An aldose reductase mutant and its application

Abstract

The present invention discloses an aldose reductase mutant and its application; the aldose reductase mutant is an aldose reductase with altered coenzyme preference, and the amino acid sequence of the aldose reductase mutant is shown in SEQ ID NO: 4, which is obtained by mutating lysine at position 270 to arginine and asparagine at position 272 to aspartic acid in the parental amino acid sequence shown in SEQ ID NO: 2. The gene of the aldose reductase mutant has a nucleotide sequence shown in SEQ ID NO: 3. By introducing this gene into Escherichia coli, a genetically engineered bacterium containing this gene is obtained, realizing the preparation of the recombinant aldose reductase mutant. The aldose reductase mutant provided by the present invention can efficiently utilize inexpensive oxidized coenzyme I (NAD+), catalyze the selective reduction of the aldehyde group at the C1-position of glucose, and achieve the green, safe and sustainable preparation of D-sorbitol.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and particularly relates to an aldose reductase mutant and its application. Background Art

[0002] D-Sorbitol, that is, hexanehexol, also known as sorbitol, with the molecular formula C6H 14 O6, is a polyol commonly present in fruits. It is widely used in fields such as medicine, food, daily chemicals, and light industry as a sweetener, humectant, texture agent, and softener, etc. At the same time, D-sorbitol is also an important platform compound for bioconversion, and a series of high-value-added bio-based chemicals and materials are derived through reaction processes such as dehydration, hydrocracking, and polymerization. Currently, the catalytic hydrogenation method is the most commonly used method for industrial production of D-sorbitol. Under high temperature (120 - 150 °C), high pressure (3 - 15 Mpa), and alkaline conditions, a metal catalyst is used to reduce the aldehyde group at the C1-position of glucose to produce D-sorbitol, and then refined D-sorbitol is obtained through methods such as membrane filtration, ion exchange, and decolorization by macroporous adsorption resin method (CN103179866A). Although the hydrogenation method has a relatively 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 technologies, the hydrogenation method faces challenges in terms of economy and sustainability in some applications. More research focuses on the development of new routes, catalyst optimization, energy efficiency improvement, and process simplification to improve production efficiency and environmental friendliness.

[0003] The electrolytic reduction method is an emerging green synthesis technology. Zhang Xuechao et al. prepared D-sorbitol by electrolysis using glucose as the raw material. Under weakly alkaline conditions, with a nickel catalyst as the cathode and Na2SO4 as the supporting electrolyte, by controlling the reaction conditions, the average conversion rate of glucose can reach 71.7%, and the average current efficiency is 60%, among which the by-product mannitol accounts for 19.7%. Compared with the catalytic hydrogenation method, the electrolytic reduction method is simpler and less polluting. Currently, this technology still needs to be deeply studied and optimized in terms of conversion rate, selectivity, electrode materials, and energy efficiency. In vitro multi-enzyme catalysis is to use cell extracts or pure enzymes to catalyze substrates to produce target products in a one-pot reaction system. Sun Yue et al. constructed a brand-new production route using in vitro multi-enzyme catalysis technology. By constructing a cascade enzyme catalysis (including phosphorylation, isomerization, hydrogenation, and dephosphorylation) and NADH regeneration system, starch was converted into D-sorbitol in a one-pot method. Through proof of concept, the yield of D-sorbitol from the bioconversion of 28 mmol / L of maltodextrin was 27.6 mmol / L, and the yield was 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 serious product inhibition and needed to be further optimized.

[0004] Aldose reductase (AR) is widely present in mammals and is a key rate-limiting enzyme in the polyol pathway, playing a crucial role in carbohydrate metabolism, redox balance, and cell protection. It can utilize the coenzyme NADPH to reduce glucose to D-sorbitol. In previous studies, we isolated an aldose reductase from Metschnikowia bicuspidata Metschnikowia bicuspidata that can selectively reduce the C1 aldehyde group of glucose to a hydroxyl group Mb AR, which strictly depends on NADPH. NADH and NADPH have the same nicotinamide fragment and adenine terminus, while NADPH has an additional 2'-phosphate group. There are differences in their stability, bioavailability, and cost. NAD + is inexpensive and highly stable, and is usually superior to NADP in biotransformation + . Altering the cofactor specificity of an enzyme often improves its industrial application in biocatalysis. Therefore, the preference of an enzyme for a specific coenzyme has become an important issue in practical applications. SUMMARY OF THE INVENTION

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an NADH-dependent aldose reductase mutant and its application in the preparation of D-sorbitol.

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

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

[0008] Another purpose of the present invention is to provide the application of the aldose reductase mutant.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] Wild-type aldose reductase derived from Metschnikowia bicuspidata Metschnikowia bicuspidata MbThe gene sequence of AR, as shown in SEQ ID NO: 1, was subjected to site-directed saturation mutagenesis and iterative saturation mutagenesis screening to obtain the gene of this aldose reductase mutant, and its nucleotide sequence is as shown in SEQ ID NO: 3:

[0011] Those skilled in the art should be aware that in the nucleic acid molecule expressing the aldose reductase mutant provided by this application, in addition to the above nucleotide fragments, it may also include nucleotide sequences such as promoters, enhancers, non-coding regions, etc., for the purpose of improving the performance of the gene in terms of expression level, expression efficiency, product activity, etc.

[0012] An aldose reductase mutant with an amino acid sequence as shown in SEQ ID NO: 4;

[0013] A recombinant vector containing the gene of the aldose reductase mutant described above. It 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. The recombinant plasmids are 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, pQE31, 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.

[0014] More preferably, the above recombinant plasmid is pET-28a(+).

[0015] Meanwhile, for expression in Bacillus subtilis, preferably, the recombinant plasmids that can be used are 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.

[0016] More preferably, the recombinant plasmid is pMA5.

[0017] A genetically engineered bacterium for producing the aldose reductase mutant, which contains the aldose reductase mutant gene or the recombinant vector of the present invention.

[0018] The host cell of the genetically engineered bacterium includes prokaryotic cells, yeast or eukaryotic cells; preferably, the prokaryotic cell is Escherichia coli E. coli cells or Bacillus subtilis. More preferably, the host cell is Escherichia coli BL21(DE3) cells.

[0019] A catalyst, which contains the aldose reductase mutant;

[0020] Optionally, the catalyst is selected from: free enzyme, free cell, immobilized enzyme or immobilized cell.

[0021] Use of the aldose reductase mutant gene, the recombinant vector, and the genetically engineered bacterium in the preparation of D-sorbitol.

[0022] A preparation method of an aldose reductase mutant, which comprises culturing the genetically engineered bacterium of the present invention to obtain the recombinantly expressed aldose reductase mutant.

[0023] The cultivation of the aldose reductase mutant of the present invention is carried out according to the following process: picking single colonies and activating them on an LB plate (containing 30 μg / mL kanamycin) at 37 °C, transferring them to fresh liquid LB the next day and culturing overnight, using the cells after two-stage activation as the seed solution and transferring them to 100 mL of fresh LB medium at a transfer amount of 4% and then culturing at 37 °C for 2-3 h until the OD 600 is about 0.8-1.0, adding IPTG to a final concentration of 0.2 mM, then culturing with shaking at 200 rpm at 16 °C, after inducing expression for 24 h, centrifuging at 8000 rpm for 15 min to collect the cells, and storing them at -80 °C.

[0024] The activity of the aldose reductase mutant (K270R / N272D) of the present invention towards coenzyme NADH reached 22.38 U / mg, achieving coenzyme-dependent inversion; it has the highest activity under the condition of pH 6.5, the enzyme activity is not affected after incubation at 30 °C for 2 h, and 65% of the enzyme activity remains after incubation at 40 °C for 2 h.

[0025] Use of the aldose reductase mutant of the present invention in the conversion of glucose to prepare D-sorbitol.

[0026] The catalytic reaction system consists of glucose at 50 - 150 g / L, sodium formate at 20 - 100 g / L, an aldose reductase mutant at 2 - 20 U / mL, formate dehydrogenase at 5 - 50 U / mL, and NAD at 0.05 - 0.5 mmol / L + , and reacts for 2 - 36 h under the conditions of pH 6.0 - 7.5, reaction temperature 25 - 35 °C, and stirring speed 100 - 500 rpm to obtain a D-sorbitol conversion solution.

[0027] The source of the formate dehydrogenase is not restricted as long as it can reduce NAD + to NADH, with the products being carbon dioxide and water. For example, Candida, Pichia, Hansenula, etc. In the preferred embodiment of the present invention, the formate dehydrogenase is derived from Candida boidinii Candida boidinii (Uniprot accession number: O13437).

[0028] Beneficial effects

[0029] Based on experimental techniques such as kinetic simulation, site-directed saturation / iterative saturation mutagenesis, and high-throughput screening, the present invention guides the directed molecular modification of key enzymes MbAR to reverse coenzyme dependence. The screened mutant K270R / N272D exhibits superior preference for the coenzyme NAD + , and the catalytic reaction reaches 66.75% in 3 h. After 24 h, the reaction is completely converted, and the space-time yield reaches 108 g L -1 d -1 , and the turnover number of the coenzyme for 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 the green synthesis of D-sorbitol. Description of the drawings

[0030] Figure 1 It is for the determination of the absorbance standard curve of NAD(P)H at 340 nm.

[0031] Figure 2 It is the coenzyme binding mode of the parent and the mutant K270R / N272D.

[0032] Figure 3 It is the effect of pH on the activities of the parent and the mutant K270R / N272D.

[0033] Figure 4 It is the effect of temperature on the activities of the parent and the mutant K270R / N272D.

[0034] Figure 5 It is the reaction progress curve of the mutant K270R / N272D.

[0035] Figure 6 It is the HPLC analysis chromatogram of glucose and D-sorbitol. Detailed implementation manners

[0036] The present invention can be better understood according to the following embodiments. However, the content described in the embodiments is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0037] Example 1: Wild-type aldose reductase MbAR Codon optimization and synthesis of the gene

[0038] 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, the codons were optimized according to the E. coli preference and the G+C content was adjusted. The optimized gene sequence was synthesized by GenScript Biotech Corporation. The gene sequence is shown in SEQ ID No: 1, and the amino acid sequence is shown in SEQ ID NO: 2. It was cloned into the vector pET-28a(+) via restriction enzymes EcoR I and Hind III and used as a template for directed evolution.

[0039] Example 2: Construction of mutant library

[0040] The present invention uses molecular docking and molecular dynamics simulation to study the binding mode of the protein and coenzyme, and selects MbThere are a total of 7 amino acid residues where AR interacts 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 mutagenesis on the selected 7 sites, and the PCR primers used are shown in Table 1. The single - point saturation mutagenesis PCR procedure is as follows: In a 20 μL reaction system, add 2 μL of KOD hot - start DNA polymerase buffer (10×), 2 μL of 2 mmol / L dNTP, 1 μL of DMSO, 0.5 μL (50 ng) of DNA template, 0.5 μL each of 100 μmol / L forward primer and reverse primer, and 1 μL of KOD DNA polymerase. Use the PCR program: 95°C for 3 min, (95°C for 30 sec, 60°C for 4.5 min, 72°C for 5 min) × 30 cycles, 72°C for 10 min, 10°C for 60 min. Add 0.5 μL of Dpn I to the obtained PCR product, digest at 37°C for 2 h to remove the template plasmid, aspirate 1 - 2 μL and transform it into Escherichia coli BL21(DE3) by electroporation, and spread it on a plate containing kanamycin, and incubate it upside - down in a 37°C incubator for 12 h to establish a single - point saturation mutant library.

[0041] Table 1 Primers used for single - point saturation mutagenesis

[0042]

[0043] Example 3: High - throughput screening of forward mutants

[0044] The transformants in the mutant library were picked and sequentially inoculated into a 96-deep well plate (primary plate) containing 300 µL of LB medium (containing 30 μg / mL kanamycin) per well. At the same time, 3 wells of wild-type clones were inoculated on each deep well plate as the parental control, and cultured overnight at 37 ºC with 250 rpm. A 96-channel manual pipetting workstation was used to aspirate 50 µL of the overnight culture and sequentially inoculate it into a secondary deep well plate containing 700 µL of self-inducing culture ZYP5052 (g / L, peptone 10, yeast extract 5, glycerol 5, glucose 0.5, lactose 2, (NH4)2SO4 3.3, KH2PO4 6.8, Na2HPO4 3.3, MgSO4 0.24). After culturing at 25 ºC with 250 rpm for 24 hours, centrifugation was carried out. A 96-channel manual pipetting workstation was used to aspirate 50 µL of the supernatant into a 96-well microplate, and 150 µL of the reaction solution composed of buffer, substrate, and coenzyme (50 mM pH 6.5 sodium phosphate, 0.2 mM NADH, 10 mM glucose) was added to each well, and the consumption of NADH was detected in batches using a microplate reader. Based on the parental samples of each deep well plate, mutants with NADH activity were picked from the library for rescreening. Glycerol with a final concentration of 10% was added to the primary deep well plate and stored at -80 ºC. In the rescreening stage, inoculation and shake flask induction expression were carried out from the glycerol bacteria, and beneficial mutants were obtained through shake flask verification. Sequencing analysis was performed on the positive mutant strains, and 2 different types of mutations occurred, as shown in Table 2, which were K270R and N272D respectively. In the second round, using the mutant pET-28a-K270R as the template, with the primers in Table 1, site-directed mutagenesis PCR, transformation, and plating were carried out, and the dominant strain K270R / N272D with double mutations was obtained through screening. Its nucleotide sequence is as shown in SEQ ID NO: 3, and the amino acid sequence is as shown in SEQ ID NO: 4.

[0045] Example 4: Fermentation culture of recombinant Escherichia coli

[0046] Wild bacteria Mb The expression of AR and mutants was carried out according to the following procedure: Single colonies were picked and activated on an LB plate (containing 30 μg / mL kanamycin) at 37 ºC. The next day, they were transferred to fresh liquid LB and cultured overnight. The bacteria after two-stage activation were used as the seed solution and transferred to 100 mL of fresh LB medium at a transfer volume of 4% and then cultured at 37 ºC for 2 - 3 h until the OD 600 was approximately 0.8 - 1.0. IPTG was added to a final concentration of 0.2 mM, and then cultured with shaking at 200 rpm at 16 ºC. After 24 h of induction expression, the bacteria were collected by centrifugation at 8000 rpm for 15 min and stored at -80 ºC.

[0047] Example 5: Recombinant protein purification

[0048] The target protein is fused with a 6×His tag at the N-terminus, and purification is carried out by nickel ion chromatography. Specific procedure: The frozen cells are resuspended in pre-cooled buffer A (20 mM sodium phosphate buffer, 500 mM NaCl, 10 mM imidazole, pH 8.0). After disruption with a high-pressure homogenizer, the cell debris is removed by centrifugation at 12,000 rpm for 30 min at 4 ºC. The supernatant is filtered through a 0.22 μm filter membrane and reserved. The supernatant is loaded onto a 5 mL Ni-NTA column installed on an AKTA and pre-equilibrated with buffer A at a flow rate of 5 mL / min. First, non-specifically bound proteins are eluted with 10 column volumes of buffer A, and then gradient elution is carried out with 0 - 100% buffer B (20 mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 8.0), and 2 mL of eluate is collected each time. The eluted samples are analyzed by 12% SDS-PAGE to determine the protein purity. After combining the pure proteins, they are concentrated with a 30 kDa ultrafiltration tube and stored in buffer C (150 mM NaCl, 1 mM DTT, 20 mM sodium phosphate, pH 6.5), and then quickly frozen in liquid nitrogen and stored at -80 °C for standby.

[0049] Example 6: Mb Enzyme activity detection of AR and mutants

[0050] Mb The activities of AR and mutants are measured by a microplate reader. One enzyme activity unit (U) is defined as the amount of enzyme required to consume 1 μmol of NAD(P)H per minute. The total reaction system contains 0.2 mM NAD(P)H, 10 mM glucose, an appropriate amount of the pure enzyme solution to be tested, and 100 mM sodium phosphate buffer at pH 6.5 to make up the total volume of 200 μL. At 30 ºC, the change in the absorbance value of NAD(P)H at a wavelength of 340 nm is detected, as shown in formula (1.1):

[0051] (Eq. 1.1)

[0052] The calculation formula for the specific activity of the enzyme is as follows:

[0053] (Eq. 1.2)

[0054] 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), which is corrected by the slope of the NAD(P)H standard curve equation here. Weigh a certain amount of NAD(P)H accurately, dilute it by different multiples to prepare NAD(P)H solutions with different concentrations, measure their absorbance values at 340 nm, and fit a linear equation (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.

[0055] Example 7: Kinetic Characterization of Mutants

[0056] Mb For the determination of the apparent kinetic parameters of AR and mutants, referring to the standard enzyme activity determination conditions in Example 6, the enzyme-catalyzed reaction rates were measured at different NAD(P)H concentrations (0 - 1 mM) respectively, and these data were non-linearly fitted by the Michaelis-Menten equation (Eq. 1.3) without inhibition using Origin Pro 2021 software to obtain the Michaelis constant K m and the maximum reaction rate V max .

[0057] (Eq. 1.3)

[0058] k cat Calculated according to formula (Eq. 1.4), where [E] is equal to the total enzyme concentration.

[0059] (Eq. 1.4)

[0060] The activities and kinetic parameters of the parent and mutants with NADH and NADPH as coenzymes are shown in Table 2. Combining mutations at sites K270 and N272, the selectivity of the mutant K270R / N272D for the coenzyme NADH increased significantly, with an activity reaching 22.38 U / mg, which is 91.87% of the parent. The K m for coenzyme NADH is 0.041 mM, K cat and the -1 is 46.87 s M K270 and N272 are the key amino acid residues affecting the coenzyme preference of 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.

[0061] Table 2 Single-site saturation and iterative saturation mutation screening results, activity determination and kinetic characterization

[0062]

[0063] Example 8: Effects of pH and temperature on parent and mutant

[0064] Effect of pH: The wild type purified in Example 5 was MbAR 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: MbAR 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.

[0065] Thermal stability study: The wild type purified in Example 5 MbAR 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: MbAR 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℃.

[0066] Example 9: Bioconversion of D-sorbitol

[0067] In a reaction system of 50 mM sodium phosphate at pH 6.5, the substrate glucose loading was 108 g / L, the co-substrate sodium formate was 50 g / L, the initial addition amount of the mutant K270R / N272D was 5 U / mL, and the addition amount of formate dehydrogenase (derived from Candida boidinii Candida boidinii , Uniprot accession number: O13437, Gene, 1995, 162(1): 99-104) was 10 U / mL. The reaction temperature was 30 ºC and the rotation speed was 400 rpm. After the system was stable, NAD with a final concentration of 0.1 mM was added + to initiate the reaction. Samples were taken at intervals during the reaction process, and the conversion progress curve of the substrate was as shown Figure 5 . The screened mutant K270R / N272D showed superior preference for the coenzyme NAD + . The catalytic reaction reached 66.75% in 3 h. After 24 h, the reaction was completely converted, and the space-time yield was 108 g L -1 d -1 , and the turnover number of the coenzyme for the product was 6000.

[0068] Detection method of the product:

[0069] Glucose and D-sorbitol were detected by high performance liquid chromatography (HPLC). The HPLC instrument 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; a differential detector was used. The liquid chromatogram is shown Figure 6 .

[0070] The above are only the preferred implementation embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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 bacteria 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.

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