Cis-epoxysuccinate hydrolase mutants and uses thereof

Abstract

This invention relates to the field of bioengineering technology, specifically to a cis-epoxysuccinate hydrolase mutant and its applications. The cis-epoxysuccinate hydrolase mutant is obtained by single-point or multi-point mutations at positions 127 and 141 of the amino acid sequence of the wild-type cis-epoxysuccinate hydrolase shown in SEQ ID NO. 1. The single-point mutation involves replacing valine at position 127 with isoleucine, or replacing aspartic acid at position 141 with threonine. The cis-epoxysuccinate hydrolase mutant obtained by this invention exhibits high thermal stability, significantly improving the service life of the cis-epoxysuccinate hydrolase in the industrial production of D(-)-tartaric acid.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a cis-epoxysuccinate hydrolase mutant and its applications. Background Technology

[0002] D-(-)-tartaric acid, also known as (2S,3S)-2,3-dihydroxysuccinic acid, is the enantiomer of natural L-(+)-tartaric acid. Due to its scarcity in nature, industrial production via extraction is not feasible. As an important chiral building block, it is widely used in the synthesis of various active compounds, such as the antitumor natural product gonofufurone, the novel fast-acting influenza drug baloxavir, and C2-diamine compounds. Currently, the production methods for D-(-)-tartaric acid include chemical resolution, bioselective consumption, and biotransformation. Chemical resolution and bioselective consumption have limitations, such as unsatisfactory optical purity, cumbersome subsequent purification steps, and low theoretical yields. In contrast, biotransformation has become the most important synthetic route.

[0003] Biotransformation utilizes cis-epoxysuccinic acid hydrolase (CESH(D)) derived from microorganisms to directly and asymmetricly hydrolyze cis-epoxysuccinic acid (or its salts) to D(-)-tartaric acid (or its salts). This method offers mild reaction conditions, high stereoselectivity, excellent product yield and optical purity, and simple post-processing, demonstrating significant cost advantages and becoming the mainstream production process. Previous studies have reported the use of CESH(D) derived from strains such as *Alcaligenes* sp. (CN202010098416.7) and *Bordetella* sp. (CN200810074166.2) to achieve this transformation. However, the industrially applied CESH(D) has significant limitations. Its thermal stability is insufficient, and its enzyme activity drops sharply above 50°C (J Microbiol Biotechnol, 2010, 20(4): 659-665), making it difficult to adapt to the industrial production environment, which is usually 55-60°C. This seriously affects the catalytic efficiency and lifespan of the biocatalyst, increasing the cost in industrial production. Therefore, improving the thermal stability of CESH(D) is one of the most important issues in the production of D(-)-tartaric acid using biocatalysis. Artificial modification of enzyme molecules through directed evolution (such as error-prone PCR) and rational / semi-rational design has become an effective strategy to solve the above-mentioned defects in enzyme properties and has greatly accelerated the development of excellent industrial enzyme preparations. This invention uses "directed evolution" to modify CESH(D) (GenBank accession number EU053208) derived from Bordetella BK-52 to improve the thermal stability of CESH(D). Summary of the Invention

[0004] To address the technical problem of low thermal stability of cis-epoxysuccinate hydrolase, this invention provides a cis-epoxysuccinate hydrolase mutant and its application scheme. This invention provides a cis-epoxysuccinate hydrolase mutant with high thermal stability, which helps to improve the service life of cis-epoxysuccinate hydrolase in the industrial production of D(-)-tartaric acid.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a cis-epoxysuccinate hydrolase mutant, which is obtained by single-point or multi-point mutation of amino acids at positions 127 and 141 of the amino acid sequence shown in SEQ ID NO.1.

[0006] Furthermore, the cis-epoxysuccinate hydrolase mutant is one of the following: (1) The valine at position 127 of the amino acid sequence shown in SEQ ID NO.1 is replaced with isoleucine, and the amino acid sequence of this mutant is shown in SEQ ID NO. 3; (2) The aspartic acid at position 141 of the amino acid sequence shown in SEQ ID NO.1 is replaced with threonine, and the amino acid sequence of the mutant is shown in SEQ ID NO.4; (3) The amino acid sequence shown in SEQ ID NO.1 is substituted with isoleucine at position 127 and substituted with threonine at position 141. The amino acid sequence of this mutant is shown in SEQ ID NO.5.

[0007] In a second aspect, the present invention provides a gene encoding a cis-epoxysuccinate hydrolase mutant as described above.

[0008] Thirdly, the present invention provides a recombinant vector or recombinant cell containing the genes described above.

[0009] Fourthly, the present invention provides the use of a cis-epoxysuccinate hydrolase mutant, recombinant vector, or recombinant cell, as described above, in the preparation of D(-) tartaric acid or its salts.

[0010] Compared with the prior art, the present invention provides a cis-epoxysuccinate hydrolase mutant and its application, which has the following beneficial effects: Compared to wild-type cis-epoxysuccinate hydrolase, the obtained single-point mutants V127I and N141T, and the two-point combination mutant V127I / N141T of cis-epoxysuccinate hydrolase exhibit significantly enhanced thermal stability without affecting the catalytic performance of cis-epoxysuccinate hydrolase. Among them, the two-point combination mutant V127I / N141T is the best, not only maintaining high catalytic activity (enzyme activity of 19.45 U / g wet cells after incubation at 37°C), but also greatly improving thermal stability. After incubation at 45°C, 55°C, and 60°C, the residual activity is maintained at 85.26%, 71.13%, and 58.22%, respectively, which is the optimal mutant obtained in this patent.

[0011] The features and advantages of the present invention will be described in detail through embodiments and in conjunction with the accompanying drawings. Attached Figure Description

[0012] Figure 1 This is a plasmid map of the recombinant plasmid pET28a-CESH[D] in an embodiment of the present invention; Figure 2 This is a comparison curve of the catalytic activity (U / g wet cells) of wild-type cis-epoxysuccinate hydrolase and its mutants after treatment at different temperatures in this embodiment of the invention. Figure 3 This is a bar chart comparing the residual enzyme activity (%) of wild-type cis-epoxysuccinate hydrolase and its mutants after different temperature treatments in this embodiment of the invention. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. The technical features of various embodiments in this invention can be combined appropriately without conflict.

[0014] The culture medium of this invention is as follows: (1) LB medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, solid medium with 2% agar powder, sterilized at 121℃.

[0015] (2) Synthetic culture medium: 20 g / L glycerol, 2.5 g / L citric acid monohydrate, 4 g / L diammonium hydrogen phosphate, 11 g / L potassium dihydrogen phosphate, pH adjusted to 6.5 with ammonia water, sterilized at 121℃.

[0016] Preparation of 1% ammonium metavanadate solution: Dissolve 108 g of sodium acetate in 400 mL of water to obtain sodium acetate solution; dissolve 10 g of ammonium metavanadate in 300 mL of 1M sodium hydroxide solution, then add sodium acetate solution and bring the volume to 1 L.

[0017] The detection methods for the corresponding parameters are described in the following embodiments: Enzyme activity assay: 0.9 mL of 1 mol / L sodium cis-epoxysuccinate solution (pH 7.0) was incubated at 37℃ for 5 min, followed by the addition of 0.1 mL of crude enzyme solution. The reaction was carried out at 37℃ for 1 h, and the tartaric acid content in the reaction solution was determined. Under the above reaction conditions, the amount of enzyme required to generate 1 mmol of tartaric acid per hour is defined as one enzyme activity unit, denoted by U.

[0018] The method for detecting tartaric acid content is as follows: Take 2.5 mL of 1% ammonium metavanadate solution into a 25 mL volumetric flask, add an appropriate amount of the above reaction solution, then add 1 mL of 1 mol / L sulfuric acid, and dilute to 25 mL with distilled water. After mixing, measure the absorbance at 480 nm and calculate the tartaric acid concentration according to the established standard curve.

[0019] Unless otherwise specified, the molecular biology experimental methods in the following examples were performed under standard conditions, referring to the conditions described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001).

[0020] Example 1: Synthesis of cis-epoxysuccinate hydrolase gene and construction of recombinant bacteria Based on the amino acid sequence of cis-epoxysuccinate hydrolase (CESH(D)) from Bordetella BK-52 (GenBank accession number EU053208, SEQ ID NO.1), the gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO. 2. The CESH(D) fragment was amplified using primers SEQ ID NO. 6 and SEQ ID NO. 7. pET-28b(+) was double-digested with Nde I and BamHI restriction endonucleases to obtain the pET-28b(+) plasmid backbone. The CESH(D) fragment was ligated to the linearized pET-28b(+) plasmid backbone using seamless cloning to construct the recombinant plasmid pET28a-CESH[D], as shown below. Figure 1 As shown, it was transformed into Escherichia coli BL21(DE3) to obtain recombinant bacteria containing CESH(D) from Bordetella BK-52.

[0021] Example 2: Directed Evolution of the CESH(D) Gene The recombinant plasmid pET28a-CESH[D], which is linked to the wild-type CESH(D) gene from Bordetella BK-52, was extracted and used to introduce replication errors into polymerase chain reaction PCR by adding divalent manganese ions, thus obtaining error-prone PCR product fragments.

[0022] The reaction system for the error-prone PCR reaction is as follows: 25 μL 2×Taq Master Mix (Vazyme), 2 μL upstream primer (SEQ ID NO.8), 2 μL downstream primer (SEQ ID NO.9), 1 μL template DNA (20 ng / μL), 1 μL 5mMnCl2 and 19 μL double-distilled water.

[0023] The error-prone PCR program is: 95℃ for 15 s, 55℃ for 15 s, 72℃ for 60 s (30 cycles).

[0024] The purified mutant fragment was double-digested with Nde I and Xba I restriction endonucleases, and then ligated to the pET-28b(+) plasmid backbone, which was also double-digested with Nde I and Xba I restriction endonucleases, using T4 ligase. The plasmid was then transformed into E. coli BL21(DE3), plated on LB agar plates containing 50 μg / mL kanamycin, and incubated at 37°C for 12 h to construct a mutant library.

[0025] Single recombinant bacteria carrying the mutant were picked and placed into 96-well plates (plate A) containing 300 μL LB medium with 50 μg / mL kanamycin. The plates were incubated at 37°C with shaking for 6 h. Then, isopropyl thio-β-D-galactoside (IPTG) was added to a final concentration of 1 mM, and the plates were incubated at 28°C with shaking for 12 h to obtain the mutant expression library. 50 μL of bacterial culture from the mutant expression library was transferred to a new 96-well plate, and 50 μL of 1 M cis-epoxysuccinate solution (pH 7.0) was added. The plates were incubated at 50°C with shaking for 1 h. The plates were then immediately placed on ice to allow them to return to room temperature. 25 μL of 1% ammonium metavanadate solution, 10 μL of 1 M sulfuric acid, and 100 μL of water were added to each well. The OD480 value was then measured using a microplate reader. Using wild-type CESH(D) (amino acid sequence as shown in SEQ ID NO.1) as a control, mutants with higher enzyme activity were selected. Plasmids containing the corresponding mutants were extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to determine the mutation sites.

[0026] Two CESH(D) mutants were obtained through screening. In mutant V127I, the codon at position 127 encoding valine (GTG) was mutated to the codon encoding isoleucine (ATT). In mutant N141T, the codon at position 141 encoding aspartic acid (AAC) was mutated to the codon encoding threonine (ACT).

[0027] Example 3: Preparation of two-point mutants Site-directed mutagenesis primers V127I-F (SEQ ID NO.10) and V127I-R (SEQ ID NO.11) were designed. Using N141T as a template, PCR amplification was performed using PrimeSTAR® Max DNA Polymerase (Takara). The purified PCR fragment was transformed into E. coli BL21(DE3), and bacteria were picked, cultured, plasmids were extracted, and sequenced for verification. Plasmids carrying the double-point mutant V127I / N141T and their recombinant expression bacteria were obtained.

[0028] Example 4: Induction and expression of cis-epoxysuccinate hydrolase and determination of crude enzyme activity The recombinant bacteria from Example 1, the recombinant bacteria containing the V127I and N141T single-point mutants from Example 2, and the recombinant bacteria containing the V127I / N141T mutant from Example 3 were inoculated into 50 mL of synthetic medium containing 50 mg / mL kanamycin and cultured in a shaker at 37°C. When the bacterial cell concentration (OD600) reached 1.1-1.2, 50 μL of 0.1 M IPTG was added for induction, and the culture was carried out in a shaker at 28°C for 18 h. 4 mL of the induced bacterial culture was collected by centrifugation at 4500 rpm for 5 min at 4°C. Different volumes of pure water were added for dilution to achieve an OD600 of 6 in the diluted solutions. The wild-type cis-epoxysuccinate hydrolase (from the recombinant strain in Example 1), V127I single-point mutant enzyme, N141T single-point mutant enzyme, and V127I / N141T combined mutant enzyme were obtained by sonication for 5 min using a cell disruptor.

[0029] Add 100 μL of cell lysis buffer to 900 μL of 1 M sodium epoxysuccinate solution, invert and mix 10 times, and react in a 37°C water bath for 1 h. After the reaction is complete, take 2.5 mL of 1% ammonium metavanadate solution into a 25 mL volumetric flask, add 250 μL of the above reaction solution, then add 1 mL of 1 mol / L sulfuric acid, and dilute to 25 mL with distilled water. Mix well, develop color at room temperature for 5 min, measure the absorbance at 480 nm, and calculate the tartaric acid concentration according to the established standard curve.

[0030] Example 5: Thermal stability analysis of wild-type cis-epoxysuccinate hydrolase and its mutants The cell lysis solutions of wild-type cis-epoxysuccinate hydrolase, single-point mutants V127I and N141T, and the combined mutant V127I / N141T obtained in Example 4 were incubated at 37°C, 45°C, 55°C, and 60°C for 30 minutes, respectively. Residual enzyme activity was then measured according to the method described in Example 4. Figure 2 As shown, the relative residual activity after each temperature treatment was calculated, as follows: Figure 3 As shown.

[0031] The results showed that the single-point mutant V127I exhibited high enzyme activity after incubation at 37°C and 45°C, reaching 22.46 U / g wet cells and 19.51 U / g wet cells, respectively, which were 1.14 times and 1.24 times that of the wild type. However, after treatment at 55°C and 60°C, its enzyme activity decreased sharply, and the trend of thermal inactivation was basically consistent with that of the wild type. The single-point mutant N141T, after incubation at 37°C, showed a decrease in enzyme activity to 15.49 U / g wet cells, but its thermostability was significantly enhanced. After incubation at 45°C, 55°C, and 60°C, the residual activity remained at 95.69%, 81.76%, and 64.19%, respectively, while the wild type retained only 80.09%, 38.49%, and 22.13% at the corresponding temperatures. The combined mutant V127I / N141T combines the advantages of two single-point mutations, maintaining not only high catalytic activity (enzyme activity of 19.45 U / g wet cells after incubation at 37°C) but also significantly improved thermal stability. After incubation at 45°C, 55°C, and 60°C, the residual activity remained at 85.26%, 71.13%, and 58.22%, respectively, making it the optimal mutant obtained in this patent.

[0032] SEQUENCE LISTING SEQ ID NO.1 MTRTKLILEARINEYMPRRGNPHVPWTPKEIGEAAAQAREAGASIVHFHARQADGSPSHDYETYAESIREIRARSDVLVHPTLGQITLGGRESRLAHIERLCLDPALKPDFAPVDLGSTNIDRYDDVEKRYETGDRVYLNNIDTLQH FSKRLRELGVKPAFIAWTVPFTRTLDAFMDMGLVDDPAYLLFELTDCGIRGGHPGTIRGLRAHTDFLPPGRQIQWTVCNKIGNLFGPAAAAIEEGGHVAIGLGDYLYPELGTPTNGEVVQTVANMARAMGREIATPAETKEILGISN SEQ ID NO.2 ATGACTCGAACCAAGTTGATACTTGAAGCTCGTATAAATGAATACATGCCGCGTCGCGGTAATCCCCATGTGCCATGGACGCCAAAGGAGATCGGTGAGGCTGCGGCACAGGCACGGGAAGCGGGTGCATCGATTGTCCATTTCCATGCCCGTCAAGCTGACGGTTCTCCCAGCCATGACTATGAAACTTATGCAGAATCGATCCGTGAGATTCGCGCACGCAGTGACGTTCTAGTACATCCGACACTGGGCCAGATCACGCTTGGAGGGAGGGAATCACGACTGGCACACATTGAGCGACTGTGCCTTGACCCAGCACTAAAACCGGACTTTGCACCGGTAGACCTGGGTAGCACGAACATCGATCGTTATGACGATGTGGAGAAGCGCTACGAGACAGGCGACCGCGTGTACTTGAACAACATCGACACGCTGCAGCACTTCAGCAAGAGACTACGAGAACTCGGGGTTAAGCCTGCGTTTATCGCTTGGACGGTCCCCTTCACCCGAACACTTGACGCCTTTATGGATATGGGTTTGGTCGACGACCCGGCATATCTGCTGTTTGAGCTCACCGACTGCGGCATCCGCGGTGGACATCCGGGAACGATACGGGGGCTGCGTGCACATACCGACTTCCTGCCACCCGGACGACAGATCCAATGGACCGTCTGCAACAAGATCGGCAACCTATTTGGCCCTGCTGCTGCGGCCATTGAGGAAGGAGGACACGTCGCAATTGGCCTAGGCGATTACCTCTATCCGGAATTGGGTACACCCACCAATGGGGAAGTCGTTCAGACCGTAGCGAATATGGCACGTGCGATGGGTCGTGAGATTGCAACGCCAGCAGAGACAAAGGAAATTCTGGGTATTAGCAACTAA SEQ ID NO.3 MTRTKLILEARINEYMPRRGNPHVPWTPKEIGEAAAQAREAGASIVHFHARQADGSPSHDYETYAESIREIRARSDVLVHPTLGQITLGGRESRLAHIERLCLDPALKPDFAPVDLGSTNIDRYDDIEKRYETGDRVYLNNIDTLQHFSKRLRELGVKPAFIAWTVPFTRTLDAFMDMGLVDDPAYLLFELTDCGIRGHPGTIRGLRAHTDFLPPGRQIQWTVCNKIGNLFGPAAAIEEGGHVAIGLGDYLYPELGTPTNGEVVQTVANMARAMGREIATPAETKEILGISN SEQ ID NO.4 MTRTKLILEARINEYMPRRGNPHVPWTPKEIGEAAAQAREAGASIVHFHARQADGSPSHDYETYAESIREIRARSDVLVHPTLGQITLGGRESRLAHIERLCLDPALKPDFAPVDLGSTNIDRYDDVEKRYETGDRVYLNTIDTLQHFSKRLRELGVKPAFIAWTVPFTRTLDAFMDMGLVDDPAYLLFELTDCGIRGHPGTIRGLRAHTDFLPPGRQIQWTVCNKIGNLFGPAAAIEEGGHVAIGLGDYLYPELGTPTNGEVVQTVANMARAMGREIATPAETKEILGISN SEQ ID NO.5 MTRTKLILEARINEYMPRRGNPHVPWTPKEIGEAAAQAREAGASIVHFHARQADGSPSHDYETYAESIREIRARSDVLVHPTLGQITLGGRESRLAHIERLCLDPALKPDFAPVDLGSTNIDRYDDIEKRYETGDRVYLNTIDTLQHFSKRLRELGVKPAFIAWTVPFTRTLDAFMDMGLVDDPAYLLFELTDCGIRGHPGTIRGLRAHTDFLPPGRQIQWTVCNKIGNLFGPAAAIEEGGHVAIGLGDYLYPELGTPTNGEVVQTVANMARAMGREIATPAETKEILGISN SEQ ID NO.6 AACTTTAAGAAGGAGATATACATATGATGACTCGAACCAAGTTGATACTTG SEQ ID NO.7 TGTCGACGGAGCTCGAATTCGGATCCTTAGTTGCTAATACCCAGAATTTCCT SEQ ID NO.8 CGACTCACTATAGGGGAATTGTGA SEQ ID NO.9 TTAGCAGCCGGATCTCAGTG SEQ ID NO.10 GTAGCGCTTCTCAATATCGTCATAACGATCGATGTTCGT SEQ ID NO.11 CCTGTCTCGTAGCGCTTCTCAAT The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cis-epoxysuccinate hydrolase mutant, characterized in that: The cis-epoxysuccinate hydrolase mutant is obtained by single-point or multi-point mutation of amino acids at positions 127 and 141 of the amino acid sequence shown in SEQ ID NO.

1.

2. The cis-epoxysuccinate hydrolase mutant according to claim 1, characterized in that: The cis-epoxysuccinate hydrolase mutant is one of the following: (1) The valine at position 127 of the amino acid sequence shown in SEQ ID NO.1 is replaced with isoleucine, and the amino acid sequence of this mutant is shown in SEQ ID NO. 3; (2) The aspartic acid at position 141 of the amino acid sequence shown in SEQ ID NO.1 is replaced with threonine, and the amino acid sequence of the mutant is shown in SEQ ID NO.4; (3) The amino acid sequence shown in SEQ ID NO.1 is substituted with isoleucine at position 127 and substituted with threonine at position 141. The amino acid sequence of this mutant is shown in SEQ ID NO.

5.

3. A gene encoding a cis-epoxysuccinate hydrolase mutant as described in claim 1.

4. A recombinant vector or recombinant cell comprising the gene as described in claim 3.

5. The use of the cis-epoxysuccinate hydrolase mutant of claim 1, the recombinant vector or recombinant cell of claim 4 in the preparation of D(-) tartaric acid or its salt.

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