Nitrilase mutant and application thereof in amide synthesis

Abstract

The invention discloses construction and application of a novel nitrilase with improved amide generation capability and a mutant thereof. The amino acid sequence of the nitrilase disclosed by the invention is as shown in SEQ ID NO: 1, and the mutant of the nitrilase is obtained by performing single mutation on the 51st site, the 162nd site and the 185th site of the sequence as shown in SEQ ID NO: 1. The nitrilase provided by the invention has excellent amide generation capability, and has important industrial application potential for green and efficient preparation of amide compounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and specifically, relates to nitrilase and mutants thereof derived from Cereibacter sphaeroides, nucleic acid molecules encoding the nitrilase, vectors and cells containing the nucleic acid molecules, and applications of the nitrilase in catalyzing the synthesis of corresponding amides from 2, 3 or 4-cyanopyridine. Background Art

[0002] Amide bonds are a very important class of chemical bonds, which are commonly found in many active molecules and drug intermediates, and have broad application prospects. Amide compounds not only play a vital role in the composition of biological systems and metabolic activities in organisms, but are also widely used as multifunctional synthons for the large-scale production of detergents, lubricants, engineering plastics, cosmetics, pesticides, and pharmaceuticals. In 2007, the American Chemical Society-Green Chemistry Institute-Pharmaceutical Roundtable (ACS GCIPR) selected the synthesis of amides as the primary challenge and listed it as a high-priority research area. It can be seen that it is of great significance to construct a green and efficient synthesis process for amide compounds.

[0003] The synthesis methods of amide compounds are mainly divided into chemical methods and biological methods. Traditional chemical methods usually require harsh conditions, long catalytic time, oxygen-free technology, luxurious ligands, high catalyst loading, toxic organic solvents and even oxidizing agents, and also require complex catalyst / product separation procedures, which hinder their application as industrial practical solutions. In contrast, microbial enzyme catalysis of organic nitriles provides a mild, environmentally friendly, and cost-effective option for the synthesis of amides. Naturally occurring nitrile conversion enzymes include nitrile hydratase and nitrile hydrolase. Nitrile hydratase catalyzes the direct hydration of nitrile substances to form amides. At present, nitrile hydratase has been successfully applied to the industrial production of amide substances, and typical representatives are the synthesis of acrylamide, nicotinamide, and 5-cyanovaleramide. However, the substrate range of nitrile hydratase is limited, and it is difficult to meet the needs of various amide bonds. In addition, its low reactivity, difficulty in recombinant expression, lack of stereoselectivity, metal ion dependence and other defects make people eager to find alternatives for amide synthesis. With the deepening of research, it was found that nitrile hydrolase has catalytic promiscuity. While converting nitrile to the main product carboxylic acid, it also produces by-product amides. If the ability of nitrilase to generate by-product amides is improved through certain strategies, it will provide a more valuable solution for the synthesis of amide substances.

[0004] Nitrilases naturally hydrolyze nitriles to produce carboxylic acids, but also exhibit the side reaction activity of producing amides. This feature has aroused the interest of researchers, and scientists have tried to improve the ability of nitrilase to produce amides through certain protein engineering strategies, and obtain "nitrile hydratase" evolved from nitrilase. For example, Kiziak et al. constructed multiple C-terminal deletion mutants of nitrilase and found that when the number of deletions was between 47-67, the mutants increased amide formation and the (R) enantiomer ratio of the acid increased. Sosedov et al. saturated the tryptophan at position 188 of Pseudomonas fluorescens EBC 191 nitrilase and screened 18 mutants with increased mandelic amide production. Similarly, they also found that mutants C163N and C163Q also enhance the production of byproduct amides. Recently, Wang et al. successfully transformed nitrilase into "nitrile hydratase" by modifying the active pocket of Nit-72W so that the amide produced by catalyzing 2-cyanopyridine accounted for more than 98% of the product.

[0005] Therefore, there is an urgent need in the art for a nitrilase that can efficiently catalyze the synthesis of the corresponding amide from 2, 3 or 4-cyanopyridine. Summary of the invention

[0006] The object of the present invention is to provide a nitrilase, which can efficiently catalyze 2, 3 or 4-cyanopyridine to synthesize the corresponding amide.

[0007] The present invention also aims to provide nucleic acid molecules encoding the nitrilase of the present invention, vectors and cells containing the nucleic acid molecules.

[0008] Another object of the present invention is to provide a method for synthesizing the corresponding amide from 2, 3 or 4-cyanopyridine by using the nitrilase.

[0009] In a first aspect, the present invention provides a nitrilase, characterized in that the nitrilase comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises the amino acid sequence shown in SEQ ID NO: 1 and has a mutation at one or more of the amino acid residue positions corresponding to positions 51, 162 and 185 of the amino acid sequence shown in SEQ ID NO: 1.

[0010] In a preferred embodiment, the nitrilase is:

[0011] a. whose amino acid sequence is as shown in SEQ ID NO: 1 and has a mutation at one or more of the amino acid residue positions 51, 162 and 185 corresponding to the amino acid sequence shown in SEQ ID NO: 1; or

[0012] b. A nitrilase derived from a having a sequence as defined in a and having one or more amino acid residues, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 3, and most preferably 1 amino acid residue substituted, deleted or added at positions other than position 51, 162 or 185, and having substantially the nitrilase function as defined in a.

[0013] In a specific embodiment, the amino acid sequence of the nitrilase is I, H, V, D, T, R or E at the 51st amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO:1;

[0014] and / or the amino acid residue at position 162 corresponding to the amino acid sequence of SEQ ID NO: 1 is H, E, N, P, C or G;

[0015] and / or the amino acid residue at position 185 corresponding to the amino acid sequence shown in SEQ ID NO: 1 is N, M, T, C, V, A, G, L, E, P or H.

[0016] In a specific embodiment, the amino acid sequence of the nitrilase is as shown in SEQ ID NO: 1 and:

[0017] a. The amino acid residue at position 51 is I, H, R or E;

[0018] b. The amino acid residue at position 162 is H or P;

[0019] c. the amino acid residue at position 185 is N, M, T, C or E; or

[0020] d. A nitrilase derived from a, wherein the amino acid sequence is as defined in any one of items ac, and one or more amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue are substituted, deleted or added at positions other than position 51, 162 or 185, and the nitrilase function as defined in any one of items ac is substantially obtained.

[0021] In a specific embodiment, the amino acid sequence of the nitrilase is as shown in SEQ ID NO: 1 and:

[0022] a. The 51st amino acid residue is I or R;

[0023] b. The amino acid residue at position 162 is H or P;

[0024] c. The amino acid residue at position 185 is N or M.

[0025] In a specific embodiment, the amino acid sequence of the nitrilase is shown in SEQ ID NO: 3, 4 or 5.

[0026] In a specific embodiment, the nitrilase is derived from Cereibacter sphaeroides.

[0027] In a preferred embodiment, the proportion of 2-pyridinecarboxamide in the process of 2-cyanopyridine to 2-pyridinecarboxamide catalyzed by the nitrilase is greater than 10%, preferably greater than 20%, 30%, 40%, 50%, 60%; and most preferably greater than 65%.

[0028] In a preferred embodiment, the proportion of 2-pyridinecarboxamide is calculated by 2-pyridinecarboxamide / (2-pyridinecarboxamide+2-pyridinecarboxylic acid).

[0029] In a preferred embodiment, the proportion of nicotinamide in the process of catalyzing 3-cyanopyridine to produce nicotinamide by the nitrilase is more than 5%, preferably more than 10%; most preferably more than 20%.

[0030] In a preferred embodiment, the proportion of niacinamide is calculated by niacinamide / (niacinamide+niacin).

[0031] In a preferred embodiment, the proportion of isonicotinamide in the process of catalyzing 4-cyanopyridine to isonicotinamide by the nitrilase is greater than 20%, preferably greater than 30%, 40%, 50%, 60%, 70%, 80%; and most preferably greater than 90%.

[0032] In a preferred embodiment, the proportion of isonicotinamide is calculated by isonicotinamide / (isonicotinamide+isonicotinic acid).

[0033] In a second aspect, the present invention provides a gene encoding the nitrilase of the first aspect.

[0034] In a third aspect, the present invention provides an expression vector comprising the encoding gene described in the second aspect.

[0035] In a preferred embodiment, the expression vector is a plasmid.

[0036] In a fourth aspect, the present invention provides a host cell, which contains the coding gene described in the second aspect or the expression vector described in the third aspect, or has the coding gene described in the second aspect or the expression vector described in the third aspect integrated into its chromosome.

[0037] In a preferred embodiment, the host cell is selected from Escherichia coli or spore bacteria.

[0038] In a fifth aspect, the present invention provides a method for preparing the nitrilase according to the first aspect, the method comprising the following steps:

[0039] 1) culturing the host cell according to the fourth aspect to induce the expression of the nitrilase;

[0040] 2) optionally isolating the nitrilase described in the first aspect from the culture obtained in 1).

[0041] In a sixth aspect, the present invention provides use of the nitrilase described in the first aspect, or the encoding gene described in the second aspect, or the expression vector described in the third aspect, or the host cell described in the fourth aspect in producing amide substances.

[0042] In a preferred embodiment, the amide substance is 2-pyridinecarboxamide, nicotinamide or isonicotinamide.

[0043] In a seventh aspect, the present invention provides a method for preparing an amide substance, the method comprising the following steps:

[0044] 1) culturing the host cell of the fourth aspect to induce the expression of the nitrilase of the first aspect; and

[0045] 2) Using the obtained nitrilase to catalyze N-heterocyclic nitrile 2, 3 or 4-cyanopyridine to generate corresponding amide substances.

[0046] In a preferred embodiment, the amide substance is 2-pyridinecarboxamide, nicotinamide or isonicotinamide.

[0047] In a preferred embodiment, the obtained nitrilase is used to catalyze N-heterocyclic nitrile 2, 3 or 4-cyanopyridine to generate corresponding amide substances. The nitrilase can be separated from the culture obtained in 1) or directly used.

[0048] In an eighth aspect, the present invention provides an immobilized enzyme, wherein the immobilized enzyme comprises the nitrilase described in the first aspect.

[0049] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The docking structure of the nitrilase Nit-0305 model and the substrate and the spatial position distribution of the key mutant residues Y51, W162, and F185 are shown. DETAILED DESCRIPTION

[0051] After extensive and in-depth research, the inventor unexpectedly discovered a new type of nitrilase Nit-0305 derived from Cereibactersphaeroides. On this basis, a mutant with significantly improved amide generation ability was constructed, which is conducive to the directional generation of the target product. The positive mutant that catalyzes 2-cyanopyridine to generate the most 2-pyridinecarboxamide in the mutant of the present invention is Y51I, and the amide content in the product generated by its catalysis is increased from 1.29% of the wild type to 66.1%. When 3-cyanopyridine is used as a substrate, the best mutant Y51R increases the proportion of nicotinamide in the product from 0 to 23.7%, realizing the generation of nicotinamide from scratch. When 4-cyanopyridine is used as a substrate, the main product generated by the catalytic mutant W162P with the most significant hydration activity is converted into isonicotinamide, and the amide content is increased from 1.38% of the wild type to 92.4%. The regulation of the main and byproducts of the nitrilase enables it to be applied to the green industrial catalytic synthesis of amides, which has important application value. The present invention was completed on this basis.

[0052] Nitrilases of the present invention

[0053] The invention starts from Nit-0305, a nitrilase derived from Cereibacter sphaeroides, and obtains a series of nitrilase mutants capable of efficiently catalyzing 2, 3 or 4-cyanopyridine to synthesize corresponding amides by molecularly modifying Nit-0305.

[0054] The present invention adopts the following technical solution:

[0055] The present invention provides a novel nitrilase mutant with significant amide generation activity derived from Cereibacter sphaeroides. The mutant is obtained by mutating the 51st, 162nd and 185th positions of the sequence shown in SEQ ID NO:1. The mutant is constructed by site-directed saturation mutagenesis. The nucleic acid sequence of the gene encoding the amino acid sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:2.

[0056] In a specific embodiment, the present invention changes the 51st amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 to I, H, V, D, T, R or E;

[0057] and / or the amino acid residue at position 162 corresponding to the amino acid sequence of SEQ ID NO: 1 is H, E, N, P, C or G;

[0058] and / or the amino acid residue at position 185 corresponding to the amino acid sequence shown in SEQ ID NO: 1 is N, M, T, C, V, A, G, L, E, P or H.

[0059] In a preferred embodiment, the nitrilase mutant of the present invention is a mutant of the amino acid sequence shown in SEQ ID NO: 1 subjected to one of the following mutations:

[0060] (1) The amino acid residue at position 51 is mutated to I, H, R, or E;

[0061] (2) amino acid residue 162 mutated to H or P;

[0062] (3) The amino acid residue at position 185 mutated to N, M, T, C or E

[0063] In a further preferred embodiment, the nitrilase mutant of the present invention is a mutant of the amino acid sequence shown in SEQ ID NO: 1 subjected to one of the following mutations:

[0064] (1) The amino acid residue at position 51 is mutated to I or R;

[0065] (2) amino acid residue 162 mutated to H or P;

[0066] (3) The amino acid residue at position 185 is mutated to N or M.

[0067] Furthermore, it is preferred that the nitrilase mutant is obtained by subjecting the amino acid sequence shown in SEQ ID NO.1 to one of the following mutations: (1) tyrosine at position 51 is mutated to isoleucine (Y51I), and the amino acid sequence is shown in SEQ ID NO.3; (2) tyrosine at position 51 is mutated to arginine (Y51R), and the amino acid sequence is shown in SEQ ID NO.4; (3) tryptophan at position 162 is mutated to proline (W162P), and the amino acid sequence is shown in SEQ ID NO.5.

[0068] In the present invention, the nitrilase of the present invention also includes mutants in which at most 20, preferably at most 10, more preferably at most 3, more preferably at most 2, and most preferably at most 1 amino acid is replaced by an amino acid with similar or similar properties compared to the specifically disclosed nitrilase. These conservatively modified mutants can be generated by, for example, making amino acid substitutions as shown in the following table.

[0069] Initial residue Representative substituted residues Preferred substitution residues Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0070] Based on the nitrilase of the present invention, the present invention also provides a gene encoding the nitrilase and a recombinant vector comprising the gene encoding the nitrilase.

[0071] The amino acid residue corresponding to position 51, 162 and / or 185 of the amino acid sequence shown in SEQ ID NO: 1

[0072] Those skilled in the art know that various mutations, such as substitution, addition or deletion, can be made to some amino acid residues in the amino acid sequence of a certain protein, but the obtained mutant can still have the function or activity of the original protein. Therefore, those skilled in the art can make certain changes to the amino acid sequence specifically disclosed in the present invention to obtain a mutant that still has the desired activity. Then, the amino acid residue in such a mutant corresponding to the amino acid residue at position 51, position 162 or position 185 of the amino acid sequence shown in SEQ ID NO: 1 may not be the amino acid residue at position 51, position 162 or position 185, but the mutant obtained in this way should still fall within the scope of protection of the present invention.

[0073] The term "corresponding to" as used herein has the meaning generally understood by those of ordinary skill in the art. Specifically, "corresponding to" means that after two sequences are aligned for homology or sequence identity, a position in one sequence corresponds to a specified position in another sequence. Therefore, with respect to "the amino acid residue corresponding to position 162 of the amino acid sequence shown in SEQ ID NO: 1", if a 6-His tag is added to one end of the amino acid sequence shown in SEQ ID NO: 1, the position 162 in the resulting mutant corresponding to the amino acid sequence shown in SEQ ID NO: 1 may be position 168; and if a few amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 are deleted, the position 162 in the resulting mutant corresponding to the amino acid sequence shown in SEQ ID NO: 1 may be position 158, and so on. For another example, if a sequence having 200 amino acid residues has a high homology or sequence identity with positions 20-220 of the amino acid sequence shown in SEQ ID NO: 1, the position 162 in the resulting mutant corresponding to the amino acid sequence shown in SEQ ID NO: 1 may be position 142. Likewise, the above description is equally applicable to "the amino acid residue corresponding to position 51 or 185 of the amino acid sequence shown in SEQ ID NO: 1".

[0074] In a specific embodiment, the homology or sequence identity may be greater than 80%, preferably greater than 90%, more preferably 95%-98%, and most preferably greater than 99%.

[0075] Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0076] Host cells

[0077] The term "host cell" used herein has the meaning generally understood by those of ordinary skill in the art, i.e., a host cell capable of producing the nitrilase of the present invention. In other words, the present invention can utilize any host cell as long as the nitrilase of the present invention can be expressed in the host cell.

[0078] For example, in a specific embodiment, the present invention utilizes a host cell containing an exogenous nitrilase encoding gene of the present invention, preferably an Escherichia coli or a strain of Glossophore. However, those skilled in the art should know that the present invention is not limited to host cells containing exogenous encoding genes. For example, the encoding gene of the nitrilase contained in the host cell of the present invention may not only be a recombinant vector or plasmid, but may also be an encoding gene of the enzyme integrated into the genome, that is, the encoding gene of the enzyme integrated into the genome may be obtained by homologous recombination by transferring into a plasmid, or may be obtained by site-directed mutagenesis of a corresponding site on the genome.

[0079] Application of the nitrilase or host cell of the present invention

[0080] The nitrilase of the present invention can efficiently catalyze the synthesis of corresponding amides from 2, 3 or 4-cyanopyridine. In a specific embodiment, the proportion of 2-pyridinecarboxamide in the process of catalyzing 2-cyanopyridine to 2-pyridinecarboxamide by the nitrilase of the present invention exceeds 10%, preferably exceeds 20%, 30%, 40%, 50%, 60%; most preferably exceeds 65%; the proportion of nicotinamide in the process of catalyzing 3-cyanopyridine to nicotinamide by the nitrilase of the present invention exceeds 5%, preferably exceeds 10%; most preferably exceeds 20%; the proportion of isonicotinamide in the process of catalyzing 4-cyanopyridine to isonicotinamide by the nitrilase of the present invention exceeds 20%, preferably exceeds 30%, 40%, 50%, 60%, 70%, 80%; most preferably exceeds 90%.

[0081] Immobilized enzyme

[0082] The term "immobilized enzyme" as used herein has the meaning conventionally understood by those of ordinary skill in the art. Specifically, the term refers to a water-soluble enzyme that has been treated by a physical or chemical method to bind the enzyme to a water-insoluble macromolecular carrier or embed the enzyme therein, so that the enzyme dissolves in water in a gel or a microcapsule of a semipermeable membrane, thereby reducing fluidity.

[0083] Immobilized enzymes still have enzymatic activity and act on substrates in a solid phase in catalytic reactions. After being immobilized, enzymes generally have increased stability, are easy to separate from the reaction system, and are easy to control and can be used repeatedly. They are easy to transport and store, and are conducive to automated production. Immobilized enzymes are an enzyme application technology that has been developed in the past decade or so, and have attractive application prospects in industrial production, chemical analysis, and medicine.

[0084] In view of the teachings herein, it is not difficult for a person skilled in the art to prepare the nitrilase of the present invention into an immobilized enzyme, and then use it to efficiently catalyze the synthesis of the corresponding amide from 2, 3 or 4-cyanopyridine.

[0085] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0086] 1. The present invention constructs a novel nitrilase with much better amide production ability than common nitrilase, thus enriching the nitrilase enzyme library;

[0087] 2. The inventors further modified the enzyme molecule Nit-0305 by site-directed mutagenesis, so that the amide generation ability of the nitrilase mutant was significantly improved, which was conducive to the directional generation of the target product amide;

[0088] 3. The present invention has important reference significance for the regulation of the main and by-products of nitrilase and the transformation of the promiscuity of other enzymes.

[0089] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0090] The formula of Luria-Bertani (LB) liquid medium is: peptone 10g / L, sodium chloride 10g / L, yeast extract 5g / L. Add a certain amount of deionized water to the three substances and stir them well. The pH value is natural. The prepared medium is sterilized at 121℃ for 20 minutes in a high-pressure steam sterilizer.

[0091] Luria-Bertani (LB) solid medium is prepared by adding 1.5% agar to LB liquid medium, and the sterilization conditions are the same as above.

[0092] Example 1. Selection of mutation sites

[0093] The inventors, relying on their rich theories and experience in this field, screened out three key residues near the catalytic triad of nitrilase Nit-0305 from Syechocystis sp. PCC6803 that may affect the ratio of main and byproduct generation: Y51, W162 and F185. Subsequently, saturation mutations were performed on these three potential sites to construct a mutant library.

[0094] Example 2. Construction of mutants

[0095] Escherichia coli carrying the pET-28a(+)-Nit-0305 recombinant plasmid was cultured in an LB liquid culture medium test tube for 10-12 hours, and the plasmid was extracted as a template for subsequent mutant construction. The primers used for mutation are shown in Table 1.

[0096] Table 1 Mutant primer information

[0097]

[0098] The PCR reaction system (20 μL) is as follows:

[0099]

[0100] PCR reaction conditions: (1) 95°C pre-denaturation for 3 min; (2) 98°C denaturation for 5 s; (3) 62°C annealing for 15 s; (4) 72°C extension for 6 min. Steps (2) to (4) were cycled 30 times; (5) a final extension at 72°C for 5 min and stored at 4°C.

[0101] After the PCR product was verified as positive by agarose gel electrophoresis, 0.5 μL of restriction endonuclease Dpn I was added and the template was removed in a 37°C water bath for 2.5 hours. Then, transformation, bacterial selection, and sequencing could be performed.

[0102] Example 3. Inducible expression of nitrilase

[0103] The correct bacterial samples were transferred to a 5 mL LB liquid culture medium tube with 1‰ v / v kanamycin resistance added, and cultured in a shaker at 37°C, 200 rpm for about 12 hours. Then they were transferred to a conical flask containing 50 mL LB liquid culture medium and cultured under the same conditions for about 2.5 hours until the OD 600 The value reaches 0.6-0.8. OD 600 When the value meets the requirements, add the inducer IPTG to a final concentration of 0.1 mM, induce for about 16-20 hours in a shaker at 20°C and 200 rpm, and then proceed to the next experiment.

[0104] After the induction culture is completed, the bacteria are harvested. The centrifuge tube with the bacterial solution is filled with deionized water and balanced. Then, it is placed in a pre-cooled 4℃ centrifuge for centrifugation (8,000rpm, 10min). After the centrifugation is completed, the supernatant is poured out, and 20mL of 0.5% NaCl solution is added to resuspend the bacteria. After balancing, centrifuge again under the same conditions. After the centrifugation is completed, the supernatant is removed as much as possible, and then placed in a -40℃ refrigerator for more than 3 hours for freeze drying. The sample is placed in the vacuum freeze dryer for about 10 hours and then taken out to obtain freeze-dried bacterial powder.

[0105] Example 4. Screening of mutants

[0106] Using the freeze-dried bacterial powder of nitrilase prepared in Example 3 as a catalyst, conversion reactions of three substrates, 2-, 3-, and 4-cyanopyridine, were carried out to screen out mutants with improved amide generation ability.

[0107] The reaction system consisted of: 40 μL 50 mM substrate (dissolved in methanol), 10 mg freeze-dried bacterial powder, 60 μL methanol, 900 μL PB buffer (100 mM pH 7.0). All components were added and mixed thoroughly, and three parallel samples were made for each reaction.

[0108] The reaction was carried out in a shaker at 25°C and 200 rpm for 12 h. The reaction of 2-cyanopyridine and 3-cyanopyridine was terminated by adding 10% (v / v) 2M HCl, and 4-cyanopyridine was quenched by heating in a 95°C water bath for ten minutes. The supernatant was then centrifuged at 14,000 rpm for 15 min and the ratio of carboxylic acid to amide in the product was analyzed by HPLC.

[0109] The HPLC detection method using 2-cyanopyridine as the substrate is: using an Extend C18 column (5μm×4.6mm×250mm) with a flow rate of 1mL / min, an aqueous solution containing 0.1% phosphoric acid and 20% methanol as the mobile phase, an ultraviolet detection wavelength of 210nm, a column temperature of 30°C, and a detection time of 10min.

[0110] The HPLC detection method when 3,4-cyanopyridine is used as the substrate is the same as follows: the chromatographic column used is Zorbax SB-C18 (5μm×4.6mm×250mm), the mobile phase is methanol and water, and the gradient elution method is adopted (Table 2). The column temperature is 30°C, and the flow rate is 1mL / min at an ultraviolet absorption wavelength of 261nm, and the injection volume of 5μL is detected.

[0111] Table 2 Gradient elution method

[0112]

[0113]

[0114] With 2-cyanopyridine as the substrate for the catalytic reaction, the mutant library constructed by the above method was screened and analyzed, and the mutants with higher 2-pyridinecarboxamide generation ability than the wild type were shown in Table 3, wherein the byproduct 2-pyridinecarboxamide accounted for = 2-pyridinecarboxamide / (2-pyridinecarboxamide+2-pyridinecarboxylic acid). As can be seen from the table, the mutant Y51I with the most improved amide generation ability increased the proportion of 2-pyridinecarboxamide in the product to 66.1%, which is equivalent to 51 times that of the wild type. The proportion of amide in the reaction product of the mutant W162H was also increased to 55.6%. The saturation mutation results of the F185 site showed that the site can significantly change the reaction specificity of the nitrilase to make it tend to generate amides. Among them, the forward mutants F185N, F185M, F185T and F185C all increased the content of 2-pyridinecarboxamide in the product to more than 50%, showing a significantly improved amide generation ability compared to the wild type.

[0115] Table 3 Nitrilase mutant 2-pyridine carboxamide production (each experiment set up 3 parallels)

[0116]

[0117] A reaction system was constructed with 3-cyanopyridine as the substrate to study the catalytic performance of the wild type and all mutants. The proportion of byproduct nicotinamide = nicotinamide / (nicotinamide + nicotinic acid). Liquid phase analysis showed that the wild-type Nit-0305 catalyzed 3-cyanopyridine to only produce nicotinic acid, without the production of byproduct nicotinamide. Therefore, the modification of the amide generation ability of Nit-0305 started from scratch. Observing the liquid phase detection results, it was found that only the four mutants shown in Table 4 could catalyze 3-cyanopyridine to produce nicotinamide. Among them, the optimal forward mutant Y51R increased the proportion of nicotinamide in the total product to 23.7%. Although the other three forward mutants W162E, F185T, and F185N also achieved the conversion of nicotinamide from nothing to something, the proportion of nicotinamide produced was less than 10%.

[0118] Table 4 Nicotinamide production by nitrilase mutants (three parallels were set for each experiment)

[0119]

[0120] The mutant library was screened with 4-cyanopyridine as substrate, and the mutants with increased isonicotinamide production are shown in Table 5, where the proportion of byproduct isonicotinamide = isonicotinamide / (isonicotinamide + isonicotinic acid). Through experimental analysis, it was found that the mutant W162P with the strongest isonicotinamide production ability made the amide proportion in the product reach 92.4%, which was 67 times higher than that of the wild type, indicating that the main and byproduct switching of 4-cyanopyridine catalyzed by nitrilase was successfully achieved. In addition, the isonicotinamide production capacity of the positive mutants Y51I and F185N was also significantly improved, and the catalytically generated isonicotinamide content was 65.4% and 65.6%, respectively.

[0121] Table 5 Isonicotinamide production by nitrilase mutants (three parallels were set for each experiment)

[0122]

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways, that is, all simple, equivalent changes and modifications made according to the claims and the contents of the specification of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

[0124] Sequence Listing

[0125] SEQ ID NO:1

[0126] MPKTVRAAAVQIAPDLASRAGTVERVLNAIAEAADKGAELIVFPETFVPWYPYFSFVLPPVQQGLEHLRLYEEAVTVPSPETHAVAEAARKRGAVVVLGVNERDHGSLYNTQLIFDADGTLKLKRRKITPTYHERMIWGQGDGAGLKVVETKVGRMGALAC WEHYNPLARYALMAQHEEIHASHFPGSLVGPIFGEQIEVTMRHHALESGCFVVNATGWLTEEQIASIHPDPKLQKGLRDGCMTCIITPEGRHAAPPLTAGEGMVIADLDMALITKRKRMMDTVGHYARPELLRLVQDARPQRPLEQILPEATPEASEAQDA

[0127] SEQ ID NO:2

[0128] ATGCCGAAAACCGTTCGCGCCGCAGCAGTTCAGATTGCACCGGATCTGGCCTCTCGTGCAGGAACAGTGGAACGTGTGCTGAATGCAATTGCCGAAGCAGCCGATAAAGGCGCCGAACTGATTGTGTTTCCGGAAACCTTTGTTCCGTGGTATCCGTATTTTAGCTTTGTGCTGCCGCCGGTTCAGCAGGGCTTAGAACATCTGCGTTTATATGAAGAAGCAGTGACCGTTCCGAGTCCGGAAACCCATGCAGTGGCAGAAGCCGCACGTAAACGCGGCGCAGTTGTTGTGTTAGGTGTTAATGAACGCGATCATGGTAGTCTGTATAATACCCAGTTAATTTTTGATGCCGATGGCACCTTAAAACTGAAACGTCGCAAAATTACCCCGACCTATCATGAACGTATGATTTGGGGTCAGGGCGATGGCGCAGGTTTAAAAGTTGTTGAAACCAAAGTGGGTCGTATGGGTGCCTTAGCCTGTTGGGAACATTATAATCCGCTGGCACGCTATGCACTGATGGCACAGCATGAAGAAATTCATGCCTCACATTTTCCGGGCTCTTTAGTGGGCCCGATTTTTGGTGAACAGATTGAAGTGACCATGCGTCATCATGCCTTAGAATCAGGCTGTTTTGTGGTGAATGCCACGGGATGGTTAACCGAAGAACAGATTGCCTCTATTCATCCCGACCCGAAATTACAGAAAGGCTTACGCGATGGCTGTATGACCTGTATTATTACCCCGGAAGGTCGTCATGCAGCCCCGCCGTTAACCGCCGGCGAAGGCATGGTTATTGCGGATCTGGATATGGCCCTGATTACCAAACGTAAACGCATGATGGATACCGTGGGCCATTATGCACGTCCGGAATTACTGCGCTTAGTTCAGGATGCACGCCCGCAGCGTCCGCTGGAACAGATTCTGCCGGAAGCCACCCCGGAAGCAAGCGAAGCCCAGGATGCATAA

[0129] SEQ ID NO:3

[0130] MPKTVRAAAVQIAPDLASRAGTVERVLNAIAEAADKGAELIVFPETFVPWIPYFSFVLPPVQQGLEHLRLYEEAVTVPSPETHAVAEAARKRGAVVVLGVNERDHGSLYNTQLIFDADGTLKLKRRKITPTYHERMIWGQGDGAGLKVVETKVGRMGALACWEHYNPLARYALMAQHEEIHASHFPGSLVGPIFGEQIEVTMRHHALESGCFVVNATGWLTEEQIASIHPDPKLQKGLRDGCMTCIITPEGRHAAPPLTAGEGMVIADLDMALITKRKRMMDTVGHYARPELLRLVQDARPQRPLEQILPEATPEASEAQDA

[0131] SEQ ID NO:4

[0132] MPKTVRAAAVQIAPDLASRAGTVERVLNAIAEAADKGAELIVFPETFVPWRPYFSFVLPPVQQGLEHLRLYEEAVTVPSPETHAVAEAARKRGAVVVLGVNERDHGSLYNTQLIFDADGTLKLKRRKITPTYHERMIWGQGDGAGLKVVETKVGRMGALACWEHYNPLARYALMAQHEEIHASHFPGSLVGPIFGEQIEVTMRHHALESGCFVVNATGWLTEEQIASIHPDPKLQKGLRDGCMTCIITPEGRHAAPPLTAGEGMVIADLDMALITKRKRMMDTVGHYARPELLRLVQDARPQRPLEQILPEATPEASEAQDA

[0133] SEQ ID NO:5

[0134] MPKTVRAAAVQIAPDLASRAGTVERVLNAIAEAADKGAELIVFPETFVPWYPYFSFVLPPVQQGLEHLRLYEEAVTVPSPETHAVAEAARKRGAVVVLGVNERDHGSLYNTQLIFDADGTLKLKRRKITPTYHERMIWGQGDGAGLKVVETKVGRMGALACPEHYNPLARYALMAQHEEIHASHFPGSLVGPIFGEQIEVTMRHHALESGCFVVNATGWLTEEQIASIHPDPKLQKGLRDGCMTCIITPEGRHAAPPLTAGEGMVIADLDMALITKRKRMMDTVGHYARPELLRLVQDARPQRPLEQILPEATPEASEAQDA

[0135] SEQ ID NO:6

[0136] CACCTGCCGGCCNNKGCCGATCACAAGGTC

[0137] SEQ ID NO:7

[0138] CTTGTGATCGGCNNMGGCCGGCAGGTGGTG

[0139] SEQ ID NO:8

[0140] GCCTTAGCCTGTNNKGAACATTATA

[0141] SEQ ID NO:9

[0142] TATAATGTTCACNNMAGGCTAAGGC

[0143] SEQ ID NO:10

[0144] ATGCCTCACATNNKCCGGGCTCTT

[0145] SEQ ID NO:11

[0146] AGAGCCCGGANNMTGTGAGGCAT。

Claims

1. A nitrilase, characterized in that The nitrilase comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises the amino acid sequence shown in SEQ ID NO: 1 and has a mutation at one or more of the amino acid residue positions corresponding to positions 51, 162 and 185 of the amino acid sequence shown in SEQ ID NO:

1.

2. The nitrilase according to claim 1, wherein The amino acid sequence of the nitrilase is I, H, V, D, T, R or E at the 51st amino acid residue corresponding to the amino acid sequence shown in SEQ ID NO: 1; and / or the amino acid residue at position 162 corresponding to the amino acid sequence of SEQ ID NO: 1 is H, E, N, P, C or G; and / or the amino acid residue at position 185 corresponding to the amino acid sequence shown in SEQ ID NO: 1 is N, M, T, C, V, A, G, L, E, P or H.

3. The nitrilase according to claim 2, wherein The amino acid sequence of the nitrilase is shown in SEQ ID NO: 1 and: a. The amino acid residue at position 51 is I, H, R or E; b. The amino acid residue at position 162 is H or P; c. the amino acid residue at position 185 is N, M, T, C or E; or d. A nitrilase derived from a, wherein the amino acid sequence is as defined in any one of items ac, and one or more amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue are substituted, deleted or added at positions other than position 51, 162 or 185, and the nitrilase function as defined in any one of items ac is substantially obtained.

4. The nitrilase according to claim 3, wherein The amino acid sequence of the nitrilase is shown in SEQ ID NO: 1 and: a. The 51st amino acid residue is I or R; b. The amino acid residue at position 162 is H or P; c. The amino acid residue at position 185 is N or M.

5. The nitrilase according to claim 4, wherein The amino acid sequence of the nitrilase is shown in SEQ ID NO: 3, 4 or 5.

6. The nitrilase according to any one of claims 1 to 5, wherein The nitrilase is derived from Cereibacter sphaeroides.

7. A gene encoding the nitrilase according to any one of claims 1 to 6.

8. An expression vector comprising the coding gene according to claim 7.

9. A host cell, comprising the coding gene of claim 7 or the expression vector of claim 8, or the coding gene of claim 7 or the expression vector of claim 8 integrated into the chromosome of the host cell.

10. The method for preparing the nitrilase according to any one of claims 1 to 6, comprising the following steps: 1) culturing the host cell of claim 9 to induce the expression of the nitrilase; 2) optionally isolating the nitrilase according to any one of claims 1 to 6 from the culture obtained in 1).

11. Use of the nitrilase according to any one of claims 1 to 6, or the encoding gene according to claim 7, or the expression vector according to claim 8, or the host cell according to claim 9 in producing amide substances.

12. A method for preparing an amide substance, the method comprising the following steps: 1) culturing the host cell of claim 9 to induce the expression of the nitrilase of any one of claims 1 to 6; and 2) Using the obtained nitrilase to catalyze N-heterocyclic nitrile 2, 3 or 4-cyanopyridine to generate corresponding amide substances.

13. An immobilized enzyme, comprising the nitrilase according to any one of claims 1 to 6.

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