CN117757765B

CN117757765B - 2-Xylitol dehydrogenase mutant and application thereof

Info

Publication number: CN117757765B
Application number: CN202311824231.XA
Authority: CN
Inventors: 沙凤; 孙科; 谭芳美
Original assignee: Suzhou Koning Polyol Co ltd
Current assignee: Suzhou Koning Polyol Co ltd
Priority date: 2023-12-27
Filing date: 2023-12-27
Publication date: 2024-08-09
Anticipated expiration: 2043-12-27
Also published as: CN117757765A

Abstract

The invention discloses a 2-xylitol dehydrogenase mutant and application thereof. The amino acid sequence of the 2-xylitol dehydrogenase mutant is shown as SEQ ID NO:4, which is set forth in SEQ ID NO:2, wherein phenylalanine at position 91 is mutated into tyrosine and phenylalanine at position 198 is mutated into valine in the parent amino acid sequence. The nucleotide sequence of the 2-xylitol dehydrogenase mutant gene is shown as SEQ ID NO: 3. The gene is introduced into escherichia coli to obtain genetically engineered bacteria containing the gene, so that the preparation of the recombinant 2-xylitol dehydrogenase mutant is realized. Compared with the wild type, the 2-xylitol dehydrogenase mutant provided by the invention has higher substrate tolerance and catalytic efficiency, can realize the preparation of high-purity D-xylulose with low cost, has space-time yield as high as 900 g L ^‑1d^‑1, and has wide industrial production prospect.

Description

2-Xylitol dehydrogenase mutant and application thereof

Technical Field

The invention relates to the technical field of biochemistry, in particular to a 2-xylitol dehydrogenase mutant and application thereof.

Background

D-xylulose (D-xylulose), a five-carbon sugar, is widely found in many organisms and plays a key role in the important metabolic processes of cellular energy production and nucleic acid synthesis. In addition, D-xylulose is also the most critical raw material for synthesizing various rare pentoses, bioethanol and furfural (futfural), a new generation of bio-based material. At present, the preparation of D-xylulose mainly comprises the following paths: ① The xylose isomerase is utilized to isomerize D-xylose ^[1], however, the catalytic reaction is limited by the inherent thermodynamic equilibrium of the isomerase, the theoretical conversion rate of the D-xylose is only about 40 percent, the physical properties of the D-xylose and the D-xylulose are highly similar, the later separation cost is high, and the yield is low; ② Glucose is converted into arabitol under the action of hypertonic resistant yeast, then acetobacter, glucose bacillus or klebsiella is adopted to oxidize the arabitol into D-xylulose, the conversion rate is 20-95%, the fermentation time is long, and the space-time yield is low (CN 102796797A, CN 101486984A); ③ D-xylose is firstly isomerized into D-xylulose, D-xylulokinase selectively phosphorylates the D-xylulose into D-xylulose-5-phosphate, the complete conversion of xylose can be dragged through coupling with isomerase, an energy ATP regeneration system is needed in the system, intermediates, products and byproducts are separated for a plurality of times, and the total yield is 61.1 percent (CN 110616239A); ④ Starting from formaldehyde which is one of the most basic organic raw materials, the biological enzyme method is utilized to catalyze formaldehyde to synthesize xylulose ^[2]. Formaldehyde polymerization is catalyzed by benzoic acid decarboxylase to produce hydroxyacetaldehyde and 1, 3-Dihydroxyacetone (DHA). The transaldolase further catalyzes the polymerization of the hydroxyaldehyde and DHA to generate xylulose, and finally realizes the enzymatic conversion from formaldehyde to xylulose. At present, the conversion rate is 4.6% in the concept verification stage.

The wild strain selectively oxidizes the hydroxyl group at the C2 position of xylitol to synthesize D-xylulose, but the intracellular 2-xylitol dehydrogenase system has low activity, low substrate load in the transformation process and low space-time yield (CN 1263944A). Therefore, in the earlier study, we excavate from thermophilic bacteria Albidovulum inexpectatum to high temperature resistant 2-xylitol dehydrogenase AiPDH capable of specifically oxidizing the hydroxyl group at the C2 position of xylitol, and through coupling water-producing NADH oxidase, a biological oxidation process of high-purity and high-concentration D-xylulose is established, the conversion rate is more than 99%, the space-time yield reaches 160g L ^-1d^-1, and compared with the traditional production method relying on isomerase, the complex operations such as later separation and the like are avoided. AiPDH the binding constant K _m for xylitol was 7.59mM, whereas when the substrate concentration was further increased, substrate inhibition was observed in AiPDH, ki was 152.95mM.

Reference to the literature

[1]Pronk J T,Bakker A W,Van Dam H E,et al.Preparation of D-xylulose from D-xylose[J].Enzyme and microbial technology,1988,10(9):537-542.

[2] Cui Bo, zhuo Bingzhao, J.Biotechnology report 2018,34 (7): 1128-1136, et al, enzymatic catalysis of formaldehyde to xylulose [ J ].

Disclosure of Invention

In view of the shortcomings of the prior art, the invention aims to provide a 2-xylitol dehydrogenase mutant which tolerates high concentration of substrate and application thereof in preparing high-purity D-xylulose.

To achieve the above object, the present invention utilizes molecular docking and molecular dynamics simulation to study the binding pattern of proteins to substrates. By describing the spatial distribution probability of xylitol on the protein surface and the active pocket under the condition of high substrate concentration (substrate: protein concentration is 40:1), 15 amino acid residues in total are selected from a reaction channel and subunit interfaces which participate in the formation of a typical non-catalytic binding conformation. Through site-directed saturation mutation and iterative saturation mutation, 2-xylitol dehydrogenase mutant F91Y/F198V is obtained through screening. The amino acid sequence of the mutant F91Y/F198V is shown in SEQ ID NO:2, wherein phenylalanine at position 91 is mutated to tyrosine and phenylalanine at position 198 is mutated to valine in the amino acid sequence of the wild-type 2-xylitol dehydrogenase AiPDH.

Another object of the present invention is to provide a method for producing the 2-xylitol dehydrogenase mutant.

It is a further object of the present invention to provide the use of the 2-xylitol dehydrogenase mutant.

The aim of the invention can be achieved by the following technical scheme:

the gene sequence of the wild type 2-xylitol dehydrogenase AiPDH from thermophilic bacteria Albidovulum inexpectatum is shown in SEQ ID NO:1, and obtaining the gene of the 2-xylitol dehydrogenase mutant through site-directed saturation mutation and iterative saturation mutation screening, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:3, shown in the following:

The skilled person will know that the nucleic acid molecule for expressing the 2-xylitol dehydrogenase mutant provided by the application can also comprise nucleotide sequences such as promoters, enhancers, non-coding regions and the like besides the nucleotide fragments so as to achieve the purpose of improving the performance of the gene in terms of expression quantity, expression efficiency, product activity and the like.

A 2-xylitol dehydrogenase mutant, the amino acid sequence of which is shown in SEQ ID NO:4, as follows:

Recombinant vector containing said 2-xylitol dehydrogenase mutant gene. It can be constructed by ligating the nucleotide sequence of the 2-xylitol dehydrogenase gene of the present invention to various vectors by a conventional method in the art, and the recombinant plasmid is selected from pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+),pQE2、pQE9、pQE30、pQE3 1、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-l、pGEX-6p-l、pGEX-6p-2、pBV220、pTrc99A、pTwin1、pEZZ18、pKK232-18、pBR322、pUC-18 or pUC-19. More preferably, the recombinant plasmid is pET-28a (+). Meanwhile, for expression in Bacillus subtilis, it is preferable that a recombinant plasmid selected from pWB980、pHT43、pBE2、pMUTIN4、pUB110、pE194、pMA5、pMK3、pMK4、pHT304、pHY300PLK、pBest502、pDG1363、pSG1154、pAX01、pSAS144、pDL、pDG148-stu、pDG641、pUCX05-bgaB、pHT01、pUB110、pTZ4、pC194、 Or (b)More preferably, the recombinant plasmid is pMA5.

A genetically engineered bacterium for producing the 2-xylitol dehydrogenase mutant, wherein the genetically engineered bacterium comprises the 2-xylitol dehydrogenase mutant gene or the recombinant vector.

The host cells of the genetically engineered bacteria comprise prokaryotic cells, yeast or eukaryotic cells; preferably, the prokaryotic cell is E.coli cell or Bacillus subtilis. More preferably, the host cell is an E.coli BL21 (DE 3) cell.

A catalyst comprising the alcohol dehydrogenase mutant of claim 1;

optionally, the catalyst is selected from: free enzyme, free cell, immobilized enzyme, or immobilized cell.

The 2-xylitol dehydrogenase mutant gene, the recombinant vector and the genetically engineered bacterium are applied to the preparation of D-xylulose.

A method for preparing a 2-xylitol dehydrogenase mutant, comprising the following steps: culturing the genetically engineered bacterium to obtain the recombinant expressed 2-xylitol dehydrogenase mutant.

The activity of the 2-xylitol dehydrogenase mutant (F91Y/F198V) reaches 16.12U/mg, which is 7.8 times that of a female parent, and the k _cat is improved by 66.7 percent compared with the female parent, so that no substrate inhibition exists; the activity is highest at pH 10.5, the enzyme activity is not affected when the temperature is kept at 50 ℃ for 2 hours, and 91.5% of the enzyme activity is still remained after the temperature is kept at 55 ℃ for 2 hours.

The application of the 2-xylitol dehydrogenase mutant in preparing D-xylulose by converting xylitol.

The catalytic reaction system is formed by reacting 100-300 g/L xylitol, 500-500 KU of 2-xylitol dehydrogenase mutant, 700-800 KU of NADH oxidase and 0.05-0.5 mmol/L NAD ⁺ for 2-36 h under the conditions of pH 7.5-9.0, reaction temperature of 30-55 ℃, stirring rotation speed of 180-1500 rpm and ventilation volume of 1-3 VVM or pure oxygen, thus obtaining D-xylulose conversion solution.

The source of NADH oxidase is not limited as long as NADH can be oxidized to NAD ⁺, and the product is water, such as Lactobacillus delbrueckii, lactococcus lactis, trichoderma harzianum, streptococcus thermophilus, etc. In a preferred embodiment of the invention the NADH oxidase is derived from Streptococcus thermophilus Streptococcus thermophilus (Uniprot accession number: Q5M3T 5).

Advantageous effects

The invention guides the directional molecular transformation of key enzyme AiPDH to release the substrate inhibition based on the experimental techniques of dynamics simulation, fixed point saturation/iteration saturation mutation, high flux screening and the like, the mutant F91Y/F198V obtained by screening shows excellent tolerance to high-concentration xylitol, in a system coupled with NADH oxidase, the catalytic reaction can reach 65.2 percent in2 hours, the conversion rate of 8 hours can reach 99.4 percent, which is far faster than that of a female parent, the space-time yield is as high as 900g L ^-1d^-1, compared with the existing preparation technique, the space-time yield is greatly improved, the later separation cost is reduced, and the invention has important industrial application value.

Drawings

FIG. 1 absorbance standard curve determination of NADH at 340 nm.

FIG. 2 effect of pH on female parent and mutant F91Y/F198V viability.

FIG. 3 effect of temperature on maternal and mutant F91Y/F198V viability.

FIG. 4 is a plot of the reaction progress of the female parent and the mutation F91Y/F198V.

FIG. 5 HPLC analysis of xylitol and D-xylulose. A) HPLC analysis spectrum of xylitol and D-xylulose standard, B) HPLC analysis spectrum of complete conversion of xylitol into D-xylulose.

Detailed Description

The invention will be better understood from the following examples. However, the description of the embodiments is merely illustrative of the invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: codon optimization and synthesis of wild type 2-xylitol dehydrogenase AiPDH gene

The gene sequence of the wild type 2-xylitol dehydrogenase AiPDH is designed by DNAMAN software, the codon is optimized and the G+C content is regulated according to the preference of E.coli without changing the amino acid sequence encoded by the original gene, the optimized gene sequence is synthesized by Suzhou Jin Wei intelligent company, the gene sequence is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID NO: 2. Cloning into vector pET-28a (+) via restriction endonucleases EcoRI and HindIII was used as a template for directed evolution.

Example 2: mutant library construction

The substrate binding pattern of AiPDH was studied from molecular docking and molecular dynamics modeling using RosettaDock and GROMACS in combination. Through describing the space distribution probability of xylitol on the protein surface and an active pocket under the condition of high substrate concentration (substrate: protein concentration is 40:1), finally, 15 amino acid residues in total formed by a reaction channel and a subunit interface participating in a typical non-catalytic binding conformation are selected for site-directed saturation mutation, so that a smaller and more efficient gene library is constructed, the screening workload is reduced, and the enzyme molecule transformation efficiency is improved. The hotspot amino acid selection is as follows: 13G,18I,37D,38I,60D,62T,87N,91F,110V,138M,140S,142A,183P,190H and F198, single point saturation mutagenesis was performed on each of the 15 selected sites using degenerate codon NNK using the PCR primers shown in Table 1. Single point saturation mutagenesis PCR procedure was as follows: mu.L of KOD hot start DNA polymerase buffer (10X), 2. Mu.L of 2mmol/L dNTPs, 1. Mu.L of DMSO, 0.5. Mu.L (50 ng) of DNA template, 100. Mu.L of forward primer and 100. Mu.L of reverse primer each 0.5. Mu.L of KOD DNA polymerase were added sequentially to the reaction system. The PCR procedure was used: 95℃for 3min, (95℃for 30sec,60℃for 4.5min,72℃for 5 min). Times.30 cycles, 72℃for 10min,10℃for 60min. The PCR product was added to 0.5. Mu.L of Dpn I, digested at 37℃for 2 hours to remove the template plasmid, 1 to 2. Mu.L was aspirated, and transformed into E.coli BL21 (DE 3) by electric shock, spread on a kanamycin-containing plate, and inverted in an incubator at 37℃for 12 hours to establish a single-point saturated mutant library.

TABLE 1 primers for Single Point saturation mutagenesis

Example 3: high throughput screening of forward mutants

Transformants in the mutant library were picked by a microbial clone selection system (Qpix HT) and sequentially inoculated into 96-well plates (primary plates) containing 300. Mu.L of LB medium (30. Mu.g/mL kanamycin) per well, while each deep plate was inoculated with 3-well wild-type clones as a female parent control, and cultured overnight at 37℃and 250rpm. Sequentially inoculating 50 μl of overnight culture solution to a secondary deep well plate containing 700 μl of self-induced culture ZYP5052 (G/L, peptone 10, yeast powder 5, glycerol 5, glucose 0.5, lactose 2,(NH₄)₂SO₄ 3.3,KH₂PO₄ 6.8,Na₂HPO₄ 3.3,MgSO₄ 0.24)) in each well by using 96 manual pipetting stations, centrifuging after culturing at 25deg.C for 24 hours, sucking 50 μl of supernatant into 96-well ELISA plates by using 96 manual pipetting stations, adding 150 μl of reaction solution (100 mM pH 8.0Tris-HCl,0.2mM NAD ⁺, 300G/L xylitol) composed of buffer solution, substrate and coenzyme into each well, respectively, and detecting NADH generation in batch in an ELISA instrument based on female parent of each deep well plate, the method comprises the steps of selecting mutants with enzyme activity higher than that of a mother average value by more than 1.2 times from a library, putting the mutants into a re-screening, adding glycerol with the final concentration of 10% into a first-stage deep hole plate, preserving at-80 ℃, inoculating shake flask from glycerol bacteria, inducing expression, and obtaining beneficial mutants through shake flask verification, carrying out sequencing analysis on 24 positive mutant strains, wherein 5 different types of mutations appear, the results are shown in a table 2, and the second round takes mutants pET-28a-F198V as templates, respectively uses primers for mutation G13S, I18L, D Q and F91V in the table 1, carries out site-directed mutagenesis PCR, conversion and plating, and obtains dominant bacteria F91Y/F198V with double mutation through screening, wherein the nucleotide sequences are shown in a table 2, and the nucleotide sequences are shown in a table 1: 3, the amino acid sequence is shown as SEQ ID NO. 4.

Example 4: fermentation culture of recombinant escherichia coli

The expression of wild fungi AiPDH and mutants were all as follows: single colonies were picked up and activated on LB plates (containing 30. Mu.g/mL kanamycin) at 37℃and transferred to fresh liquid LB for overnight next day, after two stages of activation, the thalli were transferred to 100mL of fresh LB medium as seed liquid in an additional transfer amount of 4%, and then cultured at 37℃for 2-3 hours until OD ₆₀₀ was about 0.8-1.0, IPTG was added to a final concentration of 0.2mM, and after shaking culture at 200rpm at 16℃and induced expression for 24 hours, the thalli were collected by centrifugation at 8000rpm for 15min and stored at-80 ℃.

Example 5: recombinant protein purification

The N end of the target protein is fused with a 6 XHis tag, and nickel ion chromatography is adopted for purification. The specific process comprises the following steps: the frozen cells were resuspended in pre-chilled buffer A (20 mM Tris-HCl buffer, 500mM NaCl,10mM imidazole, pH 8.0), broken by a high pressure homogenizer, centrifuged at 12 000rpm at 4℃for 30min to remove cell debris, and the supernatant was filtered through a 0.22 μm filter membrane for use. The supernatant was uploaded onto a 5mL Ni-NTA column mounted on AKTA and equilibrated with Buffer A at a flow rate of 5mL/min, and the nonspecific binding proteins were eluted first with 10 column volumes of Buffer A followed by a gradient elution with 0-100% Buffer B (20 mM Tris-HCl Buffer, 500mM NaCl,500mM imidazole, pH 8.0) and the eluate was collected every 2 mL. The eluted samples were analyzed by 12% SDS-PAGE to determine protein purity, and after combining the pure proteins, concentrated by a 30kDa ultrafiltration tube and stored in buffer C (150mM NaCl,1mM DTT,20mM Tris-HCl, pH 8), and then snap frozen in liquid nitrogen, and placed at-80℃for further use.

Example 6: aiPDH and enzyme activity detection of mutants

AiPDH and the activity of the mutants were determined using a microplate reader, and one enzyme activity unit (U) was defined as the amount of enzyme required to produce 1. Mu. Mol NADH per minute. The reaction system contains 0.5mM NAD ⁺, 1.2M xylitol, a proper amount of pure enzyme solution to be tested, 100mM Tris-HCl buffer solution with pH of 8.0, and the total volume is 200 mu L. The absorbance change of NADH at a wavelength of 340nm was measured at 30℃as shown in equation (1.1):

The specific activity of the enzyme was calculated as follows:

Wherein V is the total volume of the reaction system (200. Mu.L); d-dilution factor; v-enzyme liquid volume (5. Mu.L); c-protein concentration (mg/mL); epsilon is the molar extinction coefficient of NADH (epsilon= 6.220mM ^-1cm^-1 at 1cm optical path), where the slope of the NADH standard curve equation is used to correct, accurately weigh a certain amount of NADH, dilute different multiples to make NADH solutions of different concentrations, determine its absorbance at 340nm, and fit a linear equation (see FIG. 1). Protein quantification was determined using Bradford kit. All experiments were repeated three times with the mean as the final result.

Example 7: kinetic characterization of mutants

AiPDH and mutant apparent kinetic parameters determination, reference example 6 standard enzyme activity determination conditions, enzymatic reaction rates at different substrate concentrations (0.5-1024 mM) were determined separately and these data were used with ① inhibition by Origin Pro 2020 software: the Michaelis equation (Eq.1.3) was fitted non-linearly to find the Michaelis constant K _m and the maximum reaction speed V _max.

K _cat is calculated according to the formula (Eq.1.4), where [ E ] is equal to the total enzyme concentration.

② If inhibition is present, the Miq constant K _m、k_cat and the inhibition constant Ki are determined from the anti-competitive inhibition Miq.1.5 equation.

Wherein v is the reaction rate in the presence of substrate inhibition, μmol min ^-1mg^-1 is the substrate inhibition constant; v _max is the maximum reaction rate, μmol min ^-1mg^-1; [ S ] is the substrate concentration, mM; ki is the substrate inhibition constant, mM.

The substrate inhibition constants K _i of the mutant G13S, I, L, D and 37Q are respectively improved by 3.85-10 times compared with the parent, and the inhibition effects of F91Y and F198V are completely removed. At the same time, the affinity of the mutant is reduced, and the binding constants K _m are 7.78mM, 9.61mM, 34.26mM, 78.48mM and 41.32mM respectively, which are 1-10 times of the parent binding constant K _m; the transformation number k _cat has unobvious variation amplitude, and the F198V with the maximum amplification is improved by 36.1 percent compared with the female parent. The mutant F198V is subjected to iterative saturation mutation to obtain double-mutation dominant bacteria F91Y/F198V, the activity of the double-mutation dominant bacteria F91Y/F198V reaches 16.12U/mg, the double-mutation dominant bacteria is 7.8 times that of a female parent, and k _cat is improved by 66.7% compared with the female parent, and no substrate inhibition exists.

TABLE 2 Single point saturation and iterative saturation mutation screening results, activity assays and kinetic characterization

Example 8: influence of pH and temperature on female parent and mutant

Influence of pH: the purified wild type AiPDH and mutant F91Y/F198V of example 5 were each placed in buffers of different pH: 100mM sodium phosphate (pH 5.5-7.5), 100mM Tris-HCl (pH 7.5-9.0) and 100mM glycine-NaOH (pH 9.0-11.5), the enzyme activities of which were determined under the standard conditions of reference example 6, the highest activities were set to 100%. The results are shown in FIG. 2: aiPDH has highest activity under the condition of pH 10.5, if pH is acidic, the activity is obviously reduced, the catalytic activity is thoroughly lost when the pH is lower than 6.0, and the result also proves that the oxidation of alcohol is suitable to be carried out in an alkaline environment, which is favorable for promoting the deprotonation of AiPDH catalytic residues and the oxidation reaction of hydroxyl of an attack substrate. The pH spectrum of the mutant F91Y/F198V is consistent with that of the wild-type female parent, and the optimal pH is 10.5.

Thermal stability study: the purified wild-type AiPDH and mutant F91Y/F198V of example 5 were each incubated in a 25,30,40,45,50,55 ℃water bath for 2 hours for heat treatment, and the residual enzyme activities were determined under the standard conditions of reference example 6, with the initial enzyme activity being 100% of the highest enzyme activity. The results are shown in FIG. 3, where AiPDH derived from thermophilic bacteria Albidovuluminexpectatum has very high thermal stability. The enzyme activities of the mutants F91Y/F198 and AiPDH are not affected after the temperature is kept at 50 ℃ for 2 hours, and the enzyme activities of 90.7% and 91.5% are still respectively remained after the temperature is kept at 55 ℃ for 2 hours.

Example 9: preparation of high-purity D-xylulose by biological oxidation of xylitol

In a 100mM Tris-HCl reaction system with pH of 8.0, under pure oxygen environment, the substrate xylitol loading amount is 300g/L, the initial addition amount of AiPDH and mutant F91Y/F198V is 1g/L, the addition amount of NADH oxidase (from streptococcus thermophilus Streptococcus thermophilus, uniprot accession number is Q5M3T 5) is 1g/L, the reaction temperature is 30 ℃, and the rotating speed is 1500rpm. After the system had stabilized, the reaction was started by adding 0.5mM NAD ⁺ to the final concentration, and samples were taken at intervals during the reaction, and the conversion profile of the substrate was shown in FIG. 4. Under high substrate concentration, the xylitol conversion rate of the female parent catalytic reaction for 1h is 27.6%, the conversion speed of the system is gradually reduced, and the conversion rate of the final 8h is 60%; meanwhile, the mutant F91Y/F198V obtained by screening shows excellent tolerance against high-concentration substrates, the catalytic reaction reaches 65.2% after 2 hours, the conversion rate of 8 hours can reach 99.4%, the time-space yield is far faster than that of a female parent, and the time-space yield is as high as 900g L ^-1d^-1.

The detection method of the product comprises the following steps:

High Performance Liquid Chromatography (HPLC) was used to detect xylitol and D-xylulose. The high performance liquid chromatograph adopts Agilent 1200, and the chromatographic column is Aminex HPX-87C column (300×7.8mm) of Bio-Rad company; the mobile phase is ultrapure water; the flow rate is 0.6mL/min; the column temperature is 80 ℃; a differential detector is employed. The liquid phase diagram is shown in figure 5.

The foregoing is merely illustrative of the preferred embodiments of this invention, and it will be appreciated by those skilled in the art that variations and modifications may be made without departing from the principles of the invention, and such variations and modifications are to be regarded as being within the scope of the invention.

Claims

1. A 2-xylitol dehydrogenase mutant, characterized in that: the amino acid sequence of the 2-xylitol dehydrogenase mutant is SEQ ID NO:2, and the phenylalanine at position 91 of the amino acid sequence shown in the formula 2 is mutated into tyrosine, and the phenylalanine at position 198 is mutated into valine.

2. A 2-xylitol dehydrogenase mutant gene, characterized in that: the nucleotide sequence of the 2-xylitol dehydrogenase mutant gene is shown as SEQ ID NO: 3.

3. A recombinant vector comprising the 2-xylitol dehydrogenase mutant gene according to claim 2.

4. A genetically engineered bacterium, characterized in that: the genetically engineered bacterium comprises the 2-xylitol dehydrogenase mutant gene of claim 2 or the recombinant vector of claim 3.

5. The genetically engineered bacterium of claim 4, wherein: the host cell of the genetically engineered bacterium is an escherichia coli BL21 (DE 3) cell.

6. A catalyst comprising the 2-xylitol dehydrogenase mutant of claim 1; the catalyst is selected from: free enzyme, free cell, immobilized enzyme, or immobilized cell.

7. The use of the 2-xylitol dehydrogenase mutant gene according to claim 2, the recombinant vector according to claim 3 or the genetically engineered bacterium according to any one of claims 4 to 5 in the preparation of 2-xylitol dehydrogenase.

8. A preparation method of a 2-xylitol dehydrogenase mutant is characterized by comprising the following steps: the method comprises the following steps: culturing the genetically engineered bacterium of claim 4 or 5 to obtain a recombinant expressed 2-xylitol dehydrogenase mutant.

9. Use of the 2-xylitol dehydrogenase mutant according to claim 1 for the conversion of xylitol to prepare D-xylulose.

10. The use according to claim 9, characterized in that: the catalytic reaction system is prepared by reacting 100-300 g/L xylitol, 500-500 KU of 2-xylitol dehydrogenase mutant, 700-800 KU of NADH oxidase and 0.05-0.5 mmol/L NAD ⁺ for 2-36 h under the conditions of pH 7.5-9.0, reaction temperature 30-55 ℃, stirring rotation speed 180-1500 rpm and ventilation amount 1-3 VVM or pure oxygen, so as to obtain D-xylulose conversion solution.