CN113462677A

CN113462677A - Alpha subunit mutated nitrile hydratase mutant and application thereof

Info

Publication number: CN113462677A
Application number: CN202110867862.4A
Authority: CN
Inventors: 杨立荣; 许金玲; 周海胜; 张红玉; 吴坚平
Original assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Current assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2021-10-01
Anticipated expiration: 2041-07-29
Also published as: CN113462677B

Abstract

The invention discloses a nitrile hydratase mutant with alpha subunit mutation and application thereof. The invention remarkably improves the activity expression capacity of nitrile hydratase derived from Bordetella sp DSM12804(Bordetella petrii DSM12804) in genetically engineered bacterium E.coli BL21(DE3) through protein molecule modification. The catalytic activity of the nitrile hydratase mutant is obviously improved by taking acrylonitrile as a substrate, and the enzyme activity of unit LB shake flask fermentation liquor is the highest as that of the mutant NHAB-A2M and is 1.7 times of that of the initial NHAB; the enzyme activity of the mutant NHAB-A14M is improved by 1.4 times, the thermal stability is remarkably improved, and after heat treatment for 1 hour at 45 ℃, the residual enzyme activity is 78 percent and is 4.8 times of that of the wild type.

Description

Alpha subunit mutated nitrile hydratase mutant and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a nitrile hydratase mutant with alpha subunit mutation and application thereof.

Background

Nitrile hydratase (NHase) is a biocatalyst that catalyzes the hydration of Nitrile compounds to produce amides. Nitrile hydratase produced by microorganisms can efficiently catalyze acrylonitrile to hydrate to generate acrylamide; polyacrylamide generated by acrylamide polymerization is widely applied to the aspects of petroleum exploitation, water treatment, papermaking and the like as a resistance reducing agent, a flocculating agent and a thickening agent.

Compared with the traditional chemical catalysis method, the biological enzyme catalysis method for preparing acrylamide has the advantages of high conversion efficiency, high product purity, mild reaction conditions, environmental friendliness and the like, and has the tendency of gradually replacing the chemical method. Mitsubishi utilizes Rhodococcus rhodochrous J1(CN91101323.7) containing nitrile hydratase to catalyze acrylonitrile to produce acrylamide with annual yield of 20 million tons or more. However, rhodococcus is a wild strain, which has a long growth cycle and low production efficiency and is stable only at 10-30 ℃, so that the rhodococcus has certain limitation in practical production application. At present, the research of acrylamide production by a biological enzyme catalysis method focuses on the discovery of a high-yield nitrile hydratase strain and the improvement of the modification of the performance of nitrile hydratase. The problem of poor thermal stability of nitrile hydratase generally exists in the industrial application process, but the nitrile hydration process belongs to exothermic reaction, and the catalytic efficiency is seriously reduced in the later period of the reaction, so that the efficiency of the whole production process is not high; therefore, in the industrial catalytic process, a cooling device and other processes need to be introduced, which causes energy waste and increases the production cost.

The practical problem in acrylamide production can be solved to a certain extent by constructing a genetic engineering strain with nitrile hydratase catalytic activity and carrying out protein engineering transformation on the nitrile hydratase. The genetic engineering bacteria have strong adaptability and short fermentation period, are beneficial to realizing large-scale culture and industrial production, and can effectively reduce the production cost. Patent literature (CN104498466) discloses a Nitrile Hydratase (NHAB) gene sequence derived from Bordetella DSM12804(Bordetella petrii DSM12804), which has high catalytic activity for acrylonitrile, but its enzyme activity and thermal stability are still not satisfactory for industrialization. Therefore, the NHAB is selected for protein engineering transformation, and the nitrile hydratase mutant with improved enzyme activity and thermal stability can be obtained, and is better applied to the large-scale production of acrylamide.

Disclosure of Invention

The invention aims at nitrile hydratase derived from Bordetella DSM12804(Bordetella petrii DSM12804), carries out rational design on alpha subunit where the catalytic activity center is positioned, provides 3 nitrile hydratase mutants, not only improves the expression enzyme activity, but also greatly improves the heat stability.

An alpha subunit mutant of nitrile hydratase is obtained by mutating a wild-type alpha subunit sequence, wherein the amino acid sequence of the wild-type alpha subunit is shown as SEQ ID NO.1, and the mutation site is any one of the following groups:

(1) two-point mutation: alanine 105 to aspartic acid and serine 122 to valine;

(2) three points are mutated: alanine at position 71 is mutated into aspartic acid, alanine at position 105 is mutated into aspartic acid, and serine at position 122 is mutated into valine;

(3) fourteen-point mutation: serine at position 30 is mutated to threonine, valine at position 46 is mutated to isoleucine, alanine at position 71 is mutated to aspartic acid, alanine at position 74 is mutated to aspartic acid, alanine at position 78 is mutated to arginine, asparagine at position 79 is mutated to aspartic acid, serine at position 81 is mutated to threonine, valine at position 92 is mutated to arginine, aspartic acid at position 96 is mutated to histidine, threonine at position 97 is mutated to methionine, alanine at position 105 is mutated to aspartic acid, serine at position 122 is mutated to valine, alanine at position 133 is mutated to proline, and alanine at position 179 is mutated to glutamic acid.

The invention also discloses a nitrile hydratase mutant with the mutant alpha subunit, which comprises the alpha subunit and the beta subunit, wherein the alpha subunit is the alpha subunit mutant. The amino acid sequence of the beta subunit is shown as SEQ ID NO. 2.

The invention also discloses a gene cluster for expressing the nitrile hydratase mutant, and the gene cluster comprises gene sequences respectively encoding alpha subunit mutant, beta subunit and regulatory protein p 14K.

The gene cluster has a gene sequence of coding the alpha subunit mutant shown as any one of SEQ ID NO. 7-9, a gene sequence of coding the beta subunit shown as SEQ ID NO.10, and a gene sequence of coding the regulatory protein p14K shown as SEQ ID NO. 11.

The gene cluster has a first connecting sequence between a gene sequence for coding the alpha subunit mutant and a gene sequence for coding the beta subunit, and the sequence of the first connecting sequence is as follows: GGAGATCATC, respectively; the gene sequence coding the beta subunit and the gene sequence coding the regulatory protein p14K have a second connecting sequence, and the sequence of the second connecting sequence is as follows: and TC. The alpha subunit, the beta subunit and the coding genes corresponding to the regulatory protein p14K are independently expressed, each has an initiation codon and a termination codon, and the first connecting sequence and the second connecting sequence are non-coding sequences and belong to RBS binding sites. Wherein the alpha subunit two-point mutant nitrile hydratase is NHAB-A2M, the alpha subunit three-point mutant nitrile hydratase is NHAB-A3M, and the alpha subunit fourteen-point mutant nitrile hydratase is NHAB-A14M.

The invention also provides a recombinant expression vector containing the gene cluster.

The invention also provides a genetic engineering bacterium containing the recombinant expression vector.

The invention also provides application of the nitrile hydratase mutant in catalyzing acrylonitrile to produce acrylamide.

The invention also provides application of the genetic engineering bacteria in catalyzing acrylonitrile to produce acrylamide.

The invention has the beneficial effects that: the activity expression capacity of nitrile hydratase derived from Bordetella DSM12804(Bordetella petrii DSM12804) in genetically engineered bacterium E.coli BL21(DE3) is obviously improved through protein molecule modification. The catalytic activity of the nitrile hydratase mutant is obviously improved by taking acrylonitrile as a substrate, and the enzyme activity of unit LB shake flask fermentation liquor is the highest as that of the mutant NHAB-A2M and is 1.7 times of that of wild type NHAB; the enzyme activity of the mutant NHAB-A14M is improved by 1.4 times, the thermal stability is remarkably improved, and after heat treatment for 1 hour at 45 ℃, the residual enzyme activity is 78 percent and is 4.8 times of that of the wild type.

Drawings

FIG. 1 is a gas phase detection spectrum of acrylonitrile catalyzed by nitrile hydratase NHAB to form acrylamide.

FIG. 2 is a graph showing the results of the initial relative enzyme activities of the nitrile hydratase wild type NHAB and the mutant and the residual enzyme activity after heat treatment in a water bath at 45 ℃ for 1 hour.

Detailed Description

The experimental methods in the present invention are conventional methods unless otherwise specified, and the gene cloning procedures can be specifically described in molecular cloning protocols, compiled by J. Sambruka et al.

The invention relates to recombinant Escherichia coli with nitrile hydratase gene, wherein the carrier is pET-30a (+), and the host is Escherichia coli E.coli BL21(DE 3). Kits for preparing chemocompetent cells were purchased from TAKARA.

Reagents used in the downstream catalytic process: acrylonitrile was purchased from national drug group chemical reagents, Inc.; acetamide was purchased from Shanghai Michelin Biotech, Inc.; acrylamide was purchased from alatin reagent, inc; other commonly used reagents are available from the national pharmaceutical group chemical agents, ltd. The three-letter or one-letter expression of amino acids used in the present text uses the amino acid code specified by IUPAC (Eur. J. biochem., 138: 9-37, 1984).

Definition of enzyme Activity (U/mL): the amount of enzyme required to catalyze the formation of 1. mu. mol acrylamide from acrylonitrile as substrate per minute.

A nitrile hydratase enzyme activity standard detection system: appropriate amount of enzyme solution, 50g/L substrate, total volume is 5mL, reaction medium is 0.25M phosphate buffer solution with pH 7.5. Reacting at 28 ℃ for 5min, and detecting the generation amount of the product acrylamide by gas phase.

Detection of acrylamide: gas chromatography analysis and determination, wherein the carrier gas is nitrogen, the flow rate is 0.24mL/min, the temperature of the column furnace is 210 ℃, the detection temperature is 250 ℃, and the gasification temperature is 250 ℃. The chromatographic column is a Propack Q packed column.

Determination of thermal stability: both the naive NHAB and mutant were resuspended to OD in PB buffer pH7.5₆₀₀The residual enzyme activity was measured after heat treatment of the bacterial solution of 2 in a water bath at 45 ℃ for 1 hour, and the enzyme activity of the enzyme without heat treatment was defined as 100%, to obtain the result of thermal stability.

Example 1: construction of initial NHAB and mutant recombinant bacteria

The wild-type nitrile hydratase NHAB from Bordetella DSM12804(Bordetella petrii DSM12804) consists of an alpha subunit, a beta subunit and a regulatory protein p14K, and has the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3. Protein engineering is carried out on the alpha subunit of the catalytic active center: the alpha subunit amino acid sequence of the wild type enzyme is shown in SEQ ID NO.4, wherein the 105 th alanine of the alpha subunit of the wild type enzyme is mutated into aspartic acid, and the 122 th serine of the wild type enzyme is mutated into valine to obtain a mutant NHAB-A2M.

Furthermore, alanine at position 71 of the alpha subunit of the wild-type enzyme is mutated into aspartic acid, alanine at position 105 is mutated into aspartic acid, and serine at position 122 is mutated into valine to obtain a mutant NHAB-A3M, wherein the amino acid sequence of the alpha subunit is shown as SEQ ID NO. 5.

Furthermore, the alpha subunit of the wild-type enzyme is mutated from serine at position 30 to threonine, valine at position 46 to isoleucine, alanine at position 71 to aspartic acid, alanine at position 74 to aspartic acid, alanine at position 78 to arginine, asparagine at position 79 to aspartic acid, serine at position 81 to threonine, valine at position 92 to arginine, aspartic acid at position 96 to histidine, threonine at position 97 to methionine, alanine at position 105 to aspartic acid, serine at position 122 to valine, alanine at position 133 to proline, and alanine at position 179 to glutamic acid to obtain the mutant NHAB-A14M, wherein the alpha subunit amino acid sequence is shown in SEQ ID NO. 6.

Then, combining (using on the same vector) gene sequences SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 encoding the gene sequences SEQ ID NO.4, 5 and 6, respectively, with the wild-type beta subunit gene (SEQ ID NO.10) of nitrile hydratase derived from Bordetella DSM12804(Bordetella petrii DSM12804) and the regulatory protein p14k gene (SEQ ID NO.11), respectively, to obtain a nitrile hydratase gene cluster α - β -p14k, wherein the linkage sequence between the α subunit and the β subunit is GGAGATCATC and the linkage sequence between the β subunit and the regulatory protein p14k is TC; the gene cluster is constructed on an expression vector pET-30a (+), and is transferred into escherichia coli genetic engineering bacteria E.coli BL21(DE3) to obtain an expression NHAB-A2M strain E.coli BL21(DE3) -pET-30a (+) -NHAB-A2M, an expression NHAB-A3M strain E.coli BL21(DE3) -pET-30a (+) -NHAB-A3M and an expression NHAB-A14M strain E.coli BL21(DE3) -pET-30a (+) -NHAB-A14M.

The new biotechnology limited of Beijing Okagaku was entrusted with codon optimization and gene synthesis services, and the genes of wild type NHAB and mutant of nitrile hydratase derived from Bordetella petrii DSM12804 were cloned on pET-30a (+) plasmid, and the target gene was placed between enzyme cutting sites EcoR I and Hind III. Recombinant plasmids pET-30a (+) -NHAB and pET-30a (+) -NHAB-A2M, pET-30a (+) -NHAB-A3M and pET-30a (+) -NHAB-A14M were obtained and transformed into the genetically engineered bacterium E.coli BL21(DE3), respectively. Obtaining a strain E.coli BL21(DE3) -pET-30a (+) -NHAB-A2M for expressing NHAB-A2M, a strain E.coli BL21(DE3) -pET-30a (+) -NHAB-A3M for expressing NHAB-A3M, and a strain E.coli BL21(DE3) -pET-30a (+) -NHAB-A14M for expressing NHAB-A14M.

Example 2: microbial culture and enzyme activity determination

(1) Cultivation of microorganisms

Composition of LB liquid medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl, dissolving the peptone with deionized water, fixing the volume, and sterilizing the peptone at 121 ℃ for 20min for later use. LB solid medium (plate culture dish): adding 20g/L agar powder on the basis of LB liquid culture medium, sterilizing at 121 ℃, cooling, introducing into a culture dish, and making into a flat plate.

Coli BL21(DE3) (obtained in example 1) containing the relevant gene was inoculated into 5mL of LB liquid medium containing 50. mu.g/mL of kanamycin and shake-cultured at 37 ℃ for 12 hours. Transferred into 500mL of fresh LB liquid medium containing 50. mu.g/mL Kan, and shake-cultured at 37 ℃ to OD₆₀₀When reaching about 0.8, IPTG was added to a concentration of 0.5mM, cobalt chloride was added to a final concentration of 0.4mM, and induction culture was carried out at 18 ℃ for 16-18 hours. And after the culture is finished, centrifuging the culture solution at 4000rpm for 10min, removing the supernatant, collecting the somatic cells, and storing the somatic cells in an ultra-low temperature refrigerator at-70 ℃ for later use.

(2) Determination of enzyme Activity

The bacterial cells collected after completion of the culture were washed twice with 0.25M PB buffer (pH 7.5). The cells were then resuspended in 2 fermentation volumes of 0.25M PB buffer (pH7.5) and the cell suspension used for subsequent assays.

Definition of enzyme activity: the international conference on enzymology in 1961 stipulates that 1 unit of enzyme activity means the amount of enzyme that can convert 1. mu. mol of a substrate or 1. mu. mol of a group of interest in a substrate in 1 minute under specific conditions.

Nitrile hydratase enzyme activity determination system: the total volume was 5mL, the reaction medium was 0.25M phosphate buffer pH7.5, the substrate was 4320. mu.L 50g/L acrylonitrile, 430. mu.L OD was added₆₀₀After reacting the resulting bacterial solution at 28 ℃ for 5min, 250. mu.L of 4mol/L HCl was added to terminate the reaction.

The reaction solution was centrifuged at 12000rpm for 2min, and the amount of acrylamide produced was measured by a gas phase internal standard method by adding an equivalent amount of an internal standard (20g/L acetamide solution) to the supernatant. The gas phase analysis spectrum is shown in FIG. 1. FIG. 1 is a gas phase detection spectrum of acrylamide formation by nitrile hydratase NHAB catalyzed acrylonitrile, and the appearance sequence is substrate, internal standard substance and product, wherein the retention time of substrate acrylonitrile is 1.2min, the retention time of internal standard acetamide is 4.4min, and the retention time of product acrylamide is 7.7 min.

The crude enzyme activity of the fermentation liquor of the wild type NHAB is defined as 100 percent, and the mutant NHAB-A2M is improved by 1.7 times compared with the wild type and is the best of the three mutants; whereas the crude enzyme activity of NHAB-A14M was increased by 1.4-fold.

Example 3: thermostability assay for NHAB wild-type and mutants

Thermostability characterization of the enzymes: and respectively treating the enzyme solutions in a hot water bath at 45 ℃ for 60min, and then determining the residual enzyme activity by using an enzyme activity detection system, wherein the enzyme activity of the untreated enzyme is defined as 100%. The residual enzyme activity of the NHAB wild type is only 16% after 60min at 45 ℃, the best heat stability is the mutant NHAB-A14M, and the residual enzyme activity is 76% at 45 ℃ after 60min and is 4.75 times of that of the wild type. The thermostability of wild-type NHAB and all mutants is shown in figure 2. FIG. 2 is a graph showing the results of the initial relative enzyme activities of the nitrile hydratase wild type NHAB and the mutant and the residual enzyme activities after heat treatment in a water bath at 45 ℃ for 1 hour, wherein the initial relative enzyme activities are shown by solid columns, and the NHAB wild type is 100%; the striped bars indicate the residual enzyme activity after 1h heat treatment at 45 ℃.

Example 4: NHAB-A2M catalyzing acrylonitrile to acrylamide

The genetically engineered bacterium E.coli BL21(DE3) -pET-30a (+) -NHAB-A2M constructed in example 2 was taken, centrifuged at 4000rpm for 10min to collect the bacteria, 9.2g of wet bacteria were weighed and resuspended in 200mL of 0.25M phosphate buffer (pH7.5), hydration reaction was carried out at 20 ℃, acrylonitrile was added dropwise at the flow rate of 0.22g/min for the first 2h, and at the flow rate of 0.18g/min for the second 2 h. The dropwise addition was stopped and the reaction was continued for a total of 8 h. And then detecting the contents of acrylonitrile, acrylamide and acrylic acid in the reaction system by using gas chromatography. The substrate conversion rate is more than 89%, the acrylamide concentration is 285g/L, and no acrylic acid is generated in the reaction system.

Example 5: NHAB-A3M catalyzing acrylonitrile to acrylamide

The fermentation broth of the genetically engineered bacterium E.coli BL21(DE3) -pET-30a (+) -NHAB-A3M constructed in example 2 was centrifuged at 4000rpm for 10min to collect the mycelia, 9.2g of wet mycelia were weighed and resuspended in 200mL of 0.25M phosphate buffer (pH7.5) for hydration at 20 ℃, acrylonitrile was added dropwise at a flow rate of 0.22g/min for the first 2 hours, and then at a flow rate of 0.18g/min for the second 2 hours. The dropwise addition was stopped and the reaction was continued for a total of 8 h. And then detecting the contents of acrylonitrile, acrylamide and acrylic acid in the reaction system by using gas chromatography. The substrate conversion rate is more than 87 percent, the acrylamide concentration is 280g/L, and no acrylic acid is generated in the reaction system.

Example 6: NHAB-A14M catalyzing acrylonitrile to acrylamide

The genetically engineered bacterium E.coli BL21(DE3) -pET-30a (+) -NHAB-A14M constructed in example 2 was taken, centrifuged at 4000rpm for 10min to collect the bacteria, 9.2g of wet bacteria were weighed and resuspended in 200mL of 0.25M phosphate buffer (pH7.5), hydration reaction was carried out at 20 ℃, acrylonitrile was added dropwise at the flow rate of 0.22g/min for the first 2h, and then at the flow rate of 0.18g/min for the second 2 h. The dropwise addition was stopped and the reaction was continued for a total of 8 h. And then detecting the contents of acrylonitrile, acrylamide and acrylic acid in the reaction system by using gas chromatography. The substrate conversion rate is more than 90 percent, the acrylamide concentration is 290g/L, and no acrylic acid is generated in the reaction system.

Comparative example 1: NHAB catalyzed acrylonitrile to acrylamide

The genetically engineered bacterium E.coli BL21(DE3) -pET-30a (+) -NHAB constructed in example 2 was taken, centrifuged at 4000rpm for 10min to collect the bacterium, 9.2g of wet bacterium was weighed and resuspended in 200mL of 0.25M phosphate buffer (pH7.5) and hydrated at 20 ℃, acrylonitrile was added dropwise at the flow rate of 0.22g/min for the first 2h and at the flow rate of 0.18g/min for the last 2 h. The dropwise addition was stopped and the reaction was continued for a total of 8 h. And then detecting the contents of acrylonitrile, acrylamide and acrylic acid in the reaction system by using gas chromatography. The substrate conversion rate is more than 78 percent, the acrylamide concentration is 250g/L, and no acrylic acid is generated in the reaction system.

Sequence listing

<110> Hangzhou international scientific center of Zhejiang university

<120> alpha subunit mutant of nitrile hydratase and application thereof

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Arg Val Trp Asp Thr Thr Ala Glu Leu Arg Tyr Met Val Leu Pro Glu

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Arg Pro Ala Gly Thr Glu Gly Tyr Ser Glu Glu Gln Leu Ala Glu Leu

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Thr Tyr Glu His Lys Val Gly Pro Arg Asn Gly Ala Lys Val Val Ala

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atggggcaat cacacacaca cgaccaccat cacgacgggt accaggcacc gcctgaagac 60

attgcgctgc gggtgaaggc cttggagtct ctgctcgtcg agaaaggttt ggtcgacccg 120

gcggccatgg acgctgtggt ccaaacctat gaacacaagg tgggccctcg gaacggcgcc 180

aaggttgttg ccaaggcctg ggtggacccg gcatacaagg cgcgcttgct ggcgaatggc 240

agcgctggca ttgccgaact gggcttctct ggagtgcagg gagaagacac agtcattctg 300

gaaaacaccc ccgacgtgca caacgtcttc gtctgcaccc tgtgctcttg ctacccatgg 360

ccggtgctgg gcttgccgcc ggcctggtac aaggccgcac cctaccggtc gcgcatggtg 420

agcgacccgc gtggggtcct ggcggagttc ggtttggtga tccccaccaa caaggaaatc 480

cgcgtctggg acaccacagc cgaattgcgc tacatggtgc tgccggaaag gcccgcagga 540

accgaaggct acagcgaaga acaactggcc gaactcgtca cccgcgattc gatgatcggc 600

actggcctgc ccacccaacc caaaccttcc cactaa 636

<210> 8

<211> 636

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggggcaat cacacacaca cgaccaccat cacgacgggt accaggcacc gcctgaagac 60

attgcgctgc gggtgaaggc cttggagtct ctgctcgtcg agaaaggttt ggtcgacccg 120

gcggccatgg acgctgtggt ccaaacctat gaacacaagg tgggccctcg gaacggcgcc 180

aaggttgttg ccaaggcctg ggtggacccg gactacaagg cgcgcttgct ggcgaatggc 240

agcgctggca ttgccgaact gggcttctct ggagtgcagg gagaagacac agtcattctg 300

gaaaacaccc ccgacgtgca caacgtcttc gtctgcaccc tgtgctcttg ctacccatgg 360

ccggtgctgg gcttgccgcc ggcctggtac aaggccgcac cctaccggtc gcgcatggtg 420

agcgacccgc gtggggtcct ggcggagttc ggtttggtga tccccaccaa caaggaaatc 480

cgcgtctggg acaccacagc cgaattgcgc tacatggtgc tgccggaaag gcccgcagga 540

accgaaggct acagcgaaga acaactggcc gaactcgtca cccgcgattc gatgatcggc 600

actggcctgc ccacccaacc caaaccttcc cactaa 636

<210> 9

<211> 636

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggggcaat cacacacaca cgaccaccat cacgacgggt accaggcacc gcctgaagac 60

attgcgctgc gggtgaaggc cttggagacc ctgctcgtcg agaaaggttt ggtcgacccg 120

gcggccatgg acgctatcgt ccaaacctat gaacacaagg tgggccctcg gaacggcgcc 180

aaggttgttg ccaaggcctg ggtggacccg gactacaagg accgcttgct gcgcgacggc 240

accgctggca ttgccgaact gggcttctct ggacgccagg gagaacacat ggtcattctg 300

gaaaacaccc ccgacgtgca caacgtcttc gtctgcaccc tgtgctcttg ctacccatgg 360

ccggtgctgg gcttgccgcc ggcctggtac aaggccccgc cctaccggtc gcgcatggtg 420

agcgacccgc gtggggtcct ggcggagttc ggtttggtga tccccaccaa caaggaaatc 480

cgcgtctggg acaccacagc cgaattgcgc tacatggtgc tgccggaaag gcccgaagga 540

accgaaggct acagcgaaga acaactggcc gaactcgtca cccgcgattc gatgatcggc 600

actggcctgc ccacccaacc caaaccttcc cactaa 636

<210> 10

<211> 657

<212> DNA

<213> Bordetella (Bordetella petrii)

<400> 10

atgaacggca ttcacgacac tggcggagca catggttatg gcccggttta cagggagccg 60

aatgagccca tccttcatgg cgagtgggag ggtcgggtcc tggcattgtt tccggcgctt 120

ttcgcaaacg gcaacttcaa catcgatgag tttcgacacg gcatcgagcg catgaacccc 180

atcgactacc tgaagggaac ctactacgaa cactggatcc attccatcga aaccttgctg 240

gtcgaaaagg gtgtgctcac ggcaacggaa ctcgcgaccg gcaaggcatc tggcaagaca 300

gcgacaccgg tgctgacgcc ggtcatggtg gacggactgc tcagtaacgg agcttctgcc 360

gcccgcaagg agggggtgca ggcgcggttc gctgtgggcg acaaggttcg cgtcctcaac 420

aagcacccgg tgggccatac ccgcatgccg cgctacacgc ggggcaaagt ggggacagtg 480

gtcatcgacc atggtgtgtt cgtgacgccg gacaccgcgg cacacggaaa gggcgagcac 540

ccccagcacg tttacaccgt gagtttcacg tcggtcgaac tgtgggggca agacgcttcc 600

tcgccgaagg acacgattcg cgtcgacttg tgggatgact acctggagcc agcgtga 657

<210> 11

<211> 435

<212> DNA

<213> Bordetella (Bordetella petrii)

<400> 11

atgaaagacg aacggtttcc attgccagag ggttcgctga aggacctcga tggccctgtg 60

tttgacgagc cttggcagtc ccaggcgttt gccttggtgg tcagcatgca caaggccggt 120

ctctttcagt ggaaagactg ggccgagacc ttcaccgccg aaatcgacgc ttccccggct 180

ctgcccggcg aaagcgtcaa cgacacctac taccggcaat gggtgtcggc gctggaaaag 240

ttggtggcgt cgctggggct tgtgacgggt ggagacgtca actcgcgcgc acaggagtgg 300

aaacaggccc acctcaacac cccacatggg cacccgatcc tgctggccca tgcgctttgc 360

ccgccagcga tcgaccccaa gcacaagcac gagccacaac gctcaccgat caaggtcgtt 420

gccgcaatgg cttga 435

<210> 12

<211> 10

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggagatcatc 10

Claims

1. An alpha subunit mutant of nitrile hydratase, wherein the mutant is obtained by mutating a wild-type alpha subunit sequence, the wild-type alpha subunit sequence has an amino acid sequence shown as SEQ ID NO.1, and the mutation site is any one of the following groups:

(1) alanine 105 to aspartic acid and serine 122 to valine;

(2) alanine at position 71 is mutated into aspartic acid, alanine at position 105 is mutated into aspartic acid, and serine at position 122 is mutated into valine;

(3) serine at position 30 is mutated to threonine, valine at position 46 is mutated to isoleucine, alanine at position 71 is mutated to aspartic acid, alanine at position 74 is mutated to aspartic acid, alanine at position 78 is mutated to arginine, asparagine at position 79 is mutated to aspartic acid, serine at position 81 is mutated to threonine, valine at position 92 is mutated to arginine, aspartic acid at position 96 is mutated to histidine, threonine at position 97 is mutated to methionine, alanine at position 105 is mutated to aspartic acid, serine at position 122 is mutated to valine, alanine at position 133 is mutated to proline, and alanine at position 179 is mutated to glutamic acid.

2. An alpha subunit mutant nitrile hydratase comprising an alpha subunit and a beta subunit, wherein the alpha subunit is the alpha subunit mutant of claim 1.

3. A nitrile hydratase mutant according to claim 2 wherein the amino acid sequence of the β subunit is as shown in SEQ ID No. 2.

4. A gene cluster for expressing the nitrile hydratase mutant according to claim 2, characterized in that the gene cluster comprises gene sequences coding for the alpha subunit mutant, the beta subunit and the regulatory protein p14K, respectively.

5. The gene cluster of claim 4, wherein the gene sequence encoding the alpha subunit mutant is shown as any one of SEQ ID No. 7-9, the gene sequence encoding the beta subunit is shown as SEQ ID No.10, and the gene sequence encoding the regulatory protein p14K is shown as SEQ ID No. 11.

6. The gene cluster of claim 5, wherein a first linker sequence is present between the gene sequence encoding the alpha subunit mutant and the gene sequence encoding the beta subunit, and the first linker sequence has the sequence: GGAGATCATC, respectively; the gene sequence coding the beta subunit and the gene sequence coding the regulatory protein p14K have a second connecting sequence, and the sequence of the second connecting sequence is as follows: and TC.

7. A recombinant expression vector comprising the gene cluster of any one of claims 4 to 6.

8. A genetically engineered bacterium comprising the recombinant expression vector of claim 7.

9. Use of the nitrile hydratase mutant according to claim 3 for catalyzing the production of acrylamide from acrylonitrile.

10. The use of the genetically engineered bacterium of claim 8 in the production of acrylamide by catalyzing acrylonitrile.