CN113846040B

CN113846040B - Method for catalyzing biosynthesis of nicotinamide and acrylamide by synergizing two nitrile hydratase

Info

Publication number: CN113846040B
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: 2023-07-25
Anticipated expiration: 2041-09-10
Also published as: CN113846040A

Abstract

The invention discloses a method for efficiently catalyzing biosynthesis of nicotinamide and acrylamide by synergizing two types of nitrile hydratase, belonging to the field of bioengineering. The invention constructs a genetically engineered bacterium, which has high tolerance to nitrile substances and amide substances by high molecular weight nitrile hydratase and has similar reaction rate to low molecular weight nitrile hydratase, and can efficiently produce nicotinamide and acrylamide. The invention takes the nicotinonitrile and the acrylonitrile as substrates by a whole cell catalysis method, and adds the final concentration OD ₆₀₀ The genetically engineered bacteria with the value of 8 catalyze to generate nicotinamide and acrylamide with higher added value, the yield of the nicotinamide can reach 375-516 g/L, and the yield of the acrylamide can reach 240.8g/L.

Description

Method for catalyzing biosynthesis of nicotinamide and acrylamide by synergizing two nitrile hydratase

Technical Field

The invention relates to a method for catalyzing biosynthesis of nicotinamide and acrylamide by synergizing two types of nitrile hydratase, belonging to the field of bioengineering.

Background

Nitrile hydratase (nitrile hydratase, NHase for short) is a metalloenzyme that converts nitriles to more valuable amides by hydration. The method is mainly used for producing acrylamide, nicotinamide and the like in industrial production. Wherein, nicotinamide is a member of B vitamins and is widely applied to industries such as medicines, cosmetics, foods and the like; the acrylamide and the polymer thereof are mainly applied to industries such as coagulants, soil amendments, petroleum recovery agents and the like. Therefore, the development of the high-performance nitrile hydratase catalyst and the realization of the efficient synthesis of amide products have important industrial application value and economic value.

Nitrile hydratase polymers can be classified into Low molecular weight nitrile hydratase (Low-molecular-mass nitrile hydratase, L-NHase) and High molecular weight nitrile hydratase (High-molecular-mass nitrile hydratase, H-NHase) according to their molecular weight. L-NHase consists of B, A, E subunits, while H-NHase consists of B, A, G subunits, which have greatly different catalytic reactivity. As in patent CN104830747B, expression in E.coliR. rhodochrousJ1-derived L-NHase, recombinant L-NHase with nicotinonitrile as substrate, has a specific enzyme activity 42% higher than recombinant H-NHase (234U/mg). In terms of thermostability, H-NHase still has full activity when left at 50℃for 30 min, 50% of the activity can be preserved when left at 60℃for 1H, and L-NHase has slightly poorer stability. Wang et al report the whole cell catalysis of nicotinamide by H-NHase-containing recombinant bacteria at the final OD of the addition of the bacteria ₆₀₀ When the ratio is 8, 390 g/L nicotinamide can be synthesized in 105 min; when the concentration OD600 of the added bacteria is 160, the yield of nicotinamide is increased to 508 g/L, which is the highest value reported in the current literature. In terms of tolerance to substrates and products, L-NHase can lead to a substantial decrease in enzyme activity after incubation with a nicotinonitrile substrate or nicotinamide productThe tolerance is far lower than H-NHase. In addition, different NHases have different substrate specificities, and substrates of NHases mainly comprise aromatic substrates (including heterocyclic substrates) and aliphatic substrates. The H-NHase has higher affinity to aliphatic substrates, especially acrylonitrile; L-NHase has higher activity on nitrile substances (such as nicotinonitrile) with aromatic rings and heterocyclic rings. It can be seen that different types of nitrile hydratase each have their distinct advantages and disadvantages in catalyzing reactions.

Disclosure of Invention

Currently, nitrile hydratases having stability and/or tolerance have not been obtained by screening nitrile hydratases derived from different strains or engineering nitrile hydratases to provide more efficient nitrile hydratase cell catalysts by synergistically expressing nitrile hydratases of different molecular weights.

In order to solve the problems, the research synergistically expresses L-NHase and H-NHase, and constructs a more efficient nitrile hydratase cell catalyst so as to improve the synthesis of nicotinamide and acrylamide products, thereby providing 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 by whole-cell catalysis.

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

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

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

In one embodiment of the present invention, the E.coli includes, but is not limited to, E.coli BL21 (DE 3) and E.coli JM109.

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

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

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

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

In one embodiment of the invention, the plasmids include pacycguet plasmid and prsfcduet plasmid.

The second object of the invention is to provide a method for producing amide substances, which uses nitrile substances as substrates and uses the genetically engineered bacteria to catalyze and generate amide substances.

In one embodiment of the present invention, the nitrile material comprises isobutyronitrile, n-valeronitrile, acrylonitrile, nicotinonitrile, 2-cyanopyrazine, benzonitrile, cinnamonitrile, naphthalene carbonitrile.

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 bacterium is used as a cell catalyst to catalyze the production of nicotinamide from nicotinonitrile.

In one embodiment of the invention, the reaction conditions are such that the temperature is 20-35 ℃.

In one embodiment of the present invention, the genetically engineered bacterium has an OD ₆₀₀ 7-9, wherein the mass volume ratio of the addition amount of the substrate nicotinonitrile to the bacterial liquid is 1: (20-30), adding the substrate again until the reaction is stopped after the substrate is consumed.

In one embodiment of the invention, the method is to catalyze acrylonitrile to produce acrylamide by using the genetically engineered bacteria as cell catalysts.

In one embodiment of the present invention, the genetically engineered bacterium has an OD ₆₀₀ 7-9, wherein the mass volume ratio of the substrate acrylonitrile to the bacterial liquid is 1: (30-35), adding the substrate again until the reaction is stopped after the substrate is consumed.

The invention also protects application of the genetically engineered bacterium or the method for producing the amide substances in medicine analysis, food analysis, environmental monitoring, physical and chemical engineering and biopharmaceutical.

The beneficial effects are that: the invention constructs a recombinant bacterium for efficiently synthesizing nicotinamide or acrylamide, and carries out transformation optimization on the synergistic expression of L-NHase and H-NHase of the host strain to obtain the recombinant bacterium capable of producing nicotinamide and acrylamide by a whole-cell catalysis method and a catalysis method. The recombinant strain can take the nicotinonitrile or the acrylonitrile as a substrate, and can generate nicotinamide and acrylamide with higher added values by whole cell catalysis. The tolerance of recombinant bacteria for synergistically expressing L-NHase and H-NHase in different concentrations of nicotinonitrile, nicotinamide, acrylonitrile and acrylamide solutions is obviously improved compared with that of a strain for expressing L-NHase, and the recombinant bacteria are similar to a BAG strain for expressing H-NHase. In the production of amide substances, the recombinant bacteria for cooperatively expressing the L-NHase and the H-NHase have the yield of nicotinamide which is 2-2.8 times that of independently expressing the L-NHase and 1.6-2.2 times that of H-NHase, and the yield of the product is as high as 516g/L; the production rate of acrylamide is improved by 92.2% compared with BAG strain, and the final product concentration is improved by 19.1% compared with BAE strain.

Drawings

FIG. 1 is a schematic diagram of plasmid construction for recombinant strains.

FIG. 2 is a protein electrophoretogram of recombinant strains; m is Protein Molecular Weight Marker.

FIG. 3 is a graph of strain tolerance 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 whole cell catalytic synthesis of nicotinamide by BAE, BAG, BAG +BAE strain.

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

FIG. 6 shows whole cell catalytic synthesis of acrylamide.

Detailed Description

The following examples relate to media:

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

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

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

Plasmids referred to in the following examples:

pACYCDuet plasmid, pRSFDuet plasmid: commercial plasmids were purchased from the allied biogenic gene technologies, inc.

BAG-S/BAG plasmid: the corresponding plasmid pET-24a (+) -nhhBrbsAbsG has been disclosed in the article, see in detail: wang Z, liu Z, cui W, zhou Z. Establishment of bioprocess for synthesis of nicotinamide by recombinantEscherichia coliexpressing high-molecular-mass nitrile hydratase. Appl Biochem Biotechnol, 2017, 182(4):1458-1466。

BAE-S/BAE plasmid: has been disclosed in the article as the corresponding plasmid pET-24a (+) -BA in the article _L-NHase E, see in detail: wang Tian, programming, guo Junling, xia Yuanyuan, liu Zhongmei, zhou Zhemin. Heterologous activator of rhodococcus low molecular weight nitrile hydratase and domain function of activator. Bioengineering journal, 2020, 36 (8): 1578-1589.

The method involved in the following examples:

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

picking single colony of recombinant strain, inoculating to LB liquid culture medium containing corresponding antibiotics, shake culturing at 37deg.C and 200 rpm to OD ₆₀₀ 0.6-0.8 mM IPTG was added to the medium at a final concentration of 0.2 mM, and the culture was continued at 24℃and 200 rpm for about 20. 20 h to induce the expression of the target protein.

(2) Method for measuring cell catalytic activity (method for measuring enzyme activity):

will 100 μL OD ₆₀₀ The recombinant bacteria of 1.0 were added to 400. Mu.L of 125 mmol/L nicotinonitrile, reacted at 25℃for 10 min, and then, 500. Mu.L of acetonitrile was added to terminate the reaction. High Performance Liquid Chromatography (HPLC) detects nicotinamide content.

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

(3) Cell tolerance assay method:

will 100 μL OD ₆₀₀ The recombinant bacteria of 6.0 were added to 900. Mu.L of different concentrations of nicotinonitrile, nicotinamide, acrylonitrile and acrylamide solutions, and after 30 min at 25℃the cells were washed 2 times with 10 mmol/L KPB buffer (pH 7.4) to determine residual enzyme activities.

The relative enzyme activities are defined as: (cell catalytic activity after treatment with an organic reagent/cell catalytic activity without treatment with an organic reagent). Times.100%. Cells not treated with the organic reagent had a relative enzyme activity of 100%.

(4) Whole cell catalysis method:

resuspending the recombinant strain inducing protein expression with deionized water and adjusting absorbance OD ₆₀₀ The value is 8. Placing 50 mL bacteria liquid into a beaker, adding a reaction substrate, reacting while stirring, and controlling the reaction temperature to be 24-30 ℃. Using the time of the first addition of substrate (2 g nicotinonitrile/time or 1.5 mL acrylonitrile/time) as the starting point, 10. Mu.L of the reaction mixture was added to 990. Mu.L of acetonitrile every 2 minutes to terminate the reaction. The amount of nicotinamide or acrylamide product in the sample is analyzed by HPLC and it is determined whether there is a substrate remaining, and the substrate is continued to be added when no substrate remains. Repeating the previous stepsAnd (3) until the substrate cannot be continuously consumed.

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

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

Example 1: construction of strains that synergistically express BAG and BAE

(1) Construction of recombinant plasmids

Construction of plasmid BAG-1 expressing high molecular weight nitrile hydratase (H-NHase): taking a BAG-S/BAG plasmid expressing B, A, G subunit of H-NHase as a template, and carrying out PCR amplification by using primers PAC-BAG-F and PAC-BAG-R to obtain a BAG gene fragment; performing PCR (polymerase chain reaction) by using pACYCDuet plasmid as a template and using primers PAC-BAG-S-F and PAC-BAG-S-R to obtain a plasmid skeleton by amplification; the BAG gene fragment and the plasmid backbone were assembled by the In fusion method to construct a BAG-1 recombinant plasmid, and verified by DNA sequencing (see table 1 for primers).

Construction of plasmid pRSFDuet-BAE expressing 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 fragments; PCR was performed using pRSFDuet plasmid as a template and primers DT7-BAE-S-F and DT7-BAE-S-R to amplify the plasmid backbone; the BAE gene fragment and the plasmid backbone were assembled by the Infusion method to construct pRSFDuet-BAE recombinant plasmids (primers shown In Table 1).

Constructing a recombinant plasmid BAE-BAG for coexpression of high molecular weight and low molecular weight nitrile hydratase: performing PCR amplification by using BAG-S/BAG plasmid templates and using primers DT7-BAG-F and DT7-BAG-R to obtain BAG gene fragments; 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 by amplification; BAE-BAG recombinant plasmids were constructed by assembling BAG gene fragments and plasmid backbone by the In fusion method and verified by DNA sequencing (primers see Table 1).

Constructing a recombinant plasmid BAG-BAE for coexpression of high molecular weight and low molecular weight nitrile hydratase: performing PCR amplification by using BAG-S/BAG plasmid as a template and using primers DT7-BAG-F and DT7-BAG-2 to obtain BAG gene fragments; PCR is carried out by taking pRSFDuet-BAE plasmid as a template and utilizing primers DT7-GE-S-1 and DT7-BAG-S-R to obtain a plasmid skeleton by amplification; the BAG gene fragment and the plasmid backbone were assembled by the In fusion method to construct a BAG-BAE recombinant plasmid, and verified by DNA sequencing (see table 1 for primers).

Table 1 primers and sequences referred to herein

(2) Construction of recombinant bacteria

Transformation of BAE-S/BAE plasmidE. coliBL21 strain to obtain BAE strain; transformation of BAG-S/BAG plasmidE. coliBL21 strain to obtain BAG strain; transforming BAE-S/BAE with the BAG-1 plasmid constructed in step (1)E. coliBL21 strain obtains BAG+BAE strain; transforming the BAG-BAE plasmid constructed in the step (1)E. coliBL21 strain obtains BAG-BAE strain; transforming the BAE-BAG plasmid constructed in the step (1)E. coliBL21 strain BAE-BAG strain was obtained (FIG. 1).

The obtained 5 recombinant strains (BAE strain, BAG 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 L-NHase and H-NHase have two subunits, an alpha and a beta subunit, which are slightly different in size and an alpha subunit is similar in size. The nitrile hydratase expression of the BAE strain was significantly lower than that of the BAG strain. In the BAG+BAE double plasmid strain, expression of two types of nitrile hydratase is achieved simultaneously, wherein the expression level of L-NHase is similar to that of the BAE strain, and the expression level of H-NHase is lower than that of the BAG strain. The expression level of H-NHase in BAG-BAE strain and BAE-BAG strain is similar to that of BAG strain, and the expression level of L-NHase is lower.

Example 2: recombinant strain tolerance analysis

The tolerance of the 5 recombinant strains constructed in example 1 in different concentrations of nicotinonitrile, nicotinamide, acrylonitrile and acrylamide solutions was determined, and as can be seen from fig. 3, the tolerance of the BAG strain to 4 compounds was significantly better than the BAE strain, consistent with previous studies. The synergistic expression of L-NHase and H-NHase in BAG+BAE, BAG-BAE and BAE-BAG strains makes them tolerant to 4 compounds better than BAE strains. Wherein the BAG-BAE and BAE-BAG strains express both low molecular weight and high molecular weight nitrile hydratase on one plasmid, and their tolerance to 4 compounds reaches a level similar to that of the BAG strain. When the concentration of the nicotinonitrile in the reaction system reaches 0.8 mol/L, the residual relative enzyme activities of the BAG-BAE and the BAE-BAG strain reach 50%, and the BAE strain almost loses the enzyme activity; when the concentration of nicotinamide in the reaction system reaches 1.5 mol/L, the residual relative enzyme activities of BAG-BAE and BAE-BAG strains are close to 80%, and the BAE strains almost lose the enzyme activities; when the concentration of acrylonitrile in the reaction system reaches 2 mol/L, the residual relative enzyme activities of BAG-BAE and BAE-BAG strains reach more than 80 percent; when the acrylamide concentration in the reaction system reached 5 mol/L, the residual relative enzyme activities of BAG-BAE and BAE-BAG strains were about 60%, at which time the BAE strains lost almost the enzyme activities. Thus, the present study synergistically expresses low and high molecular weight nitrile hydratase, retaining higher tolerance of the recombinant strain to substrates and products.

Example 3: whole-cell catalytic synthesis of nicotinamide

The 5 recombinant strains constructed in example 1 were inoculated into LB medium, respectively, and shake-cultured at 37℃and 200 rpm to OD ₆₀₀ 0.6-0.8 mM IPTG with final concentration of 0.2 mM is added, and the culture is continued at 24 ℃ and 200 rpm for about 20 h to induce the expression of the target protein, thus obtaining the fermentation broth. Centrifuging the fermentation broth, removing supernatant, re-suspending thallus with deionized water, and adjusting to obtain absorbance OD ₆₀₀ Bacterial liquid with value of 8. And (3) placing 50 mL bacterial liquid in a beaker, adding a reaction substrate 2 g nicotinonitrile, reacting while stirring, controlling the reaction temperature to be 24-30 ℃, and adding the substrate (2 g nicotinonitrile) again after the substrate is consumed until the reaction is stopped and the substrate is not consumed.

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 10 minutes of the reaction; however, after the reaction was carried out for 10 minutes, since the BAE strain had poor tolerance to the product, the reaction rate was significantly reduced as the nicotinamide content in the reaction system was gradually increased, the synthesis end point was reached in a short period of time, while the bae+bag strain had better product tolerance than the BAE strain, and the post reaction rate remained at a higher level, the synthesis amount of nicotinamide as the final product was about 1.28 times that of the BAE strain, and the reaction time was shortened by about 30% as compared with the BAG strain in the case of the same amount of the product. The result shows 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 the BAE-BAG strain was fast and maintained at 7.33 g/L.min 32 min before the reaction ^-1 About, the reaction rate was substantially the same as that of the bae+bag strain; after 64 min of reaction, the reaction rate was slightly decreased due to the better tolerance of the BAE-BAG strain to the product, but the reaction could be continued for a longer time, and the final nicotinamide yield was about 2.2 times that of BAE+BAG strain and BAG strain, and 2.8 times that of BAE strain. Likewise, the final yield of BAG-BAE strain was 1.6 times that of BAE+BAG strain and BAG strain, 2 times that of BAE strain. The result shows that the L-NHase and the H-NHase are constructed on the same plasmid, thereby realizing the purposes of improving the reaction rate, prolonging the reaction time and improving the product yield.

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

Example 4: whole cell catalytic synthesis of acrylamide

The 5 recombinant strains constructed in example 1 were inoculated into LB medium, respectively, and shake-cultured at 37℃and 200 rpm to OD ₆₀₀ 0.6-0.8 mM IPTG with final concentration of 0.2 mM is added, and the culture is continued at 24 ℃ and 200 rpm for about 20 h to induce the expression of the target protein, thus obtaining the fermentation broth.Centrifuging the fermentation broth, removing supernatant, re-suspending thallus with deionized water, and adjusting to obtain absorbance OD ₆₀₀ Bacterial liquid with value of 8. Placing 50 mL bacterial liquid into a beaker, adding a reaction substrate of 1.5 mL acrylonitrile, stirring and reacting, controlling the reaction temperature to be 24-30 ℃, and adding the substrate (1.5 mL acrylonitrile) again after the substrate is consumed until the substrate is not consumed any more after the reaction is stopped.

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

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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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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. The genetically engineered bacterium is characterized in that the genetically engineered bacterium utilizes the same plasmid to co-express low molecular weight nitrile hydratase L-NHase and high molecular weight nitrile hydratase H-NHase;

the L-NHase consists of three subunits of B, A, E, the amino acid sequence of the B subunit is shown as SEQ ID NO. 1, the amino acid sequence of the A subunit is shown as SEQ ID NO. 2, the amino acid sequence of the E subunit is shown as SEQ ID NO. 3, the H-NHase consists of three subunits of B, A, G, the amino acid sequence of the B subunit is shown as SEQ ID NO. 5, the amino acid sequence of the A subunit is shown as SEQ ID NO. 6, and the amino acid sequence of the G subunit is shown as SEQ ID NO. 7.

2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium hosts escherichia coli.

3. The genetically engineered bacterium of claim 1, wherein the plasmids include pacycdat plasmid and prsfcdur plasmid.

4. A method for producing amide substances is characterized in that nicotinamide or acrylonitrile is used as a substrate, and the genetically engineered bacterium of any one of claims 1-3 is used for catalyzing and generating nicotinamide or acrylamide.

5. The method for producing an amide material according to claim 4, wherein the reaction conditions are that the temperature is 20-35 ℃, and the OD of the genetically engineered bacterium is ₆₀₀ 7-9, wherein the mass volume ratio of the substrate addition amount to the bacterial liquid is 1: (20-35), adding the substrate again until the reaction is stopped after the substrate is consumed.

6. The use of the genetically engineered bacterium of any one of claims 1 to 3 or the method for producing an amide substance of claim 4 or 5 in biopharmaceuticals.