dehydrogenase are all added in an amount of 5 to 50 g / L based on the concentration added to wet cells.

10. The method according to any one of claims 1 or 9, characterized in that In step (1), add 0.05 mM to 1 mM coenzyme NADP + .

Images