L-hydroxyproline racemase mutants and their use in the synthesis of d-hydroxyproline

Abstract

This invention discloses an L-hydroxyproline racemic enzyme mutant and its application in the synthesis of D-hydroxyproline, belonging to the field of bioengineering technology. The L-hydroxyproline racemic enzyme mutant of this invention is obtained by mutating the 241st site of the wild-type L-hydroxyproline racemic enzyme shown in SEQ ID NO.1. This mutant is unaffected by product inhibition during the synthesis of D-hydroxyproline. Using the synthesis method provided by this invention, a high D-hydroxyproline conversion rate and extremely high optical purity can be achieved, significantly improving both the yield and quality of the D-hydroxyproline product. Therefore, the L-hydroxyproline racemic enzyme mutant and synthesis method of this invention have significant application value in the synthesis of D-hydroxyproline.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to an L-hydroxyproline racemic enzyme mutant and its application in the synthesis of D-hydroxyproline. Background Technology

[0002] cis-D-hydroxyproline, also known as cis-4-hydroxy-D-proline or 4-hydroxyprolylacetic acid, is a non-natural amino acid with the molecular formula C5H9NO3. It is a hydroxylated derivative of proline and possesses a unique cis structure. This compound is widely used in the pharmaceutical, cosmetic, and food industries as a key component in collagen synthesis, contributing to skin repair and anti-aging. Furthermore, it is used in biochemical research as an important intermediate in chiral synthesis and enzymatic reactions. Its high purity and stability make it an ideal choice for high-quality raw materials.

[0003] Currently, the main synthesis methods are chemical synthesis and biosynthesis. Chemical synthesis methods include: (1) dissolving Boc-cis-D-hydroxyproline in ethanol, reacting it with lithium hydroxide, and obtaining cis-D-hydroxyproline after extraction, concentration, recrystallization, and other steps; (2) dissolving cis-D-hydroxyproline ethyl ester in ethanol, carrying out a hydrogenation reaction under the action of a catalyst, and then obtaining cis-D-hydroxyproline after acidification with acetic acid and purification with resin; (3) dissolving trans-4-hydroxyproline in acetic acid and acetic anhydride, refluxing to remove the solvent, then redissolving it in hydrogen chloride solution, refluxing to remove the solvent again, dissolving the product in ethanol and TEA, heating and adding water to obtain a dark transparent solution, and finally separating and purifying to obtain cis-D-hydroxyproline. The biosynthesis method is to use L-hydroxyproline racemic enzyme (ProR) to catalyze the isomerization of trans-L-hydroxyproline to cis-4-hydroxy-D-proline.

[0004] Although all of the above methods can synthesize cis-D-hydroxyproline, chemical synthesis methods (1) and (2) require high raw material costs and have complicated or dangerous reaction processes, which are not conducive to industrial production. Chemical synthesis method (3) obtains the target product cis-D-hydroxyproline through epimerization catalyzed by acetic acid and acetic anhydride. Although the overall operation steps are simple, the overall cycle is long and the production efficiency is low. Biological methods are limited in industrial application due to the narrow substrate spectrum of wild-type enzymes, susceptibility to product inhibition, and poor operational stability.

[0005] Therefore, there is a need in this field to develop a simple, convenient, safe, low-cost, and highly efficient method for synthesizing cis-D-hydroxyproline.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an L-hydroxyproline racemic enzyme mutant and its application in the synthesis of D-hydroxyproline. This L-hydroxyproline racemic enzyme mutant is not inhibited by the product in the synthesis of D-hydroxyproline. By using the synthesis method provided by this invention, a high yield and extremely high optical purity can be obtained.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides an L-hydroxyproline racemic enzyme mutant, which is obtained by mutating the 241st site based on the wild-type L-hydroxyproline racemic enzyme shown in SEQ ID NO.1.

[0009] Among them, wild-type L-hydroxyproline racemic enzyme is derived from Thermococcus litoralis While DSM 547 can catalyze the isomerization of trans-L-hydroxyproline to cis-4-hydroxy-D-proline, it is easily inhibited by the product, which is detrimental to industrial production. To provide a highly efficient, green, and scalable biocatalytic process, this invention starts from the wild-type L-hydroxyproline racemic enzyme gene and obtains the L-hydroxyproline racemic enzyme mutant ProR through site-directed mutagenesis targeting substrates with different spatial positions (ortho, meta, and para). It was found that only mutation at position 241 can improve the aforementioned problems.

[0010] In some embodiments, the mutation at position 241 of the L-hydroxyproline racemic enzyme mutant is as follows: tryptophan is mutated to phenylalanine, i.e., W241F. Correspondingly, the amino acid sequence of this mutant is shown in SEQ ID NO.2.

[0011] Secondly, the present invention provides biomaterials related to L-hydroxyproline racemic enzyme mutants, which are any one of the following: (1) The nucleic acid molecule encoding the above-mentioned L-hydroxyproline racemic enzyme mutant; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant expression vector containing the expression cassette described in (2); (4) A recombinant bacterium containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant expression vector described in (3).

[0012] In specific implementation schemes, the aforementioned nucleic acid molecules can be optimized according to the codon preferences of the host cell before being used to artificially synthesize gene fragments. It should be understood that all nucleic acid molecules capable of being translated into the aforementioned amino acid sequences are within the scope of protection of this invention.

[0013] In some embodiments, the nucleotide sequence of the L-hydroxyproline racemase mutant shown in SEQ ID NO.2 is shown in SEQ ID NO.4. This nucleotide sequence is based on the parental L-hydroxyproline racemase gene shown in SEQ ID NO:3, with the codon encoding tryptophan at position 281 mutated to the codon encoding phenylalanine.

[0014] In a specific implementation plan, the host cell can be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, and Bacillus licheniformis; eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells. Preferably, the host cell is Escherichia coli.

[0015] In some embodiments, the method for constructing the recombinant bacteria includes: linking the nucleic acid molecules to an expression vector, and then introducing the recombinant expression vector into the starting strain.

[0016] Thirdly, the present invention provides a method for preparing the above-mentioned L-hydroxyproline racemic enzyme mutant, the method comprising: inoculating the above-mentioned recombinant bacteria into a fermentation medium and culturing them to obtain the L-hydroxyproline racemic enzyme mutant.

[0017] In a specific implementation plan, preparing L-hydroxyproline racemic enzyme mutants using host cells refers to fermenting and culturing host cells, and the culture medium and culture conditions are well known to those skilled in the art.

[0018] Fourthly, the present invention provides the use of the above-mentioned L-hydroxyproline racemic enzyme mutant or biological material in any of the following: (1) Synthesis of D-hydroxyproline; (2) Prepare products for the synthesis of D-hydroxyproline.

[0019] Fifthly, the present invention provides a biocatalyst comprising the aforementioned L-hydroxyproline racemic enzyme mutant or recombinant bacteria. In practical applications, the biocatalyst can be the enzyme solution collected after the recombinant bacteria are broken down; or it can be the recombinant bacteria themselves, which, when added to the reaction system as a whole-cell catalyst, can also catalyze the conversion of L-hydroxyproline to D-hydroxyproline.

[0020] Sixthly, the present invention also provides a method for synthesizing optically pure D-hydroxyproline, which uses L-hydroxyproline as a substrate, adds an enzyme solution or wet bacterial cells containing the above-mentioned L-hydroxyproline racemic enzyme mutant to the reaction system, and catalyzes the synthesis of D-hydroxyproline, as shown in the synthetic route. Figure 1 As shown.

[0021] To achieve the best synthesis results, this invention also optimized the catalyst morphology and dosage, substrate concentration, pH, and temperature conditions during the synthesis reaction process.

[0022] In some embodiments, the mass ratio of wet bacterial cells to L-hydroxyproline in the reaction system is 1~5:20, and more preferably, the mass ratio of wet bacterial cells to L-hydroxyproline is 1:5. Analysis of the conversion rate after different reaction times revealed that when the bacterial cell mass accounts for 5%~25% of the substrate mass, the conversion rate increases with increasing enzyme dosage. When the bacterial cell mass accounts for 20% of the substrate mass, the conversion rate approaches its maximum value. Balancing catalytic efficiency and catalyst cost, 20% is the optimal dosage.

[0023] In some embodiments, the concentration of L-hydroxyproline in the reaction system is 25~100 g / L. A concentration of 100 g / L represents the optimal choice for both production efficiency and cost. In some embodiments, the reaction temperature is 20–40 °C. Analysis of the conversion rate after different reaction times revealed that temperature has a significant impact on the efficiency of this biocatalytic reaction, with 25–35 °C being the optimal temperature range for the reaction.

[0024] In some embodiments, sodium hydroxide solution is added during the reaction to maintain the pH of the reaction system at 8.0. Adjusting the pH during the reaction process improves reaction efficiency.

[0025] In some embodiments, the synthesis method further includes: after the reaction is completed, filtering the reaction product, collecting the filtrate and concentrating and crystallizing it to obtain crude D-hydroxyproline wet product; then recrystallizing the crude wet product, filtering and drying it again to obtain optically pure D-hydroxyproline.

[0026] Specifically, when the conversion rate reaches 45%~50%, the reaction is terminated; the reaction product is filtered, the filtrate is collected, and then concentrated under reduced pressure. The pH is adjusted to acidic with hydrochloric acid, and crystallization is carried out at room temperature. The collected crystals are the wet crude D-hydroxyproline; the wet crude product is then placed in a methanol / ethyl acetate mixed solvent, stirred at high temperature until completely dissolved, cooled to room temperature, recrystallized, filtered, and dried to obtain pure D-hydroxyproline.

[0027] In some embodiments, the volume ratio of methanol to ethyl acetate in the recrystallization mixed solvent is 1:1, and the temperature during stirring is approximately 50°C.

[0028] Through the above synthesis method, the present invention can improve the product inhibition problem that is currently prevalent, and can efficiently prepare optically pure D-hydroxyproline with extremely high purity, up to 99.96%.

[0029] The present invention has the following beneficial effects: This invention provides an L-hydroxyproline racemic enzyme mutant obtained through site-directed mutagenesis. This mutant is unaffected by product inhibition during the synthesis of D-hydroxyproline. Using the synthesis method provided by this invention, a high D-hydroxyproline conversion rate and extremely high optical purity can be achieved, significantly improving both the yield and quality of the D-hydroxyproline product. Therefore, the L-hydroxyproline racemic enzyme mutant and synthesis method of this invention have significant application value in the synthesis of D-hydroxyproline. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is the synthetic route for D-hydroxyproline in this invention; Figure 2 The results are chromatographic findings of D-hydroxyproline in Experiment Example 5. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0033] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0034] The amino acid sequence (SEQ ID NO:1) of the wild-type L-hydroxyproline racemase in this invention is as follows: MFADHVFHVVDTHTEGEPTRIVLSGVNVKGEDIIEKREYFKEHYDWIRTALLHEPRGHSDQFGAVLVPSDIADFGVIYMDTSGYLDMCGHATMGVATVLVELGIIEKKEPYTTVKLETPAGLVEAKAKVKGGVVKEVTVVDVPSFYVGEFVIEYPGRGKIKVDVAF GGNFYVIADARDLGLRVRREYIKELIPTALKLIKVANEQIKVQHPRKGVQNRINLAMLTDEPEREDSDGKNVVIWGEGSVDRSPCCTGSASRVATLYSKGILKEGDIFVHESILGTQFRIKIVGTTKIGEYTAIIPEITGSAYITKISQDIISKNDPLWKGFLLR.

[0035] Example 1 This example demonstrates the design and gene construction of an L-hydroxyproline racemic enzyme mutant, as detailed below: The amino acid sequence of the L-hydroxyproline racemase (T1ProR) gene was sent to Sangon Biotech (Shanghai) Co., Ltd. for codon optimization and synthesis in *E. coli*, and the gene was constructed into the pETDuet-1 vector. A plasmid containing the L-hydroxyproline racemase (T1ProR) gene was synthesized and named pETDuet-1-T1ProR. The plasmid pETDuet-1-T1ProR was transformed into *E. coli* competent cells BL21(DE3) for induced expression, obtaining the recombinant engineered strain pETDuet-1-T1ProR. E. coli BL21(DE3).

[0036] The mutant gene TlProR was obtained by single-point mutagenesis of wild-type L-hydroxyproline racemase ProR using a site-directed mutagenesis kit from Sangon Biotech (Shanghai) Co., Ltd. W241F (TGG-TTC) Using the wild-type hydroxyproline racemase gene plasmid pETDuet-1-ProR as a template, the following mutant primers were used for full plasmid PCR to obtain the mutant TlProR. W241F The mutant primers are as follows: W241F-F:GAGAGCATATTTCGAACTCAATT- SEQ ID NO:5; W241F-R:AATTGAGTTCGAAATATGCTCTC-SEQ ID NO:6.

[0037] PCR reaction system: 50 μL of reaction mixture containing 1 U KOD FX DNA polymerase (TOYOBO), appropriate primers (15 pmol), and template DNA was subjected to 30 cycles under the following conditions: 98℃ denaturation for 10 seconds, 50℃ annealing for 30 seconds, and 68℃ extension for a period of time, with extension time increments of 1 kbp. min 1 Calculation of elongation.

[0038] After the PCR reaction, 1 µL of DpnI was added to digest the amplification product. Each reaction mixture was gently and thoroughly mixed and incubated at 37°C for 1 h to digest the parental template. 10 µL of the digestion product was then transferred to competent *E. coli* cells for transformation. Single clones were picked and inoculated into 5 mL of LB broth containing antibiotics, and the culture was sequenced. The plasmid that was successfully sequenced was named pETDuet-1-ProR. The mutant plasmid was then transformed into competent *E. coli* cells BL21(DE3) to induce expression. The transformation method was the same as the induction method, yielding the mutant hydroxyproline racemase ProR.

[0039] Example 2 This example describes the construction of the recombinant engineered bacterium pETDuet-1-ProR-BL21(DE3) and the preparation of the L-hydroxyproline racemic enzyme mutant, as detailed below: 1. Construction of recombinant bacteria Before the experiment, the water bath was preheated to 45℃. 50 μL of competent E. coli BL21(DE3) cells were placed on ice to thaw. 2 μL of pETDuet-1-ProR plasmid was added and gently mixed. The mixture was then placed on ice for 30 min. The centrifuge tube containing the plasmid was placed in a 45℃ water bath for 30 s heat shock and then quickly returned to ice for 2 min. 450 μL of LB medium was added to the centrifuge tube in a clean bench and cultured at 37℃ and 250 r / min for 1 h. 100 μL of the transformation solution was evenly spread onto LB solid medium containing 50 μg / mL ampicillin resistance and inverted in a 37℃ incubator for 12-16 h. Single clones were picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL ampicillin resistance. The medium was shaken at 37℃ and 250 r / min for 14-16 h to obtain recombinant engineered bacteria pETDuet-1-ProR-BL21(DE3), which was then preserved in glycerol tubes.

[0040] 2. Preparation of L-hydroxyproline racemic enzyme mutant BL21(DE3) strain containing the pETDuet-1-ProR mutant plasmid was cultured at 37°C to OD0.05 in Super broth medium containing 50 mg / L ampicillin (pH 7.0, 12 g tryptone, 24 g yeast extract, 5 mL glycerol, 3.81 g / L KH2PO4, 12.5 g / L K2HPO4). 600 nm When the pH was 0.6, 1 mM IPTG was added and the cells were cultured for 6 h. The cells were then harvested by centrifugation at 30,000 rpm for 20 min for later use.

[0041] Example 3 This embodiment describes a method for preparing cis-D-hydroxyproline, as detailed below: (1) Prepare a substrate solution by dissolving 5 g of L-hydroxyproline substrate in 95 mL of 50 mM Tris-HCl buffer (pH 8.0) at a substrate concentration of 5%.

[0042] (2) Take 1 g of wet bacterial cells (equivalent to 20% of the substrate mass), resuspend them in 8 mL of suspension buffer (50 mM sodium phosphate buffer, pH 8.0, containing 300 mM NaCl and 10 mM imidazole), and sonicate them in an ice bath for 20 min to obtain a suspension of broken cells.

[0043] (3) The above-mentioned broken cell suspension was added to the substrate solution and mixed to form a reaction mixture. Catalytic conversion was carried out at 30°C. During the reaction, the pH of the system was maintained at 8.0 by adding 2 M NaOH solution. The reaction was terminated when the conversion rate reached 45%-50%.

[0044] (4) After the reaction is complete, the entire reaction mixture is filtered to remove solid residues and the clear conversion liquid is collected. The filtrate is concentrated under reduced pressure, and the pH is adjusted to 6.0 with hydrochloric acid, followed by crystallization at 25°C. The crystals are collected by filtration to obtain crude wet D-hydroxyproline.

[0045] (5) Place the wet crude product in a methanol / ethyl acetate (volume ratio 1:1) mixed solvent, stir at 50°C until completely dissolved, then cool to 20-25°C for recrystallization, filter and dry to obtain pure D-hydroxyproline.

[0046] Recombinant bacteria were constructed using wild-type L-hydroxyproline racemic enzyme, and cis-D-hydroxyproline was prepared under the same conditions. After 24 hours of reaction, the conversion rate was only 17.6%.

[0047] Experimental Example 1 To compare the effects of different catalyst forms on the conversion efficiency, this experiment used the preparation method of Example 3, except that the lysed enzyme solution was replaced with undisturbed recombinant cells to verify the difference in conversion rate between whole cells and enzyme solution. The results are shown in Table 1: Table 1. Conversion rates of whole cells and enzyme solutions

[0048] The experimental results above show that, compared with different catalyst forms, under the same reaction conditions, the catalytic efficiency of the disrupted cell catalyst is significantly higher than that of the whole cell catalyst. At each monitoring time point from 4 to 24 h, the conversion rate of the disrupted cell group was higher than that of the whole cell group. Its conversion rate after 24 h (46.8%) was close to the theoretical equilibrium endpoint and significantly better than that of the whole cell group (33.4%). This indicates that cell disruption effectively allows for sufficient contact between the enzyme and the substrate, thereby significantly improving the catalytic rate and the final conversion depth. Therefore, the disrupted cell catalyst system will be selected for subsequent process optimization.

[0049] Experiment Example 2 To compare the effects of different temperature conditions on the conversion efficiency, this experiment used the preparation method of Example 3, only varying the reaction temperature, to verify the differences in conversion rates at different temperatures. The results are shown in Table 2: Table 2 Conversion rates at different temperatures

[0050] Experiments showed that temperature significantly affects the efficiency of this biocatalytic reaction, with 25-35℃ being the optimal temperature range. Within this range, 35℃ exhibited the best overall catalytic performance, with the highest conversion rates (34.3% and 48.3%, respectively) in the later stages of the reaction (12 h and 24 h), making it the recommended temperature for achieving the maximum final conversion depth.

[0051] When the temperature is below 25℃ or above 40℃, the conversion rate at each time point decreases significantly, indicating that enzyme activity is inhibited outside this range. In particular, at 45℃, the conversion rate drops sharply to 33.2% after 24 hours, confirming that higher temperatures may lead to irreversible inactivation of the enzyme protein.

[0052] Therefore, taking into account the reaction rate, final conversion rate and operational stability, 35℃ was selected as the optimal reaction temperature.

[0053] Experimental Example 3 To compare the effect of different enzyme amounts on the conversion efficiency, this experiment used the preparation method of Example 3, only varying the amount of enzyme added, to verify the difference in conversion rate under different enzyme amounts. The results are shown in Table 3: Table 3 Conversion rates with different enzyme amounts

[0054] Within the test range of 5% to 25%, the conversion rate at each time point (8 h, 12 h, 24 h) increased monotonically with the increase of enzyme dosage. When the enzyme dosage was less than 15%, the reaction rate and the final conversion rate were significantly lower. When the enzyme dosage exceeded 15%, the increase in conversion rate slowed down significantly. The final conversion rate continued to increase with the increase of enzyme dosage, but the efficiency improvement tended to level off between 20% and 25%.

[0055] With 20% enzyme dosage, the conversion rate (46.2%) was close to the maximum value after 24 hours, and the reaction rate in the early stage was not much different from that with higher enzyme dosage. Therefore, in order to balance catalytic efficiency and catalyst cost, 20% enzyme dosage is the optimal dosage. This condition can ensure a high final conversion rate (46.2%) while making more economical use of the catalyst. Furthermore, enzyme can be added during the conversion process to promote the reaction based on the actual conversion situation.

[0056] Experiment Example 4 To compare the effect of different substrate concentrations on the conversion efficiency, this experiment used the preparation method of Example 3, only varying the substrate concentration, to verify the difference in conversion rate at different substrate concentrations. The results are shown in Table 4: Table 4. Conversion rates at different substrate concentrations

[0057] Comparing results from different substrate concentrations, increasing the substrate concentration to 10% (i.e., 100 g / L) resulted in a slight decrease in the 24-hour conversion rate (47.6%) compared to the optimal range, but significantly improved the production efficiency (yield per unit volume) of the reaction system. Considering production efficiency and cost, 100 g / L is a suitable maximum concentration choice that maximizes production output at an acceptable conversion level.

[0058] When the concentration exceeded 10%, the conversion rate decreased at all time points, indicating that the substrate inhibition effect became significant, resulting in a significant decrease in reaction rate and final conversion rate, and an increase in production cost. Even with an excess of enzyme (compared to 20% of the optimal enzyme amount), the conversion progress did not change significantly as the reaction time increased to 30 h.

[0059] Experimental Example 5 This experiment is a scaled-up experiment based on optimized conditions, as detailed below: To prepare a substrate solution, dissolve 100 g of L-hydroxyproline substrate in 900 mL of 50 mM Tris-HCl buffer (pH 8.0) according to a 1 L reaction system and a substrate concentration of 10%. Take 20 g of wet bacterial cells (equivalent to 20% of the substrate mass), resuspend them in 80 mL of suspension buffer (50 mM sodium phosphate buffer, pH 8.0, containing 300 mM NaCl and 10 mM imidazole), and sonicate on ice for 20 min to obtain a lysed cell suspension.

[0060] The above-mentioned broken cell suspension was added to the substrate solution, mixed to form a reaction mixture, and then subjected to catalytic conversion at 30°C. During the reaction, the pH of the system was maintained at 8.0 by adding 2 M NaOH solution. The reaction was terminated when the conversion rate reached 45%-50%.

[0061] After the reaction was complete, the entire reaction mixture was filtered to remove solid residue, and the clear conversion liquid was collected. The filtrate was concentrated under reduced pressure, and the pH was adjusted to 6.0 with hydrochloric acid, followed by crystallization at 25°C. The crystals were collected by filtration to obtain crude wet D-hydroxyproline.

[0062] The wet crude product was added to a methanol / ethyl acetate mixture (volume ratio 1:1) at a mass-to-volume ratio of 1:0.5 (g / mL) and stirred at 50°C until completely dissolved. The solution was then cooled to 20-25°C for recrystallization. After crystallization, the mixture was filtered, and the resulting crystals were dried to obtain 43.3 g of pure D-hydroxyproline, with a yield of 86.6% and a chiral purity of 99.96%. The chromatographic results are shown below. Figure 2 As shown.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mutant of L-hydroxyproline racemase characterized in that, The L-hydroxyproline racemic enzyme mutant was obtained by mutating site 241 based on the wild-type L-hydroxyproline racemic enzyme shown in SEQ ID NO.

1.

2. The L-hydroxyproline racemase mutant according to claim 1, characterized in that, The amino acid sequence of the L-hydroxyproline racemic enzyme mutant is shown in SEQ ID NO.

2.

3. The biological material relating to the L-hydroxyproline racemic enzyme mutant of claim 1 or 2, wherein it is any one of the following: (1) A nucleic acid molecule encoding the L-hydroxyproline racemic enzyme mutant as described in claim 1 or 2; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant expression vector containing the expression cassette described in (2); (4) A recombinant bacterium containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant expression vector described in (3).

4. A method for preparing the L-hydroxyproline racemic enzyme mutant according to claim 1 or 2, characterized in that, include: The recombinant bacteria described in claim 3 were inoculated into a fermentation medium and cultured to obtain the L-hydroxyproline racemic enzyme mutant.

5. The use of the L-hydroxyproline racemic enzyme mutant of claim 1 or 2 or the biomaterial of claim 3 in any of the following: (1) Synthesis of D-hydroxyproline; (2) Prepare products for the synthesis of D-hydroxyproline.

6. A biocatalyst, characterized in that, Includes the L-hydroxyproline racemic enzyme mutant as described in claim 1 or 2, or the recombinant bacteria as described in claim 3.

7. A method for synthesizing optically pure D-hydroxyproline, characterized in that, Using L-hydroxyproline as a substrate, an enzyme solution or wet bacterial cells containing the L-hydroxyproline racemic enzyme mutant as described in claim 1 or 2 are added to the reaction system to catalyze the synthesis of D-hydroxyproline.

8. The method according to claim 7, characterized in that, The mass ratio of wet bacterial cells to L-hydroxyproline in the reaction system is 1~5:20; Preferably, the mass ratio of wet bacterial cells to L-hydroxyproline in the reaction system is 1:5; Preferably, the concentration of L-hydroxyproline in the reaction system is 25~100 g / L.

9. The method according to claim 7, characterized in that, The reaction temperature is 20~40℃; Preferably, the reaction temperature is 25~35℃; Preferably, sodium hydroxide solution is added during the reaction to maintain the pH of the reaction system at 8.

0.

10. The method according to claim 9, characterized in that, The method further includes: after the reaction is completed, filtering the reaction product, collecting the filtrate and concentrating and crystallizing it to obtain wet crude D-hydroxyproline; then recrystallizing the wet crude product, filtering and drying it again to obtain optically pure D-hydroxyproline.

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