Allantoinase and use thereof

Abstract

This invention discloses allantoinase and its applications. The allantoinase disclosed in this invention can be better applied to the catalytic hydrolysis of IBI in the production of (… R )-IBM, and the subsequent synthesis of pregabalin.

Description

Technical Field

[0001] This invention belongs to the field of enzyme gene engineering technology, and relates to allantoinase and its applications. Background Technology

[0002] ( S Pregabalin ( S 3-Aminomethyl-5-methylhexanoic acid (Methylhexanoic acid) is a drug developed by Pfizer for the treatment of epilepsy and anxiety. It has good efficacy and low toxicity and side effects, and has always occupied an important market share.

[0003] Pregabalin is a chiral drug that can be synthesized using asymmetric methods or by racemic resolution to obtain the target chiral product. Among these methods, ( R )-3-isobutylglutaric acid monoamide (( R 3-Isobutylglutarimide (IBI) is an important chiral intermediate of pregabalin, which can be prepared by biocatalytic or alkaline hydrolysis using 3-isobutylglutarimide (IBI), followed by Hoffmann rearrangement to yield pregabalin. This route is a promising one for the synthesis of pregabalin. Literature reports that both imidases and hydantoinase can biocatalytically hydrolyze IBI to IBM, and the products exhibit high... R Type I enantioselectivity ( ee %). Among them, imidases exhibit low catalytic activity at 35℃, and are used for the batch preparation of synthetic ( R IBM is facing difficulties. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention provides allantoinase and its mutants, which differ from previously reported imidases and hydantoins, and are used in the catalytic hydrolysis of 3-isobutylglutarimide (IBI) to prepare (… R )-3-isobutylglutaric acid monoamide (( R In the reaction of )-IBM), it has a high ( R It exhibits selectivity for IBM while also possessing high catalytic activity for IBI.

[0005] Specifically, this invention first uses the sequence of imidase BpIH as a template for screening to obtain a series of wild-type allantoinases with certain structural similarities. After cloning and secondary screening, it was found that enzyme 1 (1-WP_008350018) (sequence shown in SEQ ID NO: 2) and enzyme 7 (7-WP_148578762) (sequence shown in SEQ ID NO: 4) exhibited hydrolytic activity more than twice that of imidases and higher than that of hydantoinase when using IBI as a substrate. Further testing revealed that the hydrolyzed ( RIBM's optical purity reached over 99.5%. Enzyme #1 was derived from *Carbaronia* genus of the Burkholderiaceae family. Caballeronia zhejiangensis. Enzyme No. 7 is derived from the genus *Colloidobacterium*. Zoogloea Furthermore, through directed evolution, enzyme 1 was modified to produce an allantoinase mutant Y37F / H133N / T226I with higher catalytic activity, the amino acid sequence of which is shown in SEQ ID NO: 6.

[0006] In a first aspect, the present invention provides an allantoinase having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 90%, preferably at least 99%, identity with the amino acid sequence shown in SEQ ID NO: 2.

[0007] In a second aspect, the present invention provides an allantoinase having the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, preferably at least 99%, identity with the amino acid sequence shown in SEQ ID NO: 4.

[0008] In a third aspect, the present invention provides an allantoinase mutant, which, relative to the wild-type allantoinase shown in SEQ ID NO: 2, has a tyrosine mutation at position 37 replaced by phenylalanine, a histidine mutation at position 133 replaced by asparagine, and a threonine mutation at position 226 replaced by isoleucine. This allantoinase mutant is named Y37F / H133N / T226I, and has the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 90%, preferably at least 99%, identity with the amino acid sequence shown in SEQ ID NO: 6. This allantoinase mutant can more efficiently hydrolyze IBI to generate (…). R -IBM.

[0009] The allantoinase or allantoinase mutant provided by the present invention can be artificially synthesized, or it can be obtained by first synthesizing its encoding gene and then expressing it biologically, such as by using recombinant technology to express it from prokaryotes (e.g., Escherichia coli) or eukaryotes (e.g., yeast, higher plants).

[0010] In some embodiments, the aforementioned allantoinase or allantoinase mutant is introduced into *E. coli* by transforming it into a recombinant expression vector containing its encoding gene (e.g., *E. coli*). E. coli BL21 (DE3) was used to construct a recombinant genetically engineered bacterium, which was then cultured and induced to express by adding an inducer.

[0011] In a fourth aspect, the present invention provides a nucleic acid molecule encoding any of the above-described allantoinase or allantoinase mutants.

[0012] In some embodiments, the nucleic acid molecule encoding the allantoinase described in the first aspect has the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90%, preferably at least 99%, identity with the nucleotide sequence shown in SEQ ID NO: 1.

[0013] In some embodiments, the nucleic acid molecule encoding the allantoinase described in the second aspect has the nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence having at least 90%, preferably at least 99%, identity with the nucleotide sequence shown in SEQ ID NO: 3.

[0014] In some embodiments, the nucleic acid molecule encoding the allantoinase mutant described in the third aspect has the nucleotide sequence shown in SEQ ID NO: 5 or a nucleotide sequence having at least 90%, preferably at least 99%, identity with the nucleotide sequence shown in SEQ ID NO: 5.

[0015] The nucleic acid molecules provided by this invention can usually be obtained by amplification using a PCR instrument or by artificial synthesis.

[0016] The term "identity" as used in this article can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). When the positions in the compared sequences are occupied by the same bases or amino acids, the molecules are identical at that position. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. "Sequence identity" of a polynucleotide or amino acid sequence with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0017] In a fifth aspect, the present invention provides a recombinant vector comprising any of the nucleic acid molecules described above; The recombinant vector includes a cloning vector and an expression vector, wherein the cloning vector is used to replicate the relevant gene and the expression vector is used to express the relevant gene.

[0018] In some implementations, the recombinant vector is a recombinant expression vector, for example, constructed using the pET-24a vector.

[0019] In a sixth aspect, the present invention provides a recombinant cell comprising any of the nucleic acids and / or recombinant vectors described above.

[0020] In some embodiments, the recombinant cells are induced to express any of the allantoinase or allantoinase mutants described above.

[0021] In some implementations, the method for constructing the recombinant cells includes: The recombinant expression vector is transformed into expression host cells, cultured, and induced to express by adding an inducer to obtain any of the above-described allantoinase or allantoinase mutants.

[0022] Furthermore, the recombinant expression vector is any of the recombinant expression vectors described above, and the expression host cell is a conventional host cell in the art, capable of stably replicating itself and effectively expressing the gene carried by the recombinant vector. It can be a prokaryotic cell or a eukaryotic cell, such as *Escherichia coli* or yeast, with *Escherichia coli* being preferred as the expression host. E. coli BL21 (DE3).

[0023] In some embodiments, the recombinant cells are those from Escherichia coli transformed with a recombinant expression vector. E. coli The recombinant genetically engineered bacteria obtained from BL21(DE3). The culture medium used when the recombinant genetically engineered bacteria express allantoinase or allantoinase mutants can be any culture medium in the art that allows the recombinant genetically engineered bacteria to grow and express the allantoinase or allantoinase mutants of the present invention, such as LB or TB media.

[0024] There are no special requirements for the culture method and conditions, as long as the recombinant genetically engineered strain grows normally and is induced to express allantoinase or allantoinase mutants under appropriate temperature conditions. The preferred culture method is as follows: Inoculate the recombinant bacteria into LB liquid medium containing kanamycin, and culture at 37°C and 220 rpm for 12 hours or overnight. Then, inoculate into TB medium at an inoculation rate of 0.1-10% (v / v) and culture at 37°C and 220 rpm until the bacterial culture OD... 600 When the concentration reaches 0.6-0.8, add isopropyl alcohol to a final concentration of 0.1-1.0 mM. β D Glucosinosteroids (IPTG) were used as an inducer to induce bacterial culture at 25°C for 8-24 hours. The induced bacterial culture was then centrifuged, and the bacterial cells were collected to obtain recombinant cells.

[0025] In a seventh aspect, the present invention provides a method for preparing allantoinase, comprising: 1) Culture any of the recombinant cells described above and induce the expression of any of the allantoinase or allantoinase mutants described above; Optional additions include: 2) Isolate any of the above-described allantoinase or allantoinase mutants from the culture obtained in 1).

[0026] The methods for culturing and inducing recombinant cells, and the methods for isolating allantoinase or allantoinase mutants from cultures, are conventional methods in the field.

[0027] In an eighth aspect, the present invention provides any of the above-described allantoinase, the above-described allantoinase mutant, any of the above-described nucleic acid molecules, any of the above-described recombinant vectors, and / or any of the above-described recombinant cells in the preparation ( R Application in 3-isobutylglutaric acid monoamide and / or pregabalin.

[0028] In a ninth aspect, the present invention provides a preparation ( R A method for producing 3-isobutylglutaric acid monoamide includes the following steps: using any of the above-described allantoinase, the above-described allantoinase mutant, any of the above-described nucleic acid molecules, any of the above-described recombinant vectors, and / or any of the above-described recombinant cells as catalysts, catalyzing the hydrolysis of 3-isobutylglutaric acid imide to obtain ( R )-3-isobutylglutaric acid monoamide.

[0029] Allantoinase or its mutants catalyze the hydrolysis of IBI to produce ( R The following is the synthetic route for the IBM reaction and pregabalin:

[0030] In some embodiments, the feed for the hydrolysis reaction in the above method includes: manganese chloride, 3-isobutylglutarimide, and the above-mentioned catalyst.

[0031] In some embodiments, in the above method, the amount of 3-isobutylglutarimide used in the hydrolysis reaction is (1-50) g / 100 mL, preferably (5-10) g / 100 mL; and / or The catalyst is used in an amount of (1-50) g / 100 mL, preferably (3.25-8) g / 100 mL; and / or The final concentration of manganese chloride is 0.1-2 mM, preferably 0.5-1.5 mM.

[0032] In some embodiments, in any of the methods described above, the pH of the hydrolysis reaction is 6.0-9.0, such as pH 6, pH 6.5, pH 7, pH 7.5, pH 8.0, pH 8.5, pH 9, or any value or range between these values, preferably pH 8.0-9.0.

[0033] In some embodiments, in any of the methods described above, the temperature of the hydrolysis reaction is 20-50°C, for example 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, or any value or range between these values, preferably 35-40°C.

[0034] In some embodiments, in any of the methods described above, the buffer used for the hydrolysis reaction is a conventional buffer in the art, such as Tris-HCl buffer, with a concentration of, for example, 50 mM.

[0035] It should be understood that the allantoinase or allantoinase mutant described in this invention can be used in whole-cell engineered bacteria, in unpurified crude enzyme form, or in partially or completely purified enzyme form. Furthermore, the allantoinase or allantoinase mutant of this invention can be prepared into immobilized enzymes or catalysts in immobilized cell form using immobilization techniques known in the art.

[0036] In some embodiments, in any of the methods described above, the hydrolysis reaction is carried out under stirring or agitation, for example, at a stirring speed of 50-1000 rpm.

[0037] The beneficial effects of this invention are as follows: The allantoinase and its mutants provided by this invention can efficiently catalyze the hydrolysis of IBI to generate ( R IBM, compared to imidase BpIH, due to the increased enzyme activity, can shorten the reaction time from 93 hours to less than 24 hours, while the reaction products ( R IBM maintained a success rate of over 99%. ee Value. The allantoinase and its mutants of the present invention can be better applied to the catalytic hydrolysis of IBI to produce ( R )-IBM, and the subsequent synthesis of pregabalin. Attached Figure Description

[0038] Figure 1 This is a diagram of the catalytic reaction process of wild-type allantoinase with IBI, as shown in SEQ ID NO: 2 of the present invention.

[0039] Figure 2 The results are the chiral HPLC analysis results of IBI obtained by catalytic hydrolysis of IBI by the allantoinase mutant Y37F / H133N / T226I shown in SEQ ID NO: 6 of this invention.

[0040] Figure 3 This is a comparison diagram of the catalytic reaction of the allantoinase mutant Y37F / H133N / T226I shown in SEQ ID NO: 6 of the present invention with that of the existing imidase BpIH on IBI. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0042] The enzyme activity unit (U) is defined as: the amount of enzyme required to catalyze the production of 1 micromole of IBM per minute is 1 U.

[0043] Enzyme activity assay: After the enzyme activity reaction is completed, the reaction solution is sampled and added to the high performance liquid chromatography (HPLC) mobile phase for dilution to terminate the reaction. The supernatant is collected after centrifugation at 10,000 rpm for 1 min and then the following HPLC detection is performed. HPLC detection conditions: The chromatographic column was a Cosmosil 5C18-AR-II (250 mm × 4.6 mm) column, and the column temperature was 30℃; the mobile phase was acetonitrile-water (volume ratio 4:6, pH adjusted to 2.5 with phosphoric acid), and the flow rate was 0.5 mL / min; the detection wavelength was 210 nm. The retention times of IBM and IBI were approximately 6.8 and 15.1 min, respectively.

[0044] Chiral HPLC detection conditions: chromatographic column: CHIRALPAK IC (4.6 × 250 mm, 5 μm); column temperature: 25°C; mobile phase: n-hexane-ethanol-trifluoroacetic acid (v / v 75:25:0.1); flow rate: 1.0 mL / min; detection wavelength: 210 nm. Optical purity. In the formula The peak area of ​​the isomer. for( R ) type and ( S The sum of the areas of the IBM peaks of type (). R ) type and ( S The peak times for the IBM (type 1) and (type 2) were approximately 5.5 and 6.5 minutes, respectively.

[0045] Example 1: Screening of wild-type allantoinase with relatively high activity 1. Using the sequence of imidase BpIH as a template, 33 allantoinases with certain structural similarities were found by searching on NCBI and Swissmodel websites.

[0046] 2. The nucleotide sequences of these allantoinases were artificially synthesized and used to replace the sequence between the NdeI and XhoI restriction sites of the pET-24a vector, while leaving the other sequences unchanged, to obtain the various recombinant expression plasmids. Sequencing of each recombinant expression plasmid yielded results consistent with expectations.

[0047] 3. Transform each recombinant expression plasmid into the expression host through chemical transformation. E. coli In BL21(DE3), LB solid medium containing 50 μg / mL kanamycin was spread for screening to obtain recombinant bacteria expressing various allantoinases.

[0048] 4. Inoculate each recombinant strain into 4 ml of LB liquid medium containing 50 μg / mL kanamycin and incubate overnight at 37°C and 220 rpm. Then, inoculate 0.5% (v / v) into 200 mL of TB medium and incubate at 37°C and 220 rpm. Wait for the OD of the bacterial culture to develop. 600 When the pH reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mM and induce culture at 25℃ for 14 h. Centrifuge the induced culture at 4℃ and 12000 rpm for 10 min and collect the bacterial cells. Resuspend the bacterial cells in cell disruption buffer (50 mM, pH 8.0 Tris-HCl buffer), and disrupt the cells using an ultrasonic cell disruptor at 25% power for 10 min. Then centrifuge at 4℃ and 10000 rpm for 10 min and collect the supernatant as crude enzyme solution.

[0049] 5. Allantoinase activity assay In a 5 mL enzyme activity assay reaction system, 50 mmol / L Tris-HCl (pH 8.0), 10 mmol / L LiBI, and 500 mL of crude enzyme solution were added. The reaction was carried out at 40°C and 200 rpm for 30 min. After the reaction, the mobile phase of high performance liquid chromatography (HPLC) was added for dilution to terminate the reaction. The supernatant was collected by centrifugation at 10,000 rpm for 1 min and then analyzed by HPLC to determine the enzyme activity. The results are shown in Table 1. The enzyme activity (%) in Table 1 is relative to the BpIH enzyme activity data as 100%.

[0050] Table 1

[0051] In Table 1, the amino acid sequence of BpIH is shown in SEQ ID NO: 7, and the amino acid sequence of hydantoin is shown in SEQ ID NO: 8.

[0052] Table 1 shows that enzyme 1 (1-WP_008350018, derived from Burkholderiaceae, genus Carbaronia) Caballeronia zhejiangensis ) and enzyme 7 (7-WP_148578762, derived from the genus *Colloidobacterium*) Zoogloea It achieved activity similar to that of hydantoin.

[0053] The nucleotide and amino acid sequences of enzyme No. 1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the nucleotide and amino acid sequences of enzyme No. 7 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0054] Example 2 Preparation of IBI by hydrolysis using wild-type allantoinase ( R -IBM In an 80 mL reaction system, 1 mM manganese chloride, 50 mM Tris-HCl buffer at pH 8.0, 2.6 g of wild-type allantoinase engineered bacteria (cells collected in step 4 of Example 1) as shown in SEQ ID NO: 2, and 8.0 g of IBI were added. The reaction was carried out at 40 °C and 200 rpm for 32 h.

[0055] After sampling and HPLC analysis, the product IBM (( R IBM and ( S The concentration of (-IBM total) was 21.9 g / L.

[0056] The catalytic reaction process of wild-type allantoinase with IBI shown in SEQ ID NO: 2 is illustrated in the diagram below. Figure 1 As shown.

[0057] After centrifuging the reaction solution at 4℃ and 10000 rpm for 10 min, the supernatant was adjusted to pH 3.0 with 6N HCl, and then filtered through filter paper to obtain ( R -IBM crude product. After sampling, the crude product was analyzed by chiral HPLC, and the crude product contained ( R IBM's optical purity is 99.62%. ee The value is 99.24%.

[0058] Example 3: Directed Evolution of Highly Active Allantoinase 1. Directed evolution was performed on the wild-type allantoinase shown in SEQ ID NO: 2, specifically by mutating three key amino acids at its active site and substrate channel opening. These three amino acids are Y37 / H133 / S226 in the imidase BpIH and Y37 / H133 / T226 in the wild-type allantoinase shown in SEQ ID NO: 2. Multiple-point mutations of Y37F, H133N, and T226I were performed on the wild-type allantoinase shown in SEQ ID NO: 2 to obtain the combined mutant Y37F / H133N / T226I, the amino acid sequence of which is shown in SEQ ID NO: 6, and the corresponding nucleotide sequence of which is shown in SEQ ID NO: 5.

[0059] 2. The DNA fragment shown in SEQ ID NO: 5 was used to replace the sequence between the NdeI and XhoI restriction sites in the pET-24a vector, while the remaining sequences remained unchanged, to obtain the recombinant expression plasmid pT07YHS. The recombinant expression plasmid pT07YHS was sequenced, and the results were consistent with expectations.

[0060] 3. The recombinant expression plasmid pT07YHS was transformed into the expression host via chemical transformation. E. coli In BL21(DE3), recombinant bacteria were screened by plating on LB solid medium containing 50 μg / mL kanamycin to obtain the combined mutant Y37F / H133N / T226I. E. coli BL21(DE3) / pT07YHS.

[0061] 4. Following step 4 of Example 1, a crude enzyme solution containing the combined mutant Y37F / H133N / T226I was obtained.

[0062] 5. Following step 5 of Example 1, the crude enzyme solution containing the combined mutant Y37F / H133N / T226I was subjected to enzyme activity assay. It was found that the enzyme activity of Y37F / H133N / T226I was 178% of that of the wild-type allantoinase shown in SEQ ID NO: 2.

[0063] Example 4 Preparation of IBI by hydrolysis using allantoinase mutant ( R -IBM The 100 mL reaction system contained 1 mM manganese chloride, 50 mM Tris-HCl buffer (pH 8.0), and recombinant bacteria. E. coli 8.0 g of BL21(DE3) / pT07YHS cells (cells collected in step 4 of Example 3), 5.0 g of IBI (29.55 mmol), reacted at 40°C and 220 rpm for 8 h.

[0064] After sampling and HPLC analysis, the concentration of product IBM in the reaction solution was 42.5 g / L.

[0065] After 24 h of reaction, the concentration of product IBM in the reaction solution was 53.99 g / L (total 28.84 mmol), and the conversion rate (moles of product IBM / moles of starting material IBI × 100%) was 97.60%. After centrifugation at 10,000 rpm for 10 min at 4 °C, the supernatant was adjusted to pH 3.0 with 6N HCl and filtered through filter paper to obtain (…). R -IBM crude product. After sampling, the crude product was analyzed by chiral HPLC, and the crude product contained ( R IBM's optical purity is 99.775%. ee The value is 99.55%.

[0066] The chiral HPLC analysis results of IBI obtained by catalytic hydrolysis of IBI by the allantoinase mutant Y37F / H133N / T226I shown in SEQ ID NO: 6 are as follows: Figure 2 As shown.

[0067] The comparative example was prepared by hydrolyzing IBI with imidase. R -IBM 25 g of IBI (147.74 mmol) was added to 180 ml of deionized water and stirred until homogeneous. The pH was adjusted to 8.0 with 1 mol / L NaOH, and 320 mL of imidase BpIH cell-wall breaking enzyme solution (derived from approximately 64 g of wet bacterial cells, the crude enzyme solution collected in step 4 of Example 1) was added. The reaction was carried out at 35°C and 250 rpm, with the pH maintained at 8.0 using 1 mol / L NaOH. After 93 h of reaction, the concentration of the product IBM in the reaction solution was 49.11 g / L (volume 512 mL, total 134.29 mmol), with a conversion rate of 90.9%.

[0068] The catalytic reaction comparison diagram between Example 4 and the comparative example is shown in the figure below. Figure 3 As shown. In the comparative example, the enzyme dosage was equivalent to 128 g / L of bacterial cells, and after 93 hours of reaction, 49.11 g / L of product was produced. In Example 4, the enzyme dosage was approximately 80 g / L, and the reaction time was shortened to 24 hours, producing 53.99 g / L of product. It can be seen that, compared to the imidase BpIH, the enzyme prepared in this invention (… R IBM requires less bacterial cells, significantly shortens the reaction time, and greatly improves preparation efficiency, making it more valuable for industrial applications.

[0069] Example 5: Production using the enzyme catalysis of the present invention ( RApplications of IBM in the preparation of pregabalin The product of this invention ( R IBM prepared pregabalin using the Hoffmann rearrangement effect, which can be obtained using existing patents / literature (e.g., Qi Xiuxiu, Chen Wenhua, Chen Wenqi, et al. Improvement of pregabalin synthesis process [J]. Journal of Ningxia University (Natural Science Edition), 2019, 40(4):366-369.).

[0070] The specific embodiments described in this invention are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

[0071] Although the present invention has been described in detail and specific embodiments have been cited, it will be apparent to those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the invention.

[0072] sequence SEQ ID NO: 1 ATGTCACTAGACCCAAATTATCCCAGGGATTTGATTGGTTACGGCCGCCACCCGGTTCAAGCGAACTGGCCGGGTAGAGCCCGAGTGGCGGTTCAATTTGTATTGAATTACGAGGAAGGTGGCGAGAACTGCGTCCTTCACGGCGACCCGGGCTCCGAGCAGTTTCTCTCCGAGATCGTGGGCGCAGCGGCGTATCCGAGCCGTCACATGTCTATGGAGAGCATTTATGAATACGGCAGCCGCGCGGGTGTGTGGCGTATTCTGCGTGAATTTGAGAAGCGCGGTATGCCGTTGACCATTTTTGGTGTTGGCATGGCTATCGAGCGTCACCCGGAGGTTGCGAAAGCTTTCGTGGAGCTGGGTCATGAAATCGCGTGTCATGGTTATCGCTGGATTCATTATCAAGATATGTCTCCGGAAAAAGAAGCGGAGCACATGCGCCTGGGTATGGAAGCGATCGAGCGTGTTACGGGTGTTCGTCCGCTGGGCTGGTATACGGGGCGCGATAGCCCCAACACCCATCGTCTGGTTGCTGAGTACGGCGGTTTCTTGTACGACAGCGATTATTACGGCGACGACCTGCCGTTTTGGACCGAAGTGGAAGTCACCGGTGGCGAGAAGAAGCCGCATCTGATTGTGCCGTACACCCTGGATACCAATGATATGCGTTTCGCCACCCCGCAGGGTTTCAACACTGGCGACCACTTTTTCACCTATCTGAAAGATGCATTCGACGTCTTGTACGAAGAAGGTGATGAAGCCCCTAAAATGCTGTCGATCGGCATGCACTGCCGTCTGCTGGGTCGTCCGGGCCGCTTTCGTGGTCTGCAGAAATTCTTAGACCACATCGAGAAGCACGATCGTGTGTGGGTTACGCGTCGTGTGGACATCGCCCGTCATTGGCGTGAACAGCACCCATTCGAAAAGGCTAATAACGGTACAGCAGCGCTCGAGCACCACCACCACCACCACTGA SEQ ID NO: 2 MSLDPNYPRDLIGYGRHPVQANWPGRARVAVQFVLNYEEGGENCVLHGDPGSEQFLSEIVGAAAYPSRHMSMESIYEYGSRAGVWRILREFEKRGMPLTIFGVGMAIERHPEVAKAFVELGHEIACHGYRWIHYQDMSPEKEAEHMRLGMEAIERVTGVRPLGWYTGRDSPNTHRLVAEYGGFLYDSDYYGDDLPFWTEVEVTGGEKKPHLIVPYTLDTNDMRFATPQGFNTGDHFFTYLKDAFDVLYEEGDEAPKMLSIGMHCRLLGRPGRFRGLQKFLDHIEKHDRVWVTRRVDIARHWREQHPFEKANNGTAALEHHHHHH SEQ ID NO: 3 ATGACTATGGATCCAACATATCCCAGGGACCTTGTGGGCTATGGCCGTAATCCGCCACATGCGAATTGGCCTGGTGGTGCCCGGATTGCTGTGCAGTTTGTTCTGAATTACGAGGAGGGTGGCGAGAACTGCGTTCTGCACGGCGACGCGGGTTCCGAGCAGTTCCTCTCCGAATTGTTTAACCCCCCGAGCTACCCGGCCCGTCACCTGTCTATGGAGAGCGTGTATGAATACGGCAGCCGTGTCGGTGTTTGGCGTTTCTTGCGCGAATTTGAACGCCGCGGTCTGCCGCTGACCGTTTTTGGTGTGGCTATGGCACTGGAACGTCATCCGGAATTGACGGCGGCATTCACCGAACTGGGTCATGAAATCGCCTGCCACGGCTGGCGTTGGCTGAACTACCAAAACGTTGATGAGGCGGTTGAACGCGAGCACCTGCAAACGGGTATGGCGATTATCGAGCGCTTGACTGGTGCGCGTCCGTTGGGCTGGTATACCGGTAGAGACAGCCCGAACACCCGTCGCTTGGTAGCGGATCACGGGGGCTTTCTGTATGATTCCGACTACTACGGCGACGACCTGCCACTGTGGACCGAGGTGACCAAAAGCGATGGCTCGACCACCCCGCATCTGGTGGTGCCGTATACGCTGGATACCAACGACATGCGTTTCGCCTTGCCGCAAGGTTTTAGCCATGGTGACGCGTTCTTCGACTATCTGCGTGATGCGTTCGATGTCATGTACGCAGAAGGTGACGAACGCCCGGCTATGCTGAGCGTCGGCATGCACTGCCGTCTGTTAGGCCGTCCGGCTCGCTTCAAAGCACTGCAGCGTTTCCTCGATCATATCGAGAAGCACGATCGTGTTTGGGTTTGTCGTCGCGTGGACATCGCGCGTCATTGGATTGAGCACCACCCGTTTTCTCCGCAGGCACGCctcgagCACCACCACCACCACCACTGA SEQ ID NO: 4 MTMDPTYPRDLVGYGRNPPHANWPGGARIAVQFVLNYEEGGENCVLHGDAGSEQFLSELFNPPSYPARHLSMESVYEYGSRVGVWRFLREFERRGLPLTVFGVAMALERHPELTAAFTLGHEIACHGWRWLNYQNVDEAVEREHLQTGMAIIERLTGARPLGWYTGRDSPNTRRLVADHGGFLYDSDYGDDLPLWTEVTKSDGSTTPHLVVPYTLDTNDMRFALPQGFSHGDAFFDYLRDAFDVMYAEGDERPAMLSVGMHCRLLGRPRAFKALQRFLDHIEKHDRVWVCRRVDIARHWIEHHPFSPQARLEHHHHHH SEQ ID NO: 5 ATGTCACTAGACCCAAATTATCCCAGGGATTTAATTGGCTACGGCCGCCACCCGGTCCAGGCTAACTGGCCAGGTCGGGCGCGTGTGGCCGTTCAATTCGTCTTGAATTTCGAGGAGGGTGGCGAAAACTGTGTTCTGCATGGCGACCCGGGCAGCGAACAATTTTTATCCGAGATCGTTGGCGCAGCAGCGTATCCGAGCCGTCACATGTCGATGGAAAGCATTTACGAGTACGGCTCTCGTGCTGGAGTGTGGCGTATTCTGCGCGAATTTGAGAAGCGCGGTATGCCGTTGACCATTTTTGGCGTGGGTATGGCAATCGAACGTCACCCGGAAGTGGCGAAAGCGTTTGTTGAACTGGGCCATGAAATCGCTTGCCATGGTTATCGTTGGATTAACTACCAGGACATGTCTCCGGAAAAAGAGGCGGAACACATGCGTCTCGGTATGGAAGCGATCGAGCGCGTCACGGGTGTTCGTCCGCTTGGTTGGTATACCGGCAGAGATAGCCCGAATACCCATCGTTTGGTTGCGGAGTATGGCGGTTTTCTGTACGACTCCGATTACTACGGCGACGACCTGCCGTTCTGGACGGAAGTGGAGGTGACTGGCGGTGAAAAGAAACCGCACCTGATCGTGCCGTATACCCTGGATACCAATGATATGCGTTTCGCCATCCCGCAGGGTTTTAACACCGGTGATCACTTTTTCACCTATCTGAAGGACGCATTCGATGTTTTGTACGAGGAGGGTGACGAAGCCCCTAAAATGCTGAGCATCGGCATGCACTGCCGTTTGCTGGGCCGCCCAGGTCGCTTCCGCGGTCTGCAAAAATTCCTGGACCACATCGAGAAGCACGATCGTGTGTGGGTTACGCGTCGTGTCGATATTGCTCGTCATTGGAGAGAGCAGCATCCGTTCGAGAAGGCGAACAACGGTACCGCCGCGCTCGAGCACCACCACCACCACCACTGA SEQ ID NO: 6 MSLDPNYPRDLIGYGRHPVQANWPGRARVAVQFVLNFEEGGENCVLHGDPGSEQFLSEIVGAAAYPSRMHMSMESIYEGSRAGVWRILREFEKRGMPLTIFGVGMAIERHPEVAKAFVELGHEIACHGYRWINYQDMSPEKEAEHMRLGMEAIERVTGVRPL GWYTGRDSPNTHRLVAEYGGFLYDSDYYGDDPLFWTEVEVTGGEKKPHLIVPYTLDTNDMRFAIPQGFNTGDHFFTYLKDAFDVLYEEGDEAPKMLSIGMHCRLLGRPGRFRGLQKFLDHIEKHDVRWVTRRVDIARHWREQHPFEKANNGTAALEHHHHHH SEQ ID NO: 7 MPLDPNYPRDLIGYGRHPVQANWPGRARVAVQFVLNFEEGGENCVLHGDPGSEQFLSEIVGAASFPARHMSMESIYEYGSRAGVWRILREFEKRKLPLTVFGVGMAVERHPELARAFVELGHEIACHGYRWINYQDMAPEKEAEHMRLGMEAIERVTGERPLGWYTGRDSPNTRRLVAEYGGFLYDSDYGDDLPFWMDVDVTGGAKVPQLIVPYTLDTNDMRFAIPQGFNTADHFFTYLRDAFDVLYEEGDEAPKMLSIGMHCRLLGRPGRFRALQRFLDHIEQHDRVWVTRRVDIARHWREHHPYQQNNRGAA SEQ ID NO: 8 MTKIIKNGTIVTATDTYEAHLLIKDGKIAMIGQNLEEKGAEVIDAKGCYVFPGGIDPHTHLDAPHGGTVTKDDFESGTIAAAFGGTTTIIDFCLTNKGEPLKKAIETWHNKANGAVIDYGFHLMISEITDDVLEELPKVLEEEGITSLKVFMAYKNVFQADDGTLYCTLLAAKELGALVMVHAENGDVIDYLTKKALADGNTDPIYHALTRPPELEGEATGRACQLTELAGSQLYVVHVTCAQAVEKIAEARNKGLDVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQTSFDFKGQKELGRGDFTKIPNGGPIIEDRVSILFSEGVKKGRITLNQFVDIVSTRIAKLFGLFPKKGTIVVGSDADLVIFDPNIERVISAETHHMAVDYNAFEMGVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYLKRAKYGTSTISKQSEELTI.

Claims

1. An allantoinase having the amino acid sequence shown in SEQ ID NO:

2.

2. An allantoinase having the amino acid sequence shown in SEQ ID NO:

4.

3. An allantoinase mutant having the amino acid sequence shown in SEQ ID NO:

6.

4. A nucleic acid molecule encoding the allantoinase of claim 1 or 2 or the allantoinase mutant of claim 3.

5. A recombinant vector comprising the nucleic acid molecule of claim 4.

6. A recombinant cell comprising the nucleic acid molecule of claim 4 and / or the recombinant vector of claim 5.

7. A method for preparing allantoinase, comprising: 1) The recombinant cells of claim 6 are cultured and expression of allantoinase as described in claim 1 or 2, or allantoinase mutant as described in claim 3 is induced; optionally, and 2) Isolate the allantoinase of claim 1 or 2 or the allantoinase mutant of claim 3 from the culture obtained in 1).

8. The allantoinase of claim 1 or 2, the allantoinase mutant of claim 3, the nucleic acid molecule of claim 4, the recombinant vector of claim 5, and / or the recombinant cell of claim 6 in the preparation of ( R Application in 3-isobutylglutaric acid monoamide and / or pregabalin.

9. A method for preparing ( R A method for producing 3-isobutylglutaric acid monoamide includes the following steps: Using allantoinase as described in claim 1 or 2, allantoinase mutant as described in claim 3, nucleic acid molecule as described in claim 4, recombinant vector as described in claim 5, and / or recombinant cell as described in claim 6 as catalysts, 3-isobutylglutarimide is hydrolyzed to obtain ( R )-3-isobutylglutaric acid monoamide.

10. The method according to claim 9, characterized in that: The feed for the hydrolysis reaction includes: manganese chloride, 3-isobutylglutarimide, and the catalyst; and / or The pH of the hydrolysis reaction is 6.0-9.0; and / or The hydrolysis reaction is carried out at a temperature of 20-50℃.

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