CN112322606B

CN112322606B - Nitrile hydratase mutant and application thereof

Info

Publication number: CN112322606B
Application number: CN202011307426.3A
Authority: CN
Inventors: 周哲敏; 郭军玲; 江诗进; 程中一; 刘中美; 崔文璟; 周丽; 韩来闯
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2022-05-10
Anticipated expiration: 2040-11-20
Also published as: CN112322606A; CN115322981A; CN115322981B; CN114790452B; CN114790452A

Abstract

The invention discloses a nitrile hydratase mutant and application thereof, belonging to the field of genetic engineering and enzyme engineering. The half-life of the nitrile hydratase mutant Str.t NHase-beta L48D at 65 ℃ is about 43 minutes, and compared with other NHase enzymes, the heat stability is obviously improved. By adopting the technical scheme of the invention, the substrate tolerance of the reaction taking nitrile compounds such as nicotinonitrile, acrylonitrile, benzonitrile, 2-cyanopyrazinonitrile, isobutyronitrile, n-valeronitrile, cinnamonitrile and the like as substrates is obviously improved.

Description

Nitrile hydratase mutant and application thereof

Technical Field

The invention relates to a nitrile hydratase mutant and application thereof, belonging to the field of genetic engineering and enzyme engineering.

Background

Nitrile hydratase (NHase) can be used for catalyzing 3-cyanopyridine to generate nicotinamide with higher medicinal value, wherein the nicotinamide is also called nicotinamide, is a vitamin and is widely used in industries such as feed, food, pharmacy and the like. The market demand of nicotinamide is large, more than 2000 tons are expected to be needed every year, but the production level of nicotinamide in China is not high at present, the scale is not large, and a large amount of import is needed, which is about 1000 tons per year. Therefore, the use of NHase for the production of nicotinamide has great potential. However, the reaction is a heat release process, so the high temperature in the production process can influence the exertion of the enzyme activity, mainly the high temperature influences the structure of the enzyme, and the enzyme activity is reduced, thereby leading to a large amount of energy consumption and improving the production cost. Currently, nicotinamide is mainly generated by catalyzing Rhodococcus rhodochrous J1 in industrial production in a substrate fed-batch mode, but the growth cycle of Rhodococcus rhodochrous is long, the production efficiency is not high, the yield of nicotinamide is up to 162g/L, and the yield of acrylamide is up to 300 g/L. At present, the recombinant bacteria are used for producing nicotinamide, but the concentration of the final product is lower and is only 240 g/L.

The instability of nitrile hydratase is also reflected in poor tolerance to the substrate (nitrile organic), and nitrile hydratase is easily inactivated; for example, in the process of preparing acrylamide by enzyme method, the nitrile hydratase has low tolerance to the substrate, so that the final product acrylamide can only be maintained at a low level, which affects the subsequent acrylamide concentration process. With the continuous expansion of the market, the demand of amide compounds is increasing, and therefore, how to improve the substrate tolerance of nitrile hydratase has become important research. The obtained nitrile hydratase with good substrate tolerance and high catalytic efficiency has important application value for industrial production of amides.

Disclosure of Invention

The invention aims to provide a nitrile hydratase mutant with improved stability and enzyme activity and construct an enzyme tool box capable of catalyzing various nitrile substrates with high performance.

The invention firstly provides a nitrile hydratase mutant, which is any one of (1) to (5):

(1) obtained by mutating leucine at position 37 of a nitrile hydratase beta subunit with an amino acid sequence shown as SEQ ID NO. 1;

(2) obtained by mutating phenylalanine at position 41 of beta subunit of nitrile hydratase with amino acid sequence shown as SEQ ID NO. 1;

(3) obtained by mutating tyrosine at position 46 of a beta subunit of nitrile hydratase with an amino acid sequence shown as SEQ ID NO. 1;

(4) the amino acid sequence of the leucine at the 48 th site of the beta subunit of the nitrile hydratase is shown as SEQ ID NO. 1;

(5) obtained by mutating phenylalanine at position 51 of beta subunit of nitrile hydratase with amino acid sequence shown as SEQ ID NO. 1.

In one embodiment of the present invention, the mutant is any one of (1) to (5):

(1) the amino acid sequence of the leucine at the 37 th site of the beta subunit of nitrile hydratase shown in SEQ ID NO.1 is mutated into one of proline, lysine, aspartic acid, alanine and phenylalanine, and the amino acid sequences are respectively named as: β L37P, β L37K, β L37D, β L37A, β L37F;

(2) the method is characterized in that phenylalanine at the 41 th site of a beta subunit of nitrile hydratase with an amino acid sequence shown as SEQ ID NO.1 is mutated into one of proline, lysine, aspartic acid and alanine, and the method is named as: β F41P, β F41K, β F41D, β F41A;

(3) the nitrile hydratase beta subunit 46 th tyrosine with amino acid sequence shown as SEQ ID NO.1 is respectively mutated into one of proline, lysine, aspartic acid, alanine and phenylalanine, and the amino acid sequence is respectively named as: β Y46P, β Y46K, β Y46D, β Y46A, β Y46F;

(4) the amino acid sequence of leucine at the 48 th site of the beta subunit of nitrile hydratase shown in SEQ ID NO.1 is mutated into one of proline, lysine, aspartic acid, alanine and phenylalanine, and the amino acid sequence is named as: β L48P, β L48K, β L48D, β L48A, β L48F;

(5) the amino acid sequence of phenylalanine at position 51 of the beta subunit of nitrile hydratase shown in SEQ ID NO.1 is mutated into one of proline, lysine, aspartic acid and alanine, and the mutation is named as: β F51P, β F51K, β F51D, β P51A.

In one embodiment of the invention, the nitrile hydratase is derived from Streptomyces thermoautotrophicus (Streptomyces thermoautotrophicus).

In one embodiment of the invention, the nucleotide sequence of the β subunit of nitrile hydratase is as shown in SEQ ID NO. 2.

The invention also provides a gene for coding the mutant.

The invention also provides a recombinant plasmid carrying the gene.

In one embodiment of the invention, the recombinant plasmid uses pET-24a as a starting plasmid.

The invention also provides a recombinant cell carrying the gene or the recombinant plasmid.

In one embodiment of the present invention, the recombinant cell is a bacterial or fungal expression host.

In one embodiment of the invention, the expression host is E.coli.

In one embodiment of the invention, the expression host is e.coli BL 21.

The present invention also provides a method for improving nitrile hydratase tolerance, which comprises mutating nitrile hydratase according to any one of the following (1) to (5):

(1) respectively mutating leucine at 37 th position of nitrile hydratase beta subunit with amino acid sequence shown as SEQ ID NO.1 into any one of proline, lysine, aspartic acid, alanine and phenylalanine;

(2) respectively mutating phenylalanine at the 41 th site of a beta subunit of nitrile hydratase with an amino acid sequence shown as SEQ ID NO.1 into any one of proline, lysine, aspartic acid and alanine;

(3) respectively mutating tyrosine at 46 th site of nitrile hydratase beta subunit shown in SEQ ID NO.1 into any one of proline, lysine, aspartic acid, alanine and phenylalanine;

(4) respectively mutating leucine at 48 th site of nitrile hydratase beta subunit with amino acid sequence shown as SEQ ID NO.1 into any one of proline, lysine, aspartic acid, alanine and phenylalanine;

(5) phenylalanine at position 51 of a nitrile hydratase beta subunit with an amino acid sequence shown as SEQ ID NO.1 is mutated into any one of proline, lysine, aspartic acid and alanine.

In one embodiment of the invention, the tolerance is substrate tolerance, and the substrate is a nitrile compound.

In one embodiment of the present invention, the nitrile compound is one or more selected from the group consisting of nicotinonitrile, acrylonitrile, benzonitrile, 2-cyanopyrazinonitrile, isobutyronitrile, n-valeronitrile, and cinnamonitrile.

The present invention also provides a method for increasing nitrile hydratase activity, comprising mutating nitrile hydratase according to any one of the following (1) to (5):

The invention also provides a method for preparing the nitrile hydratase mutant, which comprises the steps of inoculating the recombinant cells into an LB culture medium, and culturing at 35-37 ℃ to OD₆₀₀When the expression level is 0.6-0.8, adding an inducer IPTG to induce for 12-18h at 25 ℃, and expressing the nitrile hydratase mutant enzyme.

In one embodiment of the invention, the method is to inoculate the genetically engineered bacteria in LB expression medium containing kanamycin, and culture the bacteria at 37 ℃ and 200r/min in a shaking way until OD is reached₆₀₀When the concentration is 0.6-0.8, adding inducer IPTG to 0.1mM, Co²⁺And inducing the enzyme to 0.1mg/L at 25 ℃ for 12 to 18 hours to express the nitrile hydratase mutant enzyme.

In one embodiment of the invention, the recombinant cell is a host of escherichia coli BL 21.

In one embodiment of the invention, the recombinant cell uses a pET series plasmid as a vector.

In one embodiment of the invention, the vector is pET24a (+).

In one embodiment of the present invention, the method further comprises collecting the cells of the genetically engineered bacteria, disrupting the cells, collecting the supernatant, membrane-filtering the supernatant, and separating the supernatant with a Strep Trap HP column to obtain a nitrile hydratase mutant.

The invention also provides the application of the mutant, the gene, the recombinant plasmid or the recombinant cell in preparing products containing nicotinamide, acrylamide, hydrocinnamamide, pyrazinamide, valeramide and isobutyramide.

Advantageous effects

(1) The half-life of the nitrile hydratase mutant Str.t NHase-beta L48D at 65 ℃ is 43 minutes, and compared with other NHase enzymes, the heat stability is obviously improved.

(2) The invention provides a nitrile hydratase mutant for improving substrate tolerance, and by adopting the technical scheme of the invention, the substrate tolerance of the nitrile hydratase mutant is obviously improved for the reaction taking nitrile compounds as substrates; the following examples only show the best mutation results: when nicotinonitrile is used as a substrate, the specific enzyme activity of the nitrile hydratase mutant beta L48D is 7 times that of wild enzyme;

when acrylonitrile is used as a substrate, the specific enzyme activity of the nitrile hydratase mutant beta L48F is 2.5 times that of wild enzyme;

when benzonitrile is taken as a substrate, the specific enzyme activity of the nitrile hydratase mutant beta L48K is 3 times that of wild enzyme;

when 2-cyanopyrazine is taken as a substrate, the specific enzyme activity of the nitrile hydratase mutant beta L48D is 3.7 times of that of wild enzyme;

when isobutyronitrile is used as a substrate, the specific enzyme activity of the nitrile hydratase mutant beta L48P is 1.8 times that of wild enzyme;

when n-valeronitrile is taken as a substrate, the specific enzyme activity of the nitrile hydratase mutant beta Y46K is 4.9 times that of wild enzyme;

when cinnamonitrile was used as a substrate, the specific enzyme activity of the nitrile hydratase mutant β Y46A was 7.8 times that of the wild enzyme.

Therefore, the nitrile hydratase mutant provided by the invention not only has good enzymological properties, but also can provide selection for efficiently catalyzing various nitrile substrates, and is beneficial to subsequent industrial production.

Drawings

FIG. 1: the nitrile hydratase mutant Str.t NHase-. beta.L 48D was assayed for thermal stability at 65 ℃.

FIG. 2: and (3) measuring the specific enzyme activity of the nitrile hydratase by using nicotinonitrile as a substrate.

FIG. 3: and (3) measuring the specific enzyme activity of the nitrile hydratase by using acrylonitrile as a substrate.

FIG. 4: and (3) determining the specific enzyme activity of the nitrile hydratase by using benzonitrile as a substrate.

FIG. 5: and (3) determining the specific enzyme activity of the nitrile hydratase by using 2-cyanopyrazine nitrile as a substrate.

FIG. 6: and (3) determining the specific enzyme activity of the nitrile hydratase by using isobutyronitrile as a substrate.

FIG. 7: and (3) measuring the specific enzyme activity of the nitrile hydratase by using n-valeronitrile as a substrate.

FIG. 8: and (3) determining the specific enzyme activity of the nitrile hydratase by using cinnamonitrile as a substrate.

Detailed Description

The media involved in the following examples are as follows:

LB liquid medium: 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl.

2YT liquid medium: 16g/L of peptone, 10g/L of yeast extract and 5g/L of NaCl.

The solutions referred to in the following examples are as follows:

binding buffer: 20mmol/L Na₂HPO₄、280mmol/L NaCl、6mmol/L KCl。

The detection methods referred to in the following examples are as follows:

detection of nitrile hydratase Activity:

the mobile phase was water, as detected by HPLC: acetonitrile 1: 2; the chromatographic column is a C18 column, and the wavelength: and detecting the product generation amount under the optimal detection wavelength of the product.

Nitrile hydratase reaction system:

the substrate was 490. mu.L of a substrate solution of an appropriate concentration, 10. mu.L of a pure enzyme solution of an appropriate concentration was added thereto, the reaction was terminated with 500. mu.L of acetonitrile at a temperature of 25 ℃ for 10 minutes, and the supernatant was collected and passed through a 0.22 μm membrane to prepare a sample for liquid phase assay.

Definition of enzyme activity (U): the amount of enzyme required to convert a nitrile substrate to 1. mu. mol/L of the corresponding amide per minute is defined as 1U.

Specific enzyme activity (U/mg): enzymatic activity per mg of NHase.

Definition of relative enzyme activity: the enzyme activity of the mutant enzyme was defined as 100% when it was reacted at 25 ℃ for 10 minutes at pH 7.4.

The detection method of the thermal stability comprises the following steps: the wild enzyme and the mutant are respectively treated in KPB buffer solution with the pH value of 7.4 at the temperature of 65 ℃ for 30min, 60min, 120min, 180min and 240min, and then the residual enzyme activity is measured, so that the thermal stability result is obtained.

Example 1: construction of nitrile hydratase mutant

The method comprises the following specific steps:

1. (1) construction of mutant β L48D:

the NHase gene (nucleotide sequence is shown in SEQ ID NO. 3) of nitrile hydratase is chemically synthesized, and the gene is cloned at the Nde I and EcoR I enzyme cutting sites of pET24a plasmid and completed by Jinzhi Zhi Suzhou to obtain pET24a-NHase recombinant plasmid. Using pET24a-NHase as a template, carrying out PCR by using primers shown in Table 1 under the conditions shown in Table 2 to obtain a PCR product, transforming E.coli JM109 competent cells of the obtained PCR product, sending the E.coli JM109 competent cells to Suzhou Jinzhi sequencing, and obtaining a plasmid with a correct sequencing result, namely a recombinant plasmid pET24 a-beta L48D carrying a coding mutant gene; respectively transforming E.coli BL21 strains by recombinant plasmids pET24 a-beta L48D and pET24a-NHase for expression, selecting transformants for verification, and obtaining the following products after the verification is correct: recombinant strains E.coli BL21/pET24 a-beta L48D and E.coli BL21/pET24 a-NHase.

TABLE 1 mutant primers

The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 1min, annealing at 58 deg.C for 30s, and extension at 72 deg.C for 2 min; (repeat for 30 cycles); extension at 72 ℃ for 10 min.

The PCR product is identified by an agarose gel electrophoresis method, and then the PCR product is purified, digested and transferred into escherichia coli BL21 competent cells.

(2) Recombinant Escherichia coli E.coli BL21/pET24 a-beta L48D and E.coli BL21/pET24a-Nhase were inoculated to 5mL LB liquid medium containing 50. mu.g/mL kanamycin, respectively, and cultured overnight at 37 ℃ with shaking at 200r/min to obtain seed solutions.

The seed solutions were inoculated at 1% (v/v) into 100mL of 2YT liquid medium containing 50. mu.g/mL kanamycin, and cultured at 37 ℃ with shaking at 200r/min to OD₆₀₀Adding 0.1mM IPTG inducer to 0.6-0.8, and Co concentration of 0.1mg/L²⁺And (3) inducing the ionic solution at 25 ℃ for 12-18h to obtain fermentation liquor, and centrifuging the fermentation liquor at the rotating speed of 12000r to collect thalli.

(3) Concentrating the bacterial cells obtained in step (2) with binding buffer solution 5 times, ultrasonically disrupting to obtain cell disruption solution, centrifuging cell disruption solution 12000r for 40min, collecting supernatant to obtain crude enzyme solution containing nitrile hydratase mutant β L48D and crude enzyme solution containing nitrile hydratase wild enzyme WT, respectively filtering the crude enzyme solutions with 0.22 μm filter, balancing 1mL strep Trap HP column with binding buffer solution of 10 column volumes, washing off non-specifically adsorbed protein with binding buffer solution of 15 column volumes, and washing off with 20mM Na of 8 column volumes₂HPO₄280mM NaCl, 6mM KCl and 2.5mM desthiobiotin buffer solution to elute protein, collecting samples, namely pure enzyme solution containing nitrile hydratase mutant beta L48D and pure enzyme solution containing nitrile hydratase wild enzyme WT, and analyzing and identifying by SDS-PAGE.

2. By using the primers shown in Table 3, according to the method of the above step 1, a pure enzyme solution containing nitrile hydratase mutant β L37P, a pure enzyme solution of β L37K, a pure enzyme solution of β L37D, a pure enzyme solution of β L37A, a pure enzyme solution of β L37F, a pure enzyme solution of β F41P, a pure enzyme solution of β F41K, a pure enzyme solution of β F41D, a pure enzyme solution of β F41A, a pure enzyme solution of β Y46P, a pure enzyme solution of β Y46K, a pure enzyme solution of β Y46D, a pure enzyme solution of β Y46A, a pure enzyme solution of β Y46F, a pure enzyme solution of β L P, a pure enzyme solution of β L48 9, a pure enzyme solution of β L48A, a pure enzyme solution of β L F, a pure enzyme solution of β F51P, a pure enzyme solution of β F8651, a pure enzyme solution of β F D, a pure enzyme solution of β L8651, a pure enzyme solution of β P8651, and a pure enzyme solution of β P867 were obtained, respectively.

TABLE 3 mutant primers

Example 2: half-life of nitrile hydratase mutant

The nitrile hydratase still has good thermal stability after mutation. In this example, the half-life of the mutant β L48D was measured as follows:

mu.L of the mutant enzyme purified in example 1 at a concentration of 0.5mg/ml was added to 500. mu.L of the buffer reaction system, and the mixture was treated in a metal bath at 65 ℃ for 0min, 30min, 60min, 120min, 180min and 240min, respectively, to determine the residual enzyme activity.

As shown in fig. 1, the half-lives of the mutant enzyme β L48D were found to be at 65 ℃ for the mutant: and (6) 43 min.

The results show that: the enzyme activity is improved, and the product still has good thermal stability.

Example 3: determination of substrate nicotinonitrile enzyme activity by nitrile hydratase

mu.L of a pure enzyme solution of the wild enzyme and the mutant obtained in example 2 at a concentration of 0.5mg/ml to 490. mu.L of 200mM nicotinonitrile solution was added to obtain a reaction system, the reaction system was reacted at 25 ℃ for 10 minutes, and then the reaction was terminated with 500. mu.L of acetonitrile to obtain a reaction solution, and the reaction solution was collected and passed through a 0.22 μm membrane as a sample for liquid phase assay to detect the nitrile hydratase specific enzyme activity. The reaction results are shown in fig. 2 and table 4:

table 4: specific enzyme activity of different nitrile hydratase mutants on substrate nicotinonitrile

Sample name	Specific activity (U/mg)
		WT	73.49
L37P	101.95
		L37K	84.62
L37D	135.42
		L37A	88.73
L37F	265.68
		F41P	150.19
F41K	178.60
		F41D	161.44
F41A	119.42
		Y46P	117.70
Y46K	187.11
		Y46D	89.54
Y46A	85.01
		Y46F	67.71
L48P	368.29
		L48K	346.30
L48D	481.22
		L48A	441.12
L48F	209.28
		F51P	177.59
F51K	106.44
		F51D	95.23
F51A	78.66

Example 4: enzyme activity assay of substrate acrylonitrile by nitrile hydratase

mu.L of a pure enzyme solution of the wild enzyme and the mutant obtained in example 2 at a concentration of 0.5mg/ml to 490. mu.L of 200mM nicotinonitrile solution was added to obtain a reaction system, the reaction system was reacted at 25 ℃ for 10 minutes, and then the reaction was terminated with 500. mu.L of acetonitrile to obtain a reaction solution, and the reaction solution was collected and passed through a 0.22 μm membrane as a sample for liquid phase assay to detect the nitrile hydratase specific enzyme activity. The reaction results are shown in fig. 3 and table 5:

table 5: specific enzyme activity of different nitrile hydratase mutants on substrate acrylonitrile

Sample name	Specific activity (U/mg)
		WT	550.84
L37P	655.14
		L37K	310.33
L37D	1066.17
		L37A	639.98
L37F	766.27
		F41P	1004.49
F41K	1050.65
		F41D	794.84
F41A	740.70
		Y46P	599.21
Y46K	1053.44
		Y46D	119.72
Y46A	594.74
		Y46F	311.72
L48P	641.77
		L48K	742.03
L48D	994.78
		L48A	1019.06
L48F	1322.05
		F51P	507.42
F51K	385.06
		F51D	555.66
F51A	604.92

Example 5: enzyme activity assay of substrate benzonitrile by nitrile hydratase

mu.L of a pure enzyme solution of the wild enzyme and the mutant obtained in example 2 at a concentration of 0.1mg/ml was added to 490. mu.L of 50mM nicotinonitrile solution to obtain a reaction system, the reaction system was reacted at 25 ℃ for 10 minutes, and then the reaction was terminated with 500. mu.L of acetonitrile to obtain a reaction solution, and the reaction solution was collected and passed through a 0.22 μm membrane as a sample for liquid phase assay to detect the nitrile hydratase specific enzyme activity. The reaction results are shown in fig. 4 and table 6:

table 6: specific enzyme activity of different nitrile hydratase mutants on substrate benzonitrile

Example 6: enzyme activity determination of substrate 2-cyanopyrazine by nitrile hydratase

mu.L of a pure enzyme solution of the wild enzyme and the mutant obtained in example 2 at a concentration of 0.1mg/ml was added to 490. mu.L of 100mM nicotinonitrile solution to obtain a reaction system, the reaction system was reacted at 25 ℃ for 10 minutes, and then the reaction was terminated with 500. mu.L of acetonitrile to obtain a reaction solution, and the reaction solution was collected and passed through a 0.22 μm membrane as a sample for liquid phase assay to detect the nitrile hydratase specific enzyme activity. The reaction results are shown in fig. 5 and table 7:

table 7: specific enzyme activity of different nitrile hydratase mutants on substrate 2-cyanopyrazine

Example 7: enzyme activity assay of nitrile hydratase on substrate isobutyronitrile

mu.L of a pure enzyme solution of the wild enzyme and the mutant obtained in example 2 at a concentration of 0.1mg/ml to 490. mu.L of 200mM nicotinonitrile solution was added to obtain a reaction system, the reaction system was reacted at 25 ℃ for 10 minutes, and then the reaction was terminated with 500. mu.L of acetonitrile to obtain a reaction solution, and the reaction solution was collected and passed through a 0.22 μm membrane as a sample for liquid phase assay to detect the nitrile hydratase specific enzyme activity. The reaction results are shown in fig. 6 and table 8:

table 8: specific enzyme activity of different nitrile hydratase mutants on substrate isobutyronitrile

Sample name	Specific activity (U/mg)
		WT	521.49
L37P	414.19
		L37K	443.49
L37D	446.00
		L37A	356.51
L37F	215.61
		F41P	490.75
F41K	693.47
		F41D	421.02
F41A	360.10
		Y46P	411.14
Y46K	666.52
		Y46D	525.08
Y46A	339.25
		Y46F	198.90
L48P	954.42
		L48K	139.41
L48D	796.63
		L48A	649.08
L48F	576.12
		F51P	7.32
F51K	278.69
		F51D	8.74
F51A	7.57

Example 8: enzyme activity assay of nitrile hydratase on substrate n-valeronitrile

mu.L of a pure enzyme solution of the wild enzyme and the mutant obtained in example 2 at a concentration of 0.5mg/ml was added to 490. mu.L of 100mM nicotinonitrile solution to obtain a reaction system, the reaction system was reacted at 25 ℃ for 10 minutes, and then the reaction was terminated with 500. mu.L of acetonitrile to obtain a reaction solution, and the reaction solution was collected and passed through a 0.22 μm membrane as a sample for liquid phase assay to detect the nitrile hydratase specific enzyme activity. The reaction results are shown in fig. 7 and table 9:

table 9: specific enzyme activity of different nitrile hydratase mutants on substrate n-valeronitrile

Sample name	Specific activity (U/mg)
		WT	21.91
L37P	9.69
		L37K	9.99
L37D	9.69
		L37A	12.29
L37F	23.98
		F41P	25.32
F41K	37.88
		F41D	22.30
F41A	25.91
		Y46P	30.80
Y46K	104.67
		Y46D	29.15
Y46A	22.44
		Y46F	19.67
L48P	10.78
		L48K	10.41
L48D	16.60
		L48A	31.39
L48F	34.24
		F51P	12.01
F51K	42.91
		F51D	1.38
F51A	5.13

Example 9: enzyme activity determination of nitrile hydratase on substrate cinnamonitrile

mu.L of a pure enzyme solution of the wild enzyme and the mutant obtained in example 2 at a concentration of 0.1mg/ml to 490. mu.L of 5mM nicotinonitrile solution was added to obtain a reaction system, the reaction system was reacted at 25 ℃ for 10 minutes, and then the reaction was terminated with 500. mu.L of acetonitrile to obtain a reaction solution, and the reaction solution was collected and passed through a 0.22 μm membrane as a sample for liquid phase assay to detect the nitrile hydratase specific enzyme activity. The reaction results are shown in fig. 8 and table 10.

Table 10: specific enzyme activity of different nitrile hydratase mutants on substrate cinnamonitrile

Sample name	Specific activity (U/mg)
		WT	16.50
L37P	55.37
		L37K	2.18
L37D	22.82
		L37A	56.46
L37F	1.50
		F41P	50.40
F41K	8.89
		F41D	14.4
F41A	6.90
		Y46P	31.62
Y46K	78.63
		Y46D	19.90
Y46A	128.55
		Y46F	9.57
L48P	43.97
		L48K	0.52
L48D	15.19
		L48A	27.02
L48F	7.07
		F51P	40.24
F51K	20.43
		F51D	54.23
F51A	82.87

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> nitrile hydratase mutant and application thereof

<130> BAA201052A

<160> 3

<170> PatentIn version 3.3

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Met Asn Gly Val His Asp Leu Gly Gly Thr Asp Gly Leu Gly Thr Ile

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Gly Pro Glu Glu Asn Glu Pro Val Phe His Ser Glu Trp Glu Lys Val

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Val Phe Ala Leu Leu Pro Ala Thr Phe Ala Ala Gly Tyr Tyr Asn Leu

35 40 45

Asp Gln Phe Arg His Gly Ile Glu Gln Met His Pro Val Glu Tyr Leu

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Ser Ser Arg Tyr Tyr Glu His Trp Leu His Thr Ile Thr His His Ala

65 70 75 80

Ile Arg Val Gly Ala Ile Asp Pro Asp Glu Leu Asp Glu Arg Thr Arg

85 90 95

Tyr Tyr Arg Asp Asn Pro Asp Ala Pro Leu Pro Asp Arg Arg Asn Pro

100 105 110

Glu Leu Leu Lys Leu Met Glu Thr Ile Val Ala Gln Gly Ser Ser Ala

115 120 125

Arg Arg Pro Leu Asp Ser Lys Pro Arg Phe Ser Ile Gly Asp Arg Val

130 135 140

Arg Val Ala Asp Asp His Pro Phe Gly His Thr Arg Arg Ala Arg Tyr

145 150 155 160

Ile Arg Gly Lys Val Gly Val Ile Asp Arg Val His Gly Thr Phe Ile

165 170 175

Tyr Pro Asp Thr Ala Ala Arg Gly Glu Gly Asp Asp Pro Gln Trp Val

180 185 190

Tyr Ser Val Arg Phe Asp Ala Lys Glu Leu Trp Gly Glu Gln Tyr Ala

195 200 205

Asp Ala Asn Gly Ser Val Tyr Phe Asp Val Trp Glu Pro Tyr Ile Asp

210 215 220

Arg Val

225

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atgaacggtg ttcacgacct gggtggtacc gacggtctgg gtaccatcgg tccggaagaa 60

aacgaaccgg ttttccactc tgaatgggaa aaagttgttt tcgctctgct gccggctacc 120

ttcgctgctg gttactacaa cctggaccag ttccgtcacg gtatcgaaca gatgcacccg 180

gttgaatacc tgtcttctcg ttactacgaa cactggctgc acaccatcac ccaccacgct 240

atccgtgttg gtgctatcga cccggacgaa ctggacgaac gtacccgtta ctaccgtgac 300

aacccggacg ctccgctgcc ggaccgtcgt aacccggaac tgctgaaact gatggaaacc 360

atcgttgctc agggttcttc tgctcgtcgt ccgctggact ctaaaccgcg tttctctatc 420

ggtgaccgtg ttcgtgttgc tgacgaccac ccgttcggtc acacccgtcg tgctcgttac 480

atccgtggta aagttggtgt tatcgaccgt gttcacggta ccttcatcta cccggacacc 540

gctgctcgtg gtgaaggtga cgacccgcag tgggtttact ctgttcgttt cgacgctaaa 600

gaactgtggg gtgaacagta cgctgacgct aacggttctg tttacttcga cgtttgggaa 660

ccgtacatcg accgtgtt 678

<210> 3

<211> 1825

<212> DNA

<213> Artificial sequence

<400> 3

atgaacggtg ttcacgacct gggtggtacc gacggtctgg gtaccatcgg tccggaagaa 60

aacgaaccgg ttttccactc tgaatgggaa aaagttgttt tcgctctgct gccggctacc 120

ttcgctgctg gttactacaa cctggaccag ttccgtcacg gtatcgaaca gatgcacccg 180

gttgaatacc tgtcttctcg ttactacgaa cactggctgc acaccatcac ccaccacgct 240

atccgtgttg gtgctatcga cccggacgaa ctggacgaac gtacccgtta ctaccgtgac 300

aacccggacg ctccgctgcc ggaccgtcgt aacccggaac tgctgaaact gatggaaacc 360

atcgttgctc agggttcttc tgctcgtcgt ccgctggact ctaaaccgcg tttctctatc 420

ggtgaccgtg ttcgtgttgc tgacgaccac ccgttcggtc acacccgtcg tgctcgttac 480

atccgtggta aagttggtgt tatcgaccgt gttcacggta ccttcatcta cccggacacc 540

gctgctcgtg gtgaaggtga cgacccgcag tgggtttact ctgttcgttt cgacgctaaa 600

gaactgtggg gtgaacagta cgctgacgct aacggttctg tttacttcga cgtttgggaa 660

ccgtacatcg accgtgtttg gagccacccg cagttcgaaa agtaaaagga gatatagata 720

tgtctacctc tcagtctccg ccgccgatct ctgaatcttt cccgaaatct gaagaagaaa 780

tcgctgctcg tgttaaagct ctggaatctc tgctgatcga aaaaggtgtt ctgaccaccg 840

aagttgttga ccgtatcgct gaaatctacg aacacgaagt tggtccgcac ctgggtgcta 900

aagttgttgc tcgtgcttgg gttgacccgg aattcaaaaa acgtctgctg gctgacgctt 960

ctgctgcttg ccgtgaactg cacatcggtg gtctgcaggg tgaagacatg gttgttgttg 1020

aaaacaccga ctctgttcac aacgttgttg tttgcaccct gtgctcttgc tacccgtggc 1080

cggttctggg tctgccgccg aactggtaca aatacccggc ttaccgtgct cgtatcgttc 1140

gtgaaccgcg taccgttctg cgtgaagaat tcggtctgga cctgccggaa tctgttgaaa 1200

tccgtgtttg ggactcttct gctgaactgc gttactgggt tctgccgcag cgtccggctg 1260

gtaccgaaca cctgtctgaa gaacagctgg ctgctctggt tacccgtgac tctatgatcg 1320

gtgttggtct gccgcgttct ccgcaggaag gttaaaagga gatatagata tgaaagctat 1380

gacctctacc gctcgtgacg ttcgtcagcg tttcctgcag gacgtttctc aggaccgtgc 1440

taaagttgaa cagctgctgg accagctgcc ggaaggtgct gctatcccga aaaaatgcgg 1500

tgaagcttct ttcgacaaag cttgggaaat ccgtgctttc gctctggctg ttgctgctca 1560

ccaggttggt cagtacgaat ggtctgaatt ccagcgtgaa ctgatcggtg ctatctctcg 1620

ttgggaatct accgctgctg accagccgtg gcgttactac gaccgttggc tggaagctct 1680

ggaatctctg ctggctgctt ctggtctggt taccaaatct gaactggacg accgtacccg 1740

taaagttctg gctaccccgc gtgacacctc tcaccagcac gctcgtcgtg acccggttgc 1800

tgttgactct ggtaaccacg cttaa 1825

Claims

1. A nitrile hydratase mutant, wherein the nucleotide sequence of the wild nitrile hydratase gene is as shown in SEQ ID No.3, and wherein the nitrile hydratase mutant is:

the amino acid sequence is shown as SEQ ID NO.1, wherein leucine at the 48 th site of the beta subunit of the nitrile hydratase is respectively mutated into any one of proline, lysine, aspartic acid, alanine and phenylalanine.

2. A gene encoding the mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. A recombinant cell carrying the gene of claim 2, or the recombinant plasmid of claim 3.

5. The recombinant cell of claim 4, wherein E.coli is the expression host.

6. A method for improving the nitrile compound tolerance of nitrile hydratase, wherein the nucleotide sequence of the wild nitrile hydratase gene is shown as SEQ ID NO.3, comprises mutating nitrile hydratase according to the following method:

respectively mutating leucine at the 48 th site of a beta subunit of nitrile hydratase with an amino acid sequence shown as SEQ ID NO.1 into any one of proline, lysine, aspartic acid, alanine and phenylalanine; the nitrile compound is: one or more of nicotinonitrile, acrylonitrile, benzonitrile and 2-cyanopyrazinonitrile.

7. A method for improving the nitrile compound tolerance of nitrile hydratase, wherein the nucleotide sequence of the wild nitrile hydratase gene is shown as SEQ ID NO.3, comprises mutating nitrile hydratase according to the following method:

leucine at the 48 th site of a beta subunit of nitrile hydratase with an amino acid sequence shown as SEQ ID NO.1 is mutated into any one of proline, aspartic acid, alanine and phenylalanine respectively, and the nitrile compound is isobutyronitrile.

8. A method for improving the nitrile compound tolerance of nitrile hydratase, wherein the nucleotide sequence of the wild nitrile hydratase gene is shown as SEQ ID NO.3, comprises mutating nitrile hydratase according to the following method:

leucine at the 48 th site of a beta subunit of nitrile hydratase with an amino acid sequence shown as SEQ ID NO.1 is mutated into alanine or phenylalanine respectively, and the nitrile compound is n-valeronitrile.

9. A method for improving the nitrile compound tolerance of nitrile hydratase, wherein the nucleotide sequence of the wild nitrile hydratase gene is shown as SEQ ID NO.3, comprises mutating nitrile hydratase according to the following method:

leucine at the 48 th site of a beta subunit of nitrile hydratase with an amino acid sequence shown as SEQ ID NO.1 is mutated into proline or alanine respectively, and the nitrile compound is cinnamonitrile.

10. Use of the mutant of claim 1, or the gene of claim 2, or the recombinant plasmid of claim 3, or the recombinant cell of claim 4 or 5 for the preparation of a product containing nicotinamide, acrylamide, phenylacrylamide and pyrazinamide.

11. Use of a nitrile hydratase mutant, or a gene encoding such a mutant, or a recombinant plasmid comprising such a mutant, or a recombinant cell carrying a gene encoding such a mutant, or a recombinant plasmid comprising such a mutant, in the preparation of a product comprising valeramide, wherein the nucleotide sequence of the wild type nitrile hydratase gene is as shown in SEQ ID No.3 and the nitrile hydratase mutant is: the leucine at the 48 th site of the beta subunit of nitrile hydratase with the amino acid sequence shown as SEQ ID NO.1 is mutated into alanine or phenylalanine respectively.

12. Use of a nitrile hydratase mutant, or a gene encoding such a mutant, or a recombinant plasmid comprising such a mutant, or a recombinant cell carrying a gene encoding such a mutant, or a recombinant cell carrying a recombinant plasmid comprising such a mutant, for the preparation of a product comprising isobutyramide, wherein the nucleotide sequence of the wild type nitrile hydratase gene is as shown in SEQ ID No.3, and wherein the nitrile hydratase mutant is: leucine at the 48 th site of the beta subunit of nitrile hydratase with the amino acid sequence shown as SEQ ID NO.1 is mutated into any one of proline, aspartic acid, alanine and phenylalanine.