Hydantoinase mutant, immobilized hydantoinase and application of immobilized hydantoinase in synthesis of pregabalin intermediate
By immobilizing the hein enzyme by site-directed mutation and covalent binding method, the problems of low enzyme activity, poor stability and inability to synthesise pregabalin chiral intermediates by biological enzymes were solved, and efficient and low-cost synthesis of pregabalin intermediates were achieved.
Patent Information
- Application Number
- CN202510754309.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-06
- Publication Date
- 2025-08-29
AI Technical Summary
The existing biological enzyme method for synthesizing pregabalin chiral intermediates has problems such as low enzyme activity, poor stability, non-recyclable, complex reaction system, low product yield and high production cost.
By performing site-directed mutation of the heinase of Bacillus stearothermophilus NS1122A strain, a new heinase mutant was formed, and the covalent binding method was used to immobilize the heinase, improving its catalytic activity and stability, simplifying the post-treatment process, and realizing the reused use of the enzyme.
The efficient catalysis of immobilized heinase was achieved, with the enzyme activity recovery rate greater than 50%, the substrate concentration was 170g/L, the reaction time was 6-12 hours, the immobilized heinase was reused 17 times, the conversion rate was greater than 98%, and the product ee value was greater than 99%, which reduced production costs.
Smart Images
Figure BDA0005438257120000051 
Figure BDA0005438257120000081 
Figure BDA0005438257120000091
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biocatalytic synthesis, and in particular relates to a hydantoinase mutant, an immobilized hydantoinase and applications in the synthesis of a pregabalin intermediate. The pregabalin intermediate is (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid. Background Art
[0002] Pregabalin is a structural analog of the important inhibitory neurotransmitter γ-aminobutyric acid (GABA) and a chiral drug. The pharmacological activity of the S-isomer is 10 times that of the R-isomer. It was developed and produced by Pfizer Inc. in the United States under the trade name Lyrica. In July 2004, the European Union approved its use for the treatment of partial epileptic seizures. Since December 2004, it has been approved by the U.S. FDA for the treatment of neuropathic pain associated with diabetic peripheral neuropathy and postherpetic neuralgia, as an adjunctive treatment for partial epileptic seizures in adults, and for the treatment of fibromyalgia and pain associated with spinal cord injury. It was approved for marketing in China in 2010. Pregabalin has excellent development potential and is one of the world's best-selling analgesics.
[0003] Currently, the synthesis methods of pregabalin mainly include fully chemical synthesis and chemical-enzymatic synthesis. The first-generation manufacturing process of pregabalin uses isovaleraldehyde and diethyl malonate as starting materials. Knoevenagel condensation and Michael addition are used to obtain the key intermediate. Then, hydrolysis, decarboxylation, and reduction are carried out in a single pot to obtain racemic pregabalin. Finally, the chiral resolution agent (S)-(+)-mandelic acid is added. The resolved product undergoes a three-step recrystallization process to obtain S-configuration pregabalin with an ee value of 100%. The total yield of this synthesis process is 25-29%. Although this synthetic route is cost-effective, the other configuration after the late chiral resolution cannot be recycled, resulting in low atomic utilization.
[0004] Compared with the fully chemical synthesis method, the synthesis route of pregabalin involving hydantoinase is short, the atomic utilization rate reaches 100%, and it is green and environmentally friendly. However, the bio-enzymatic method for preparing pregabalin chiral intermediates has problems such as low free enzyme activity, poor stability, non-recyclability, and high protein content in the reaction system, which leads to cumbersome post-reaction processing steps, low product yield, and high production cost.
[0005] In the prior art, patent CN117448309A discloses a method for preparing immobilized hydantoinase and its application in pregabalin intermediates. This application utilizes immobilization technology to immobilize hydantoinase, and uses the immobilized hydantoinase to catalyze the hydrolysis of 3-isobutylglutarimide, a pregabalin intermediate, to (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid. This method increases the substrate concentration to 140 g / L without affecting its catalytic activity. It also increases the maximum number of uses of the immobilized enzyme, reduces the impact on the product purification process, achieves bio-enzyme catalyst recycling, further simplifies the post-processing process, and improves yield. However, when using immobilized hydantoinase as a catalyst to prepare chiral intermediates of the pregabalin drug in this technology, the batch conversion time is long, and hydantoinase with enhanced catalytic activity is required to improve the efficiency of the enzymatic conversion. Summary of the Invention
[0006] Based on the above-mentioned defects and deficiencies in the prior art, the present invention aims to provide a hydantoinase mutant, an immobilized hydantoinase, and its application in the synthesis of pregabalin intermediates. The present invention addresses the problems existing in the hydantoinase-catalyzed synthesis of pregabalin chiral intermediates by modifying the enzyme using molecular engineering techniques to obtain a hydantoinase mutant with enhanced catalytic activity. Another object of the present invention is to immobilize hydantoinase using covalent bonding to improve its stability, facilitate its recovery and reuse, and simplify post-processing, reduce production costs, and improve yield. The present invention provides a method for preparing immobilized hydantoinase. By screening different immobilization carriers and optimizing immobilization conditions, an immobilized hydantoinase with minimal loss of catalytic activity and unchanged stereoselectivity is obtained. The immobilized hydantoinase is then applied to the synthesis of pregabalin chiral intermediates, achieving the maximum number of batches of the immobilized enzyme while ensuring substrate conversion and product stereoselectivity.
[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0008] One of the technical solutions of the present invention provides a hydantoinase mutant. The present invention is a hydantoinase mutant modified based on the natural hydantoinase of the Bacillus stearothermophilus NS1122A strain. The mutant is obtained by site-directed mutagenesis of the hydantoinase of the amino acid sequence shown in SEQ ID No. 2; the mutant is a derivative protein composed of a new amino acid sequence formed by replacing any one or any two amino acids of serine at position 38, glycine at position 66, glutamic acid at position 135, or cysteine at position 432 in the amino acid sequence shown in SEQ ID No. 2. Furthermore, the amino acid sequence of the hydantoinase mutant is selected from any one of the following:
[0009] (1) replacing serine at position 38 in the amino acid sequence shown in SEQ ID No. 2 with valine;
[0010] (2) replacing glycine at position 66 in the amino acid sequence shown in SEQ ID No. 2 with alanine;
[0011] (3) replacing glutamic acid at position 135 in the amino acid sequence shown in SEQ ID No. 2 with aspartic acid;
[0012] (4) replacing cysteine at position 432 in the amino acid sequence shown in SEQ ID No. 2 with valine;
[0013] (5) in the amino acid sequence shown in SEQ ID No. 2, the serine at position 38 was replaced with valine, and the glycine at position 66 was replaced with alanine;
[0014] (6) in the amino acid sequence shown in SEQ ID No. 2, the serine at position 38 is replaced by valine, and the glutamic acid at position 135 is replaced by aspartic acid;
[0015] (7) in the amino acid sequence shown in SEQ ID No. 2, the serine at position 38 was replaced with valine, and the cysteine at position 432 was replaced with valine;
[0016] (8) In the amino acid sequence shown in SEQ ID No. 2, glycine at position 66 is replaced with alanine, and glutamic acid at position 135 is replaced with aspartic acid;
[0017] (9) In the amino acid sequence shown in SEQ ID No. 2, glycine at position 66 was replaced with alanine, and cysteine at position 432 was replaced with valine;
[0018] (10) In the amino acid sequence shown in SEQ ID No. 2, glutamic acid at position 135 is replaced by aspartic acid, and cysteine at position 432 is replaced by valine.
[0019] Furthermore, by genetic engineering means, the gene fragment corresponding to the above amino acid sequence is combined with a cloning vector (preferably a plasmid vector) to obtain a recombinant expression vector (recombinant plasmid), which is transformed into a competent host cell to obtain a genetically engineered bacterium (recombinant expression transformant) that produces hydantoinase. The genetically engineered bacterium is induced to express the target hydantoinase, and the bacterial cells are collected and broken before being combined with an immobilized carrier to prepare an immobilized enzyme, thereby increasing the reuse rate of the enzyme.
[0020] Furthermore, the present invention provides a nucleic acid encoding the hydantoinase mutant, a recombinant expression vector comprising the nucleic acid, a recombinant expression transformant comprising the recombinant expression vector, a hydantoinase mutant catalyst, and an immobilized hydantoinase. The hydantoinase mutant catalyst comprises: (1) a crude enzyme solution obtained by culturing the recombinant expression transformant, isolating and crushing transformant cells containing the hydantoinase mutant, or a pure enzyme solution obtained by purifying and separating the crude enzyme solution; (2) a crude enzyme solution obtained by culturing the recombinant expression transformant, isolating and crushing transformant cells containing the hydantoinase mutant, and freeze-drying the crude enzyme solution to obtain crude enzyme powder.
[0021] Furthermore, the hydantoinase mutant catalyst can be used to prepare immobilized hydantoinase.
[0022] The second technical solution of the present invention provides an immobilized hydantoinase.
[0023] The third technical solution of the present invention provides a method for preparing immobilized hydantoinase. The present invention uses a covalent bonding method to immobilize hydantoinase, which can achieve efficient preparation of immobilized hydantoinase. The specific preparation method is as follows:
[0024] (1) Preparation of wet cells: Recombinant Escherichia coli containing the target gene for hydantoinase was inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C, 200 rpm, and shaken for 10-12 h. The seed solution was transferred to 500 mL of LB medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculum and cultured in a shaker at 37°C, 200 rpm. When the cell concentration in the culture medium was appropriate, IPTG was added to a final concentration of 0.2 mM and cultured at 16°C for 22-24 h. The cells containing hydantoinase were collected by centrifugation at 10,000 rpm for 5 min.
[0025] (2) Preparation of crude enzyme solution: wet bacterial cells were suspended in potassium phosphate buffer at pH 6.0-8.0 to obtain a bacterial suspension, and the cells were broken and centrifuged in an ice bath to obtain a crude hydantoinase enzyme solution.
[0026] (3) Preparation of resin:
[0027] Amino resin activation: Wash an appropriate amount of amino resin three times with potassium phosphate buffer (pH 6.0-8.0), filter dry, add 2% glutaraldehyde solution to the pretreated amino resin, shake at 200 rpm at 15-35°C for 1-10 hours, filter, and wash five times with pH 6.0-8.0 buffer to remove residual glutaraldehyde. Filter dry again to obtain activated amino resin.
[0028] Epoxy resin equilibration: Take an appropriate amount of epoxy resin and wash it three times with potassium phosphate buffer at pH 6.0-8.0, then filter and dry to obtain a well-equilibrated epoxy resin. There is no need to add glutaraldehyde solution to activate the resin surface groups.
[0029] (4) Preparation of immobilized enzyme: Add activated amino resin or equilibrated epoxy resin to the hydantoinase solution at a resin addition amount of 1 g / 10 mL crude enzyme solution; place in a shaker at 15-35°C and oscillate at 200 rpm for 1-10 h, filter, and wash three times with a pH 6.0-8.0 buffer solution, and filter again to obtain immobilized hydantoinase.
[0030] Furthermore, the temperature during the preparation of the immobilized enzyme in step (4) is 15-35°C, preferably 30°C;
[0031] The pH of the KPB buffer is 6.0-8.0, preferably 7.0;
[0032] The protein concentration is 30-90 mg 蛋白 / g 树脂 , preferably 75mg 蛋白 / g 树脂 ;
[0033] The immobilization time is 1-10 hours, preferably 5 hours;
[0034] The resin in the present invention is an amino resin or an epoxy resin. The amino resin is selected from any one of LX1000NH, LXTE703, LXTE704, or XJGD29. The epoxy resin is selected from any one of LX1000HFA, HFA001, LXTE609, or XJGD89. More preferably, the epoxy resin is LX1000HFA.
[0035] A fourth technical solution of the present invention provides the use of the aforementioned hydantoinase mutant and immobilized hydantoinase in pregabalin chiral drug intermediates.
[0036] Furthermore, the hydantoinase catalyzes the synthesis of the chiral intermediate (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid from the precursor 3-isobutylglutarimide, and the reaction formula is as follows:
[0037]
[0038] Specifically, the application is as follows: hydantoinase is used as a catalyst, 3-isobutylglutarimide is used as a substrate, and manganese chloride tetrahydrate is used as an enzyme activator; hydrolysis is carried out in a 100 mM sodium carbonate-sodium bicarbonate buffer solution with a pH of 8.0-8.5; the total reaction volume is 50 mL, the reaction temperature is controlled at 40-50° C. in a water bath, and magnetic stirring is applied; the generated hexanoic acid is neutralized with 1 M sodium hydroxide during the reaction process, and the reaction pH is controlled at 8.0-8.5 using an automatic potentiometric titrator to prepare the pregabalin chiral drug intermediate (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.
[0039] In the catalytic reaction of immobilized hydantoinase, the dosage of manganese chloride is 0.2 g / L, the addition amount of immobilized hydantoinase is 100 g / L, the substrate concentration is 170 g / L, and the reaction time is 6-12 h.
[0040] Compared with the prior art, the present invention has at least the following beneficial effects:
[0041] The immobilized hydantoinase provided by the present invention has an enzyme activity recovery rate of greater than 50%. The immobilized hydantoinase is used as a catalyst to prepare a pregabalin chiral drug intermediate. The substrate concentration is 170 g / L, the reaction time for each batch is 6-12 hours, and the immobilized hydantoinase is reused 17 times. The conversion rate is greater than 98%, and the ee value of the product (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid is greater than 99%.
[0042] Compared with the free enzyme process, immobilized hydantoinase can enhance its stability, improve its tolerance to mechanical shear, avoid excessive cell protein from being free in the reaction solution, simplify post-processing steps, increase product yield, reduce production costs, realize enzyme recovery and repeated reuse, and has application value in industrial continuous production. DETAILED DESCRIPTION
[0043] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.
[0044] Unless otherwise specified, all raw materials in the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well known to those skilled in the art.
[0045] Example 1: Construction of hydantoinase mutants
[0046] 1 μL of the recombinant pET-28a(+) plasmid containing the nucleotide sequence shown in SEQ ID No. 1 was taken, and 1 μL of each upstream primer and downstream primer (such as the upstream primer 38V-F shown in SEQ ID No. 3 and the downstream primer 38V-R shown in SEQ ID No. 4, or the upstream primer 66A-F shown in SEQ ID No. 5 and the downstream primer 66A-R shown in SEQ ID No. 6, or the upstream primer 135D-F shown in SEQ ID No. 7 and the downstream primer 135D-R shown in SEQ ID No. 8, or the upstream primer C432V-F shown in SEQ ID No. 9 and the downstream primer C432V-R shown in SEQ ID No. 10) was added, 7 μL of deionized water, and 10 μL of DNA polymerase. Different mutants were constructed by whole-plasmid PCR amplification. After the PCR process was completed, 1 μL of restriction endonuclease Dpn was added to the reaction solution. I, and incubated in a 37°C water bath for 2 hours to remove methylated plasmid templates and reduce false positive clones. The recombinant plasmid was transformed into competent E. coli BL21(DE3) cells. After a 30-minute ice bath, the cells were heat-shocked in a 42°C water bath for 90 seconds. After another 2-minute ice bath, the bacterial solution was evenly spread onto a plate containing LB solid medium containing kanamycin resistance and incubated at 37°C for 12 hours to obtain colonies. A single colony from the solid plate was picked and inoculated into 4 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured for 10-12 hours. In a clean bench, 500 μL of the bacterial solution and 500 μL of 50% (w / w) glycerol were pipetted into cryovials and mixed thoroughly. The strain name and storage time were recorded and the cells were stored in a -80°C freezer. The remaining bacterial solution was sequenced to verify the successful construction of an engineered E. coli strain expressing the hydantoinase mutant, i.e., a recombinant E. coli strain expressing the hydantoinase mutant.
[0047] Example 2: Whole cell culture of recombinant Escherichia coli producing hydantoinase and preparation of crude enzyme solution
[0048] Take out the recombinant E. coli containing hydantoinase from the -80℃ freezer, thaw at room temperature, and inoculate 20μL of glycerol into a 4mL LB tube containing 50μg / mL kanamycin. Incubate at 37℃, 200rpm, and shake overnight for 12h. Transfer the above bacterial solution to 100mL LB liquid medium containing 50μg / mL kanamycin and shake at 37℃ until the OD 600=0.6-0.8, IPTG was added to a final concentration of 0.2 mM. After induction culture at 16°C for 22 h, the bacterial culture was removed and centrifuged at 10,000 rpm for 5 min in a low-temperature high-speed centrifuge to collect the cells. The wet cells were resuspended in potassium phosphate buffer (pH 7.0) to a cell concentration of 100 g / L and ultrasonically disrupted in an ice bath for 15 min (ultrasonic power 30%, alternating between on for 2 seconds and off for 3 seconds). After disruption, the disrupted liquid was centrifuged to obtain the supernatant, which was the crude hydantoinase solution.
[0049] Example 3: Production of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid from hydantoinase crude enzyme solution
[0050] 8 mL of sodium carbonate-sodium bicarbonate (100 mM, pH 8.5) buffer was added to 1 mL of 10 mM MnCl2·H2O, 0.17 g of 3-isobutylglutarimide, and 1 mL of the crude hydantoinase solution prepared according to Example 2. The mixture was reacted at 40°C for 4 h. The amount of product generated was detected by liquid chromatography. The results are shown in Table 1.
[0051] The product conversion rate detection method in the present invention is liquid chromatography, and the detection conditions and parameters are set as follows:
[0052] Instrument: SHIMADZU LC-2030Plus
[0053] Column: Shim-pack GIST C18, 5 μm, 4.6 × 250 mm
[0054] Detection wavelength: 210nm
[0055] Flow rate: 0.5 mL / min
[0056] Column temperature: 30°C
[0057] Injection volume: 10 μL
[0058] Detection time: 17.5 minutes
[0059] Mobile phase: Acetonitrile was used as mobile phase A and 1‰ phosphoric acid water was used as mobile phase B. Isocratic elution was performed at a ratio of mobile phase A:mobile phase B = 40%:60%.
[0060] The method for selective detection of product enantiomers in the present invention is liquid chromatography, and the detection conditions and parameters are set as follows:
[0061] Instrument: SHIMADZU LC-2010A HT
[0062] Chromatographic column: CHIRALPAK AD-H, 5μm, 4.6×250mm
[0063] Detection wavelength: 210nm
[0064] Flow rate: 0.5 mL / min
[0065] Column temperature: 35°C
[0066] Injection volume: 10 μL
[0067] Detection time: 20 minutes
[0068] Mobile phase: n-hexane (1‰ TFA) was used as mobile phase A, ethanol (1.5‰ TFA) was used as mobile phase B, and isocratic elution was performed at a ratio of mobile phase A:mobile phase B = 88%:12%.
[0069] Table 1 Comparison of catalytic conversion rates of different hydantoinase mutants
[0070]
[0071] In the table, as shown in S38V, S represents the amino acid before mutation, 38 represents the mutation site, and V represents the amino acid after mutation; as shown in S38VG66A, it indicates that there are two mutation sites, S38V and G66A.
[0072] The catalytic activity of hydantoinase after amino acid mutations at positions 66 and 432 was the most ideal, and this hydantoinase mutant was selected for immobilization in the subsequent examples.
[0073] Example 4: Carrier Activation
[0074] (1) Activation of amino resin: Wash the amino resin three times with potassium phosphate buffer (50 mM, pH 7.5) and filter dry. Weigh 20 g of the resin and add 80 mL of 2% glutaraldehyde potassium phosphate buffer. Oscillate at 25°C and 200 rpm for 2 h. After filtration, wash the resin five times with deionized water to remove residual glutaraldehyde and store at 4°C until use.
[0075] (2) Equilibration of epoxy resin: Wash the epoxy resin three times with potassium phosphate buffer (50 mM, pH 7.5) and filter dry. Do not use glutaraldehyde solution for group activation and store at 4°C until use.
[0076] Example 5: Preparation of immobilized enzyme
[0077] 1 g of the amino resin or epoxy resin activated in Example 4 was weighed, and 10 mL of the crude enzyme solution of the hydantoinase G66AC432V mutant was added. The mixture was shaken at 200 rpm at 25° C. for 10 h. After removal, the mixture was filtered and washed three times with 50 mM potassium phosphate buffer at pH 7.5. After filtration, the immobilized hydantoinase was obtained.
[0078] Example 6: Comparison of the activity of hydantoinase immobilized on different resins
[0079] Immobilized hydantoinase was prepared using the different immobilization resins provided in Table 2 according to the method described in Example 5. The activity of the immobilized hydantoinase was assayed as follows: 900 μL of sodium carbonate-sodium bicarbonate (100 mM, pH 8.5) buffer (800 μL of buffer when using crude enzyme solution), 100 μL of 10 mM MnCl2·H2O, 170 mg of 3-isobutylglutarimide, and 10 mg of immobilized hydantoinase or 100 μL of crude hydantoinase solution prepared according to Example 2 were added to a 2 mL centrifuge tube. The reaction was incubated at 40°C for 20 min, and the product yield was determined by liquid chromatography. Enzyme activity was defined as the amount of enzyme required to produce 1 μmol of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in 1 min under optimal conditions, and the results are shown in Table 3.
[0080] Table 2 Different resin models
[0081]
[0082] Table 3 Comparison of catalytic activity of hydantoinase immobilized on different resins
[0083]
[0084] As can be seen from Table 3, the enzyme activity recovery rate when amino resin is used as the immobilization carrier of hydantoinase is significantly lower than that of epoxy resin. The enzyme activity recovery rate when epoxy resin LX1000HFA is used as the carrier for immobilizing hydantoinase is higher, and the immobilization effect of HFA001 is second. Therefore, resin LX1000HFA is selected for the immobilization of hydantoinase.
[0085] Example 7: Immobilization of Hydantoinase
[0086] KPB buffer (50 mM, pH 7.5) was used as the buffer for resuspension of the cells. 10 mL of crude enzyme solution of the hydantoinase G66AC432V mutant was prepared according to the method described in Example 2. 1 g of the equilibrated epoxy resin LX1000HFA was added and fixed at 15°C and 200 rpm for 10 h. The solution was removed and filtered, washed three times with KPB buffer (50 mM, pH 7.5), and filtered again to obtain immobilized hydantoinase. The enzyme activity recovery rate of the immobilized enzyme was 27.7%.
[0087] Example 8: Immobilization of hydantoinase
[0088] KPB buffer (50 mM, pH 6.0) was used as the buffer for resuspension of the cells. 10 mL of crude enzyme solution of the hydantoinase G66AC432V mutant was prepared according to the method described in Example 2. 1 g of the equilibrated epoxy resin LX1000HFA was added and fixed at 30°C and 200 rpm for 10 h. The solution was removed and filtered, washed three times with KPB buffer (50 mM, pH 6.0), and filtered again to obtain immobilized hydantoinase. The enzyme activity recovery rate of the immobilized enzyme was 43.8%.
[0089] Example 9: Immobilization of Hydantoinase
[0090] KPB buffer (50 mM, pH 8.0) was used as the buffer for resuspension of the cells. 10 mL of crude enzyme solution of the hydantoinase G66AC432V mutant was prepared according to the method described in Example 2. 1 g of the equilibrated epoxy resin LX1000HFA was added and fixed at 30°C and 200 rpm for 10 h. The solution was removed and filtered, washed three times with KPB buffer (50 mM, pH 8.0), and filtered again to obtain immobilized hydantoinase. The enzyme activity recovery rate of the immobilized enzyme was 29.8%.
[0091] Example 10: Immobilization of hydantoinase
[0092] KPB buffer (50 mM, pH 7.0) was used as the buffer for resuspension of the cells. 10 mL of crude enzyme solution of the hydantoinase G66AC432V mutant was prepared according to the method described in Example 2. 1 g of the equilibrated epoxy resin LX1000HFA was added and fixed at 30°C and 200 rpm for 5 h. The solution was removed and filtered, washed three times with KPB buffer (50 mM, pH 7.0), and filtered again to obtain immobilized hydantoinase. The enzyme activity recovery rate of the immobilized enzyme was 51.3%.
[0093] Example 11: Immobilization of hydantoinase
[0094] KPB buffer (50 mM, pH 7.0) was used as the buffer for resuspension of the cells. 10 mL of crude enzyme solution of the hydantoinase G66AC432V mutant was prepared according to the method described in Example 1. 1 g of the equilibrated epoxy resin LX1000HFA was added and fixed at 30°C and 200 rpm for 1 h. The solution was removed and filtered, washed three times with KPB buffer (50 mM, pH 7.0), and filtered again to obtain immobilized hydantoinase. The enzyme activity recovery rate of the immobilized enzyme was 41.3%.
[0095] Based on the enzyme activity recovery results of Examples 7 to 11, it can be seen that the recovery rate in Example 10 is the highest, so the immobilized enzyme prepared in this example was used for subsequent experiments.
[0096] Example 12: Production of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid by immobilized hydantoinase
[0097] To a 100 mL reaction flask were added 8.5 g of the substrate 3-isobutylglutarimide, 9.9 mg of MnCl2·H2O (as an enzyme activator), 5 g of the immobilized hydantoinase prepared as in Example 10, and 50 mL of 100 mM sodium carbonate-sodium bicarbonate buffer (pH 8.5). The reaction was incubated at 40°C for 8 h. During the reaction, the pH was maintained at 8.5 using 1 M sodium hydroxide solution. The conversion rate was 100% after 8 h, and the product ee value was greater than 99%. The ee value represents the enantiomeric excess.
[0098] The results show that the immobilized hydantoinase provided by the present invention has excellent catalytic conversion efficiency and selectivity.
[0099] Example 13: Reuse of immobilized hydantoinase
[0100] The immobilized hydantoinase prepared in Example 10 was used for continuous conversion according to the conversion scheme described in Example 12. After each conversion, the immobilized enzyme obtained by filtration was used in the next batch of catalytic reactions. The batch conversion rates are shown in Table 4. As can be seen from the data in the table, the immobilized hydantoinase was reused 17 times to achieve essentially complete conversion of the substrate, which is higher than the reuse rate of the free enzyme (the free enzyme can only be used once and cannot be recycled) without a significant decrease in the conversion rate.
[0101] Table 4 Batch conversion rate of immobilized hydantoinase
[0102]
[0103] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.
[0104] The sequence information involved in the present invention is as follows:
[0105] SEQ ID No. 1 (nucleotide sequence of the parent hydantoinase):
[0106]
[0107] SEQ ID No. 2 (amino acid sequence of the parent hydantoinase):
[0108] MTKLIKNGTI VTATDIYEAD LLIQDGKIAV IGRNLDESGA EVIDATGCYVFPGGIDPHTHLDEPHGGTVT KDDFESGTIA AAFGGTTTIIDFCLTNKGEPLKKAIETWHN KATGKAVIDY GFHLMISEITDDVLEELPKV IEEEGITSFKVFMAYKDVFQ ADDGTLYRTL VAAKELGALVMVHAENGDVIDYLTKKALEDGHTDPIYHA LTRPPELEGE ATGRACQLTE LAGSQLYVVHVSCAQAVEKIAEARNKGLNVW GETCPQYLVL DQSYLEKPNF EGAKYVWSPP LREKWHQEVLWNALKNGQLQTLGSDQTSFD FKGQKELGRG DFTKIPNGGP IIEDRVSILFSEGVKKGRIT LNQFVDIVST RIAKLFGLFPKKGTIAVGAD ADLVIFDPTVERVISAETHH MAVDYNPFEG MKVTGEPVSV LCRGEFVVRDKQFVGKPGYGQYVKRAKYGA LMADQDVVKM S
[0109] Site-directed mutagenesis primer sequence list
[0110]
Claims
1. A hydantoinase mutant, characterized in that: The amino acid sequence of the hydantoinase mutant is selected from any one of the following: (1) replacing serine at position 38 in the amino acid sequence shown in SEQ ID No. 2 with valine; (2) replacing glycine at position 66 in the amino acid sequence shown in SEQ ID No. 2 with alanine; (3) replacing glutamic acid at position 135 in the amino acid sequence shown in SEQ ID No. 2 with aspartic acid; (4) replacing cysteine at position 432 in the amino acid sequence shown in SEQ ID No. 2 with valine; (5) in the amino acid sequence shown in SEQ ID No. 2, the serine at position 38 was replaced with valine, and the glycine at position 66 was replaced with alanine; (6) in the amino acid sequence shown in SEQ ID No. 2, the serine at position 38 is replaced by valine, and the glutamic acid at position 135 is replaced by aspartic acid; (7) in the amino acid sequence shown in SEQ ID No. 2, the serine at position 38 was replaced with valine, and the cysteine at position 432 was replaced with valine; (8) In the amino acid sequence shown in SEQ ID No. 2, glycine at position 66 is replaced with alanine, and glutamic acid at position 135 is replaced with aspartic acid; (9) In the amino acid sequence shown in SEQ ID No. 2, glycine at position 66 was replaced with alanine, and cysteine at position 432 was replaced with valine; (10) In the amino acid sequence shown in SEQ ID No. 2, glutamic acid at position 135 is replaced by aspartic acid, and cysteine at position 432 is replaced by valine.
2. A nucleic acid encoding the hydantoinase mutant according to claim 1.
3. A recombinant expression vector, characterized in that: Comprising the nucleic acid of claim 3.
4. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector according to claim 4.
5. A hydantoinase mutant catalyst, characterized in that: Is any of the following forms: (1) culturing the recombinant expression transformant according to claim 4, isolating and disrupting the transformant cells containing the hydantoinase mutant according to claim 1 to obtain a crude enzyme solution, or subjecting the crude enzyme solution to purification and separation operations to obtain a pure enzyme solution; (2) Cultivating the recombinant expression transformant according to claim 4, isolating and disrupting the transformant cells containing the hydantoinase mutant according to claim 1 to obtain a crude enzyme solution, and freeze-drying the crude enzyme solution to obtain a crude enzyme powder.
6. An immobilized hydantoinase, characterized in that: The immobilized hydantoinase comprises an immobilization carrier and a hydantoinase loaded on the carrier; the hydantoinase is the hydantoinase mutant according to claim 1; the immobilization carrier is an amino resin or an epoxy resin; The amino resin is selected from any one of LX1000NH, LXTE703, LXTE704 or XJGD29, and the epoxy resin is selected from any one of LX1000HFA, HFA001, LXTE609 or XJGD89.
7. A method for preparing immobilized hydantoinase according to claim 6, characterized in that: The following steps are involved: The amino resin is activated or the epoxy resin is balanced for later use; the activated amino resin or the balanced epoxy resin is added to the hydantoinase solution, and the solution is shaken in a shaker. After the shaking is completed, the solution is washed and filtered to obtain the immobilized hydantoinase.
8. The method for preparing immobilized hydantoinase according to claim 7, wherein: The resin addition amount is 1g / 10mL enzyme solution; the concentration of hydantoinase mutant protein in the enzyme solution is 30-90mg 蛋白 / g 树脂 The shaking temperature is 15-35 ° C; the shaking time is 1-10h; the solvent in the hydantoin enzyme solution or the washing KPB buffer with a pH of 6.0-8.0; The steps of activating the amino resin are as follows: taking the amino resin, washing it, filtering it to dryness, activating it with glutaraldehyde solution, and filtering it again to obtain the activated amino resin; The steps of balancing the epoxy resin are as follows: taking the epoxy resin, washing it, and filtering it to obtain the balanced epoxy resin.
9. Use of the hydantoinase mutant according to claim 1, or the hydantoinase mutant catalyst according to claim 5, or the immobilized hydantoinase according to claim 6, characterized in that: Hydantoinase mutants or immobilized hydantoinase catalyze the selective hydrolysis of 3-isobutylglutarimide to (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.
10. The use according to claim 9, characterized in that During the catalytic reaction, MnCl2 is added as an enzyme activator.