Aldose reductase mutant and application thereof

Abstract

The invention discloses an aldose reductase mutant and application thereof. The aldose reductase mutant is an aldose reductase with changed coenzyme preference, the amino acid sequence of the aldose reductase mutant is shown as SEQ ID NO: 4, and the amino acid sequence of the aldose reductase mutant is obtained by mutating 270-site lysine into arginine and mutating 272-site asparagine into aspartic acid in a female parent amino acid sequence shown as SEQ ID NO: 2. The nucleotide sequence of the aldose reductase mutant gene is as shown in SEQ ID NO: 3. The gene is introduced into escherichia coli to obtain a genetically engineered bacterium containing the gene, so that the preparation of the recombinant aldose reductase mutant is realized. The aldose reductase mutant provided by the invention can efficiently utilize cheap oxidized coenzyme I (NAD < + >) to catalyze selective reduction of a C1-site aldehyde group of glucose, so that green, safe and sustainable preparation of D-sorbitol is realized.

Description

Technical Field

[0001] The invention relates to the technical field of biochemistry, and in particular to an aldose reductase mutant and application thereof. Background Art

[0002] D-Sorbitol, also known as hexane hexol, is also called sorbitol, with a molecular formula of C6H 14 O6, a polyol commonly found in fruits, is widely used in medicine, food, daily chemicals, light industry and other fields as a sweetener, moisturizer, texture agent and softener. At the same time, D-sorbitol is also an important biotransformation platform compound, and a series of high value-added bio-based chemicals and materials are derived through dehydration, hydrogenolysis, polymerization and other reaction processes. At present, catalytic hydrogenation is the most commonly used method for industrial production of D-sorbitol. Under high temperature (120~150℃), high pressure (3~15 Mpa) and alkaline conditions, the aldehyde group of glucose C1 is reduced to D-sorbitol using a metal catalyst, and then refined D-sorbitol is obtained by membrane filtration, ion exchange and macroporous adsorption resin decolorization (CN103179866A). Although the hydrogenation method has a high conversion rate in the industrial production of D-sorbitol, it still faces challenges such as poor catalyst selectivity, high energy consumption, harsh reaction conditions, by-product generation and environmental impact. With the rise of green production technology, the hydrogenation method faces challenges in economy and sustainability in some applications. More research focuses on the development of new pathways, catalyst optimization, energy efficiency improvement and process simplification to improve production efficiency and environmental friendliness.

[0003] Electrolytic reduction is an emerging green synthesis technology. Zhang Xuechao et al. prepared D-sorbitol by electrolysis using glucose as raw material. Under weak alkaline conditions, nickel catalyst was used as cathode and Na2SO4 was used as supporting electrolyte. By controlling the reaction conditions, the average conversion rate of glucose can reach 71.7%, and the average current efficiency is 60%, of which the by-product mannitol accounts for 19.7%. Compared with catalytic hydrogenation, electrolytic reduction is simpler and less polluting. At present, this technology still needs to be further studied and optimized in terms of conversion rate, selectivity, electrode materials and energy efficiency. In vitro multi-enzyme catalysis is the use of cell extracts or pure enzymes to catalyze substrates to generate target products in a one-pot reaction system. Sun Yue et al. used in vitro multi-enzyme catalysis technology to construct a new production pathway. By constructing cascade enzyme catalysis (including phosphorylation, isomerization, hydrogenation and dephosphorylation) and NADH regeneration system, starch was converted into D-sorbitol in one pot. Through proof of concept, the bioconversion of 28 mmol / L maltodextrin to D-sorbitol yielded 27.6 mmol / L, with a yield of 98.6%. Unfortunately, when the substrate concentration was increased by 10 times, the yield of D-sorbitol was only 65.1 mmol / L, and the multi-enzyme cascade system still had severe product inhibition, which needed further optimization.

[0004] Aldose reductase (AR) is widely present in mammals and is a key rate-limiting enzyme in the polyol pathway. It plays a key role in sugar metabolism, redox balance and cell protection. It can reduce glucose to D-sorbitol using the coenzyme NADPH. Metschnikowia bicuspidata Aldose reductase that selectively reduces the C1-aldehyde group of glucose to a hydroxyl group Mb AR and is strictly dependent on NADPH. NADH and NADPH have the same nicotinamide fragment and adenine end, but NADPH has an additional 2'-phosphate group. The two differ in stability, bioavailability, and cost. + Low price, high stability, usually better than NADP in biotransformation + Changing the cofactor specificity of an enzyme can often improve the industrial application of the enzyme in biocatalysis. Therefore, the preference of the enzyme for a specific coenzyme has become an important issue in practical applications. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a NADH-dependent aldose reductase mutant and its use in the preparation of D-sorbitol.

[0006] To achieve the above objectives, the present invention uses molecular docking and molecular dynamics simulation to study the binding mode of protein and coenzyme. Mb AR interacts with the C2-phosphate group of NADPH in a total of 7 amino acid residues. The aldose reductase mutant K270R / N272D was screened by site-directed saturation mutagenesis and iterative saturation mutagenesis. The amino acid sequence of the mutant K270R / N272D is the same as that of the wild-type aldose reductase shown in SEQ ID NO: 2. Mb In the amino acid sequence of AR, lysine at position 270 mutated to arginine, and asparagine at position 272 mutated to aspartic acid.

[0007] Another object of the present invention is to provide a method for preparing the aldose reductase mutant.

[0008] Another object of the present invention is to provide applications of the aldose reductase mutant.

[0009] The purpose of the present invention can be achieved through the following technical solutions: Derived from Metschnikova bicuspidatum Metschnikowia bicuspidata Wild-type aldose reductase MbThe gene sequence of AR is shown in SEQ ID NO: 1. After site-directed saturation mutagenesis and iterative saturation mutagenesis screening, the gene of the aldose reductase mutant is obtained, and its nucleotide sequence is shown in SEQ ID NO: 3: Those skilled in the art should be aware that the nucleic acid molecules expressing the aldose reductase mutants provided in the present application may include, in addition to the above nucleotide fragments, nucleotide sequences such as promoters, enhancers, and non-coding regions to achieve the purpose of improving the expression level, expression efficiency, product activity and other aspects of the gene performance.

[0010] An aldose reductase mutant, the amino acid sequence of which is shown in SEQ ID NO: 4; A recombinant vector comprising the aldose reductase mutant gene. The recombinant vector can be constructed by connecting the nucleotide sequence of the aldose reductase gene of the present invention to various vectors by conventional methods 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-1, pGEX-6p-1, pGEX-6p-2, pBV220, pTrc99A, pTwin1, pEZZ18, pKK232-18, pBR322, pUC-18 or pUC-19.

[0011] More preferably, the above-mentioned recombinant plasmid is pET-28a(+).

[0012] At the same time, for expression in Bacillus subtilis, preferably, the recombinant plasmid that can be used is 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, φ1 or φ105.

[0013] More preferably, the above-mentioned recombinant plasmid is pMA5.

[0014] A genetically engineered bacterium for producing the aldose reductase mutant, wherein the genetically engineered bacterium comprises the aldose reductase mutant gene or the recombinant vector of the present invention.

[0015] The host cells of the genetically engineered bacteria include prokaryotic cells, yeast or eukaryotic cells; preferably, the prokaryotic cells are Escherichia coli E. coli More preferably, the host cell is an Escherichia coli BL21 (DE3) cell.

[0016] A catalyst comprising the aldose reductase mutant; Optionally, the catalyst is selected from: free enzyme, free cell, immobilized enzyme or immobilized cell.

[0017] The application of the aldose reductase mutant gene, the recombinant vector and the genetically engineered bacteria in the preparation of D-sorbitol.

[0018] A method for preparing an aldose reductase mutant comprises culturing the genetically engineered bacteria of the invention to obtain a recombinantly expressed aldose reductase mutant.

[0019] The aldose reductase mutant of the present invention is cultured according to the following process: a single colony is picked on an LB plate (containing 30 µg / mL kanamycin) and activated at 37°C, and the next day it is transferred to fresh liquid LB for overnight culture. The bacteria after two-stage activation are used as seed liquid and transferred to 100 mL of fresh LB medium at a transfer volume of 4%, and then cultured at 37°C for 2-3 hours until the OD reaches 0. 600 The p-value was about 0.8~1.0, IPTG was added to a final concentration of 0.2 mM, and then cultured at 16 ºC with shaking at 200 rpm. After inducing expression for 24 hours, the bacteria were collected by centrifugation at 8000 rpm for 15 min and stored at -80 ºC.

[0020] The activity of the aldose reductase mutant (K270R / N272D) of the present invention for the coenzyme NADH reaches 22.38 U / mg, realizing the coenzyme-dependent reversal; the activity is highest under the condition of pH 6.5, the enzyme activity is not affected after being incubated at 30°C for 2 hours, and 65% of the enzyme activity is still retained after being incubated at 40°C for 2 hours.

[0021] The aldose reductase mutant of the invention is used for converting glucose to prepare D-sorbitol.

[0022] The catalytic reaction system is 50~150 g / L glucose, 20~100 g / L sodium formate, 2~20 U / mL aldose reductase mutant, 5~50 U / mL formate dehydrogenase and 0.05~0.5 mmol / L NAD + , react for 2~36 h at pH 6.0~7.5, reaction temperature 25~35°C, and stirring speed 100~500 rpm to obtain D-sorbitol conversion solution.

[0023] The source of formate dehydrogenase is not limited as long as it can reduce NAD + The formate dehydrogenase is derived from Candida boidinii. Candida boidinii (Uniprot accession number: O13437).

[0024] Beneficial Effects

[0025] The present invention is based on experimental techniques such as kinetic simulation, site-directed saturation / iterative saturation mutagenesis and high-throughput screening to guide the key enzyme MbB Directed molecular engineering reverses coenzyme dependence. The mutant K270R / N272D obtained by screening targets the coenzyme NAD + The catalytic reaction showed excellent preference, reaching 66.75% in 3 h. After 24 hours, the reaction conversion was complete, and the space-time yield reached 108 g L -1 d -1 The number of cyclic conversions of the coenzyme to the product is 6000. The mutant K270R / N272D can be used as a new biocatalyst to replace the traditional hydrogenation method in industrial production to achieve green synthesis of D-sorbitol. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The absorbance value of NAD(P)H at 340 nm was used as the standard curve.

[0027] Figure 2 The coenzyme binding modes of the parent and mutant K270R / N272D.

[0028] Figure 3 The effect of pH on the activity of the parent plant and mutant K270R / N272D.

[0029] Figure 4 The effect of temperature on the activity of the maternal plant and the mutant K270R / N272D.

[0030] Figure 5 This is the reaction progress curve of the mutation K270R / N272D.

[0031] Figure 6 HPLC analysis spectrum of glucose and D-sorbitol. DETAILED DESCRIPTION

[0032] The present invention can be better understood according to the following examples. However, the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0033] Example 1: Wild-type aldose reductase MbB Codon optimization and synthesis of genes Wild-type aldose reductase Mb The gene sequence of AR was designed using DNAMAN software without changing the amino acid sequence encoded by the original gene. E. coli The codons were optimized and the G+C content was adjusted according to the preference of the gene sequence. The optimized gene sequence was synthesized by Suzhou Genewise Co., Ltd. The gene sequence is shown in SEQ ID No: 1 and the amino acid sequence is shown in SEQ ID NO: 2. EcoR I and Hind III was cloned into the vector pET-28a(+) and used as a template for directed evolution.

[0034] Example 2: Construction of mutant library The present invention uses molecular docking and molecular dynamics simulation to study the binding mode of protein and coenzyme, and selects MbAR has 7 amino acid residues that interact with the C2-phosphate group of NADPH, namely Gln219 (Q219), Lys270 (K270), Ser271 (S271), Asn272 (N272), Lys273 (K273), Arg276 (R276), and Asn280 (N280). The degenerate codon NNK was used to perform single-point saturation mutations on the selected 7 sites, and the PCR primers used are shown in Table 1. The single-point saturation mutation PCR program is as follows: 2 μL KOD hot start DNA polymerase buffer (10×), 2 μL 2 mmol / L dNTP, 1 μL DMSO, 0.5 μL (50 ng) DNA template, 100 μmol / L forward primer and reverse primer, 0.5 μL each, and 1 μkL KOD DNA polymerase were added to the 20 μL reaction system. The PCR program was as follows: 95℃ 3 min, (95℃ 30sec, 60℃ 4.5 min, 72℃ 5 min) × 30 cycles, 72℃ 10 min, 10℃ 60 min. The obtained PCR product was added with 0.5 μL Dpn I, digested at 37℃ for 2 h to remove the template plasmid, 1~2 μL was taken and transformed into Escherichia coli BL21 (DE3) by electroporation, and spread on a plate containing kanamycin, and inverted in a 37ºC incubator for 12 h to establish a single-site saturation mutant library.

[0035] Table 1 Primers used for single-site saturation mutagenesis

[0036] Example 3: High-throughput screening of forward mutants Transformants from the mutant library were picked and sequentially inoculated into 96 deep-well plates (primary plates) containing 300 µL LB medium (containing 30 μg / mL kanamycin) per well. At the same time, 3 wells of wild-type clones were inoculated in each deep-well plate as the mother control and cultured overnight at 37ºC, 250 rpm. 50 µL of overnight culture medium was pipetted into each well of a secondary deep-well plate containing 700 µL of auto-induced culture ZYP5052 (g / L, peptone 10, yeast powder 5, glycerol 5, glucose 0.5, lactose 2, (NH4)2SO43.3, KH2PO46.8, Na2HPO43.3, MgSO40.24) using a 96-channel manual pipetting workstation. After incubation at 25°C, 250 rpm for 24 hours, the plate was centrifuged and 50 µL of supernatant was pipetted into a 96-well ELISA plate using a 96-channel manual pipetting workstation. 150 µL of reaction solution consisting of buffer, substrate, and coenzyme (50 mM pH 6.5 sodium phosphate, 0.2 mM NADH, 10 mMglucose) was added to each well and the plate was placed in an ELISA reader to batch detect NADH consumption. Based on the parent of each deep-well plate, mutants with NADH activity were selected from the library for rescreening. Glycerol with a final concentration of 10% was added to the primary deep-well plate and stored at -80ºC. In the rescreening stage, shake flasks were inoculated from glycerol bacteria to induce expression, and beneficial mutants were obtained after shake flask verification. Sequencing analysis was performed on the positive mutant strains, and two different types of mutations appeared. The results are shown in Table 2, namely K270R and N272D. In the second round, the mutant pET-28a-K270R was used as a template, and the primers in Table 1 were used for site-directed mutagenesis PCR, transformation, and plate coating. The dominant bacteria K270R / N272D with double mutations were obtained through screening. Its nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

[0037] Example 4: Fermentation culture of recombinant Escherichia coli wild fungi Mb The expression of AR and mutants was performed according to the following process: a single colony was picked and activated on an LB plate (containing 30 µg / mL kanamycin) at 37 ºC, and the next day it was transferred to fresh liquid LB for overnight culture. The bacteria after two-stage activation were used as seed liquid and transferred to 100 mL fresh LB medium at a transfer volume of 4% and then cultured at 37 ºC for 2-3 h until the OD reached 0. 600 The p-value was about 0.8~1.0, IPTG was added to a final concentration of 0.2 mM, and then cultured at 16 ºC with shaking at 200 rpm. After inducing expression for 24 hours, the bacteria were collected by centrifugation at 8000 rpm for 15 min and stored at -80 ºC.

[0038] Example 5: Recombinant protein purification The target protein was fused with a 6×His tag at the N-terminus, and nickel ion chromatography was used for purification. Specific process: frozen cells were resuspended in pre-cooled buffer A (20 mM sodium phosphate buffer, 500 mM NaCl, 10 mM imidazole, pH 8.0), broken by high-pressure homogenizer, centrifuged at 12 000 rpm at 4 ºC for 30 min to remove cell debris, and the supernatant was filtered through a 0.22 μm filter membrane for use. The supernatant was loaded onto a 5 mL Ni-NTA column installed on AKTA and pre-equilibrated with buffer A at a flow rate of 5 mL / min. Non-specific binding proteins were first eluted with 10 column volumes of buffer A, followed by gradient elution with 0~100% buffer B (20mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 8.0), and the eluate was collected every 2 mL. The eluted samples were analyzed by 12% SDS-PAGE to determine the protein purity. The pure proteins were combined and concentrated using a 30 kDa ultrafiltration tube and stored in buffer C (150 mM NaCl, 1 mM DTT, 20 mM sodium phosphate, pH 6.5), then quickly frozen in liquid nitrogen and stored at -80°C for use.

[0039] Embodiment 6: Mb Enzyme activity detection of AR and its mutants Mb The activity of AR and mutants was measured by microplate reader. One unit of enzyme activity (U) was defined as the amount of enzyme required to consume 1 μmol NAD(P)H per minute. The total reaction system contained 0.2 mM NAD(P)H, 10 mM glucose, an appropriate amount of pure enzyme solution to be tested, and 100 mM sodium phosphate buffer at pH 6.5 to make up the total volume to 200 μL. At 30 ºC, the absorbance change of NAD(P)H at a wavelength of 340 nm was detected, as shown in formula (1.1): (Eq. 1.1) The specific activity of the enzyme was calculated as follows: (Eq. 1.2)

[0040] Where V is the total volume of the reaction system (200 μL); D is the dilution factor; v is the volume of the enzyme solution (5 μL); C is the protein concentration (mg / mL); ε is the molar extinction coefficient of NAD(P)H (when the optical path is 1 cm, ε = 6.220 mM -1 cm -1 ), the slope of the NAD(P)H standard curve equation was used for correction. A certain amount of NAD(P)H was accurately weighed, and NAD(P)H solutions of different concentrations were prepared by diluting them at different times. The absorbance at 340 nm was measured and the linear equation was fitted (see Figure 1 ). Protein quantification was determined using a Bradford kit. All experiments were repeated three times, and the mean value was used as the final result.

[0041] Example 7: Kinetic characterization of mutants Mb The apparent kinetic parameters of AR and its mutants were determined by referring to the standard enzyme activity determination conditions in Example 6, and the enzymatic reaction rates at different NAD(P)H concentrations (0-1 mM) were measured respectively. These data were fitted nonlinearly using the Origin Pro 2021 software using the Michaelis-Menten equation (Eq. 1.3) without inhibition to obtain the Michaelis constant. K m and maximum reaction speed V max .

[0042] (Eq. 1.3) k cat Calculated according to the formula (Eq. 1.4), here [E] is equal to the total enzyme concentration.

[0043] (Eq. 1.4)

[0044] The activities and kinetic parameters of the parent and mutants with NADH and NADPH as coenzymes are shown in Table 2. By combining mutations at the K270 and N272 sites, the selectivity of the mutant K270R / N272D for the coenzyme NADH increased significantly, with an activity of 22.38 U / mg, which is 91.87% of that of the parent. K m 0.041mM, K cat 46.87s -1 K270 and N272 are the impact M The key amino acid residues for bAR coenzyme preference are shown in Figure 2 , (1) The K270 site, mutated from lysine to arginine, introduced a guanidine group with greater steric hindrance and positive charge, which not only reduced the binding pocket of the phosphate group and increased the difficulty of the coenzyme NADPH to bind to the protein, but also formed hydrogen bonds with the 2'- and 3'-hydroxyl groups of adenosine ribose in NADH, making the mutant K270R NADH-dependent and the coenzyme preference reversed. (2) The N272 site, mutated from asparagine to aspartic acid, introduced a negatively charged carboxyl group at the end of the side chain. This charge change will produce charge repulsion with the phosphate group in NADPH, further reducing the binding stability of NADPH.

[0045] Table 2 Single-site saturation and iterative saturation mutation screening results, activity determination and kinetic characterization

[0046] Example 8: Effects of pH and temperature on parent and mutant Effect of pH: The wild type purified in Example 5 was MbB The mutant K270R / N272D was placed in buffers with different pH values: 100 mM sodium phosphate (pH 5.5-7.5), 100 mM Tris-HCl (pH 7.5-9.5), and the enzyme activity was determined under the standard conditions of Example 6, with the highest activity set at 100%. The results are shown in Figure 3 As shown: MbB The activity was highest at pH 6.5, and more than 90% of the activity was retained at pH 5.5-7.0. The pH spectrum of the mutant K270R / N272D was consistent with that of the wild-type parent, and the optimal pH was also 6.5.

[0047] Thermal stability study: The wild type purified in Example 5 MbB The mutant K270R / N272D was heat treated at 20, 25, 30, 35, and 40°C in a water bath for 2 hours, and the residual enzyme activity was measured under the standard conditions of Example 6, with the initial enzyme activity being the highest enzyme activity of 100%. The results are shown in Figure 4 show: MbB Under the condition of 25-35℃, the enzyme activity is still very high, and its optimum temperature is 30℃, but if the temperature continues to rise, the enzyme activity will be greatly affected. For example, incubated at 40℃ for 2 hours, only 65% ​​of the enzyme activity is retained. When the temperature is too low, the molecular movement is slow, which reduces the catalytic activity of the enzyme. When the temperature is too high, the protein may denature, thereby seriously inhibiting the activity of the enzyme. The thermal stability of the mutant K270R / N272D is consistent with that of the wild-type parent, and the optimum reaction temperature is 30℃.

[0048] Example 9: Bioconversion of D-sorbitol In the pH 6.5 50 mM sodium phosphate reaction system, the substrate glucose loading was 108 g / L, the cosubstrate sodium formate was 50 g / L, the initial addition amount of mutant K270R / N272D was 5 U / mL, and formate dehydrogenase (from Candida boidinii) was 1. Candida boidinii , Uniprot accession number: O13437, Gene, 1995, 162(1): 99-104) The addition amount was 10U / mL, the reaction temperature was 30 ºC, and the rotation speed was 400 rpm. After the system was stable, a final concentration of 0.1 mM NAD was added +Start the reaction, take samples at intervals during the reaction, and the conversion curve of the substrate is as follows: Figure 5 The mutant K270R / N272D obtained by screening targets the coenzyme NAD + The reaction showed excellent preference, reaching 66.75% in 3 h. After 24 hours, the reaction conversion was complete, with a space-time yield of 108 g L -1 d -1 , the number of cyclic conversions of the coenzyme to the product is 6000.

[0049] Product detection method: High performance liquid chromatography (HPLC) was used to detect glucose and D-sorbitol. The HPLC instrument used was Agilent 1260, and the chromatographic column was Aminex HPX-87C column (300 × 7.8 mm) from Bio-Rad, USA. The mobile phase was ultrapure water, the flow rate was 0.6 mL / min, the column temperature was 80 ºC, and a differential detector was used. The liquid phase spectrum is shown in Figure 6 .

[0050] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An aldose reductase mutant, the amino acid sequence of which is shown in SEQ ID NO:

4.

2. An aldose reductase mutant gene, encoding the aldose reductase mutant according to claim 1, wherein the nucleotide sequence is shown in SEQ ID NO:

3.

3. A recombinant vector comprising the aldose reductase mutant gene according to claim 2.

4. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria comprises the aldose reductase mutant gene according to claim 2 or the recombinant vector according to claim 3.

5. The genetically engineered bacterium according to claim 4, characterized in that: The host cell of the genetically engineered bacteria is Escherichia coli BL21 (DE3) cell.

6. A catalyst, characterized in that It comprises the aldose reductase mutant according to claim 1; and the catalyst is selected from: one of free enzyme, free cell, immobilized enzyme or immobilized cell.

7. Use of the aldose reductase mutant gene according to claim 2, the recombinant vector according to claim 3 or the genetically engineered bacteria according to any one of claims 4 to 5 in the preparation of aldose reductase.

8. A method for preparing an aldose reductase mutant, characterized in that: Cultivate the genetically engineered bacteria according to claim 4 or 5 to obtain a recombinantly expressed aldose reductase mutant.

9. Use of the aldose reductase mutant according to claim 1 in converting glucose to prepare D-sorbitol.

10. The use according to claim 9, characterized in that: The catalytic reaction system for converting glucose to D-sorbitol is 50-150 g / L glucose, 20-100 g / L sodium formate, 2-20 U / mL aldose reductase mutant, 5-50 U / mL formate dehydrogenase, and 0.05-0.5 mmol / L NAD + , react for 2~36 h at pH 6.0~7.5, reaction temperature 25~35°C, and stirring speed 100~500 rpm to obtain D-sorbitol conversion solution.

Images