A nitrilase and its use in methionine synthesis

Abstract

The present application relates to the technical field of biological catalysis, and particularly relates to a nitrilase and its use in methionine synthesis. The present application provides a protein selected from any one of the following: (1) compared with the amino acid sequence shown in SEQ ID NO. 2, the protein comprises the following mutation sites: F188A and Q193Y; (2) the protein has at least 80% or more identity with the protein in (1) and retains the mutation sites F188A and Q193Y, and the obtained protein has nitrilase activity; the protein as the nitrilase mutant ScNLE-F188A / Q193Y can efficiently catalyze 2-amino-4-methylthiobutyronitrile to generate 2-amino-4-methylthiobutyric acid, and the production efficiency is as high as 73.9 kg methionine / kg DCW / h, which provides a potential efficient biological catalyst for industrial production of methionine by biological enzyme method.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, and in particular to a nitrilase and application thereof in methionine synthesis. Background Art

[0002] Methionine, also known as 2-amino-4-methylthiobutyric acid, is an amino acid, the building block of protein. In animals, methionine is classified as an essential amino acid, meaning it cannot be synthesized naturally and must be obtained through the diet. It is the only essential amino acid that contains sulfur and plays a vital role in animal growth and development. In the livestock industry, especially poultry farming, methionine is often a key growth factor, as it is insufficient in common feed ingredients such as corn and soybean meal. Therefore, additional methionine is often added to poultry feed to meet their nutritional needs.

[0003] The main methods for methionine synthesis include chemical synthesis, microbial fermentation, and enzyme catalysis. Although chemical synthesis is a mature process, it is subject to significant environmental pollution, high energy consumption, and numerous byproducts. Microbial fermentation also suffers from low production yields, making it unsuitable for industrial production. Enzyme catalysis, however, has gradually become the mainstream method for methionine synthesis due to its high efficiency, environmental friendliness, and high specificity.

[0004] Caoda and Rhone-Poulenc have both studied the process of hydrolyzing 2-amino-4-methylthiobutyric acid using nitrilase, as shown in Formula (I). The production efficiency of nitrilase biocatalysts derived from sources such as Alcaligenes faecalis, Gordon terrae, and Rhodococcus sp. is only 0.6-0.9 kg. 蛋氨酸 / kg DCW Patent document CN115851684A discloses a novel nitrilase ScNLE and its mutant ScNLE-F188A, which catalyzes the hydrolysis of 2-amino-4-methylthiobutyronitrile to produce methionine with a maximum production efficiency of 29.8 kg. 蛋氨酸 / kg DCW / h. This significant increase in production efficiency is primarily due to the unique enzymatic properties of ScNLE and its mutant, ScNLE-F188A. Compared to traditional nitrilases, ScNLE and its mutants exhibit higher substrate affinity and catalytic activity, enabling them to complete more catalytic reactions in a shorter time.

[0005]

[0006] Although the hydrolysis reaction efficiency of the key enzyme nitrilase, ScNLE-F188A, has been improved in the aforementioned biocatalytic production of methionine using nitrilase, further improving the reaction efficiency remains a primary goal for industrial methionine production. To overcome this challenge, the present invention is directed toward improving and optimizing nitrilase. By using genetic engineering techniques to perform directed evolution of nitrilase, mutants with higher catalytic activity and stability are screened, thereby significantly increasing the efficiency of methionine production. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a nitrilase and its use in methionine synthesis, wherein the nitrilase catalyzes the synthesis of methionine (i.e., 2-amino-4-methylthiobutyric acid) with higher production efficiency.

[0008] To this end, the present invention provides the following technical solutions:

[0009] The present invention provides a protein selected from any one of the following:

[0010] (1) Compared with the amino acid sequence shown in SEQ ID NO. 2, the protein includes the following mutation sites: F188A and Q193Y;

[0011] (2) A protein having nitrilase activity obtained by having at least 80% identity with the protein described in (1) and retaining the mutation sites F188A and Q193Y.

[0012] The present invention provides a nucleic acid molecule encoding the protein.

[0013] The present invention provides a recombinant vector comprising the nucleic acid molecule.

[0014] The present invention provides a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

[0015] The present invention provides a method for preparing a protein, comprising:

[0016] 1) culturing the recombinant cells and inducing expression of the protein;

[0017] 2) Isolating the protein from the culture obtained in 1).

[0018] The present invention provides a nitrilase, comprising the protein, the nucleic acid molecule, the recombinant vector, the recombinant cell or the protein prepared by the protein preparation method.

[0019] The present invention provides use of the nitrilase in preparing methionine or catalyzing the hydrolysis of nitrile.

[0020] The invention provides a method for preparing methionine, comprising: using the nitrilase as a catalyst to catalyze the hydrolysis reaction of a substrate 2-amino-4-methylthiobutyronitrile.

[0021] Optionally, the pH of the hydrolysis reaction is 5-9;

[0022] And / or, the temperature of the hydrolysis reaction is 30-50°C.

[0023] Optionally, the final concentration of the substrate 2-amino-4-methylthiobutyronitrile is 30-300 mM.

[0024] The technical solution of the present invention has the following advantages:

[0025] 1. The present invention provides a protein selected from any one of the following: (1) compared with the amino acid sequence shown in SEQ ID NO. 2, the protein includes the following mutation sites: F188A and Q193Y; (2) having at least 80% identity with the protein described in (1) and retaining the mutation sites F188A and Q193Y, thereby obtaining a protein having nitrilase activity; the above protein as the nitrilase mutant ScNLE-F188A / Q193Y can efficiently catalyze 2-amino-4-methylthiobutyronitrile to produce 2-amino-4-methylthiobutyric acid with a production efficiency of up to 73.9 kg methionine / kgDCW / h, providing a potential high-efficiency biocatalyst for the industrial production of methionine by bioenzymatic method.

[0026] 2. The present invention provides a method for preparing methionine, comprising: using nitrilase (ScNLE-F188A / Q193Y) as a catalyst to catalyze the hydrolysis reaction of the substrate 2-amino-4-methylthiobutyronitrile. The reaction conditions are mild, easy to control, and the production efficiency is high, which has broad application prospects.

[0027] Furthermore, in the reaction system, the substrate 2-amino-4-methylthiobutyronitrile is added to a buffer solution containing nitrilase, and the efficient reaction can be ensured by controlling parameters such as pH value and temperature. DETAILED DESCRIPTION

[0028] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0029] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0030] The hydrolysis reaction efficiency of the existing key enzyme nitrilase is still insufficient to meet the needs of industrialized methionine production. Therefore, the present invention improves and optimizes nitrilase. By using genetic engineering techniques to perform directed evolution of nitrilase, mutants with higher catalytic activity and stability are screened, thereby significantly improving the production efficiency of methionine.

[0031] In a first aspect, an embodiment of the present invention discloses a protein selected from any one of the following:

[0032] (1) Compared with the amino acid sequence shown in SEQ ID NO. 2, the protein includes the following mutation sites: F188A and Q193Y;

[0033] (2) A protein having nitrilase activity obtained by having at least 80% identity with the protein described in (1) and retaining the mutation sites F188A and Q193Y.

[0034] The amino acid sequence shown in SEQ ID NO.2 is the original sequence of the nitrilase ScNLE from Shewanella corallii (its nucleotide sequence is shown in SEQ ID NO:1). The protein provided by the present invention is obtained by site-directed mutagenesis of the wild-type nitrilase ScNLE (SEQ ID NO.2) by introducing F188A (mutating phenylalanine at position 188 to alanine) and Q193Y (mutating glutamine at position 193 to tyrosine). The amino acid sequence of the protein is shown in SEQ ID NO.6. The protein, as the nitrilase mutant ScNLE-F188A / Q193Y, has high catalytic activity and can efficiently catalyze the hydrolysis of 2-amino-4-methylthiobutyronitrile to produce methionine.

[0035] In some embodiments, the protein may be a protein having nitrilase activity, wherein the protein has at least 80% identity to the amino acid sequence of SEQ ID NO. 6 and retains the mutations F188A and Q193Y. The identity to the amino acid sequence of SEQ ID NO. 6 may be at least 80%, 83%, 85%, 88%, 90%, 93%, 95%, 97%, 99%, 99.1%, 99.2%, or 99.3%. In a preferred embodiment, the protein is SEQ ID NO. 6 or an amino acid sequence having at least 90%, more preferably 95%, or even more preferably at least 99% identity thereto.

[0036] In some embodiments, the enzyme of the present invention can be substituted, deleted, and / or added to the amino acid sequence of SEQ ID NO. 6, while retaining the mutation sites F188A and Q193Y, to obtain a protein having nitrilase activity. The number of amino acids substituted, deleted, or added can be any number, such as 1, 5, 10, 15, or more, such that the sequence identity of the modified amino acid sequence to its respective corresponding original sequence is 80%, 83%, 85%, 88%, 90%, 93%, 95%, 97%, 99%, 99.1%, 99.2%, or 99.3% or more.

[0037] As defined herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences.

[0038] In certain embodiments, the above-mentioned proteins can be obtained by artificial synthesis, or by synthesizing their encoding genes and then preparing them through a biological expression process, for example, expressing them from prokaryotes (such as Escherichia coli) using recombinant technology.

[0039] In some embodiments, the above-mentioned protein is obtained by transforming a recombinant vector containing its encoding gene into an Escherichia coli expression host (such as E. coli BL21 (DE3)) to construct a recombinant genetically engineered bacterium, then culturing the strain and adding an inducer to induce expression.

[0040] In a second aspect, an embodiment of the present invention provides a nucleic acid molecule encoding the protein.

[0041] As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably and include DNA molecules or RNA molecules. DNA molecules can be single-stranded or double-stranded.

[0042] Given the redundancy of the genetic code, numerous nucleic acid sequences exist that can be used to encode the proteins of the present invention. Therefore, once a specific amino acid sequence has been determined, those skilled in the art can produce any number of different nucleic acids by simply modifying one or more codons without altering the amino acid sequence of the encoded protein. Codon optimization can be used to select more preferred polynucleotides based on the preferences of the host cell used in the actual production process.

[0043] The nucleic acid molecules can be obtained by conventional methods, such as PCR amplification or artificial synthesis, etc. Currently, the polynucleotide sequences can be obtained completely by chemical synthesis.

[0044] In some preferred embodiments, but not limiting, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.5.

[0045] In a third aspect, an embodiment of the present invention provides a recombinant vector comprising the nucleic acid molecule.

[0046] In some embodiments, the recombinant vector may further comprise regulatory elements, such as a promoter, a terminator, and a selection marker, to regulate the expression of the nucleic acid molecule.

[0047] In some embodiments, the recombinant vector is a plasmid vector.

[0048] In some preferred embodiments, the recombinant vector is capable of stably replicating and efficiently expressing the nucleic acid molecule in a host cell. The construction of the recombinant vector can be achieved by conventional molecular biology techniques, for example, cloning the nucleic acid molecule into an appropriate vector and transforming it into a host cell for screening and identification.

[0049] In some preferred embodiments, but not limiting, the recombinant vector can be constructed by pET-26b(+) to obtain the recombinant vector pET-26b(+)-ScNLE-F188A / Q193Y.

[0050] In a fourth aspect, an embodiment of the present invention provides a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

[0051] In some embodiments, the induced recombinant cells are capable of expressing the above-mentioned proteins.

[0052] In some embodiments, the method for constructing the recombinant cell comprises the following steps:

[0053] The recombinant vector is transformed into an expression host cell, and the expression is induced by culturing and applying an inducer to obtain the above protein.

[0054] Furthermore, the recombinant vector refers to any of the above-mentioned types of recombinant vectors, and the expression host cell is a conventional host cell in the art. The key is that the recombinant vector can stably replicate itself and the gene it carries can be effectively expressed. These host cells can be prokaryotic cells or eukaryotic cells, such as Escherichia coli and yeast, among which E. coli BL21 (DE3) is the preferred expression host.

[0055] In some preferred embodiments, but not limiting, the recombinant cell is E. coli BL21(DE3) / pET-26b(+)-ScNLE-F188A / Q193Y, which is a recombinant genetically engineered bacterium obtained by transforming the recombinant vector pET-26b(+)-ScNLE-F188A / Q193Y into E. coli BL21(DE3). The culture medium used for protein expression in the recombinant genetically engineered bacterium can be any culture medium known in the art that can grow the recombinant genetically engineered bacterium and express the nitrilase mutant of the present invention, such as LB culture medium.

[0056] The culture process of recombinant cells does not require special conditions. It is only necessary to ensure that the genetically engineered strain can grow normally and induce the expression of the target protein at an appropriate temperature. The recommended culture steps are as follows: First, inoculate the recombinant strain E. coli BL21 (DE3) / pET-26b (+) -ScNLE-F188A / Q193Y into a test tube containing LB liquid culture medium containing kanamycin, and then culture it at 30-37 ° C and 100-300 rpm for 10-14 hours. The commonly used parameters are 37 ° C and 220 rpm for 12 hours. Then, transfer the bacterial liquid to a 500mL triangular shake flask containing 50-150mL (common parameters are 100ml) of LB liquid culture medium containing kanamycin with an inoculum volume of 1-2% (v / v), and continue to culture it at 30-37 ° C (commonly 37 ° C) and 100-300 rpm (commonly 220 rpm). When the OD of the culture medium reaches 0. 600 When the value reaches 0.6-0.8, 100-500 μM isopropyl β-D-thiogalactopyranoside (IPTG) is added as an inducer, followed by induction at 16-25°C (usually 16°C) for 16-24 hours. Finally, the bacterial precipitate is collected by centrifugation and washed with physiological saline to obtain recombinant cells.

[0057] In a fourth aspect, an embodiment of the present invention provides a method for preparing a protein, comprising:

[0058] 1) culturing the recombinant cells and inducing expression of the protein;

[0059] 2) Isolating the protein from the culture obtained in 1).

[0060] Among them, the methods of culturing and inducing recombinant cells and the methods of isolating proteins from the culture are conventional methods in the art.

[0061] In a fifth aspect, an embodiment of the present invention provides a nitrilase, comprising the protein, the nucleic acid molecule, the recombinant vector, the recombinant cell, or the protein prepared by the protein preparation method.

[0062] In some embodiments, the specific use form of the nitrilase depends on the requirements of the application scenario. For example, in the form of engineered bacteria whole cells, the nitrilase can directly participate in the reaction, without the need for complicated separation and purification steps, thus simplifying the operating process. In the form of crude enzyme or partially purified enzyme, the concentration of the enzyme can be increased to a certain extent, thereby improving the reaction efficiency. For completely purified enzymes, higher catalytic activity and selectivity can be obtained, which is suitable for occasions with higher product purity requirements. In addition, immobilized enzymes or immobilized cells prepared by immobilization technology can not only improve the stability of the enzyme, but also can be easily recycled and reused, reducing production costs.

[0063] In a sixth aspect, an embodiment of the present invention provides use of the nitrilase in preparing methionine or catalyzing the hydrolysis of nitrile.

[0064] In some embodiments, it is specifically used to catalyze the hydrolysis of 2-amino-4-methylthiobutyronitrile to produce methionine.

[0065] In a seventh aspect, an embodiment of the present invention provides a method for preparing methionine, comprising: using the nitrilase as a catalyst to catalyze the hydrolysis reaction of the substrate 2-amino-4-methylthiobutyronitrile.

[0066] In some embodiments, the temperature of the hydrolysis reaction is 30-50°C, such as 30°C, 40°C, 50°C, or a value or range between any two of these values, preferably 40°C.

[0067] In some embodiments, in any of the above methods, the pH of the hydrolysis reaction is 5-9, such as pH 5, pH 6, pH 7, pH 8, pH 9, or a value or range between any two of these values, wherein pH 8 is preferred;

[0068] The reaction materials include: 2-amino-4-methylthiobutyronitrile and ammonia;

[0069] The final concentration of 2-amino-4-methylthiobutyronitrile is 30-300 mM, for example, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, or a value or range between any two of these values, preferably 300 mM.

[0070] The amount of ammonia added is 1.5-10 equivalents of the theoretical yield of 2-amino-4-methylthiobutyric acid, for example, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 equivalents, preferably 3 equivalents.

[0071] In some embodiments, in any of the above methods, the buffer used in the hydrolysis reaction is a conventional buffer in the art, such as Tris-HCl buffer, and its concentration is preferably 50-300 mM, more preferably 100 mM.

[0072] In some embodiments, any of the above methods comprises using any of the above recombinant cells to catalyze the hydrolysis of 2-amino-4-methylthiobutyronitrile to produce 2-amino-4-methylthiobutyric acid;

[0073] Specifically, lyophilized cells or lyophilized powders of the aforementioned recombinant cells can be used as catalysts to achieve whole-cell catalytic production of 2-amino-4-methylthiobutyric acid. In this process, the dosage of lyophilized cells or lyophilized powder ranges from 0.005 to 0.05 g / g of 2-amino-4-methylthiobutyronitrile. For example, the dosage can be 0.005, 0.01, 0.02, 0.03, 0.04, or 0.05 g / g of 2-amino-4-methylthiobutyronitrile, or any combination or range therebetween. In particular, 0.01 g / g of 2-amino-4-methylthiobutyronitrile is a recommended dosage. The lyophilized powder is produced by disrupting any of the aforementioned recombinant cells to obtain a cell disrupted liquid, which is then freeze-dried. Lyophilized cells are obtained by directly freeze-drying the recombinant cells.

[0074] It should be understood that the nitrilase mutant ScNLE-F188A / Q193Y of the present invention can be used in the form of whole engineered bacterial cells, unpurified crude enzyme, partially purified enzyme, or completely purified enzyme. The nitrilase mutant ScNLE-F188A / Q193Y of the present invention can also be prepared as an immobilized enzyme or immobilized cell catalyst using immobilization techniques known in the art.

[0075] In some embodiments, in any of the above methods, the hydrolysis reaction is carried out under stirring or shaking, for example, under stirring at 100-500 rpm.

[0076] Shewanella corallii was obtained from the German Collection of Microorganisms (DSMZ) with the strain collection number DSM 21332.

[0077] pET-26b(+) is a product of Novagen, with the catalog number being 69862-3CN.

[0078] The sequences involved in the following examples are shown in the table below:

[0079] Table 1. Sequence

[0080]

[0081]

[0082] The underlines in SEQ ID NO. 7 to 8 in the above table correspond to the mutation sites of F188A and Q193Y.

[0083] Example 1 Construction of recombinant vector pET-26b(+)-ScNLE

[0084] This embodiment provides a method for constructing a recombinant vector pET-26b(+)-ScNLE, comprising the following steps:

[0085] (1) Extract genomic DNA of Shewanella corallii DSM 21332.

[0086] (2) Using the genomic DNA of Shewanella corallii DSM 21332 in step (1) as a template and ScNLE-FP (SEQ ID NO: 3) and ScNLE-RP (SEQ ID NO: 4) as primers, PCR amplification was performed to obtain a fragment containing the nitrilase gene ScNLE, wherein the nucleotide sequence of the nitrilase gene is shown in SEQ ID NO: 1, and the amino acid sequence of the nitrilase ScNLE encoded thereby is shown in SEQ ID NO: 2.

[0087] (3) The gene fragment was obtained by using EcoRI and HindIII to double-enzyme cut the fragment containing the nitrilase ScNLE gene obtained in step (2); the vector fragment was obtained by double-enzyme cutting pET-26b(+); the enzyme-cut gene fragment and the vector fragment were connected to obtain a recombinant expression plasmid, which was named pET-26b(+)-ScNLE, and the plasmid was sent for sequencing, and the result was consistent with the expectation.

[0088] Example 2 Construction of mutant recombinant vector pET-26b(+)-ScNLE-F188A / Q193Y

[0089] The present example provides a method for constructing a recombinant vector pET-26b(+)-ScNLE-F188A / Q193Y, comprising the following steps:

[0090] The recombinant vector pET-26b(+)-ScNLE constructed in Example 1 was used as a template, and ScNLE-F188A / Q193Y-FP (SEQ ID NO: 7) and ScNLE-F188A / Q193Y-RP (SEQ ID NO: 8) were used as primers for whole plasmid PCR to introduce site-directed mutations, and the PCR reaction system was as follows:

[0091] Table 2, PCR reaction system

[0092] Components Concentration / dosage volume pET-26b(+)-ScNLE 100ng 1 μL ScNLE-F188A / Q193Y-FP 10 pmol of each primer 1 μL ScNLE-F188A / Q193Y-RP 10 pmol of each primer 1 μL PrimeSTAR Max Premix (2x) (commercially available) 2x 25 μL <![CDATA[ddH2O]]> / 22 μL

[0093] The PCR reaction program was as follows: 95℃ for 5min; 98℃ for 10sec, 55℃ for 5sec, 72℃ for 35sec, 30 cycles; 72℃ for 5min; 4℃ for storage.

[0094] The mutant recombinant expression plasmid pET-26b(+)-ScNLE-F188A / Q193Y was obtained, and the plasmid was sent for sequencing, and the result was consistent with the expectation, wherein the nucleotide sequence of the nitrilase ScNLE mutant gene is shown as SEQ ID NO: 5, and the amino acid sequence of the nitrilase mutant ScNLE-F188A / Q193Y encoded thereby is shown as SEQ ID NO: 6.

[0095] Example 3 Construction of mutant ScNLE-F188A / Q193Y expression strain

[0096] The mutant recombinant expression plasmid pET-26b(+)-ScNLE-F188A / Q193Y obtained in Example 2 was chemically transformed into the expression host E. coli BL21(DE3), and the cells were screened by coating LB solid medium containing 50 μg / mL kanamycin to obtain recombinant bacteria E. coli BL21(DE3) / pET-26b(+)-ScNLE-F188A / Q193Y expressing the mutant ScNLE-F188A / Q193Y.

[0097] Example 4 Preparation of mutant enzyme ScNLE-F188A / Q193Y

[0098] The recombinant E. coli BL21(DE3) / pET-26b(+)-ScNLE-F188A / Q193Y obtained in Example 3 was inoculated into a test tube containing LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm for 12 hours. The inoculum was then transferred to a 500 mL Erlenmeyer shake flask containing 100 mL of LB medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculum and cultured at 37°C and 220 rpm until the OD 600nm =0.6-0.8, add the inducer IPTG to a final concentration of 300 μM, and culture at 16°C for 20 hours. Centrifuge the induced culture at 9000 rpm for 10 minutes, collect the bacterial pellet, and wash with physiological saline to obtain resting cells. The resting cells can be directly lyophilized to obtain lyophilized cells, or ultrasonically disrupted and lyophilized to obtain lyophilized powder, which can be stored at 4°C.

[0099] Example 5 Preparation of 2-amino-4-methylthiobutyric acid catalyzed by mutant enzyme

[0100] The mutant enzyme ScNLE-F188A / Q193Y prepared in Example 4 was used as a biocatalyst in the preparation reaction of 2-amino-4-methylthiobutyric acid.

[0101] A 200 mL reaction system was prepared by adding 300 mM 2-amino-4-methylthiobutyronitrile (2-amino-4-methylthiobutyronitrile), 0.08 g lyophilized cells (equivalent to 0.01 g / g 2-amino-4-methylthiobutyronitrile), and adding ammonia (3 times the theoretical yield of 2-amino-4-methylthiobutyric acid) in the form of aqueous ammonia. The reaction was stirred at 200 rpm at 40°C, pH 8.0 for 1.5 hours.

[0102] After the reaction, 1 mL of the reaction solution was centrifuged to remove the biocatalyst, and the supernatant was analyzed by high-performance liquid chromatography to determine the concentration of 2-amino-4-methylthiobutyric acid. Calculations showed that when the mutant enzyme ScNLE-F188A / Q193Y was used as the biocatalyst, the conversion rate of the substrate 2-amino-4-methylthiobutyronitrile (conversion rate = (initial amount of reactants - remaining amount of reactants) / initial amount of reactants × 100%) was 99%, and the production efficiency of the product 2-amino-4-methylthiobutyric acid (production efficiency = amount of product generated / (reaction time * amount of catalyst)) was 73.9 kg. 蛋氨酸 / kg DCW From the above, it can be concluded that the nitrilase ScNLE-F188A / Q193Y of the present invention has higher catalytic activity, requires less addition amount, has higher production efficiency of 2-amino-4-methylthiobutyric acid, and has lower production cost.

[0103] The HPLC detection method is:

[0104] An Agilent high-performance liquid chromatograph and an Aglient ZORBAX SB-Aq column (size 4.6×150 mm) were used; the mobile phase was ethanol:water:trifluoroacetic acid = 5:95:0.04 (volume ratio); the flow rate was 1.0 mL / min; the excitation and emission wavelengths of the fluorescence detector were 330 nm and 465 nm, respectively; the column temperature was 20°C; all samples were centrifuged at 10,000 rpm and filtered through a 0.22 μm organic filter membrane, and the sample volume was 10 μL.

[0105] The elution time of the 2-amino-4-methylthiobutyronitrile standard was 8.9 min, and the elution time of the 2-amino-4-methylthiobutyric acid standard was 3.1 min.

[0106] The elution time of 2-amino-4-methylthiobutyronitrile in the reaction solution was 8.9 min, and the elution time of 2-amino-4-methylthiobutyric acid was 3.1 min.

[0107] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A nitrilase mutant, characterized in that Based on the amino acid sequence shown in SEQ ID NO.2, the following mutations were introduced: F188A and Q193Y.

2. A nucleic acid molecule, characterized in that A nucleic acid molecule encoding the nitrilase mutant according to claim 1.

3. A recombinant vector, characterized in that Comprising the nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that Comprising the nucleic acid molecule according to claim 2 or the recombinant vector according to claim 3.

5. A method for preparing a nitrilase mutant, characterized in that: include: 1) culturing the recombinant cell of claim 4 and inducing expression of the nitrilase mutant of claim 1; 2) Isolating the nitrilase mutant according to claim 1 from the culture obtained in 1).

6. Use of the nitrilase mutant according to claim 1 in preparing methionine or catalyzing the hydrolysis of nitrile.

7. A method for preparing methionine, characterized in that: include: The nitrilase mutant according to claim 1 is used as a catalyst to catalyze the hydrolysis reaction of the substrate 2-amino-4-methylthiobutyronitrile.

8. The method for preparing methionine according to claim 7, wherein The pH of the hydrolysis reaction is 5-9; And / or, the temperature of the hydrolysis reaction is 30-50°C.

9. The method for preparing methionine according to claim 7 or 8, characterized in that: The final concentration of the substrate 2-amino-4-methylthiobutyronitrile is 30-300 mM.

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