CN113846040A

CN113846040A - Method for efficiently catalyzing biosynthesis of nicotinamide and acrylamide by cooperating with two kinds of nitrile hydratase

Info

Publication number: CN113846040A
Application number: CN202111060419.2A
Authority: CN
Inventors: 周哲敏; 赵凯阳; 黄微煜; 蔡宋佳; 陈员庆; 吴超城; 周丽; 程中一; 崔文璟; 刘中美
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2021-12-28
Anticipated expiration: 2041-09-10
Also published as: CN113846040B

Abstract

The invention discloses a method for efficiently catalyzing biosynthesis of nicotinamide and acrylamide by cooperating with two kinds of nitrile hydratase, belonging to the field of bioengineering. The invention constructs a genetic engineering bacterium, has high tolerance of high molecular weight nitrile hydratase to nitrile substances and amide substances, has similar reaction rate to low molecular weight nitrile hydratase, and can efficiently produce nicotinamide and acrylamide. The invention uses nicotinonitrile and acrylonitrile by a whole-cell catalysis methodTo the substrate, add the final concentration OD₆₀₀The genetically engineered bacterium with the value of 8 catalyzes and generates nicotinamide and acrylamide with higher added values, the yield of the nicotinamide can reach 375-516 g/L, and the yield of the acrylamide can reach 240.8 g/L.

Description

Method for efficiently catalyzing biosynthesis of nicotinamide and acrylamide by cooperating with two kinds of nitrile hydratase

Technical Field

The invention relates to a method for efficiently catalyzing biosynthesis of nicotinamide and acrylamide by cooperating with two kinds of nitrile hydratase, belonging to the field of bioengineering.

Background

Nitrile hydratase (NHase) is a metalloenzyme that can convert nitriles into more valuable amides by hydration. The method is mainly used for producing acrylamide, nicotinamide and the like in industrial production. Wherein, the nicotinamide is a member of B vitamins and is widely applied to industries such as medicine, cosmetics, food and the like; acrylamide and its polymer are mainly used in coagulant, soil improver, petroleum recovering agent and other industries. Therefore, the development of a high-performance nitrile hydratase catalyst for realizing the high-efficiency synthesis of the amide product has important industrial application value and economic value.

Nitrile hydratase multimers can be classified into Low-molecular-mass nitrile hydratases (L-NHases) and High-molecular-mass nitrile hydratases (H-NHases) according to their molecular weights. The L-NHase is composed of B, A, E three subunits, while the H-NHase is composed of B, A, G three subunits, and the catalytic reaction performance of the three subunits is greatly different. In Escherichia coli, as in patent CN104830747BThe recombinant L-NHase expresses the L-NHase from R.rhodochrous J1, and the specific enzyme activity of the recombinant L-NHase with nicotinonitrile as a substrate is 42 percent higher than that of the recombinant H-NHase (234U/mg). In terms of thermostability, H-NHase still has complete activity when placed at 50 ℃ for 30min, 50% of the activity can be preserved when placed at 60 ℃ for 1H, and L-NHase is slightly less stable. Wang et al reported that recombinant bacteria containing H-NHase were used to catalyze nicotinonitrile to synthesize nicotinamide in whole cells, and OD was added to the final concentration of added bacteria₆₀₀When the concentration is 8, 390g/L nicotinamide can be synthesized at 105 min; when the added bacteria concentration OD600 is 160, the yield of nicotinamide is improved to 508g/L, which is the highest value reported in the literature at present. In the aspect of the tolerance to substrates and products, the L-NHase can cause great reduction of enzyme activity after being incubated with a nicotinonitrile substrate or a nicotinamide product, and the tolerance is far lower than that of the H-NHase. In addition, different NHases have different substrate specificities, and the substrates of the NHases mainly comprise aromatic substrates (including heterocyclic substrates) and aliphatic substrates. The affinity of H-NHase to fatty substrates is higher, especially acrylonitrile; and the L-NHase has higher activity on nitriles (such as nicotinonitrile) with aromatic rings and heterocyclic rings. It can be seen that different types of nitrile hydratases each have their distinct advantages and disadvantages in the catalytic reaction.

Disclosure of Invention

At present, nitrile hydratase with stability and/or tolerance is obtained mainly by screening nitrile hydratase derived from different strains or modifying the nitrile hydratase, and nitrile hydratase with different molecular weights is not synergistically expressed to provide a more efficient nitrile hydratase cell catalyst.

In order to solve the problems, the research synergistically expresses the L-NHase and the H-NHase, constructs a more efficient nitrile hydratase cell catalyst, aims to improve the synthesis of products of nicotinamide and acrylamide, and provides reference for industrial application.

The invention provides a genetically engineered bacterium for efficiently synthesizing nicotinamide and acrylamide and a method for synthesizing nicotinamide and acrylamide through whole-cell catalysis.

The first object of the present invention is to provide a genetically engineered bacterium which coexpresses a low molecular weight nitrile hydratase L-NHase and a high molecular weight nitrile hydratase H-NHase.

In one embodiment of the present invention, L-NHase and H-NHase are expressed on two plasmids, respectively, or on the same plasmid.

In one embodiment of the present invention, the genetically engineered bacterium is a host escherichia coli.

In one embodiment of the present invention, the Escherichia coli includes, but is not limited to, Escherichia coli BL21(DE3) and Escherichia coli JM 109.

In one embodiment of the invention, the L-NHase is composed of B, A, E subunits, the B subunit has the amino acid sequence shown in SEQ ID NO. 1, the A subunit has the amino acid sequence shown in SEQ ID NO. 2, and the E subunit has the amino acid sequence shown in SEQ ID NO. 3.

In one embodiment of the invention, the nucleotide sequence encoding the L-NHase gene is shown as SEQ ID NO. 4.

In one embodiment of the invention, the H-NHase is composed of B, A, G subunits, the B subunit has the amino acid sequence shown in SEQ ID NO. 5, the A subunit has the amino acid sequence shown in SEQ ID NO. 6, and the G subunit has the amino acid sequence shown in SEQ ID NO. 7.

In one embodiment of the invention, the nucleotide sequence encoding the H-NHase gene is shown as SEQ ID NO. 8.

In one embodiment of the invention, the plasmids include the pACYCDuet plasmid and the pRSFDuet plasmid.

The second purpose of the invention is to provide a method for producing amide substances, which takes nitrile substances as substrates and utilizes the genetic engineering bacteria to catalyze and produce the amide substances.

In one embodiment of the invention, the nitrile includes isobutyronitrile, n-valeronitrile, acrylonitrile, nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, naphthonitrile.

In one embodiment of the present invention, the amide-based substance includes isobutyramide, valeramide, acrylamide, nicotinamide, pyrazinamide, naphthoyl, benzamide, cinnamamide.

In one embodiment of the invention, the genetically engineered bacteria are used as cell catalysts to catalyze nicotinonitrile to produce nicotinamide.

In one embodiment of the invention, the reaction is carried out at a temperature of from 20 to 35 ℃.

In one embodiment of the present invention, OD of the genetically engineered bacterium₆₀₀7-9, wherein the mass-volume ratio of the addition amount of the substrate nicotinonitrile to the bacterial liquid is 1: (20-30), and adding the substrate again after the substrate is completely consumed until the reaction is stopped.

In one embodiment of the invention, the method is to use the genetically engineered bacteria as a cell catalyst to catalyze acrylonitrile to produce acrylamide.

In one embodiment of the present invention, OD of the genetically engineered bacterium₆₀₀7-9, wherein the mass-volume ratio of substrate acrylonitrile to bacterial liquid is 1: (30-35), and adding the substrate again after the substrate is completely consumed until the reaction is stopped.

The invention also protects the application of the genetically engineered bacterium or the method for producing the amide substance in drug analysis, food analysis, environmental monitoring, physical and chemical engineering and biological pharmacy.

Has the advantages that:

1. the invention constructs a recombinant bacterium for efficiently synthesizing nicotinamide or acrylamide, and modifies and optimizes the host strain cooperatively expressing L-NHase and H-NHase to obtain the recombinant bacterium capable of producing nicotinamide and acrylamide by a whole-cell catalysis method and the catalysis method. The recombinant strain can be used for producing nicotinamide and acrylamide with higher added value through whole-cell catalysis by taking nicotinonitrile or acrylonitrile as a substrate.

2. The tolerance of the recombinant bacteria which synergistically express the L-NHase and the H-NHase in the solutions of nicotinonitrile, nicotinamide, acrylonitrile and acrylamide with different concentrations is obviously improved compared with that of the bacterial strain which expresses the L-NHase, and the bacterial strain is similar to a BAG bacterial strain which expresses the H-NHase.

3. In the production of amide substances, the yield of nicotinamide produced by the recombinant bacteria which synergistically express the L-NHase and the H-NHase is 2-2.8 times that of the recombinant bacteria which independently express the L-NHase and 1.6-2.2 times that of the recombinant bacteria which independently express the H-NHase, and the product yield is up to 516 g/L; the production rate of acrylamide was increased by about 92.2% compared to BAG strain and the final product concentration was increased by 19.1% compared to BAE expressing strain.

Drawings

FIG. 1 is a schematic diagram of the plasmid structure of the recombinant strain.

FIG. 2 is a protein electrophoretogram of the recombinant strain; m is Protein molecular weight Marker.

FIG. 3 shows the tolerance of the strains to organic solvents; (a) the organic solvent is nicotinonitrile; (b) the organic solvent is nicotinamide; (c) the organic solvent is acrylonitrile; (d) the organic solvent is acrylamide.

FIG. 4 shows the whole-cell catalytic synthesis of nicotinamide by BAE, BAG + BAE strains.

FIG. 5 shows the whole-cell catalytic synthesis of nicotinamide by BAE-BAG, BAG-BAE, BAE-T7BAG strains.

FIG. 6 shows the whole-cell catalytic synthesis of acrylamide.

Detailed Description

The media referred to in the examples below:

LB liquid medium: weighing 10g of Tryptone (Tryptone), 5g of Yeast powder (Yeast extract) and 10g of sodium chloride (NaCl) in a beaker by using an electronic balance, adding deionized water in the beaker to a constant volume of 1L, and finally performing moist heat sterilization at 121 ℃ for 20min in a high-pressure steam sterilization pot.

LB solid medium: 20g of agar powder is weighed and added into 1L of LB liquid culture medium, and then the mixture is placed in a high-pressure steam sterilization pot for moist heat sterilization at 121 ℃ for 20 min.

Corresponding antibiotics are selectively added into the culture medium according to the resistance genes carried on the plasmids, and the addition amount of the antibiotics is as follows: kanamycin to a final concentration of 50. mu.g/mL, chloramphenicol to a final concentration of 34. mu.g/mL.

Plasmids referred to in the following examples:

pacycuet plasmid, pRSFDuet plasmid: commercial plasmids were purchased from the International Biogene technology Ltd of the Union of Beijing village.

BAG-S/BAG plasmid: it is disclosed in the article as corresponding plasmid pET-24a (+) -nhhBrbsArbsG, see in detail: wang Z, Liu Z, Cui W, Zhou Z. expression of biological processes for synthesizing by microorganisms Escherichia coli expressing high-molecular-mass nitrile hydrate, 2017,182(4):1458 and 1466.

BAE-S/BAE plasmid: is disclosed in the article as the corresponding plasmid pET-24a (+) -BA_L-NHaseE, see details in: queen sweet memory, Cheng, Guo Jun Ling, summer Yuan Tss, Liu Zhong Mei, West philosophy of West philosophy, Rhodococcus low molecular weight nitrile hydratase heterologous activation and activator domain function. Biotechnology proceedings, 2020,36(8): 1578-.

The methods referred to in the following examples:

(1) the method for inducing expression of the recombinant protein comprises the following steps:

selecting recombinant strain single colony, inoculating into LB liquid culture medium containing corresponding antibiotic, and shake culturing at 37 deg.C and 200rpm to OD₆₀₀0.6-0.8, adding IPTG with final concentration of 0.2mM into the culture medium, and culturing at 24 deg.C and 200rpm for about 20 hr to induce the expression of the target protein.

(2) The method for measuring the catalytic activity of the cells (enzyme activity measurement method) comprises the following steps:

100 μ L of OD₆₀₀The recombinant strain of 1.0 is added into 400 mu L of 125mmol/L nicotinonitrile, and after the reaction is carried out for 10min at 25 ℃, 500 mu L of acetonitrile is added to stop the reaction. Detecting the content of nicotinamide by High Performance Liquid Chromatography (HPLC).

The unit enzyme activity is defined as: the amount of nicotinamide produced by nicotinonitrile (U/mL) catalyzed per minute per milliliter of cells at 25 ℃.

(3) Cell tolerance assay methods:

100 μ L of OD₆₀₀Adding the recombinant strain of 6.0 into 900 μ L of nicotinonitrile, nicotinamide, acrylonitrile and acrylamide solution with different concentrations, standing at 25 deg.C for 30min, washing the cells with 10mmol/LKPB buffer solution (pH 7.4) for 2 times, and measuring the residual enzyme activity of the cells.

The relative enzyme activity is defined as: (cell catalytic activity after treatment with organic agent/cell catalytic activity without treatment with organic agent). times.100%. The relative enzyme activity of the cells which are not treated by the organic reagent is 100 percent.

(4) The whole-cell catalysis method comprises the following steps:

resuspending the recombinant strain for inducing protein expression with deionized water, and adjusting the absorbance OD₆₀₀The value was 8. 50mL of bacterial liquid is taken and placed in a beaker, a reaction substrate is added, the reaction is carried out while stirring, and the reaction temperature is controlled to be 24-30 ℃. Starting from the time of the first addition of the substrate (2g of nicotinonitrile/time or 1.5mL of acrylonitrile/time), 10. mu.L of the reaction mixture was added to 990. mu.L of acetonitrile at 2min intervals to terminate the reaction. The samples were analyzed by HPLC for the amount of nicotinamide or acrylamide product and it was determined whether there was a substrate remaining and addition of substrate was continued until no substrate remained. The previous steps are repeated until the substrate cannot continue to be consumed.

(5) The method for detecting the contents of nicotinonitrile, nicotinamide, acrylonitrile and acrylamide comprises the following steps:

detecting the contents of nicotinonitrile, nicotinamide, acrylonitrile and acrylamide by using a C18 chromatographic column and high performance liquid chromatography. The mobile phase is a mixed solution of water and acetonitrile (water: acetonitrile in volume ratio of 2: 1). The detection wavelength was set at 220nm and the column temperature was 40 ℃. The flow rate was set to 0.8mL/min and the injection volume was 10. mu.L.

Example 1: construction of strains synergistically expressing BAG and BAE

(1) Construction of recombinant plasmid

Construction of plasmid BAG-1 expressing high molecular weight nitrile hydratase (H-NHase): carrying out PCR amplification by using primers PAC-BAG-F and PAC-BAG-R by taking a BAG-S/BAG plasmid expressing B, A, G subunits of H-NHase as a template to obtain a BAG gene segment; carrying out PCR by using the pACYCDuet plasmid as a template and using primers PAC-BAG-S-F and PAC-BAG-S-R to amplify to obtain a plasmid skeleton; BAG gene fragments and a plasmid skeleton are assembled by an In fusion method to construct BAG-1 recombinant plasmids, and DNA sequencing verification is carried out (primers are shown In Table 1).

Construction of plasmid pRSFDuet-BAE expressing a Low molecular weight nitrile hydratase (L-NHase): taking BAE-S/BAE plasmid expressing B, A, E subunit of L-NHase as a template, and carrying out PCR amplification by using primers DT7-BAE-F and DT7-BAE-R to obtain BAE gene fragment; PCR was performed using pRSFDuet plasmid as template and primers DT7-BAE-S-F and DT7-BAE-S-R to amplify the plasmid backbone; BAE gene fragment and plasmid backbone were assembled by In fusion method to construct pRSFDuet-BAE recombinant plasmid (primers shown In Table 1).

Construction of a recombinant plasmid BAE-BAG co-expressing high and low molecular weight nitrile hydratases: carrying out PCR amplification by using a BAG-S/BAG plasmid template and primers DT7-BAG-F and DT7-BAG-R to obtain a BAG gene fragment; PCR is carried out by taking pRSFDuet-BAE recombinant plasmid as a template and utilizing primers DT7-BAG-S-F and DT7-BAG-S-R to obtain a plasmid skeleton; BAE-BAG recombinant plasmids were constructed by assembling BAG gene fragments and plasmid backbone using In fusion method and verified by DNA sequencing (primers shown In Table 1).

Construction of a recombinant plasmid BAG-BAE co-expressing high and low molecular weight nitrile hydratase: using BAG-S/BAG plasmid as template, utilizing primer DT7-BAG-F and DT7-BAG-2 to make PCR amplification so as to obtain BAG gene fragment; PCR is carried out by taking pRSFDuet-BAE plasmid as a template and utilizing the primers DT7-GE-S-1 and DT7-BAG-S-R to obtain a plasmid skeleton through amplification; BAG gene fragments and a plasmid skeleton are assembled by an In fusion method to construct BAG-BAE recombinant plasmids, and DNA sequencing verification is carried out (primers are shown In Table 1).

TABLE 1 primers and sequences referred to herein

(2) Construction of recombinant bacteria

Transformation of the BAE-S/BAE plasmid into e.coli BL21 strain to obtain BAE strain; transforming E.coli BL21 strain with BAG-S/BAG plasmid to obtain BAG strain; transforming E.coli BL21 strain with BAE-S/BAE and BAG-1 plasmid constructed in step (1) to obtain BAG + BAE strain; transforming the BAG-BAE plasmid constructed in the step (1) into E.coli BL21 strain to obtain BAG-BAE strain; coli BL21 strain was transformed with the BAE-BAG plasmid constructed in step (1) to obtain BAE-BAG strain (FIG. 1).

The 5 recombinant strains obtained (BAE strain, BAG + BAE strain, BAG-BAE strain, BAE-BAG strain) were cultured and induced to express nitrile hydratase, followed by whole cell SDS-PAGE electrophoresis. As shown in FIG. 2, it is known that both L-NHase and H-NHase have two subunits, alpha and beta, with the beta subunit being slightly different in size and the alpha subunit being close in size. BAE strains express significantly less nitrile hydratase than BAG strains. In the BAG + BAE double plasmid strain, the expression of two types of nitrile hydratase is simultaneously realized, wherein the expression level of L-NHase is similar to that of BAE strain, and the expression level of H-NHase is lower than that of BAG strain. The expression level of H-NHase in BAG-BAE strain and BAE-BAG strain is similar to that of BAG strain, while the expression level of L-NHase is lower.

Example 2: recombinant strain tolerance assay

The 5 recombinant strains constructed in example 1 were tested for their tolerance in solutions of nicotinonitrile, nicotinamide, acrylonitrile and acrylamide at different concentrations, and as can be seen from fig. 3, the BAG strain was significantly better tolerant to 4 compounds than the BAE strain, consistent with previous studies. The BAG + BAE, BAG-BAE and BAE-BAG strains express L-NHase and H-NHase synergistically, so that the tolerance of the BAE strains to 4 compounds is better than that of the BAE strains. Wherein, BAG-BAE and BAE-BAG strains express low molecular weight nitrile hydratase and high molecular weight nitrile hydratase on a plasmid, and the tolerance of the strains to 4 compounds reaches the similar level of the BAG strains. When the concentration of nicotinonitrile in the reaction system reaches 0.8mol/L, the residual relative enzyme activity of BAE-BAE and BAE-BAG strains reaches 50%, and the BAE strains almost lose the enzyme activity; when the concentration of nicotinamide in the reaction system reaches 1.5mol/L, the residual relative enzyme activity of BAE-BAE and BAE-BAG strains approaches 80%, and the BAE strains almost lose enzyme activity; when the concentration of acrylonitrile in the reaction system reaches 2mol/L, the residual relative enzyme activity of BAG-BAE and BAE-BAG strains reaches more than 80 percent; when the concentration of acrylamide in the reaction system reaches 5mol/L, the residual relative enzyme activity of BAE-BAE and BAE-BAG strains is about 60%, and the BAE strains almost lose the enzyme activity. Thus, the present study synergistically expressed low and high molecular weight nitrile hydratases, retaining the high substrate and product tolerance of the recombinant strain.

Example 3: whole cell catalytic synthesis of nicotinamide

The 5 recombinant strains constructed in example 1 were inoculated in LB medium, respectively, and cultured with shaking at 37 ℃ and 200rpm to OD₆₀₀Adding IPTG (0.6-0.8 mM) to the culture medium, and culturing at 24 deg.C and 200rpm for about 20 hr to induce expression of target protein to obtain fermentation liquid. Centrifuging the fermentation liquid, discarding the supernatant, resuspending the thallus with deionized water, and adjusting to obtain the absorbance OD₆₀₀Bacterial liquid with value of 8. And (3) putting 50mL of bacterial liquid into a beaker, adding a reaction substrate of 2g of nicotinonitrile, reacting while stirring, controlling the reaction temperature to be 24-30 ℃, and adding the substrate (2g of nicotinonitrile) again after the substrate is consumed until the reaction is stopped and the substrate is not consumed any more.

As a result, as shown in FIG. 4, the reaction rates of the BAG + BAE strain and the BAE strain were substantially the same for the first 10min of the reaction; however, after 10min, because the BAE strain has poor tolerance to the product, the reaction rate is obviously reduced along with the gradual rise of the nicotinamide content in the reaction system, the synthesis end point is reached in a shorter time, while the BAE + BAG strain has better product tolerance than the BAE strain, and the later reaction rate is still kept at a higher level, the synthesis amount of the final product nicotinamide is about 1.28 times of that of the BAE strain, and under the condition of the same product amount, the reaction time is shortened by about 30 percent compared with that of the BAG strain. The results show that the purposes of improving the reaction rate, shortening the reaction time and improving the product yield are realized by cooperatively expressing the L-NHase and the H-NHase.

As shown in FIG. 5, the reaction rate of BAE-BAG strain was very fast, maintained at 7.33 g/L.min, in the first 32min of the reaction^-1On the left and right, the reaction rate with BAE + BAG strain is basically the same; after 64min of reaction, the reaction rate decreased slightly due to better tolerance of BAE-BAG strain to the product, but could continue to react for a longer time, and the final yield of nicotinamide was about 2.2 times that of BAE + BAG strain and 2.8 times that of BAE strain. Likewise, the final yield of BAG-BAE strain is 1.6 times that of BAE + BAG strain and 2 times that of BAE strain. The results show that the L-NHase and the H-NHase are constructed on the same plasmid, and the purposes of improving the reaction rate, prolonging the reaction time and improving the product yield are achieved.

The L-NHase and the H-NHase are expressed synergistically, the BAE-BAG strain can generate 400g/L nicotinamide after reacting for 70min, and meanwhile, the concentration of a final nicotinamide product can be increased to 516 g/L; the BAG-BAE strain can produce 300g/L nicotinamide after reacting for 70min, and meanwhile, the concentration of the final nicotinamide product can be increased to 375g/L, and the results show that the aims of improving the catalytic rate, prolonging the reaction time and improving the product yield are successfully achieved.

Example 4: whole cell catalytic synthesis of acrylamide

The 5 recombinant strains constructed in example 1 were inoculated in LB medium, respectively, and cultured with shaking at 37 ℃ and 200rpm to OD₆₀₀Adding IPTG (0.6-0.8 mM) to the culture medium, and culturing at 24 deg.C and 200rpm for about 20 hr to induce expression of target protein to obtain fermentation liquid. Centrifuging the fermentation liquid, discarding the supernatant, resuspending the thallus with deionized water, and adjusting to obtain the absorbance OD₆₀₀Bacterial liquid with value of 8. And (3) putting 50mL of bacterial liquid into a beaker, adding 1.5mL of acrylonitrile as a reaction substrate, reacting while stirring, controlling the reaction temperature to be 24-30 ℃, and adding the substrate (1.5mL of acrylonitrile) again after the substrate is consumed until the reaction is stopped and the substrate is not consumed any more.

As shown in FIG. 6, the rate of acrylamide synthesis by BAE strain catalyzed acrylonitrile is significantly higher than that by BAG strain. The co-expression of the L-NHase and the H-NHase ensures that the catalytic reaction rate of the BAE + BAG strain, the BAE-BAG strain and the BAG-BAE strain is close to that of the BAE strain, and the average reaction rate is improved by about 92.2 percent compared with that of the BAG strain. Wherein the reaction time of the BAE-BAG strain and the BAG-BAE strain is prolonged to 150min, the concentration of a final product is improved by 19.1 percent compared with that of the BAE strain and 18.2 percent compared with that of the BAG strain, and the result shows that the synergistic expression of the L-NHase and the H-NHase can obviously improve the catalytic rate of catalyzing acrylonitrile to synthesize acrylamide and the yield of acrylamide.

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

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ccgtatctgc taccggcctg gagccacccg cagttcgaaa agtgaaagga gatatagata 720

tgaccgccca caatcccgtc cagggcacgt tgccacgatc gaacgaggag atcgccgcac 780

gcgtgaaggc catggaggcc atcctcgtcg acaagggcct gatctccacc gacgccatcg 840

accacatgtc ctcggtctac gagaacgagg tcggtcctca actcggcgcc aagatcgtcg 900

cccgcgcctg ggtcgatccc gagttcaagc agcgcctgct caccgacgcc accagcgcct 960

gccgtgaaat gggcgtcggc ggcatgcagg gcgaagaaat ggtcgtgctg gaaaacaccg 1020

gcacggtcca caacatggtc gtatgtacct tgtgctcgtg ctatccgtgg ccggttctcg 1080

gcctgccacc caactggtac aagtaccccg cctaccgcgc ccgcgctgtc cgcgaccccc 1140

gaggtgtgct ggccgaattc ggatataccc ccgaccctga cgtcgagatc cggatatggg 1200

actcgagtgc cgaacttcgc tactgggtcc tgccgcaacg cccagccggc accgagaact 1260

tcaccgaaga acaactcgcc gacctcgtca cccgcgactc gctcatcggc gtatccgtcc 1320

ccaccacacc cagcaaggcc tgaaaggaga tatagatatg ccccgactca acgaacaacc 1380

ccacccgggt ctcgaagcca acctcggcga cctggtacag aatctgccgt tcaacgaacg 1440

aatcccccgc cgctccggcg aggtcgcctt cgatcaggcc tgggagatcc gcgccttcag 1500

cattgccacc gcattgcatg gccagggccg attcgaatgg gacgaattcc agtcccgcct 1560

gatcgagtcg atcaaacagt gggaagccga acacgccacc accgagcagt ggagttacta 1620

cgagcgttgg atgctcgcac tcgaagagct gctgcacgac aagggatttg tcgcaggcga 1680

ggaactcgcg caccgtaccg agcaggtgct ggcaacgccc gccggcgccc atcaccagca 1740

cgccgtgcgt gatcccatcg ccgtgcacgc catcggcaca cgcaccactg actccgacgg 1800

gtga 1804

<210> 5

<211> 237

<212> PRT

<213> Artificial sequence

<400> 5

Met Asp Gly Ile His Asp Thr Gly Gly Met Thr Gly Tyr Gly Pro Val

1 5 10 15

Pro Tyr Gln Lys Asp Glu Pro Phe Phe His Tyr Glu Trp Glu Gly Arg

20 25 30

Thr Leu Ser Ile Leu Thr Trp Met His Leu Lys Gly Ile Ser Trp Trp

35 40 45

Asp Lys Ser Arg Phe Phe Arg Glu Ser Met Gly Asn Glu Asn Tyr Val

50 55 60

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

65 70 75 80

Arg Ile Leu Val Ala Asp Lys Ile Ile Thr Glu Glu Glu Arg Lys His

85 90 95

Arg Val Gln Glu Ile Leu Glu Gly Arg Tyr Thr Asp Arg Lys Pro Ser

100 105 110

Arg Lys Phe Asp Pro Ala Gln Ile Glu Lys Ala Ile Glu Arg Leu His

115 120 125

Glu Pro His Ser Leu Ala Leu Pro Gly Ala Glu Pro Ser Phe Ser Leu

130 135 140

Gly Asp Lys Ile Lys Val Lys Ser Met Asn Pro Leu Gly His Thr Arg

145 150 155 160

Cys Pro Lys Tyr Val Arg Asn Lys Ile Gly Glu Ile Val Ala Tyr His

165 170 175

Gly Cys Gln Ile Tyr Pro Glu Ser Ser Ser Ala Gly Leu Gly Asp Asp

180 185 190

Pro Arg Pro Leu Tyr Thr Val Ala Phe Ser Ala Gln Glu Leu Trp Gly

195 200 205

Asp Asp Gly Asn Gly Lys Asp Val Val Cys Val Asp Leu Trp Glu Pro

210 215 220

Tyr Leu Ile Ser Ala Trp Ser His Pro Gln Phe Glu Lys

225 230 235

<210> 6

<211> 203

<212> PRT

<213> Artificial sequence

<400> 6

Met Ser Glu His Val Asn Lys Tyr Thr Glu Tyr Glu Ala Arg Thr Lys

1 5 10 15

Ala Ile Glu Thr Leu Leu Tyr Glu Arg Gly Leu Ile Thr Pro Ala Ala

20 25 30

Val Asp Arg Val Val Ser Tyr Tyr Glu Asn Glu Ile Gly Pro Met Gly

35 40 45

Gly Ala Lys Val Val Ala Lys Ser Trp Val Asp Pro Glu Tyr Arg Lys

50 55 60

Trp Leu Glu Glu Asp Ala Thr Ala Ala Met Ala Ser Leu Gly Tyr Ala

65 70 75 80

Gly Glu Gln Ala His Gln Ile Ser Ala Val Phe Asn Asp Ser Gln Thr

85 90 95

His His Val Val Val Cys Thr Leu Cys Ser Cys Tyr Pro Trp Pro Val

100 105 110

Leu Gly Leu Pro Pro Ala Trp Tyr Lys Ser Met Glu Tyr Arg Ser Arg

115 120 125

Val Val Ala Asp Pro Arg Gly Val Leu Lys Arg Asp Phe Gly Phe Asp

130 135 140

Ile Pro Asp Glu Val Glu Val Arg Val Trp Asp Ser Ser Ser Glu Ile

145 150 155 160

Arg Tyr Ile Val Ile Pro Glu Arg Pro Ala Gly Thr Asp Gly Trp Ser

165 170 175

Glu Glu Glu Leu Thr Lys Leu Val Ser Arg Asp Ser Met Ile Gly Val

180 185 190

Ser Asn Ala Leu Thr Pro Gln Glu Val Ile Val

195 200

<210> 7

<211> 104

<212> PRT

<213> Artificial sequence

<400> 7

Met Ser Glu Asp Thr Leu Thr Asp Arg Leu Pro Ala Thr Gly Thr Ala

1 5 10 15

Ala Pro Pro Arg Asp Asn Gly Glu Leu Val Phe Thr Glu Pro Trp Glu

20 25 30

Ala Thr Ala Phe Gly Val Ala Ile Ala Leu Ser Asp Gln Lys Ser Tyr

35 40 45

Glu Trp Glu Phe Phe Arg Gln Arg Leu Ile His Ser Ile Ala Glu Ala

50 55 60

Asn Gly Cys Glu Ala Tyr Tyr Glu Ser Trp Thr Lys Ala Leu Glu Ala

65 70 75 80

Ser Val Val Asp Ser Gly Leu Ile Ser Glu Asp Glu Ile Arg Glu Arg

85 90 95

Met Glu Ser Met Ala Ile Ile Asp

100

<210> 8

<211> 1669

<212> DNA

<213> Artificial sequence

<400> 8

atggatggta ttcatgatac cggtggcatg accggctatg gtccggtgcc gtatcagaaa 60

gatgaaccgt ttttccatta tgaatgggaa ggccgtaccc tgagtattct gacctggatg 120

catctgaaag gtattagctg gtgggataaa agtcgctttt tccgtgaaag catgggtaat 180

gaaaattatg tgaatgaaat ccgtaacagt tactataccc attggctgag cgcagcagaa 240

cgtattctgg ttgccgataa aattattacc gaagaagaac gcaaacatcg tgtgcaggaa 300

attctggaag gtcgttatac cgatcgtaaa ccgagccgta aatttgatcc ggcccagatt 360

gaaaaagcaa ttgaacgcct gcatgaaccg catagcctgg ccctgccggg tgccgaaccg 420

agttttagtc tgggtgacaa aattaaggtg aaaagcatga atccgctggg tcatacccgt 480

tgcccgaaat atgtgcgtaa taagattggc gaaattgtgg catatcatgg ttgccagatc 540

tatccggaaa gtagcagcgc cggtctgggc gatgatccgc gccctctgta taccgttgcc 600

tttagtgccc aggaactgtg gggtgacgat ggtaatggca aagatgttgt ttgtgtggat 660

ctgtgggaac cgtatctgat tagcgcatgg agccacccgc agttcgaaaa gtaaaaggag 720

atatagatat gagtgaacat gttaacaagt acaccgaata tgaagcacgt accaaagcaa 780

ttgaaaccct gctgtatgaa cgcggcctga ttaccccggc cgcagtggat cgtgttgtga 840

gctattatga aaatgaaatt ggtccgatgg gcggtgcaaa agtggttgca aaaagttggg 900

tggatccgga atatcgcaaa tggctggaag aagatgcaac cgccgccatg gccagcctgg 960

gttatgcagg tgaacaggcc catcagatta gcgccgtgtt taatgatagc cagacccatc 1020

atgtggtggt gtgcaccctg tgcagttgtt atccgtggcc ggtgctgggt ctgccgccgg 1080

catggtataa aagtatggaa tatcgcagtc gcgttgttgc cgatccgcgt ggcgtgctga 1140

aacgtgattt tggttttgat attccggatg aagttgaagt gcgtgtttgg gatagcagta 1200

gcgaaattcg ttatattgtt attccggaac gcccggcagg taccgatggc tggagtgaag 1260

aagaactgac caaactggtg agtcgtgata gcatgattgg tgttagcaat gccctgaccc 1320

cgcaggaagt gattgtttaa aaggagatat agatatgagc gaagataccc tgaccgatcg 1380

cctgccggca accggcaccg cagcacctcc tcgtgataat ggtgaactgg tttttaccga 1440

accgtgggaa gcaaccgcat ttggcgttgc cattgcactg agcgatcaga aaagttatga 1500

atgggaattt ttccgccagc gtctgattca tagcattgca gaagccaatg gttgcgaagc 1560

ctattatgaa agttggacca aagcactgga agcaagtgtt gttgatagtg gcctgattag 1620

cgaagatgaa attcgtgaac gtatggaaag tatggccatt attgattaa 1669

Claims

1. A genetically engineered bacterium is characterized in that the genetically engineered bacterium coexpresses a low-molecular-weight nitrile hydratase L-NHase and a high-molecular-weight nitrile hydratase H-NHase.

2. The genetically engineered bacterium of claim 1, wherein the L-NHase consists of B, A, E subunits, the B subunit has the amino acid sequence shown as SEQ ID NO. 1, the A subunit has the amino acid sequence shown as SEQ ID NO. 2, the E subunit has the amino acid sequence shown as SEQ ID NO. 3, the H-NHase consists of B, A, G subunits, the B subunit has the amino acid sequence shown as SEQ ID NO. 5, the A subunit has the amino acid sequence shown as SEQ ID NO. 6, and the G subunit has the amino acid sequence shown as SEQ ID NO. 7.

3. The genetically engineered bacterium of claim 1, wherein said genetically engineered bacterium is a host escherichia coli.

4. The genetically engineered bacterium of claim 1, wherein the L-NHase and the H-NHase are expressed on two plasmids, respectively, or on the same plasmid.

5. The genetically engineered bacterium of claim 1, wherein the plasmids comprise the pacycuet plasmid and the pRSFDuet plasmid.

6. A method for producing amide substances, which is characterized in that nitrile substances are used as substrates, and the amide substances are generated by catalysis of the genetically engineered bacteria of any one of claims 1 to 5.

7. The method according to claim 6, wherein the nitrile compound includes isobutyronitrile, n-valeronitrile, acrylonitrile, nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, and naphthonitrile.

8. The method for producing amides of claim 7, wherein the genetically engineered bacteria of any one of claims 1 to 5 is used as a whole-cell catalyst to catalyze nicotinonitrile or acrylonitrile.

9. The method for producing amides as claimed in claim 8, wherein the reaction is carried out at 20-35 deg.C and OD of genetically engineered bacteria₆₀₀7-9, wherein the mass-volume ratio of the substrate addition amount to the bacterial liquid is 1: (20-35), and adding the substrate again after the substrate is completely consumed until the reaction is stopped.

10. Use of the genetically engineered bacterium according to any one of claims 1 to 5 or the method for producing an amide-based substance according to any one of claims 6 to 9 in drug analysis, food analysis, environmental monitoring, physicochemical engineering, and biopharmaceutical applications.