Nitrilase and application thereof in methionine synthesis

Abstract

The invention relates to the technical field of biological catalysis, in particular to nitrilase and application thereof in methionine synthesis. The protein provided by the invention is selected from any one of the following items: (1) compared with an amino acid sequence as shown in SEQ ID NO.2, the protein comprises the following mutation sites: F188A and Q193Y; (2) a protein with nitrilase activity, which has at least more than 80% of identity with the protein in (1) and retains the mutation sites F188A and Q193Y; as a nitrilase mutant ScNLE-F188A / Q193Y, the protein can efficiently catalyze 2-amino-4-methylthio butyronitrile to generate 2-amino-4-methylthio butyric acid, the production efficiency reaches up to 73.9 kg methionine / kg DCW / h, and a potential efficient biocatalyst is provided for industrial production of methionine prepared by a biological enzyme method.

Description

Technical Field

[0001] The 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, which is the basic unit of protein. In animals, methionine is classified as an essential amino acid, which means that animals cannot synthesize it themselves and must obtain it through diet. It is the only essential amino acid that contains sulfur and plays a vital role in the growth and development of animals. In the livestock industry, especially poultry farming, methionine is often a key factor limiting its growth because common feed ingredients such as corn and soybean meal do not contain enough of it. Therefore, it is usually necessary to add additional methionine to the feed to meet the nutritional needs of poultry.

[0003] The main methods for methionine synthesis include chemical synthesis, microbial fermentation and enzyme catalysis. Although the chemical synthesis method is mature, it has problems such as large environmental pollution, high energy consumption and many by-products. The production process of microbial fermentation has low yield and lacks industrial production value. The enzyme catalysis method has gradually become the mainstream method for methionine synthesis due to its advantages such as high efficiency, environmental protection and strong specificity.

[0004] Caoda Company and Rhone-Poulenc Company have studied the process of preparing 2-amino-4-methylthiobutyric acid by hydrolysis of 2-amino-4-methylthiobutyronitrile catalyzed by nitrilase, and the reaction is shown in formula (I). The production efficiency of nitrilase biocatalysts from Alcaligenes faecalis, Gordaon terrae, Rhodococcus sp. is only 0.6-0.9 kg 蛋氨酸 / kg DCW / h, which is still at a relatively low level. 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 improvement in production efficiency is mainly due to the unique enzymatic properties of ScNLE and its mutant ScNLE-F188A. Compared with traditional nitrilase, ScNLE and its mutants show higher substrate affinity and catalytic activity, thus being able 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 above-mentioned technical route of biocatalytic preparation of methionine using nitrilase, further improving the reaction efficiency is still the main goal of industrial production of methionine. In order to overcome this problem, the present invention is directed to the improvement and optimization of nitrilase. Through genetic engineering technology, nitrilase is directed to evolve to screen out mutants with higher catalytic activity and stability, thereby significantly improving the production efficiency of methionine. 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 vector of 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 the 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 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 a substrate 2-amino-4-methylthiobutyronitrile to perform a hydrolysis reaction.

[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. A protein provided by the present invention is 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) 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 a 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, with mild reaction conditions, easy control, high production efficiency, and 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, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope 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 the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0030] Since the hydrolysis reaction efficiency of the existing key enzyme nitrilase is still difficult to meet the needs of industrial production of methionine, the present invention improves and optimizes the nitrilase. The nitrilase is subjected to directed evolution by genetic engineering technology to screen out mutants with higher catalytic activity and stability, 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 the above SEQ ID NO.2 is the original sequence of the nitrilase ScNLE of Shewanella corallii (its nucleotide sequence is shown in SEQ ID NO:1). The protein provided by the present invention is obtained by site-directed mutation F188A (mutating the phenylalanine at position 188 to alanine) and Q193Y (mutating the glutamine at position 193 to tyrosine) based on the wild-type nitrilase ScNLE (SEQ ID NO.2), and the amino acid sequence of the protein is shown in SEQ ID NO.6. The protein, as a nitrilase mutant ScNLE-F188A / Q193Y, has high catalytic activity and can efficiently catalyze the hydrolysis of 2-amino-4-methylthiobutyronitrile to prepare methionine.

[0035] In some embodiments, the protein may be a protein having nitrilase activity by having at least 80% identity with the amino acid sequence shown in SEQ ID NO.6 and retaining the mutation sites F188A and Q193Y. The identity with the amino acid sequence shown in SEQ ID NO.6 may be at least 80%, 83%, 85%, 88%, 90%, 93%, 95%, 97%, 99%, 99.1%, 99.2%, 99.3% or more. In a preferred embodiment, the sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 90%, more preferably 95%, and even more preferably at least 99% sequence identity therewith.

[0036] In some embodiments, the enzyme of the present invention can replace, delete and / or add at least one amino acid on the basis of the amino acid sequence shown in SEQ ID NO.6, and retain the mutation sites F188A and Q193Y, to obtain a protein with nitrilase activity. The number of the above-mentioned substituted, deleted or added amino acids can be any value, such as 1, 5, 10, 15 and above, so that the sequence identity of the changed amino acid sequence and its respective corresponding original sequence can be 80%, 83%, 85%, 88%, 90%, 93%, 95%, 97%, 99%, 99.1%, 99.2%, 99.3% or more.

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

[0038] In certain embodiments, the above-mentioned protein can be obtained by artificial synthesis, or by synthesizing its encoding gene and then preparing it through a biological expression process, for example, expressing it from a prokaryotic organism (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 genetic engineering 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] In view of the redundancy of the genetic code, there are many nucleic acid molecule sequences that can be used to encode the proteins involved in the present invention. Therefore, when a specific amino acid sequence has been determined, a person skilled in the art can simply modify one or more codons without changing the amino acid sequence of the encoded protein to prepare any number of different nucleic acids. According to the preference of the host cell used in the actual preparation process, a more preferred polynucleotide can be selected through codon optimization.

[0043] The nucleic acid molecule can be obtained by conventional methods, such as PCR amplification or artificial synthesis, etc. At present, the polynucleotide sequence 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 can stably replicate and efficiently express the nucleic acid molecule in the 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, thereby obtaining 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, yeast, etc., among which Escherichia coli expression host E.coli BL21 (DE3) is preferred.

[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 when the recombinant genetically engineered bacterium expresses the protein can be a culture medium 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, just 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. Next, transfer the bacterial solution to a 500mL triangular shake flask containing 50-150mL (commonly used parameters are 100ml) of LB liquid culture medium containing kanamycin at an inoculum volume of 1-2% (v / v), and continue to culture at 30-37°C (commonly used 37°C) and 100-300 rpm (commonly used 220 rpm). When the OD of the culture solution reaches 400,000, the culture temperature is 400 °C and the temperature is 400 °C. 600 When the value reaches 0.6-0.8, 100-500 μM isopropyl βD-thiogalactoside (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 the expression of the protein;

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

[0060] Among them, the method of culturing and inducing the recombinant cells and the method of separating the protein from the culture are both 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 a protein prepared by the method for preparing the protein.

[0062] In some embodiments, the specific use form of the nitrilase depends on the needs of the application scenario. For example, in the form of engineered bacteria whole cells, the nitrilase can directly participate in the reaction without complicated separation and purification steps, simplifying the operation 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 fully purified enzymes, higher catalytic activity and selectivity can be obtained, which is suitable for occasions with higher product purity requirements. In addition, the immobilized enzyme or immobilized cell 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 a 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, wherein 40°C is preferred.

[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, wherein 300 mM is preferred.

[0070] The amount of ammonia added is 1.5-10 times 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 times the equivalent, preferably 3 times the equivalent.

[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 reaction of 2-amino-4-methylthiobutyronitrile to prepare 2-amino-4-methylthiobutyric acid;

[0073] Specifically, the lyophilized cells or lyophilized powder of the above-mentioned recombinant cells can be used as catalysts to achieve the whole-cell catalytic production of 2-amino-4-methylthiobutyric acid. In this process, the amount of lyophilized cells or lyophilized powder used ranges from 0.005 to 0.05 g / g 2-amino-4-methylthiobutyronitrile, for example, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05 g / g 2-amino-4-methylthiobutyronitrile, or any combination or range between these values. In particular, 0.01 g / g 2-amino-4-methylthiobutyronitrile is a recommended amount. As for the lyophilized powder, it is prepared by crushing any of the above-mentioned recombinant cells to obtain a cell crushing liquid, and then freeze-drying the cell crushing liquid. The 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 engineered bacteria whole cells, unpurified crude enzymes, partially purified enzymes or completely purified enzymes. The nitrilase mutant ScNLE-F188A / Q193Y of the present invention can also be prepared into catalysts in the form of immobilized enzymes or immobilized cells 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 is from the German Collection of Microorganisms (DSMZ), and the strain collection number is DSM 21332.

[0077] pET-26b(+) is a product of Novagen, with the product 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 using ScNLE-FP (SEQ ID NO: 3) and ScNLE-RP (SEQ ID NO: 4) as primers, PCR amplification is performed to obtain a fragment containing the nitrilase gene ScNLE, wherein the nucleotide sequence of the nitrilase gene is as shown in SEQ ID NO: 1, and the amino acid sequence of the nitrilase ScNLE encoded by it is as shown in SEQ ID NO: 2.

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

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

[0089] This embodiment 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 to perform full plasmid PCR to introduce site-directed mutations. The PCR reaction system was as follows:

[0091] Table 2. PCR reaction system

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

[0093] The PCR reaction program was as follows: 95°C for 5 min; 98°C for 10 sec, 55°C for 5 sec, 72°C for 35 sec, 30 cycles; 72°C for 5 min; and stored at 4°C.

[0094] The mutant recombinant expression plasmid pET-26b(+)-ScNLE-F188A / Q193Y was obtained, and the plasmid was sent for sequencing. The results were consistent with expectations. The nucleotide sequence of the nitrilase ScNLE mutant gene was shown in SEQ ID NO:5, and the amino acid sequence of the nitrilase mutant ScNLE-F188A / Q193Y encoded by it was shown in 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 transferred into the expression host E. coli BL21(DE3) by chemical transformation, and LB solid culture medium containing 50 μg / mL kanamycin was applied for screening 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. Then, the inoculum was transferred to a 500 mL triangular shake flask containing 100 mL LB medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculum volume, and cultured at 37°C and 220 rpm until the OD 600nm =0.6-0.8, add the inducer IPTG with a final concentration of 300μM, induce and culture at 16℃ for 20 hours, centrifuge the induced culture solution at 9000rpm for 10 minutes, collect the bacterial precipitate, wash with physiological saline to obtain resting cells. The resting cells are directly freeze-dried to obtain freeze-dried cells, or freeze-dried after ultrasonic disruption to obtain freeze-dried powder, and stored at 4℃.

[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] 200mL reaction system: 300mM substrate 2-amino-4-methylthiobutyronitrile, 0.08g lyophilized cells (equivalent to 0.01g / g 2-amino-4-methylthiobutyronitrile), ammonia in the form of aqueous ammonia with a final concentration of 3 times the theoretical yield of 2-amino-4-methylthiobutyric acid equivalents, 100mM Tris-HCl buffer, stirred at 40°C, pH 8.0, 200rpm for 1.5 hours.

[0102] After the reaction is completed, 1 mL of the reaction solution is centrifuged to remove the biocatalyst, and the supernatant is analyzed by high performance liquid chromatography to detect the concentration of 2-amino-4-methylthiobutyric acid. According to calculations, when the mutant enzyme ScNLE-F188A / Q193Y is 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%) is 99%, and the production efficiency of the product 2-amino-4-methylthiobutyric acid (production efficiency = amount of product generated by the reaction / (reaction time * amount of catalyst)) is 73.9 kg 蛋氨酸 / kg DCW / h. It can be concluded from the above that the nitrilase ScNLE-F188A / Q193Y of the present invention has higher catalytic activity, less addition amount, higher production efficiency of 2-amino-4-methylthiobutyric acid and lower production cost.

[0103] The HPLC detection method is:

[0104] Agilent high performance liquid chromatograph and Aglient ZORBAX SB-Aq chromatographic column (size 4.6×150 mm) were used; mobile phase: ethanol: water: trifluoroacetic acid = 5:95:0.04 (volume ratio); flow rate: 1.0 mL / min; the excitation and emission wavelengths of the fluorescence detector were 330 nm and 465 nm, respectively; column temperature: 20°C; all samples were centrifuged at 10000 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 the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A protein, characterized in that Select any 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) 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.

2. A nucleic acid molecule, characterized in that A nucleic acid molecule encoding the protein of claim 1.

3. A recombinant vector, characterized in that: A vector 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 protein, characterized in that: include: 1) culturing the recombinant cell according to claim 4, and inducing the expression of the protein according to claim 1; 2) Isolating the protein according to claim 1 from the culture obtained in 1).

6. A nitrilase, characterized in that A protein comprising the protein of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, the recombinant cell of claim 4 or a protein prepared by the method for preparing the protein of claim 5.

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

8. A method for preparing methionine, characterized in that: include: The nitrilase according to claim 6 is used as a catalyst to catalyze the hydrolysis reaction of the substrate 2-amino-4-methylthiobutyronitrile.

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

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

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