CN116656656A - Nitrile hydratase mutant, bacterial strain and application thereof in catalyzing aromatic nitrile compounds to synthesize amide compounds - Google Patents

Nitrile hydratase mutant, bacterial strain and application thereof in catalyzing aromatic nitrile compounds to synthesize amide compounds Download PDF

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CN116656656A
CN116656656A CN202310125922.4A CN202310125922A CN116656656A CN 116656656 A CN116656656 A CN 116656656A CN 202310125922 A CN202310125922 A CN 202310125922A CN 116656656 A CN116656656 A CN 116656656A
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pfnh
nitrile hydratase
mutant
nitrile
compounds
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王亚军
徐浩博
李树芳
徐成龙
郑裕国
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Zhejiang University of Technology ZJUT
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Zhejiang University of Technology ZJUT
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
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    • C12Y402/01084—Nitrile hydratase (4.2.1.84)
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C12R2001/39—Pseudomonas fluorescens

Abstract

The invention discloses a nitrile hydratase mutant, a bacterial strain and application thereof in catalyzing aromatic nitrile compounds to synthesize amide compounds, wherein the nitrile hydratase PfNH mutant is obtained by carrying out single mutation on the 86 th site of a nitrile hydratase PfNH amino acid sequence shown in SEQ ID NO. 9. The substrate preference of the nitrile hydratase PfNH mutant PfNH-Q86W is obviously changed, the catalytic performance of aromatic nitriles with larger molecular weight such as 4-cyanopyridine, benzonitrile and 2, 6-difluorobenzonitrile is obviously improved compared with that of a wild type, and the comparison enzyme activity is respectively improved by 2.56 times, 9.35 times and 2.29 times; the catalytic performance of aliphatic nitriles with smaller molecular weight, such as isobutyronitrile and n-valeronitrile, is reduced compared with the wild type, and the specific enzyme activities are respectively 0.38 times and 0.80 times of the wild type. The catalytic activity of the mutant to the aromatic nitrile compound is obviously improved, and the mutant has industrial application prospect.

Description

Nitrile hydratase mutant, bacterial strain and application thereof in catalyzing aromatic nitrile compounds to synthesize amide compounds
Field of the art
The invention relates to an encoding gene of pseudomonas fluorescens Pseudomonas fluorescens nitrile hydratase PfNH and application of a mutant thereof in biocatalysis synthesis of amide compounds.
(II) background art
Amides are a class of compounds with high industrial added value that can be important synthetic precursors for rubber, nylon polymers, cosmetics and textiles. The traditional industrial production of the amide compound is mainly prepared by hydration of nitrile chemical method, the chemical method is generally harsh in reaction condition, high temperature and high pressure are needed, and the participation of chemical catalyst is needed, so that the reaction byproducts are more and the yield is low. The bioconversion method has the advantages of mild reaction conditions, less byproducts, less pollution and the like, and accords with the development direction of green chemistry. Currently, biocatalysis has gradually replaced traditional chemical methods for industrial production of amides.
Nitrile hydratase (Nitrile hydratase, EC 4.2.1.84) is capable of catalyzing the hydration of nitriles to the corresponding amides. Nitrile hydratase has been successfully used in the large-scale industrial preparation of bulk chemicals such as acrylamide and nicotinamide since the 70 s of the last century. The polymer polyacrylamide of acrylamide has very wide application and is often used as a lubricant, a clay stabilizer and a thickener; nicotinamide is widely used in medicine, food, feed additives and pharmaceutical intermediates. Along with the continuous and deep research, the nitrile hydratase is widely applied to the production of medical and pesticide intermediates, such as the synthesis of isobutyramide, 2, 6-difluorobenzamide, 4-pyridine formamide, benzamide and the like, and has great application value and potential. Isobutyramide is an important intermediate for synthesizing an AIDS therapeutic drug ritonavir; 2, 6-difluorobenzamide is an important intermediate for the pesticide benzoylurea; 4-pyridine carboxamide is a cephalosporin intermediate; benzamide and its derivative anthranilamide are important intermediates of vorinostat as anticancer drugs, etc.
Nitrile hydratase acts as a metalloenzyme. Nitrile hydratase is mainly classified into iron type nitrile hydratase (Fe-NHase) and cobalt type nitrile hydratase (Co-NHase) depending on the metal ions contained. Fe-NHase and Co-NHase exhibit different enzymatic properties in addition to the bound metal ions. The substrate preference of two nitrile hydratases is different, and Fe-NHase is generally preferred to catalyze aliphatic nitrile compounds with smaller molecular weight, and Co-NHase has higher substrate affinity for aromatic nitrile compounds with larger molecular weight.
(III) summary of the invention
The invention provides a nitrile hydratase PfNH mutant, a bacterial strain and application thereof in catalyzing aromatic nitrile compounds to synthesize amide compounds, and the invention separates a pseudomonas fluorescens (Pseudomonas fluorescens) ZJUT001 with nitrile hydratase activity through enrichment culture, clones a coding gene of iron ion dependent nitrile hydratase PfNH from a P.fluoroscens ZJUT001 genome, constructs the nitrile hydratase PfNH mutant with obviously improved catalytic activity on the aromatic nitrile compounds through a gene mutation technology, and is used for catalyzing the aromatic nitrile compounds to synthesize the amide compounds. The invention overcomes the defect that the existing iron ion dependent nitrile hydratase has low catalytic efficiency on aromatic nitrile compounds.
The technical scheme adopted by the invention is as follows:
the invention provides a nitrile hydratase PfNH mutant, which is obtained by single mutation at the 86 th position of a nitrile hydratase PfNH amino acid sequence shown in SEQ ID NO. 9. The nitrile hydratase PfNH comprises an alpha subunit and a beta subunit, the amino acid sequence of the alpha subunit is shown as SEQ ID NO.5, and the amino acid sequence of the beta subunit is shown as SEQ ID NO. 6.
Further, the nitrile hydratase PfNH mutant is obtained by mutating glutamine at position 86 of the amino acid sequence shown in SEQ ID NO.9 to tryptophan (Q86W, designated as M1).
The nucleotide sequence of the gene encoding the nitrile hydratase PfNH is shown as SEQ ID NO.4, pfNH consists of alpha subunit (SEQ ID NO.5, corresponding nucleotide sequence is shown as SEQ ID NO. 1) and beta subunit (SEQ ID NO.6, corresponding nucleotide sequence is shown as SEQ ID NO. 2) and regulatory protein (SEQ ID NO.7, corresponding nucleotide sequence is shown as SEQ ID NO. 3). Wherein the regulatory protein gene is derived from the nitrile hydratase gene cluster of the P.fluoscensZJUT001 genome, and can express the regulatory protein (SEQ ID NO. 7). The regulatory protein plays an important role in the expression and assembly process of the nitrile hydratase, and can ensure the correct expression of the nitrile hydratase.
The invention also provides a coding gene of the nitrile hydratase PfNH mutant, a recombinant vector containing the coding gene, and recombinant genetic engineering bacteria constructed by the recombinant vector. The recombinant vector adopts plasmid pET-28a (+), pET-28b (+), and the recombinant genetically engineered bacterium adopts E.coliBL21 (DE 3) as host bacterium.
The invention also provides an application of the nitrile hydratase PfNH mutant in catalyzing aromatic nitrile compounds to synthesize amide compounds, and the application method specifically comprises the following steps: wet thalli or purified enzyme extracted from wet thalli obtained by inducing and culturing recombinant genetic engineering bacteria containing nitrile hydratase PfNH mutant coding genes are used as catalysts, aromatic nitrile compounds are used as substrates, a reaction system is formed by taking a pH7.0 and 50mM sodium phosphate buffer solution as a reaction medium, the reaction is carried out at 30 ℃ and 600-800 rpm, the reaction is finished, and the reaction solution is separated and purified, thus obtaining the amide compounds.
Further, in the transformation system, the final concentration of the substrate is 10 to 100g/L (preferably 100 g/L), the catalyst is used in an amount of 0.1 to 20g/L (DCW is dry weight of cells, preferably 6 g/L) based on the total dry weight of wet cells or 0.01 to 1mg/L (preferably 0.05 mg/L) based on the purified enzyme protein content.
Further, the aromatic nitrile compound includes benzonitrile, 4-cyanopyridine, 2, 6-difluorobenzonitrile.
Further, the wet cell is prepared as follows: inoculating recombinant genetic engineering bacteria containing the gene encoding the nitrile hydratase PfNH mutant into LB liquid medium containing kanamycin with a final concentration of 50 mug/mL, and culturing at 37 ℃ and 180rpm for 10 hours to obtain seed liquid; inoculating the seed solution into LB liquid medium containing kanamycin with final concentration of 50 μg/mL at 1.0% (v/v) by volume, culturing at 37deg.C and 180rpm to OD 600 The value is 0.6 to 0.8, isopropyl thiogalactoside and FeSO are added into the culture solution 4 After culturing for 14 hours at 18℃at final concentrations of 0.2mM and 0.5mM, respectively, the nitrile-containing hydratase wet cell was obtained by centrifugation at 8000rpm at 4℃for 10 minutes.
Further, the purified enzyme was prepared as follows: the wet cells were resuspended in pH7.0, 50mM sodium phosphate buffer at 100g/L and sonicated on an ice-water mixture for 15min under sonication conditions: the power is 200W, the mixture is crushed for 1s and is suspended for 3s, the crushed mixture is taken and centrifuged for 10min at 8000rpm and 4 ℃, the supernatant is collected, and after being filtered by a microfiltration membrane of 0.22 mu m, the protein is purified by a nickel affinity column, and the purification operation is as follows: (1) the nickel affinity column was equilibrated with pH7.0, 50mM sodium phosphate buffer containing 300mM NaCl until baseline stabilized; (2) loading at a flow rate of 1.0mL/min followed by washing unbound impurities with 300mM naci, 20mM imidazole in pH7.0, 50mM sodium phosphate buffer at a flow rate of 1.0mL/min until baseline is stable; (3) then eluting the target protein with a pH7.0 containing 300mM NaCl and 500mM imidazole and a 50mM sodium phosphate buffer solution at a concentration of 1.0 mL/min; and (3) collecting effluent when the ultraviolet absorption detection value rises upwards relative to the base line, stopping collecting when the ultraviolet absorption detection value returns to the base line, placing the collected target protein on ice for storage, dialyzing (the molecular weight cut-off of a dialysis bag is 10 kDal) for 12 hours at the temperature of 50mM pH7.0 sodium phosphate buffer solution and collecting the cut-off, thus obtaining the purified enzyme.
The invention also provides pseudomonas fluorescens (Pseudomonas fluorescens) ZJUT001 for extracting the nitrile hydratase PfNH gene, which is preserved in China center for type culture Collection, with a preservation date of 2022, 12 months and 5 days, a preservation number of CCTCC No. M20221873, a preservation address of Wuhan, university of Wuhan, post code: 430072.
the invention relates to a preparation method of a nitrile hydratase PfNH mutant, which is characterized in that a site-directed saturation mutation technology is adopted to mutate a nitrile hydratase PfNH gene (SEQ ID NO. 4), and the obtained mutant plasmid is transferred into E.coliBL21 (DE 3) competent cells in a heat shock mode to obtain a mutant strain. The specific method comprises the following steps: in the first step, recombinant E.coliBL21 (DE 3)/pET-28 b (+) -pfnh was activated and plasmid pET-28b (+) -pfnh was extracted and stored at-20 ℃. And secondly, screening an amino acid residue Q86 at the inlet of a substrate channel, carrying out site-directed saturation mutation by taking pET-28b (+) -pfnh as a template plasmid, obtaining a mutant plasmid, and transforming. All mutants were evaluated for catalytic performance and the dominant mutant, pfNH-Q86W (designated M1), was selected.
The culture medium used for inoculation, transfer and induction of the nitrile hydratase PfNH and mutants thereof of the invention is preferably LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, and distilled water to adjust the pH to 7.0. The culture method and the culture conditions are not particularly limited, and may be appropriately selected according to the general knowledge in the art depending on the type of host, the culture method and the like.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention separates a strain P.fluoroscens ZJUT001 with nitrile hydratase activity through enrichment culture; cloning to obtain the encoding gene of new type iron ion dependent nitrile hydratase PfNH, constructing recombinant expression vector pET-28b (+) -PfNH, and successfully implementing heterologous expression in colibacillus BL21 (DE 3).
(2) The nitrile hydratase PfNH mutant with obviously improved substrate catalytic activity is obtained through site-directed saturation mutation, wherein the substrate preference of the mutant PfNH-Q86W is obviously changed, the catalytic performance of aromatic nitrile compounds with larger molecular weight such as 4-cyanopyridine, benzonitrile and 2, 6-difluorobenzonitrile is obviously improved compared with that of a wild type, and the comparative enzyme activities are respectively improved by 2.56 times, 9.35 times and 2.29 times; the catalytic performance of aliphatic nitriles with smaller molecular weight, such as isobutyronitrile and n-valeronitrile, is reduced compared with the wild type, and the specific enzyme activities are respectively 0.38 times and 0.80 times of the wild type. The invention overcomes the defect of poor catalytic activity of wild nitrile hydratase PfNH on aromatic nitrile compounds such as 4-cyanopyridine, benzonitrile and 2, 6-difluorobenzonitrile.
(3) The invention uses the nitrile hydratase PfNH mutant from P.fluoroonscens ZJUT001 for synthesizing the amide compound by biocatalysis of the aromatic nitrile compound for the first time, the catalytic activity of the mutant PfNH-Q86W on the aromatic nitrile substrate is obviously improved, and the defect that the catalytic activity of the existing iron type nitrile hydratase on the aromatic nitrile compound is poor is overcome. For the mutant PfNH-Q86W, when the feeding amount of the substrate benzonitrile can reach 100g/L, the reaction can be completed within 10min, the conversion rate is more than 99%, and the good industrial application prospect is shown.
(IV) description of the drawings
FIG. 1 is a standard curve for detecting acrylonitrile and acrylamide concentrations by HPLC.
FIG. 2 shows colony morphology (A) of P.fluoroscens ZJUT001 and photographs under an optical microscope (B).
FIG. 3 is a P.fluoroscens ZJUT001 genomic agarose gel electrophoresis, lane M representing a standard DNA molecule, and lane 1 representing genomic DNA.
FIG. 4 is a 16S rDNA agarose gel electrophoresis of P.Fluorescens ZJUT001, lane M representing a standard DNA molecule, and lane 1 representing a PCR amplification product.
FIG. 5 is a P.fluoroscens ZJUT001 phylogenetic tree.
FIG. 6 is an agarose gel electrophoresis of a fragment of the nitrile hydratase PfNH gene, lane M representing a standard DNA molecule, and lane 1 representing the nitrile hydratase PfNH gene.
FIG. 7 is a SDS-PAGE electrophoresis of nitrile hydratase PfNH and its mutant pure enzyme; m: a standard protein molecule; lane 1: the nitrile hydratase PfNH-WT purified enzyme; lane 2: mutant PfNH-M1 purified enzyme.
FIG. 8 is a standard curve for detecting the concentration of benzonitrile and benzamide by HPLC.
FIG. 9 is a high performance liquid chromatography detection chart of a conversion solution of benzonitrile to benzamide catalyzed by the nitrile hydratase PfNH mutant M1 in example 5.
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
the culture medium used in the invention comprises the following components:
LB medium: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, and distilled water to adjust the pH to 7.0.
Enrichment medium: glucose 10g/L MgSO 4 0.2g/L，K 2 HPO 4 1g/L，KH 2 PO 4 1g/L，FeSO 4 ·7H 2 O 0.002g/L，CoCl 2 ·6H 2 O 0.002g/L，CaCl 2 0.015g/L, 2g/L of 4-cyanopyridine (sole nitrogen source), and distilled water as solvent.
Plate medium was isolated: glucose 10g/L MgSO 4 0.2g/L，K 2 HPO 4 1g/L，KH 2 PO 4 1g/L，FeSO 4 ·7H 2 O 0.002g/L，CoCl 2 ·6H 2 O 0.002g/L，CaCl 2 0.015g/L, 2g/L of 4-cyanopyridine (unique nitrogen source), 20g/L of agar and distilled water as solvent.
Seed culture medium: glucose 10g/L, yeast powder 3g/L, naCl 1g/L, K 2 HPO 4 0.3g/L，KH 2 PO 4 0.3g/L，MgSO 4 0.2g/L, and distilled water as solvent.
Fermentation medium: glucose 10g/L, yeast powder 5g/L, peptone 2g/L, naCl 1g/L, mgSO 4 0.2g/L，K 2 HPO 4 1g/L，KH 2 PO 4 1g/L，FeSO 4 ·7H 2 O 0.03g/L，CoCl 2 ·6H 2 O 0.03g/L，CaCl 2 0.05g/L, caprolactam (inducer) 1g/L, and distilled water as solvent.
Inorganic salt culture medium: glucose 10g/L, NH 4 NO 3 1.3g/L，NaCl 1g/L，MgSO 4 0.2g/L，K 2 HPO 4 1g/L，KH 2 PO 4 1g/L，CaCl 2 0.05g/L, caprolactam (inducer) 1g/L, and distilled water as solvent.
Example 1: screening and species identification of nitrile hydratase-producing microorganism strain
(1) Enrichment culture
1g of soil sample collected near a chemical plant is weighed and evenly dispersed in 10mL of distilled water, 2mL of soil sample suspension is removed after evenly mixing and added into 100mL of sterilized enrichment medium, and the mixture is placed in a shaking table at 28 ℃ for culturing for 4 to 5 days at 180 rpm; transferring 2mL of bacterial liquid into 100mL of sterilized enrichment medium, and culturing in a shaking table at 28 ℃ for 4-5 days; enrichment culture is carried out again until the culture solution is turbid.
(2) Seed culture
Diluting the bacterial liquid enriched for 3 times, coating the bacterial liquid on a separation plate, placing the separation plate in a 28 ℃ incubator for culture until single bacterial colonies grow, picking the bacterial strains with different growth forms into a seed culture medium, and placing the bacterial strains in a 28 ℃ table for culture at 180rpm for 1 day.
(3) Fermentation culture
The seed solution was inoculated to the fermentation medium at an inoculum size of 1.0% (v/v) by volume, and after 1 day of culture in a shaker at 28℃at 180rpm, the bacterial solution was centrifuged at 12000rpm for 10 minutes, and the bacterial cells were collected.
(4) Strain screening
0.5g of wet cells was weighed and mixed well in 10mL of sodium phosphate buffer pH7.0, and acrylonitrile was added to a final concentration of 1g/L, and reacted at 28℃and 800rpm for 30 minutes. After the reaction is completed, the reaction solution is centrifuged at 12000rpm for 2min, the supernatant is taken and filtered by a microfiltration membrane of 0.22 mu m, the peak area of the product acrylamide is detected by High Performance Liquid Chromatography (HPLC) of the permeate, and the acrylamide content in the conversion solution is calculated according to a standard curve (figure 1) of the acrylamide concentration and the peak area.
Through multiple rounds of enrichment culture and separation screening, more than 50 strains with different forms are obtained. Finally, a strain which generates 115mg/L acrylamide under the above reaction conditions is obtained by screening and is designated as a strain ZJUT001.
The liquid phase detection method comprises the following steps: c18 reverse phase chromatography column (4.6X250 mm, welchrom). The mobile phase composition was acetonitrile: water=1:9 (v/v), flow rate was 0.6mL/min, detection wavelength was 210nm, column temperature was 40 ℃, and acrylamide and acrylonitrile retention times were 5.4min, 10.5min, respectively.
(5) Identification of strains
And (3) carrying out strain morphology, physiological and biochemical identification and 16SrDNA sequence analysis on the strain ZJUT001 obtained by screening.
Strain morphology: the colonies were grown on LB solid medium at 30℃for 24 hours, and presented white round protrusions, smooth and moist surface, clean edges (FIG. 2A), and the microscopic morphology of the strain was rod-shaped (FIG. 2B).
And (3) physiological and biochemical identification: the strain ZJUT001 is subjected to physiological and biochemical identification by utilizing a Biolog (GENIII) automatic microorganism identification system, and the physiological and biochemical identification comprises carbon source utilization detection and chemosensitivity evaluation. The results of the detection are shown in tables 1 and 2.
TABLE 1 availability of Strain ZJUT001 to 71 carbon sources
Injecting + and positive; negative of
TABLE 2 evaluation of chemosensitivity of Strain ZJUT001
Injecting + and positive; negative of
Determination of 16S rDNA sequence and phylogenetic tree analysis:
the genome DNA of the strain ZJUT001 is extracted by adopting a bacterial genome extraction kit (Optimus Praeparata) and detected by 1% agarose gel electrophoresis, and the extracted genome DNA is a single band with the size of more than 12000bp (shown in figure 3).
TABLE 3.16S rDNA general primers
Primer name Primer sequence (5 '. Fwdarw.3')
27F AGAGTTTGATCCTGGCTCAG
1492R TACGGCTACCTTGTTACGACTT
The 16S rDNA sequence of strain ZJUT001 was amplified using genomic DNA as a template and 27F/1492R as a primer (Table 3). The PCR reaction system and the reaction conditions are as follows:
PCR reaction System (50. Mu.L): 1. Mu.L of forward primer (100. Mu.M), 1. Mu.L of reverse primer (100. Mu.M), 25. Mu.L of 2 XPhanta buffer, 1. Mu.L of dNTP mixture (10 mM each), 1. Mu.L of template, 1. Mu.L of DNA polymerase and 20. Mu.L of ultrapure water.
The PCR procedure was as follows: pre-denaturation at 95℃for 5min, followed by 30 cycles (denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 2 min) and final extension at 72℃for 10min.
The PCR amplification product was detected by 1% agarose gel electrophoresis to obtain a fragment of about 1500bp (FIG. 4), and the 16S rDNA sequence of the strain ZJUT001 was confirmed to be 1434bp (SEQ ID NO. 8) in full length by sequencing.
SEQ ID NO.8
gtggcgcgactaccatgcaagtcgagcggtagagagaagcttgcttctcttgagagcggcggacgggtgagtaatgcctaggaatctgcctggtagtgggggataacgttcggaaacggacgctaataccgcatacgtcctacgggagaaagcaggggaccttcgggccttgcgctatcagatgagcctaggtcggattagctagttggtgaggtaatggctcaccaaggcgacgatccgtaactggtctgagaggatgatcagtcacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattggacaatgggcgaaagcctgatccagccatgccgcgtgtgtgaagaaggtcttcggattgtaaagcactttaagttgggaggaagggcagttacctaatacgtgattgttttgacgttaccgacagaataagcaccggctaactctgtgccagcagccgcggtaatacagagggtgcaagcgttaatcggaattactgggcgtaaagcgcgcgtaggtggttagttaagttggatgtgaaatccccgggctcaacctgggaactgcattcaaaactgactgactagagtatggtagagggtggtggaatttcctgtgtagcggtgaaatgcgtagatataggaaggaacaccagtggcgaaggcgaccacctggactgatactgacactgaggtgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgtcaactagccgttgggagccttgagctcttagtggcgcagctaacgcattaagttgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggccttgacatccaatgaactttctagagatagattggtgccttcgggaacattgagacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgtaacgagcgcaacccttgtccttagttaccagcacgttatggtgggcactctaaggagactgccggtgacaaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacggcctgggctacacacgtgctacaatggtcggtacagagggttgccaagccgcgaggtggagctaatcccataaaaccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgctagtaatcgcgaatcagaatgtcgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaccagaagtagctagtctaaccttcggggggacggtaccacggtgatcagta。
The resulting sequence was aligned with sequences in GenBank by BLAST analysis and strain sequences close to the strain ZJUT001 sequence were searched for phylogenetic analysis. Phylogenetic tree was constructed using MEGA7 (fig. 5). Through homology analysis, the strain ZJUT001 has highest homology with Pseudomonas fluorescens, combines physiological and biochemical characteristics and morphological characteristics, and is identified as Pseudomonas fluorescens (Pseudomonas fluorescens), named as Pseudomonas fluorescens (Pseudomonas fluorescens) ZJUT001, and is preserved in China center for type culture Collection, with a preservation date of 2022, 12 months and a preservation number of CCTCC No. M20221873.
(6) Metal ion type of p.fluoroenszjut001 nitrile hydratase
In order to determine the metal ion type of the P.Fluorescens ZJUT001 nitrile hydratase, a control group and three experimental groups (experimental group 1, experimental group 2 and experimental group 3) are arranged, wherein the control group adopts an inorganic salt culture medium without any iron ion or cobalt ion, and 0.002g/L FeSO is added into the inorganic salt culture medium respectively 4 ·7H 2 O (experimental group 1); 0.002g/L CoCl 2 ·6H 2 O (experimental group 2); 0.002g/L FeSO 4 ·7H 2 O and 0.002g/L CoCl 2 ·6H 2 O (real)Test group 3). Deionized water was used for the medium formulation to exclude ionic interference. P. fluoscenszjut 001 was inoculated into each group of medium and after incubation at 30 ℃ for 24h, the acrylamide content was measured using the HPLC method described in step (4). The results showed that the control group and the experimental group 2 added only with cobalt ions hardly detected nitrile hydratase activity, while the experimental group 1 added only with iron ions and the experimental group 3 added both with iron and cobalt ions showed remarkable nitrile hydratase activity, in which the fermentation broth of the experimental group 2 reached 27U/mL in enzyme activity, indicating that p.fluoscenszjut 001 nitrile hydratase belongs to the iron ion-dependent group. Sequencing results showed that the α subunit of PfNH contained the highly conserved amino acid sequence "-vcslact-" of iron-type nitrile hydratase, further proving that p.fluoscenszjut001 nitrile hydratase is an iron ion-dependent nitrile hydratase.
Example 2: construction of P.Fluorescens ZJUT001 nitrile hydratase PfNH Gene clone and recombinant plasmid
(1) Cloning of nitrile hydratase PfNH and regulatory protein genes thereof
The genome of P.fluoiscensZJUT001 was extracted using a bacterial genome extraction kit (Optimago), and PCR primers NH-F and NH-R were designed according to the homologous nitrile hydratase sequence in P.fluoiscensL 111 (GenBank accession number: CP 015638.1) reported in NCBI, in combination with analysis of P.fluoiscensL 111 genome, as shown in Table 4.
TABLE 4 nitrile hydratase and regulatory protein Gene cloning primer design
Primer name Primer sequence (5 '. Fwdarw.3')
NH-F ATGAGTACATCGATTTCCACGAC
NH-R TCAAGTCGGTTGAACCGCCAT
PCR cloning of the gene encoding nitrile hydratase PfNH by using P.Fluorescens ZJUT001 genome DNA as a template and NH-F and NH-R as primers, and the PCR reaction system and reaction conditions are as follows:
PCR reaction System (50. Mu.L): 1. Mu.L of forward primer (100. Mu.M), 1. Mu.L of reverse primer (100. Mu.M), 25. Mu.L of 2 XPhanta buffer, 1. Mu.L of dNTP mixture (10 mM each), 1. Mu.L of template, 1. Mu.L of DNA polymerase and 20. Mu.L of ultrapure water.
The PCR procedure was as follows: pre-denaturation at 95℃for 5min, followed by 30 cycles (denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 2 min) and final extension at 72℃for 10min.
The PCR amplified product was detected by 1% agarose gel electrophoresis, and the product was a single band with a size of about 2500bp (FIG. 6). Obtaining a nitrile hydratase PfNH gene fragment containing a regulatory protein gene, wherein the total length of the gene is 2564bp, and the gene fragment is formed by sequentially connecting an alpha subunit coding gene (SEQ ID NO. 1) and a beta subunit coding gene (SEQ ID NO. 2) and a regulatory protein coding gene (SEQ ID NO. 3); wherein the nucleotide sequence of the nitrile hydratase PfNH gene is shown as SEQ ID NO.4, and the amino acid sequence is shown as SEQ ID NO. 9. The regulatory protein coding gene is derived from a nitrile hydratase gene cluster of a pseudomonas fluorescens (P.fluoscens) genome, can express the regulatory protein (SEQ ID NO. 7), plays an important role in the expression and assembly process of the nitrile hydratase, and can ensure the correct expression of the nitrile hydratase, thereby obtaining the active nitrile hydratase. The target fragment was purified and recovered using a DNA purification recovery kit (Axygen Biotechnology Co., hangzhou).
SEQ ID NO.4
atgagtacatcgatttccacgaccgcgacaccttcgacacccggcgagagggcatgggccttgtttcaagtgctcaagagcaaggaactcattccagagggctatgtcgagcagctcactcaattgatggcccatgactggagcccggagaacggcgctcgcgtggtcgccaaggcatgggtcgatccgcagttccgggcgctgctgctcaaggacggaacagccgcttgcgcgcagttcggctacaccggcccacaaggcgaatacatcgtcgccctggaagatacaccgggggtgaagaacgtcattgtctgcagcctgtgctcctgcaccaactggccggtcctcggcctgccgcccgagtggtacaagggctttgagtttcgtgcgcgcctggtccgggaggggcgcaccgtactgcgcgagctggggacggagttgccgagcgacacggtcatcaaagtctgggataccagcgccgaaagccgttacctggtgttgccgcaaaggcctgaaggctctgagcacatgagtgaagaacagcttcaacagctggtgaccaaagacgtgctgattggcgtcgccctgccacgcgttggctgagaaaaaacaactcatcatcgttcaacttgcggagttttcattatggatggctttcacgatctcggcggtttccaaggctttggcaaagtaccgcacaccatcaacagcctcagctacaaacaggttttcaagcaggactgggaacacctggcctatagcttgatgtttgtcggcgttgaccagttgaaaaagttcagcgtggacgaagtgcgtcacgccgtcgaacgcctggacgttcgccagcatgtcggcacccagtactacgaacgctacatcatcgcgaccgccacgctgctggtggaaacgggcgttatcacccaggcggagctcgatcaggcattgggttcccacttcaagctggcgaaccccgcccatgcgacaggtcgcccggcgatcaccggcaggccgcccttcgaagtgggcgatcgggttgtggttcgagacgaatatgtggcggggcatatccgcatgccggcctacgtgcgcggtaaggaaggcgtggtcctgcaccgcacctcagagcagtggcccttccccgacgccattggccacggcgacttgagcgcagcccatcagcctacctaccacgtcgagtttcgcgtgaaagatctatggggtgacgcggcagatgacggttacgtcgtggtcgatcttttcgaaagctacttggataaggcccccggtgcccaagcggtgaacgcatgattgaaggcgcccaggcgggccgactgccggtgacggtcctttccggcttcctcggcgccggcaaaaccaccctgctcaacgctatcctgcgcaatcgccaaggactgcgggtcgcggtcatcgtcaacgacatgagcgaagtcaacctcgatgccgaatcggtgcagcgcgacgtctcgctgcaccgtggccgcgatgagttgatcgagatgagcaatggctgcatttgctgcaccctgcgcgccgacctgctggaacagatcagtgacctggcacgtcagcagcgcttcgattacctgcttatcgaatccacggggatctccgaaccgatgccggtcgcggagaccttcgccttcctcgacaccgaaggcttcagcctcagcgagctggcacgccttgatacactggtgacggtggtcgatggcagccaatttcaggcgctgctggaatcgacggataccgttgcccgcgccgacaccgaggcgcacacgtccacgcgtcacctggcggatcttctgatcgaacaggtggagtacgccaacgtcattctggtcaacaagcgggacctgatcgacgagcccggctaccaggcagtgcatgcgatcctcgctggcctcaacccgagcgcgcggatcatgccgatggctcacgggaacgtcgcgctgtccagcctcctcgacacccatctgtttgatttacccagcctcgcagcctcgccgggctggatgcgtaaaatggaagctaccgatacgccggcctcggagtcggatacctatggcgtgacatcctgggtctaccgggagcgcgcgccttttcacccccagcgcttgctcgagtttctacagaagccctggcacaacggtcgcttgttacgcagcaaaggttacttctggctcgccagccgccacctggaaatcggcctgctggcgcaaagcggcaagcagttccagtgggattatgtcgggcgctggtggaacttcatcgagccatcgcaatggccgcgggacgaatatcggttgcagggcatcatggccaagtgggacagcgttgtcggcgactgccgacaggagctggtcttcatcggccaggggctcgacacccgcgtcttgcagcgcgaactcgaccattgtttgctgagtgcccaggaaatagccgccggcccactggcctggcaggccctgccggcggcgacggcctttggcaccgaggccttatcggcacgccccacgccccccatggcggttcaaccgacttga。
SEQ ID NO.9
MSTSISTTATPSTPGERAWALFQVLKSKELIPEGYVEQLTQLMAHDWSPENGARVVAKAWVDPQFRALLLKDGTAACAQFGYTGPQGEYIVALEDTPGVKNVIVCSLCSCTNWPVLGLPPEWYKGFEFRARLVREGRTVLRELGTELPSDTVIKVWDTSAESRYLVLPQRPEGSEHMSEEQLQQLVTKDVLIGVALPRVGMDGFHDLGGFQGFGKVPHTINSLSYKQVFKQDWEHLAYSLMFVGVDQLKKFSVDEVRHAVERLDVRQHVGTQYYERYIIATATLLVETGVITQAELDQALGSHFKLANPAHATGRPAITGRPPFEVGDRVVVRDEYVAGHIRMPAYVRGKEGVVLHRTSEQWPFPDAIGHGDLSAAHQPTYHVEFRVKDLWGDAADDGYVVVDLFESYLDKAPGAQAVNA。
(2) Construction of recombinant vector pET-28b (+) -pfnh and recombinant engineering bacterium E.coli BL21 (DE 3)/pET-28 b (+) -pfnh
The recombinant plasmid is constructed by connecting the vector and the target gene through homologous recombination. First, the gene sequence of nitrile hydratase PfNH was analyzed, the appropriate cloning sites (Nco I and Xho I) of expression vector pET-28b (+) were selected, primers were designed as shown in Table 5, and a linearized vector fragment with homology arms was amplified using pET-28b (+) as a template, and the PCR reaction system and reaction conditions were as follows:
PCR reaction System (50. Mu.L): 1. Mu.L of forward primer (100. Mu.M), 1. Mu.L of reverse primer (100. Mu.M), 25. Mu.L of 2 XPhanta buffer, 1. Mu.L of dNTP mixture (10 mM each), 1. Mu.L of template, 1. Mu.L of DNA polymerase and 20. Mu.L of ultrapure water.
The PCR procedure was as follows: pre-denaturation at 95℃for 5min, followed by 30 cycles (denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 3 min) and final extension at 72℃for 10min.
TABLE 5 linearization vector primer design
The PCR amplified products were detected by 1% agarose gel electrophoresis, and the linearized vector fragment was purified and recovered using a DNA purification recovery kit (Axygen Biotechnology Co., hangzhou).
The linearized vector fragment and the target gene fragment were ligated by recombination using ClonExpress Ultra One Step Cloning Kit C (Nanjinozan Biotechnology Co., ltd.) and diluted to 50 ng/. Mu.L in concentration according to the reaction system of Table 6, prepared on ice, mixed well, transferred to a water bath at 50℃for recombination, incubated for 5min, and then placed on ice for cooling to convert the recombinant product into E.coli BL21 (DE 3) competent cells.
TABLE 6 one-step cloning reaction System
After colony PCR verification, positive transformants are selected, sent to the Hangzhou qing department biotechnology Co Ltd for sequencing, and the verified genetically engineered bacteria are E.coliBL21 (DE 3)/pET-28 b (+) -PfNH, marked as female parent PfNH-WT and stored in a refrigerator at the temperature of minus 80 ℃.
Example 3: construction of a library of nitrile hydratase PfNH mutants
The preparation of the nitrile hydratase PfNH mutant library is realized by a round of site-directed saturation mutation, the recombinant plasmid pET-28b (+) -PfNH constructed in example 2 is used as a template, the upstream and downstream primers designed in Table 7 are adopted, and the 86 th glutamine of the amino acid sequence of the nitrile hydratase PfNH shown in SEQ ID NO.9 is mutated into the rest 19 amino acids by saturation mutation PCR, so that dominant strains are screened.
The PCR reaction system and the reaction conditions are as follows:
PCR reaction System (50. Mu.L): 1. Mu.L of forward primer (100. Mu.M), 1. Mu.L of reverse primer (100. Mu.M), 25. Mu.L of 2 XPhanta buffer, 1. Mu.L of dNTP mixture (10 mM each), 1. Mu.L of template, 1. Mu.L of DNA polymerase and 20. Mu.L of ultrapure water.
The PCR procedure was as follows: pre-denaturation at 95℃for 5min, followed by 30 cycles (denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 5 min) and final extension at 72℃for 10min.
The Dpn I enzyme digested PCR product was transformed into E.coli BL21 (DE 3) competent cells, spread on LB solid medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃for 10 hours at 180 rpm. A series of nitrile hydratase mutant strains were obtained by selecting a single clone, inoculating the single clone into LB liquid medium containing 50. Mu.g/mL kanamycin, and culturing the single clone at 37℃and 180rpm for 12 hours.
TABLE 7 site-directed saturation mutagenesis primer design for nitrile hydratase
Example 4: inducible expression of nitrile hydratase
The mother strain PfNH-WT constructed in example 2 and the mutant strain constructed in example 3 were inoculated into LB liquid medium containing kanamycin at a final concentration of 50. Mu.g/mL, cultured at 37℃and 180rpm for 10 hours, inoculated into LB liquid medium containing kanamycin at a final concentration of 50. Mu.g/mL at 1.0% (v/v), and cultured at 37℃and 180rpm for 2 hours to OD, respectively 600 The value is 0.6 to 0.8, isopropyl thiogalactoside (IPTG) and FeSO are added into the culture solution 4 The final concentrations were 0.2mM and 0.5mM, respectively, and the culture was induced at 18℃and 180rpm for 14 hours, and the fermentation broth was collected and centrifuged at 8000rpm for 10 minutes at 4℃to collect wet cells. The cells obtained above can be used for the preparation of purified enzymes.
Example 5: mutant library screening
The wet cell of the mutant strain induced to express in example 4 was added to a 50mM sodium phosphate buffer solution at pH7.0 and resuspended to obtain a mutant strain suspension. Under the same conditions, a control strain E.coli BL21 (DE 3)/pET-28 b (+) -pfnh wet cell was used to prepare a control strain suspension.
Preparing a reaction system of 0.5mL by using mutant strain bacterial suspension and control strain bacterial suspension as catalysts and using substrate benzonitrile and phosphate buffer solution, wherein the input amount of wet bacterial bodies of the reaction system is 0.2g DCW/L based on the total dry bacterial body amount, and the final concentration of the substrate is 10g/L. The reaction was stopped at 800rpm at 30℃for 2min, followed by the addition of 0.5mL of pure acetonitrile. Removing thalli by centrifugation, filtering supernatant by using a 0.22 mu m microfiltration membrane, detecting and analyzing the content of the product benzamide by using HPLC, and calculating the concentration of the product benzamide according to a benzamide analysis standard curve (figure 8). The enzyme activities of the PfNH-WT and the mutants were calculated according to the definition of the enzyme activities in example 7, and the relative enzyme activities of the mutants were calculated by defining the enzyme activities of the wild-type PfNH-WT units as 100%, and the experimental results are shown in Table 8. The results showed that the mutant PfNH-Q86W (PfNH-M1) was the most active.
Unit cell enzyme activity: the unit of activity per gram of dry cell is designated U/g.
The liquid phase detection method comprises the following steps: c18 reverse phase chromatography column (4.6X250 mm, welchrom). The mobile phase composition was acetonitrile, water=1:1 (v/v), flow rate 1mL/min, detection wavelength 230nm, column temperature 40 ℃, benzamide and benzonitrile retention times 3.0min, 6.7min, respectively. The high performance liquid chromatography detection chart of the reaction liquid for synthesizing the benzamide by converting the mutant PfNH-Q86W into the benzonitrile is shown in figure 9.
TABLE 8 catalytic Properties of PfNH-WT and mutants thereof
Example 6: protein purification of nitrile hydratase PfNH and mutants thereof
Wet cells containing nitrile hydratase PfNH-WT prepared in the manner of example 4, wet cells containing nitrile hydratase mutant PfNH-M1 were resuspended in sodium phosphate buffer pH7.0, 50mM, respectively, at an amount of 100g/L, sonicated on an ice-water mixture for 15min, sonicated conditions: the power is 200W, the mixture is crushed for 1s and is suspended for 3s, the crushed mixture is taken and centrifuged for 10min at 8000rpm and 4 ℃, the supernatant is collected and filtered by a microfiltration membrane of 0.22 mu m, and the filtrate is purified by a nickel affinity column to purify protein, and the purification operation is as follows: (1) the nickel affinity column was equilibrated with pH7.0, 50mM sodium phosphate buffer containing 300mM NaCl until baseline stabilized; (2) loading at a flow rate of 1.0mL/min, followed by washing unbound impurities with 300mM NaCl, 20mM imidazole in pH7.0, 50mM sodium phosphate buffer at a flow rate of 1.0mL/min until baseline is stable; (3) then eluting the target protein with pH7.0 containing 300mM NaCl and 500mM imidazole, and 50mM sodium phosphate buffer solution with the concentration of 1.0 mL/min; and (3) collecting effluent when the ultraviolet absorption detection value rises upwards relative to the base line, stopping collecting when the ultraviolet absorption detection value returns to the base line, placing the collected target protein on ice for preservation, dialyzing (the cut-off molecular weight of a dialysis bag is 10 kDal) for 12 hours at 4 ℃ by using 50mM (mM) and pH7.0 sodium phosphate buffer solution, and taking the cut-off liquid as purified enzyme to respectively obtain the maternal nitrile hydratase PfNH-WT purified enzyme and the mutant PfNH-M1 purified enzyme.
Protein subunit molecular weights were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The parent nitrile hydratase PfNH-WT pure enzyme was prepared using the purification conditions described above and the results of the electrophoresis are shown in FIG. 7. The expression level of the mutant is not significantly changed compared with that of the parent nitrile hydratase PfNH-WT, so that the improvement of the enzyme activity of the mutant is not caused by the increase of the expression level of the enzyme, but is related to the enhancement of the self-catalytic activity of the enzyme.
Example 7: specific enzyme activity determination of nitrile hydratase PfNH-WT and mutant PfNH-Q86W on benzonitrile
The enzyme activities of nitrile hydratase are as follows: the amount of enzyme required to catalyze the formation of 1. Mu. Mol of the corresponding amide per minute of nitrile substrate at 30℃and pH 7.0.
Specific enzyme activity of nitrile hydratase (U/mg): the enzyme activity of the nitrile hydratase per milligram.
Standard conditions for enzyme activity detection: the protein concentration of the purified enzyme prepared in example 6 was diluted to 0.05mg/mL with 50mM sodium phosphate buffer, pH7.0, and 250. Mu.L of the diluted purified enzyme solution was removed to a 2mL EP tube and placed in a metal bath at 30 ℃. To the EP tube, 250. Mu.L of benzonitrile (20 g/L), 800rpm, 30℃were added, the reaction was terminated by adding 500. Mu.L of pure acetonitrile, the cells were removed by centrifugation, and the supernatant was filtered with a 0.22 μm microfiltration membrane. The filtrate was checked for benzamide content using HPLC as described in example 5. The specific enzyme activities of the nitrile hydratase PfNH-WT and the mutant PfNH-Q86W to benzonitrile were calculated to be 22.28.+ -. 0.95U/mg and 230.54.+ -. 3.05U/mg, respectively.
Example 8: specific enzyme activity determination of nitrile hydratase PfNH-WT and mutant PfNH-Q86W on 4-cyanopyridine
With 50mM, pH7.0Sodium phosphate buffer the protein concentration of the purified enzyme prepared in example 6 was diluted to 0.05mg/mL, respectively, and 250. Mu.L of the diluted purified enzyme solution was removed to a 2mL EP tube and placed on a metal bath at 30 ℃. To the EP tube, 250. Mu.L of 4-cyanopyridine (20 g/L), 800rpm, 30℃were added, the reaction was terminated by adding 500. Mu.L of pure acetonitrile, the cell was removed by centrifugation of the reaction solution, and the supernatant was filtered with a 0.22 μm microfiltration membrane. The filtrate was tested for peak area of 4-pyridinecarboxamide by HPLC, and r was measured according to the standard curve equation y=0.213 x-0.02 for peak area and concentration (mg/L) of 4-pyridinecarboxamide 2 =0.999, obtaining 4-pyridinecarboxamide content. The specific enzyme activities of the nitrile hydratase PfNH-WT and mutant PfNH-Q86W to 4-cyanopyridine were calculated to be 78.28.+ -. 1.75U/mg and 278.40.+ -. 15.78U/mg, respectively, according to the enzyme activity definition of example 7.
The liquid phase detection method comprises the following steps: c18 reverse phase chromatography column (4.6X250 mm, welchrom). The mobile phase composition was acetonitrile: water=1:9 (v/v), the flow rate was 1mL/min, the detection wavelength was 230nm, the column temperature was 40 ℃, and the retention times of 4-pyridinecarboxamide and 4-cyanopyridine were 3.7min and 10.6min, respectively.
Example 9: specific enzyme activity determination of nitrile hydratase PfNH-WT and mutant PfNH-Q86W on 2, 6-difluorobenzonitrile
The protein concentration of the purified enzyme prepared in example 6 was diluted to 0.05mg/mL with 50mM sodium phosphate buffer, pH7.0, and 250. Mu.L of the diluted purified enzyme solution was removed to a 2mLEP tube and placed on a metal bath at 30 ℃. 250. Mu.L of 2, 6-difluorobenzonitrile (20 g/L) was added to the EP tube, reacted at 800rpm and 30℃for 2min, then 500. Mu.L of pure acetonitrile was added to terminate the reaction, the cell was removed by centrifugation of the reaction solution, and the supernatant was filtered with a 0.22 μm microfiltration membrane. The filtrate was tested for peak area of 2, 6-difluorobenzamide by HPLC, and r was measured according to standard curve equation y=0.126 x-0.2 for peak area and concentration (mg/L) of 2, 6-difluorobenzamide 2 =0.999, obtaining 2, 6-difluorobenzamide content. The specific enzyme activities of the nitrile hydratase PfNH-WT and mutant PfNH-Q86W against 2, 6-difluorobenzonitrile were calculated to be 12.13.+ -. 0.44U/mg and 39.94.+ -. 0.93U/mg, respectively, according to the enzyme activity definition of example 7.
The liquid phase detection method comprises the following steps: c18 reverse phase chromatography column (4.6X250 mm, welchrom). The mobile phase composition was acetonitrile, water=1:1 (v/v), flow rate 1mL/min, detection wavelength 230nm, column temperature 40 ℃, retention time 2, 6-difluorobenzamide and 2, 6-difluorobenzonitrile 2.4min, 7.1min respectively.
Example 10: specific enzyme activity determination of nitrile hydratase PfNH-WT and mutant PfNH-Q86W on isobutyronitrile
The protein concentration of the purified enzyme prepared in example 6 was diluted to 0.05mg/mL with 50mM sodium phosphate buffer, pH7.0, and 250. Mu.L of the diluted purified enzyme solution was removed to a 2mL EP tube and placed on a metal bath at 30 ℃. To the EP tube, 250. Mu.L of isobutyronitrile (20 g/L), 800rpm, 30℃were added and the reaction was terminated by adding 500. Mu.L of pure acetonitrile, and the reaction solution was centrifuged to remove the cells, and the supernatant was filtered with a 0.22 μm microfiltration membrane. The filtrate was subjected to gas chromatography to determine the peak area of isobutyramide, and r was represented by the standard curve equation y=343.5x+75, based on the peak area and concentration (g/L) of isobutyramide 2 =0.994, and the isobutyramide content was obtained. The specific enzyme activities of the nitrile hydratase PfNH-WT and mutant PfNH-Q86W against isobutyronitrile were calculated to be 157.22.+ -. 15.57U/mg and 59.04.+ -. 3.26U/mg, respectively, according to the enzyme activity definition of example 7.
Gas chromatography method: BGB-174 capillary chiral chromatography column (30X 0.25mm ID,0.25 μm film, ea), injector and detector temperatures were set at 260 ℃, temperature program: 5min at 40 ℃;30 ℃/min;160 ℃ for 10min. The retention times of isobutyronitrile and isobutyramide were 5.4min and 10.5min, respectively.
Example 11: specific enzyme activity determination of nitrile hydratase PfNH-WT and mutant PfNH-Q86W on n-valeronitrile
The protein concentration of the purified enzyme prepared in example 6 was diluted to 0.05mg/mL with 50mM sodium phosphate buffer, pH7.0, and 250. Mu.L of the diluted purified enzyme solution was removed to a 2mL EP tube and placed on a metal bath at 30 ℃. To the EP tube, 250. Mu.L of 4-cyanopyridine (20 g/L), 800rpm, 30℃were added, the reaction was terminated by adding 500. Mu.L of pure acetonitrile, the cell was removed by centrifugation of the reaction solution, and the supernatant was filtered with a 0.22 μm microfiltration membrane. Detecting the peak area of n-valeramide by gas chromatography of the filtrate, and according to the standard curve equation of the peak area and the concentration (g/L) of n-valeramide, y=293 x-44.5, R 2 =0.993, giving n-valeramide content. Enzyme activity assay according to example 7As a result, the specific enzyme activities of the nitrile hydratase PfNH-WT and the mutant PfNH-Q86W against n-valeronitrile were calculated to be 34.85.+ -. 0.16U/mg and 27.84.+ -. 1.38U/mg, respectively.
Gas chromatography method: BGB-174 capillary chiral chromatography column (30X 0.25mm ID,0.25 μm film, ea), injector and detector temperatures were set at 260 ℃, temperature program: 100 ℃ for 5min;20 ℃/min; and at 260 ℃ for 10min. The retention times of n-valeronitrile and n-valeramide were 4.7min and 9.2min, respectively.
Example 12: determination of the thermal stability of the nitrile hydratase PfNH and its variants
1. Half-life period
Half-life (t) 1/2 ) Refers to the time required for 50% decrease in enzyme activity at a specific temperature, and is an important parameter for characterizing the thermostability of the enzyme. The purified enzyme of nitrile hydratase PfNH and its mutants prepared in the method of example 6 was diluted to a protein concentration of 1.0mg/mL with a 50mM sodium phosphate buffer solution at pH7.0, and incubated at 30℃for each time period, 25.0. Mu.L of the incubated enzyme solution was taken out, and the residual enzyme activity was measured under the conditions of the enzyme activity detection standard of example 7, and the half-life was determined according to the heat inactivation equation (A t ＝A 0 e -kd t ) Calculation of k in the thermal deactivation equation d For the kinetic constant of deactivation A 0 And A t Representing the initial enzyme activity and the enzyme activity at time t, respectively. In ln (A) t /A 0 ) On the ordinate, incubation time t is plotted on the abscissa, and experimental data is fitted to k d Using the formula ln2/k d Calculating the half-life t 1/2 . The results are shown in Table 9, where the half-life of mutant PfNH-Q86W was slightly reduced compared to the wild type.
TABLE 9 nitrile hydratase PfNH and its mutants half-life at 30 ℃
2、T m
T m The melting temperature of a protein is indicated as the temperature corresponding to 50% unfolding of the protein. The thermal denaturation process of proteins is closely related to the change of their spatial conformation,T m the value can reflect the trend of protein conformation change in the temperature change process, and is an important index for measuring the thermal stability of the protein. The melting temperature of nitrile hydratase and mutants thereof were analyzed using a Chirascan Circular Dichroism (CD) spectrometer. First, purified enzyme of nitrile hydratase PfNH and its mutant prepared in the method of example 6 was diluted to a protein concentration of 0.1mg/mL with 50mM phosphate buffer solution, 200. Mu.L was then loaded into a 10mM quartz cuvette, and T of nitrile hydratase PfNH and its mutant was measured by CD circular dichroism m The measurement was performed. Continuously collecting melting curves of nitrile hydratase PfNH and its mutant at 180-260nm wavelength and 20-65deg.C, and calculating T by using round dichromatic analyzer with software Global 3 m The results are shown in Table 10, T of mutant PfNH-Q86W m The values were slightly decreased, but the magnitude of the decrease was not significant. In combination with the half-life assay results of table 9, it was shown that mutation site Q86W had little effect on the thermal stability of the nitrile hydratase PfNH.
TABLE 10 melting temperature of nitrile hydratase PfNH and mutants thereof
Example 13: nitrile hydratase mutant PfNH-Q86W for catalyzing benzonitrile to generate benzamide
In a 10mL reaction system, the nitrile hydratase mutant PfNH-Q86W wet thalli prepared in the method of example 4 is resuspended by a sodium phosphate buffer solution with the pH of 7.0 and 50mM, the total dry weight of the wet thalli is added to be 6g DCW/L, the feeding amount of substrate benzonitrile is 100g/L, the reaction system is formed by taking the sodium phosphate buffer solution with the pH of 7.0 and 50mM as a reaction medium, and the reaction is carried out at 30 ℃ and 800 rpm. After sampling, the concentration of benzonitrile and benzamide was determined by HPLC as described in example 5, and PfNH-Q86W was able to convert benzonitrile completely to the product benzamide in 10min.

Claims (10)

1. A nitrile hydratase PfNH mutant is characterized in that the nitrile hydratase PfNH mutant is obtained by single mutation at position 86 of the nitrile hydratase PfNH amino acid sequence shown in SEQ ID NO. 9.
2. The mutant nitrile hydratase PfNH of claim 1, wherein the mutant nitrile hydratase PfNH is a mutation of glutamine 86 of the amino acid sequence shown in SEQ ID No.9 to tryptophan.
3. A gene encoding the nitrile hydratase PfNH mutant according to claim 1.
4. A recombinant genetically engineered bacterium comprising the coding gene of claim 3.
5. Use of the nitrile hydratase PfNH mutant of claim 1 for catalyzing synthesis of amides from aromatic nitriles.
6. The application according to claim 5, wherein the method of application is: wet thalli or purified enzyme extracted from wet thalli obtained by inducing and culturing recombinant genetic engineering bacteria containing nitrile hydratase PfNH mutant genes are used as catalysts, aromatic nitrile compounds are used as substrates, a reaction system is formed by taking a pH7.0 and 50mM sodium phosphate buffer solution as a reaction medium, the reaction is carried out at 30 ℃ and 600-800 rpm, the reaction is finished, and the reaction solution is separated and purified, thus obtaining the amide compounds.
7. The method according to claim 6, wherein the final substrate concentration in the conversion system is 10 to 100g/L, and the catalyst is used in an amount of 0.1 to 20g DCW/L based on the total dry weight of the wet cells or 0.01 to 1mg/L based on the purified enzyme protein content.
8. The use according to claim 6, wherein the aromatic nitrile compound comprises benzonitrile, 4-cyanopyridine, 2, 6-difluorobenzonitrile.
9. The use according to claim 6, wherein the wet cells are prepared byThe preparation method comprises the following steps: inoculating recombinant genetic engineering bacteria containing the nitrile hydratase PfNH mutant gene into LB liquid medium containing kanamycin with final concentration of 50 mug/mL, and culturing at 37 ℃ and 180rpm for 10 hours to obtain seed liquid; inoculating the seed solution into LB liquid medium containing kanamycin with final concentration of 50 μg/mL at 1.0% by volume, culturing at 37deg.C and 180rpm to OD 600 The value is 0.6 to 0.8, isopropyl thiogalactoside and FeSO are added into the culture solution 4 After culturing at 18℃for 14 hours at final concentrations of 0.2mM and 0.5mM, respectively, the cells were centrifuged at 8000rpm at 4℃for 10 minutes to obtain wet cells containing nitrile hydratase.
10. Pseudomonas fluorescens (Pseudomonas fluorescens) ZJUT001 for extracting the nitrile hydratase PfNH gene of claim 1, which is preserved in China center for type culture Collection, with a preservation date of 2022, 12 months and 5 days, and a preservation number of CCTCC NO: M20221873, with a preservation address of Wuhan, university of Wuhan, post code: 430072.
CN202310125922.4A 2023-02-10 2023-02-10 Nitrile hydratase mutant, bacterial strain and application thereof in catalyzing aromatic nitrile compounds to synthesize amide compounds Pending CN116656656A (en)

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