Xylose reductase XR31 mutant and application thereof

Abstract

The invention relates to the technical field of enzyme engineering, in particular to a xylose reductase XR31 mutant and application thereof. Specifically, the invention provides a novel xylose reductase mutant aiming at the defect of poor thermal stability of the existing wild type xylose reductase (SEQ ID NO: 2), and particularly provides a xylose reductase mutant which is obtained by performing single or multiple site amino acid mutation on the 21st site, the 46th site, the 128th site, the 168th site and the 205th site of a xylose reductase sequence shown as SEQ ID NO: 2, compared with wild type xylose reductase, the obtained xylose reductase mutant has the advantages that the catalytic activity and the thermal stability on substrate xylose are obviously improved, the xylose reductase mutant can stably work under a high-temperature condition, and substrate conversion in industrial application is more efficiently realized.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to the XR31 mutant of xylose reductase and its applications. Background Technology

[0002] Xylitol is a pentose sugar alcohol that is naturally found in fruits and vegetables (such as strawberries and spinach). It has a similar sweetness to sucrose (about 95% of sucrose) but is low in calories (2.4 kcal / g, compared to 4 kcal / g for sucrose) and its metabolism does not depend on insulin. Therefore, it can be widely used in food, medical, and chemical industries as a diabetes-friendly sweetener, a sucrose substitute, and a biodegradable material for producing xylitol-based polyesters.

[0003] Currently, the main methods for preparing xylitol include: Traditional chemical processes primarily use lignocellulose (such as corn cobs and sugarcane bagasse) as raw material, which is acid-hydrolyzed to produce xylose, and then catalytically hydrogenated to xylitol using Ni / Al2O3 as a catalyst under high temperature and pressure. Although this method is mature, lignocellulose contains multiple components such as xylose, glucose, and arabinose, and the Ni / Al2O3 catalyst lacks selectivity during catalysis, thus generating other sugar alcohols during the catalytic process. To obtain high-purity xylitol, pretreatment is required to separate and purify hemicellulose to obtain xylose or to further purify the sugar alcohol product, which is complex and costly. Furthermore, nickel, as a heavy metal catalyst, can cause serious pollution. Emerging green processes, such as microbial catalysis, utilize genetically engineered microorganisms (such as yeast)... Candida tropicalis ,bacteria Escherichia coli This method expresses xylose reductase and xylitol dehydrogenase to convert xylose into xylitol. It effectively improves the selectivity of xylitol catalytic production, avoids heavy metal catalyst pollution, and, due to its green and environmentally friendly characteristics, shows broad application prospects, becoming a research hotspot both domestically and internationally. However, in industrial applications, especially under high-temperature conditions, the thermal stability of natural xylose reductase is poor, which to some extent limits its widespread application in industrial production.

[0004] Thermostability of xylose reductase refers to its ability to maintain its activity and structure under high-temperature conditions. Thermal stability is influenced by various factors, including the enzyme's amino acid sequence, three-dimensional structure, interactions with other molecules, and the solution environment. The significance of studying thermostability lies in the fact that improving the thermostability of xylose reductase can help achieve higher conversion efficiency and product yield in industrial applications, especially in biotechnological processes requiring high-temperature treatment. Therefore, developing a novel xylose reductase with high thermostability has significant industrial importance and application value. Summary of the Invention

[0005] This invention addresses the poor thermostability of existing xylose reductases by providing a novel xylose reductase mutant. Specifically, it is obtained by making single or multiple amino acid mutations at positions 21, 46, 128, 168, and 205 of the xylose reductase sequence shown in SEQ ID NO: 2. The resulting xylose reductase mutant exhibits significantly improved catalytic activity and thermostability towards the substrate xylose compared to the original xylose reductase. It can operate stably under high-temperature conditions, enabling more efficient substrate conversion in industrial applications.

[0006] In this regard, the present invention includes, but is not limited to, the following technical solutions: In one aspect, the present invention provides a xylose reductase mutant, characterized in that the mutant has five or fewer substitutions relative to SEQ ID NO: 2 and has less than 100% sequence identity with SEQ ID NO: 2, wherein the substitutions are selected from: G21P (glycine at position 21 is mutated to proline), G21S (glycine at position 21 is mutated to serine), G21C (glycine at position 21 is mutated to cysteine), G21Y (glycine at position 21 is mutated to tyrosine), G21K (glycine at position 21 is mutated to lysine), G21E (glycine at position 21 is mutated to glutamic acid), D46P (aspartic acid at position 46 is mutated to proline), D46G (aspartic acid at position 46 is mutated to glycine), and D46S (aspartic acid at position 46 is mutated to serine). D46T (aspartic acid at position 46 is replaced with threonine), D46E (aspartic acid at position 46 is replaced with glutamic acid), P128I (proline at position 128 is replaced with isoleucine), P128W (proline at position 128 is replaced with tryptophan), P128S (proline at position 128 is replaced with serine), P128T (proline at position 128 is replaced with threonine), P128R (proline at position 128 is replaced with arginine), P128E (proline at position 128 is replaced with glutamic acid), V168A (valine at position 168 is replaced with alanine), V168L (valine at position 168 is replaced with valine). The mutations at position 168 and 168 are: V168I (valine at position 168 isoleucine), V168W (valine at position 168 is tryptophan), V168P (valine at position 168 is proline), V168S (valine at position 168 is serine), V168T (valine at position 168 is threonine), V168Y (valine at position 168 is tyrosine), V168N (valine at position 168 is asparagine), V168H (valine at position 168 is histidine), and V168R (valine at position 168 is arginine). V168E (valine at position 168 is replaced by glutamic acid), R205L (arginine at position 205 is replaced by leucine), R205F (arginine at position 205 is replaced by phenylalanine), R205P (arginine at position 205 is replaced by proline), R205C (arginine at position 205 is replaced by cysteine), R205N (arginine at position 205 is replaced by asparagine), R205Q (arginine at position 205 is replaced by glutamine), R205H (arginine at position 205 is replaced by histidine), and R205D (arginine at position 205 is replaced by aspartic acid), or combinations thereof.

[0007] In one aspect, the substitutes described in this invention are selected from: G21Y, D46E, P128S, V168I and R205H, or combinations thereof.

[0008] In another aspect, the present invention provides a xylose reductase mutant obtained by performing the following mutation on SEQ ID NO: 2: G21Y; D46E; P128S; V168I; R205H; G21Y+D46E; G21Y+P128S; D46E+V168I; G21Y+D46E+P128S; G21Y+V168I+R205H; D46E+V168I+R205H; G21Y+D46E+P128S+V168I; G21Y+P128S+V168I+R205H; G21Y+D46E+P128S+V168I+R205H; G21P; G21S; G21C; G21K; G21E; D46P; D46G; D46S; D46T; P128I; P128W; P128T; P128R; P128E; V168A; V168L; V168I; V168W; V168P; V168S; V168T; V168Y; V168N; V168H; V168R; V168E; R205L; R205F; R205P; R205C; R205N; R205Q; R205H; or R205D.

[0009] In one aspect, the xylose reductase mutant of the present invention is obtained by performing the following mutation on SEQ ID NO: 2: G21Y; D46E; P128S; V168I; R205H; G21Y+D46E; G21Y+P128S; D46E+V168I; G21Y+D46E+P128S; G21Y+V168I+R205H; D46E+V168I+R205H; G21Y+D46E+P128S+V168I; G21Y+P128S+V168I+R205H; or G21Y+D46E+P128S+V168I+R205H.

[0010] In the technical solution of the present invention concerning xylose reductase mutants, "+" represents the simultaneous presence of two or more mutation forms. For example, G21Y+D46E represents a xylose reductase mutant obtained by mutating the xylose reductase sequence shown in SEQ ID NO: 2 as follows: G21Y and D46E; G21Y+D46E+P128S+V168I+R205H represents a xylose reductase mutant obtained by mutating the xylose reductase sequence shown in SEQ ID NO: 2 as follows: G21Y, D46E, P128S, V168I, and R205H.

[0011] In another aspect, the present invention provides a polynucleotide encoding the xylose reductase mutant described in the present invention.

[0012] In another aspect, the present invention provides a recombinant vector comprising the polynucleotides described herein.

[0013] In one aspect, the vector described in this invention is pET-28a. The expression vector of this invention may also be replaced with other suitable expression vectors conventionally used in the art.

[0014] In another aspect, the present invention provides a host cell characterized in that it comprises the polynucleotide or the recombinant vector described in the present invention.

[0015] In one aspect, the host cell described in this invention is a fungal cell, a bacterial cell, or a plant cell. Preferably, the host cell is a bacterial cell, and more preferably, the bacterial cell is an *Escherichia coli* cell. It should be noted that the plant cell described in this invention is not intended to protect any specific plant, and this invention does not disclose any method for developing plant cells into a complete plant. The plant cell described in this invention is only used as an engineered cell for expressing the enzyme mutant of this invention.

[0016] In one aspect, the host cell of the present invention is a bacterial cell, preferably, the bacterial cell is an *Escherichia coli* cell. More preferably, the host cell is *Escherichia coli* (…). E. coli BL21(DE3) cells.

[0017] In one aspect, the Escherichia coli cells of the present invention are E. coli BL21(DE3) cells.

[0018] In another aspect, the present invention provides the application of the xylose reductase mutant, polynucleotide, recombinant vector, or host cell described herein in the production of xylitol. In one aspect, xylose is used as a substrate, and crude enzyme solution obtained after induction expression of a recombinant engineered bacterium containing a xylose reductase mutant encoding gene is used as a catalyst to catalyze a reduction reaction to produce xylitol.

[0019] In one aspect, the system for the reduction reaction to produce xylitol also includes a coenzyme cycle; said coenzyme cycle includes a coenzyme, glucose, and glucose dehydrogenase, wherein the coenzyme includes NADP. + And NADPH.

[0020] In one respect, the concentration of the substrate xylose is 50–800 mM.

[0021] In another aspect, the present invention also provides a method for preparing xylitol, using xylose as a substrate, NADPH as a coenzyme, and the xylose reductase mutant as a catalyst to catalyze a reduction reaction to produce xylitol. The cells expressing the xylose reductase mutant can be the host cells described in this invention.

[0022] In one respect, the reaction is carried out in a phosphate buffer solution with a pH of 6 to 9 (preferably, pH 7.5).

[0023] In one respect, the catalytic reaction temperature is controlled at 20-60℃, preferably 20-50℃.

[0024] In one aspect, the concentration of the substrate xylose is 50-800 mM.

[0025] In one aspect, the amount of catalyst used in the reaction system is 20-80 μL, and the substrate concentration is 50 mM.

[0026] In one aspect, the catalyst used in the reaction system is the crude enzyme solution after cell disruption.

[0027] In another aspect, the present invention provides a method for producing the xylose reductase mutant described herein, comprising the following steps: (1) The host cells of the present invention are cultured under suitable conditions for expressing the xylose reductase mutant; and (2) The xylose reductase mutant was recovered.

[0028] In one aspect, the present invention also provides a method for preparing xylitol, the method comprising: mixing the xylose reductase mutant of the present invention or the host cell of the present invention with xylose and NADPH under suitable conditions to generate xylitol.

[0029] Those skilled in the art can routinely confirm the suitable conditions for expressing the xylose reductase mutant and the suitable conditions for generating xylitol as described in this invention.

[0030] In one aspect, the preparation method of crude enzyme solution of xylose reductase mutant is as follows: Engineered bacteria containing the xylose reductase mutant encoding gene are inoculated into liquid LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 12 h. Then, at a 2% (v / v) inoculation rate, they are transferred to fresh liquid LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking until the bacterial cell concentration (OD600) reaches 0.4-0.8. IPTG is then added to the medium to a final concentration of 0.5 mM, and the culture is induced at 18°C ​​for 16 h. The fermentation broth is centrifuged at 4000 rpm for 10 min, and the bacterial cells are collected. The washed bacterial cells are resuspended in 100 mM pH 7.5 phosphate buffer, and lysozyme solution to a final concentration of 1.5 g / L is added to disrupt the cells and obtain the crude enzyme solution of xylose reductase mutant.

[0031] In this invention, the source is Gaeumannomyces tritici R3-111a-1 The base sequence of xylose reductase has been codon-optimized for the expression host Escherichia coli, resulting in the nucleotide sequence shown in SEQ ID NO: 1, whose encoded amino acid sequence is shown in SEQ ID NO: 2.

[0032] SEQ ID NO: 1: ATGGCGGCAATCCCGAACGTTAAACTGAACTCTGGTAAAGAAATGCCGCTGGTTGGTTTCGGTCTGTGGAAAGTTCCGAACGATGTTTGCGCGGATGTTGTTTACAACGCGATTAAAGTTGGTTACCGTCTGTTCGATGGTGCGTGCGATTACGGTAACGAAGTTGAATGCGGTCAGGGTGTTGCGCGTGCGATTAAAGAAGGTATCGTTAAACGTGAAGATCTGTTCATCGTTTCTAAACTGTGGAACACCTTCCACGATCACGATCGTGTTGAACCGATCGTTCGTCGTGGTCTGGCGGATTGGGGTATCGATTACTTCGATCTGTACCTGATCCACTTCCCGGTTGCGCTGGAATACGTTGATCCGGCGGTTCGTTACCCGCCGGGTTGGCACTACGATGCGGAAGGTAAAGAAATGCGTCGTTCTAAAGCGACCATCCAGGATACCTGGACCGCGATGGAAAAACTGGTTGATGCGGGTCTGGCGCGTTCTATCGGTGTTTCTAACTTCCAGTCTCAGCTGCTGTACGATCTGCTGCGTTACGCGCGTATCCCGCCGGCGACCCTGCAGGTTGAAATGCACCCGTACAACGTTCAGCAGGATCTGGTTCGTCTGTGCGCGAACGAAGGTATCCAGCTGACCGCGTACTCTTCTCTGGGTCCGGCGTCTTTCGTTGAATTCCACTTCCCGCACGCGGATCGTATGACCCCGCTGCTGCAGCACCCGACCATCCTGGATGTTGCGAAAAAAGTTGGTAAAACCGCGGCGCAGGTTCTGCTGCGTTGGTCTACCCAGCGTGGTATCGCGGTTATCCCGAAAACCACCTCTGAAGAACAGCAGCGTCAGAACCTGGATATCCTGGCGTGGGATATCGCGGCGGATGATCTGGCGAAAATCACCGCGCTGGATCTGAAAACCCGTTTCAACGAACCGGTTAACTACTTCCCGGGTTCTACCCTGTACATCTTCGGTTAA SEQ ID NO: 2: MAAIPNVKLNSGKEMPLVGFGLWKVPNDVCADVVYNAIKVGYRLFDGACDYGNEVECGQGVARAIKEGIVKREDLFIVSKLWNTFHDHDRVEPIVRRGLADWGIDYFDLYLIHFPVALEYVDPAVRYPPGWHYDAEGKEMRRSKATIQDTWTAMEKLVDAGLA RSIGVSNFQSQLLYDLLRYARIPPATLQVEMHPYNVQQDLVRLCANEGIQLTAYSSLGPASFVEFHFPHADRMTPLLQHPTILDVAKKVGKTAAQVLLRWSTQRGIAVIPKTTSEEQQRQNLDILAWDIAADDLAKITALDLKTRFNEPVNYFPGSTLYIFG* The beneficial effects of the present invention include, but are not limited to, the following: This invention uses a semi-rational design method to Gaeumannomyces tritici R3-111a-1 The xylose reductase XR31 was modified to obtain a novel xylose reductase mutant with excellent thermostability. This mutant can catalyze the reduction reaction of xylose to xylitol using xylose as a substrate and NADPH as a coenzyme. This invention enhances hydrophobic interactions and improves thermostability by mutating amino acids near the substrate-binding channel of the xylose reductase, and also improves the enzyme-substrate binding affinity. The xylose reductase mutant provided by this invention can be applied to the bioconversion of xylitol using mixed sugar substrates and hemicellulose hydrolysate as raw materials. Its excellent thermostability can improve the conversion efficiency and product yield in high-temperature processes for xylitol bioconversion, reduce production costs, and is of great significance for promoting the green manufacturing of xylitol. Detailed Implementation

[0033] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0034] Reagents used in upstream genetic engineering operations: The restriction endonucleases, Primer STAR DNA polymerase, DNA ligase, and recombinase used in the embodiments of this invention were all purchased from TaKaRa; the genome extraction kit, plasmid extraction kit, and DNA recovery and purification kit were purchased from Axygen. E. coliBL21 (DE3) and plasmids were purchased from Novagen; DNA markers, low molecular weight standard proteins, and agarose gel electrophoresis reagents were purchased from Beijing TransGen Biotech Co., Ltd.; primer synthesis and gene sequencing were performed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. Please refer to the product instructions for the usage of the above reagents.

[0035] Example 1: Construction of wild-type enzyme engineered bacteria The National Coalition Building Institute (NCBI) database was searched for keywords such as D-xylosereductase, and the amino acid sequence encoding xylose reductase (Sequence ID: XP_009224392.1) was selected. Based on the codon preference of E. coli, the amino acid sequence was converted into a nucleotide sequence (i.e., SEQ ID NO:1), and the gene was synthesized and integrated into the multiple cloning site of the expression vector pET-28a. No I and Not Between I and II. Finally, the constructed plasmid was introduced into E. coli BL21(DE3) to construct an engineered strain of wild-type xylose reductase.

[0036] Example 2: Construction of mutant enzyme I. Activation of engineered bacteria and plasmid extraction All engineered bacteria (obtained in Example 1) were activated and cultured using LB medium with the following formulation: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, dissolved in deionized water and brought to a final volume. The culture was then sterilized at 121°C for 20 min and set aside for use. The solid culture medium was LB medium with 2% agar added.

[0037] The preserved engineered bacterial glycerol tubes were inoculated into test tubes containing 10 mL of LB medium and cultured at 37°C and 220 rpm for 12 h. After obtaining the cultured bacterial cells, plasmids were extracted according to the instructions of the Axygen plasmid extraction kit. The obtained plasmids can be used directly for point mutagenesis or stored long-term at -20°C.

[0038] II. Site-directed gene mutation Gene mutations were obtained using whole-plasmid PCR. Single-site site-directed mutagenesis primers were designed using the online tool PrimerX (www.bioinformatics.org / primerx / cgi-bin / protein_3.cgi), and usage instructions can be found on the website.

[0039] PCR amplification system: DNA polymerase 25 μL 1 μL of upstream primer 1 μL of downstream primer 1 μL of plasmid template ddH2O2 2 μL.

[0040] PCR amplification conditions: 1) Pre-denaturation: 98℃ for 5 min; 2) Denaturation: 98℃ for 30 s; Annealing: 60℃ for 30 s; Extension: 72℃ for 90 s; 30 cycles in total; 3) Post-extension: 72℃ for 10 min; 4) Store at 4℃.

[0041] After PCR amplification, the amplification products were detected by 1% agarose gel electrophoresis. The results showed that the amplification products were single bands, each approximately 6000 bp in size. The amplification products were then purified and recovered using a DNA purification kit; the specific steps were described in the kit's instructions.

[0042] III. Construction of mutant engineered bacteria Use the purified gene fragment Dpn The wild-type pET28a plasmid template was removed by digestion with an I restriction enzyme (Takara, Code No. 1235A). Then, the digested linear plasmid was recombined with the amplification product using a recombinase to obtain a circular plasmid. The recombinant product (i.e., the circular plasmid) was transformed into... E. coli BL21(DE3) competent cells were plated, and single colonies were picked and cultured in 96-well plates containing LB medium. Enzyme activity and thermostability were tested for unknown mutations. Positive mutants were cultured in test tubes containing 10 mL of LB medium to further verify whether thermostability was improved. Mutants with improved thermostability were sequenced for verification. After confirmation, sterile glycerol was added to a final concentration of 25%, and the cells were numbered and stored at -80℃ for later use.

[0043] IV. Construction of multi-point mutant engineered bacteria The plasmids of the best positive mutants from each round were extracted as templates. Based on the Iterative Saturation Mutation (ISM) strategy, saturation mutant libraries of new key amino acid sites were constructed, plated, and cultured according to the construction protocol of the mutant engineered bacteria in Example 2. Similarly, the screened transformants were re-screened and verified by enzyme activity and thermostability assays, and then sequenced for verification. For mutants that passed verification, sterile glycerol at a final concentration of 25% was added, numbered, and stored at -80℃ for later use.

[0044] Example 3: Cultivation of bacterial cells and preparation of crude enzyme solution and determination of enzyme activity I. Culture of mutant bacteria LB liquid culture medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in deionized water and brought to a final volume, sterilized at 121℃ for 20 min, ready for use.

[0045] Transformation plates containing xylose reductase mutants were inoculated by picking single colonies and transferring them to 0.3 mL of LB liquid medium in 96-well plates containing 50 μg / mL kanamycin. The plates were then incubated at 37°C with shaking for 12 h. Finally, a 2% (v / v) inoculum was transferred to 0.5 mL of fresh LB liquid 96-well plates also containing 50 μg / mL kanamycin and incubated at 37°C with shaking until OD (outlet count) reached. 600 When the concentration reaches approximately 0.6, add IPTG to a final concentration of 0.5 mM, and induce culture at 18°C ​​for 16 h. After culture, centrifuge the culture medium at 4000 rpm for 10 min, discard the supernatant, collect the bacterial cells, and store in an ultra-low temperature freezer at -80°C.

[0046] II. Preparation of crude enzyme solution After the culture was completed, the bacterial cells were collected and washed twice with 100 mM pH 7.5 phosphate buffer. Then, the bacterial cells were resuspended in 100 μL pH 7.5 phosphate buffer and repeatedly frozen and thawed at -80℃ 2-3 times. 100 μL of lysozyme solution with a final concentration of 1.5 g / L was added to resuspend the cells, and the mixture was shaken at 37℃ for 3 h to obtain the crude xylose reductase enzyme solution.

[0047] III. Mutant Enzyme Activity Assay Xylose reductase activity was determined spectrophotometrically at a wavelength of 340 nm, based on the oxidation level of NADPH at 30°C. The enzyme activity of xylose reductase was measured using a standard reaction system containing 200 μL (pH 7.5) of xylose substrate, 200 μM NADPH, 50 mM substrate, 100 mM phosphate buffer, and an appropriate amount of enzyme solution (the volume of crude enzyme solution was adjusted according to enzyme activity, generally 20–80 μL). The enzyme activity of xylose reductase catalyzing xylose was calculated using the following formula. Simultaneously, the conversion rate was calculated based on the amount of xylose consumed.

[0048]

[0049] k1: Slope of the NADPH standard curve k2: Slope of the curve showing the change in absorbance of the reaction sample over time (per minute) V: Total volume of the reaction system v: Volume of added enzyme solution X: Crude enzyme solution dilution factor Enzyme activity is defined as: 1 unit (U) is the amount of enzyme required to consume 1 mmol of NADPH in 1 min under reaction conditions at 30℃. Unit: U / L.

[0050] Method for obtaining the slope (k1) of the NADPH standard curve: Prepare NADPH standard solutions with different concentration gradients, and detect the absorbance at 340 nm using a spectrophotometer. Plot the NADPH standard curve with concentration as the x-axis and absorbance as the y-axis, and obtain the slope through linear fitting.

[0051] Method for obtaining the slope (k2) of the absorbance curve of the reaction sample changing with time (per minute): After the reaction system is prepared, it is placed in a spectrophotometer for detection. Each detection is 1 minute apart, and the corresponding absorbance data is recorded. The reaction progress curve is plotted with reaction time as the x-axis and absorbance value as the y-axis, and the slope is obtained by linear fitting.

[0052] Example 4: XR31 Single-Point Saturation Mutation Experiment The crude enzyme solution of the XR31 mutant was prepared as described in Example 3, incubated at 40℃ for 2 h, and the enzyme activity retention was measured. The highest enzyme activity before static incubation was taken as 100%, and the relative enzyme activity of the enzyme solution after static incubation was calculated. The results are shown in Table 1.

[0053] Table 1 Results of single-point mutagenesis experiment on xylose reductase

[0054] As shown in Table 1, multiple potential amino acid mutation sites can be identified by saturation mutation of candidate amino acids to improve the thermostability of xylose reductase. Among them, the five single-point mutations G21Y, D46E, P128S, V168I, and R205H significantly improved the thermostability of xylose reductase.

[0055] Example 5: Thermal stability experiment Based on the Iterative Saturation Mutation (ISM) strategy, a saturation mutation library of new key amino acid sites was established, plated, and mutant culture was carried out according to the construction scheme of mutant engineered bacteria in Example 2. Crude enzyme solution of mutant was prepared according to Example 3.

[0056] The crude enzyme solution to be tested was placed incubated at 30℃, 40℃, 50℃, and 60℃ for 2 hours, and then the enzyme activity was measured. The enzyme activity detection method was the same as in Example 3. The highest enzyme activity before static incubation was taken as 100%, and the relative enzyme activity and xylose conversion rate of the enzyme solution to be tested under different temperature conditions were calculated. The results (as shown in Table 2) showed that the enzyme activity of wild-type XR31 (SEQ ID NO: 2) decreased to below 60% after incubation at 50℃ for 2 hours, while its optimal mutant G21Y / D46E / P128S / V168I / R205H still retained 81.9% enzyme activity after incubation at 50℃ for 2 hours.

[0057] Table 2. Retention of xylose reductase and mutant enzyme activities after incubation at different temperatures for 2 h.

[0058] Table 3. Conversion rates of xylose reductase and mutants after 2 h of incubation.

[0059] Experimental results showed that among the various xylose reductase mutants, the thermostability of 5 single-point mutants and 9 combined mutants was significantly improved, as shown in Table 3. Compared with the wild type (SEQ ID NO: 2), the xylose reductase mutants maintained considerable activity after 2 h of incubation, and the conversion rate of the catalytic reaction was increased by 2 to 5 times compared with the original strain.

[0060] Example 6: Catalytic production of xylitol from wild-type XR31 and mutants under high-temperature conditions Wild-type XR31 (SEQ ID NO: 2) and the crude enzyme solution of the mutant were cultured as described in Example 3. In a 1 mL reaction system, 0.5 mL of 30 g / L xylose dissolved in phosphate buffer (100 mM, pH 7.5), along with 0.5 mM GDH and 0.1 M glucose, were added. The mixture was incubated at 50°C for 6 h, and then boiled for 10 min to terminate the enzyme reaction. The xylose content in the reaction system was determined using a BOXBIO xylose assay kit. The procedure was performed according to the kit instructions.

[0061] After 6 hours of reaction, the conversion rate of xylitol produced by the crude enzyme solution of wild-type XR31 was 28.4%, while the conversion rates of the crude enzyme solutions of mutants were all improved. Among them, the mutant G21Y / D46E / P128S / V168I / R205H had the highest conversion rate of 90.3%, which was nearly 2 times higher than that of wild-type XR31. This indicates that at higher temperatures, the thermostability of mutants, especially mutant G21Y / D46E / P128S / V168I / R205H, is significantly higher than that of wild-type XR31.

[0062] The results above demonstrate that a mutant enzyme with significantly improved thermostability was successfully obtained through targeted molecular modification of XR31 xylose reductase (SEQ ID NO: 2). This mutant enzyme can efficiently catalyze the reduction reaction to xylitol using xylose as a substrate and NADPH as a coenzyme. Compared with the wild-type enzyme, the mutant enzyme exhibits significantly enhanced catalytic activity towards xylose under high-temperature conditions. This characteristic not only improves the conversion efficiency and product yield in the high-temperature process for industrial xylitol production but also reduces production costs, which is of great significance for promoting the green manufacturing of xylitol. This invention provides a new, efficient, economical, and environmentally friendly biocatalytic production route for xylitol suitable for industrial applications, with broad application prospects.

Claims

1. A xylose reductase mutant, characterized in that, The mutant has 5 or fewer substitutions relative to SEQ ID NO: 2 and is similar to SEQ ID NO:

2. Having less than 100% sequence identity, wherein the substitution is selected from: G21P, G21S, G21C, G21Y, G21K, G21E, D46P, D46G, D46S, D46T, D46E, P128I, P128W, P128S, P128T, P128R, P128E, V168A, V168L, V168I, V168W, V168P, V168S, V168T, V168Y, V168N, V168H, V168R, V168E, R205L, R205F, R205P, R205C, R205N, R205Q, R205H and R205D, or combinations thereof.

2. The xylose reductase mutant according to claim 1, characterized in that, The substituted material is selected from G21Y, D46E, P128S, V168I and R205H, or combinations thereof.

3. A xylose reductase mutant, characterized in that, The mutant was obtained by performing any of the following mutations on SEQ ID NO: 2: G21Y; D46E; P128S; V168I; R205H; G21Y+D46E; G21Y+P128S; D46E+V168I; G21Y+D46E+P128S; G21Y+V168I+R205H; D46E+V168I+R205H; G21Y+D46E+P128S+V168I; G21Y+P128S+V168I+R205H; G21Y+D46E+P128S+V168I+R205H; G21P; G21S; G21C; G21K; G21E; D46P; D46G; D46S; D46T; P128I; P128W; P128T; P128R; P128E; V168A; V168L; V168I; V168W; V168P; V168S; V168T; V168Y; V168N; V168H; V168R; V168E; R205L; R205F; R205P; R205C; R205N; R205Q; R205H; or R205D.

4. A polynucleotide, characterized in that, Encode the xylose reductase mutant according to any one of claims 1-3.

5. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, It contains the polynucleotide according to claim 4 or the recombinant vector according to claim 5.

7. The host cell according to claim 6, characterized in that, The host cell is a fungal cell, a bacterial cell, or a plant cell. Preferably, the host cell is a bacterial cell, and more preferably, the bacterial cell is an Escherichia coli cell.

8. The use of the xylose reductase mutant according to any one of claims 1-3, the polynucleotide according to claim 4, the recombinant vector according to claim 5, or the host cell according to any one of claims 6-8 in the production of xylitol.

9. A method for producing a xylose reductase mutant according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Culture host cells according to any one of claims 6-8 under conditions suitable for expressing the xylose reductase mutant; and (2) The xylose reductase mutant was recovered.

10. A method for preparing xylitol, characterized in that, The method comprises: mixing xylose reductase mutant according to any one of claims 1-3 or host cell according to claim 6 or 7 with xylose and NADPH under suitable conditions to generate xylitol.

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