CN111057695B

CN111057695B - Nitrilase and preparation method and application thereof

Info

Publication number: CN111057695B
Application number: CN201911328262.XA
Authority: CN
Inventors: 李文均; 熊梦洁; 田野; 董雷; 鲜文东; 肖敏
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2021-10-08
Anticipated expiration: 2039-12-20
Also published as: CN111057695A

Abstract

The invention discloses a microbial nitrilase, and a preparation method and application thereof. Specifically, the amino acid sequence is disclosed as SEQ ID NO: 1 as a nitrilase; the nucleotide sequence is shown as SEQ ID NO: 2 as the nitrilase coding gene. The invention provides the SEQ ID NO: the protein shown in 1 has nitrilase activity, can effectively catalyze phenylacetonitrile, adiponitrile, butenenitrile, n-valeronitrile and the like to generate corresponding products such as phenylacetic acid, adipic acid, butenoic acid, n-valeric acid and the like, and has very high industrial application potential.

Description

Nitrilase and preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, and particularly relates to a novel nitrilase and application thereof.

Background

Nitrile compounds are important organic chemical raw materials and are toxic substances with pungent smells. In the process of synthesizing rubber and refining petroleum, sewage contains various nitrile compounds, and people tend to treat the nitrile compounds in the sewage by adopting a biological method which is harmless to the environment compared with by-products in the process of treating the nitrile compounds by a physical method and a chemical method. Compared with physical methods and chemical methods, the biological method has the characteristics of mild reaction conditions, stable operation and low cost. Since the 19 th century, biological treatment of organic matter in wastewater has become the dominant method in countries around the world. The metabolic function of the microorganism is utilized to degrade organic matters such as nitrile, phenols and the like in the wastewater into water, carbon dioxide and the like, thereby achieving the purification effect of the wastewater.

Nitrilases (Nitrilase, EC 3.5.5.1) are an important class of industrial enzymes belonging to the Nitrilase superfamily. Not only can degrade nitrile pollutants, but also can convert cheap nitrile compounds into intermediates of products with high added values, such as industry, food, medicine and the like, for example: acrylic acid, nicotinic acid, chiral mandelic acid, and the like. Since the 60's of the 20 th century, the american scientist Robinson and colleagues discovered the first nitrilase in the world, one continued to isolate and identify nitrilases from a variety of sources, such as: rhodococcus, Alcaligenes, Pseudomonas, Acinetobacter, and the like.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a novel nitrilase and application thereof.

The first purpose of the invention is to provide a polypeptide with an amino acid sequence shown as SEQ ID NO: 1 as a nitrilase.

The second purpose of the invention is to provide a nucleotide sequence shown as SEQ ID NO: 2 as the nitrilase coding gene.

A third object of the present invention is to provide the amino acid sequence as set forth in SEQ ID NO: 1 in catalyzing the hydrolysis of nitrile.

The fourth purpose of the invention is to provide an application of the recombinant vector in preparing n-pentanoic acid by using n-valeronitrile as a substrate for nitrile hydrolysis.

The fifth purpose of the invention is to provide the application of the engineering bacteria in preparing the n-pentanoic acid by using the n-valeronitrile as a substrate for nitrile hydrolysis.

In order to achieve the purpose, the invention is realized by the following technical scheme:

nitrile degrading bacteria (Nitrilorkaliphilus) ANL-iso2 (accession number DSM 45188) is a strain with nitrilase activity, and can hydrolyze various nitrile compounds such as phenylacetonitrile, adiponitrile, butenenitrile and n-valeronitrile to generate corresponding acid substrates. Therefore, the nitrile degrading bacteria (Nitrilliuptor alkal iphilus) ANL-iso2 (accession number DSM 45188) nitrilase gene was cloned and its expression in E.coli was highly efficient.

The invention provides an amino acid sequence derived from nitrile degrading bacteria (Nitrilorkaliphilus) ANL-iso2 (deposit number DSM 45188) as shown in SEQ ID NO: 1 shows nitrilase (Nit3) activity. In order to realize the soluble heterologous expression of nitrilase in prokaryotes such as escherichia coli and the like, a gene engineering conventional operation is carried out, and a synthetic method is adopted to obtain a polypeptide corresponding to the sequence shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, respectively.

The invention therefore claims the following:

the amino acid sequence is shown as SEQ ID NO: 1 as a nitrilase.

The nucleotide sequence is shown as SEQ ID NO: 2 as the nitrilase coding gene.

The amino acid sequence as set forth in SEQ ID NO: 1 for use in the hydrolysis of nitrile.

Preferably, one or more of phenylacetonitrile, adiponitrile, butenenitrile and n-valeronitrile are used as the substrate.

Preparing phenylacetic acid by using phenylacetonitrile as a substrate and using the nitrilase; preparing adipic acid by using adiponitrile as a substrate and the nitrilase; preparing butenoic acid by using butenenitrile as a substrate and using the nitrilase; and (3) taking n-valeronitrile as a substrate, and preparing n-pentanoic acid by using the nitrilase.

Preferably, the temperature for hydrolyzing nitrile by taking n-valeronitrile as a substrate is 25-65 ℃.

More preferably, the temperature for the hydrolysis of nitrile using n-valeronitrile as a substrate is 45 ℃.

Preferably, the pH value of the hydrolysis of the nitrile by using the n-valeronitrile as a substrate is 3-12.

More preferably, the pH for the hydrolysis of nitrile with n-valeronitrile as substrate is 8.0.

The invention also claims an application of the recombinant vector in preparation of n-pentanoic acid by using n-valeronitrile as a substrate for nitrile hydrolysis, wherein the recombinant vector contains a nucleotide sequence shown as SEQ ID NO: 2.

The invention also claims an application of the engineering bacteria in preparation of n-pentanoic acid by using n-valeronitrile as a substrate to carry out nitrile hydrolysis, wherein the engineering bacteria carry the recombinant vector.

The preparation method of the thallus of the engineering bacteria comprises the following steps: inoculating the engineering bacteria to an LB liquid culture medium containing 50 mu g/mL ampicillin, culturing at 37 ℃ for 12h, then inoculating the engineering bacteria to a fresh LB liquid culture medium containing 50 mu g/mL ampicillin in an inoculation amount with a volume concentration of 2%, and culturing at 37 ℃ until the bacterial concentration OD₆₀₀The value was 0.5, IPTG was added to the LB liquid medium to a final concentration of 0.5mM, induction culture was carried out at 15 ℃ for 20 hours, the culture was centrifuged at 4 ℃ and 8000rpm for 5 minutes, the supernatant was discarded, and the cells containing the recombinant nitrilase were collected.

The nitrilase coding gene Nit3 is connected with an expression vector pCold I DNA, and an expression recombinant plasmid pCold I DNA-Nit3 containing the nitrilase gene Nit3 is constructed. The expression recombinant plasmid pCold I DNA-Nit3 was transformed into E.coli BL21(DE3) strain to obtain recombinant E.coli BL21(DE3)/pCold I DNA-Nit3 containing recombinant plasmid pCold I DNA-Nit 3. Taking the recombinant bacteria as an enzyme source for biocatalysis. The recombinant strain BL21(DE3)/pCold I DNA-Nit3 shows high nitrilase activity, the optimum reaction temperature for purifying nitrilase is 45 ℃, the optimum reaction pH is 8.0, and the nitrilase can effectively convert substrate n-valeronitrile into n-pentanoic acid.

The preparation method of the nitrilase purification liquid of the recombinant strain comprises the following steps: collecting thalli in fermentation liquor, utilizing PB Buffer solution to resuspend and break cells, carrying out solid-liquid separation to obtain supernatant fluid, obtaining cell-free extract, preparing a sample for loading through a 0.45 mu m filter membrane, firstly Washing a Ni-NTA agarose gel column to balance with a Binding/Washing Buffer, Washing the column at the flow rate of 2mL/min, then loading the sample at the flow rate of 1mL/min, after complete adsorption, eluting with an elute Buffer at the flow rate of 2mL/min, and collecting eluent in each stage, namely nitrilase purified liquid.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides the SEQ ID NO: the protein shown in 1 has nitrilase activity, can effectively catalyze n-valeronitrile to be converted into n-pentanoic acid, and the engineering bacteria prepared by using the protein has high nitrilase activity, short fermentation period and high catalysis efficiency, and is suitable for the requirement of industrial production of n-pentanoic acid.

Drawings

FIG. 1 is a SDS-PAGE pattern of the separation and purification of recombinant nitrilase; lane 1 (left to right): cell-free extract supernatant, lane 2: purified nitrilase eluate, lane 3: purified nitrilase eluate, lane 4: and (3) standard protein molecular weight Marker.

FIG. 2 shows the optimum reaction temperature for the recombinant nitrilase (reaction using n-valeronitrile as a substrate).

FIG. 3 shows the optimum reaction pH for the recombinant nitrilase (reaction using n-valeronitrile as a substrate).

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 acquisition of nitrilase

First, experiment method

The data retrieval of NCBI database is utilized to obtain the genome data (NCBI Reference Sequence: NZ _ KQ033901.1) of nitrile group degrading bacteria (Nitrilorkaliphilus) ANL-iso2 (deposition number DSM 45188), and the genome is screened according to the prediction and annotation of functional genes to obtain a nitrilase gene (Nit 3). According to the amino acid sequence of the nitrilase, codon optimization is carried out according to the preferred codon of escherichia coli, and enzyme cutting sites Nde I and Xba I are designed according to the characteristics of expression vector pCold I DNA.

Second, experimental results

The nitrilase gene Nit3 (nucleotide sequence shown in SEQ ID: 2) was synthesized by a general procedure of genetic engineering in a total synthesis manner, and the amino acid sequence of the encoded enzyme is shown in SEQ ID: 1.

Example 2 construction of recombinant expression vector pCold I DNA-Nit3 and construction of recombinant engineering bacteria

First, experiment method

The fragment containing the Nit3 gene (nucleotide sequence shown in SEQ ID: 2) was subjected to double digestion and recovery treatment with Nde I and Xba I restriction enzymes, and ligated with a commercial vector pCold I DNA treated with the same restriction enzymes at 16 ℃ for 6h using T4 DNA ligase to construct an intracellular recombinant expression vector pCold I DNA-Nit 3. The constructed intracellular expression vector pCold I DNA-Nit3 is transformed into E.coli BL21(DE3) recipient bacteria, coated on an LB agar plate containing ampicillin (the final concentration is 50 mu g/mL), cultured overnight at 37 ℃, and randomly picked out from colonies growing on the plate the next day, cloned and extracted to carry out agarose gel electrophoresis identification.

Second, experimental results

Obtain recombinant gene engineering bacteria E.coli BL21(DE3)/pCold I DNA-Nit 3.

Example 3 preparation of cells containing nitrilase Nit3

First, experiment method

The genetically engineered bacterium E.coli BL21(DE3)/pCold I DNA-Nit3 constructed in example 2 was inoculated into LB liquid medium containing 50. mu.g/mL ampicillin, cultured at 37 ℃ for 12 hours, inoculated into fresh LB liquid medium containing 50. mu.g/mL ampicillin in a 2% inoculum size (v/v), and cultured at 37 ℃ until the cell concentration OD₆₀₀About 0.5, adding IPTG with the final concentration of 0.5mM into LB liquid culture medium, inducing and culturing at 15 ℃ for 20h, centrifuging the culture solution at 4 ℃ and 8000rpm for 5min, discarding the supernatant, and collecting wet thalli containing recombinant nitrilase, namely Escherichia coli BL21(DE3)/pCold I DNA-Nit3 wet thalli containing intracellular expression of recombinant nitrilase. And then SDS-PAGE electrophoresis is carried out to verify the size and the expression condition of the target protein.

Second, experimental results

SDS-PAGE shows that the Escherichia coli BL21(DE3) containing pCold I DNA-Nit3 can efficiently express target protein with the size of 40000 daltons by induction.

EXAMPLE 4 isolation and purification of recombinant nitrilase and enzymatic characterization

First, experiment method

The fermentation broth of the recombinant bacteria was centrifuged at 8000rpm for 5min at 4 ℃ to remove the supernatant. The cells collected by centrifugation were suspended in PB buffer (0.2M, pH7.4) to prepare a suspension. And (3) crushing the bacterial suspension in an ice bath by using an ultrasonic crusher, working for 30min under the power of 200W, and stopping working for 2s and 4s in each cycle. The sample was stained with crystal violet and observed under a microscope until complete lysis of the cells was indicated. Centrifuging the cell disruption solution at 4 deg.C and 12,000rpm for 10min to obtain supernatant as cell-free extractive solution. Filtering the cell-free extract with a 0.45-micrometer filter membrane to prepare a sample, firstly Washing a Ni-NTA agarose gel column with a Binding/Washing Buffer until the balance is achieved, Washing the column at the flow rate of 2mL/min, then loading the sample at the flow rate of 1mL/min, after complete adsorption, eluting with an Elution Buffer at the flow rate of 2mL/min, collecting the eluates at each stage, obtaining a nitrilase purification solution, and then carrying out SDS-PAGE electrophoresis to detect the expression and purification conditions of the target protein. And carrying out degradation reaction by using purified liquid of nitrilase by using n-valeronitrile as a substrate.

Second, experimental results

The expression and purification of the target protein by SDS-PAGE electrophoresis are shown in FIG. 1, and the apparent molecular weight is 42000 daltons.

When n-valeronitrile was used as a substrate, the optimum reaction temperature for nitrilase was 45 ℃ (as shown in FIG. 2), and the optimum reaction pH was 8.0 (as shown in FIG. 3). Under the optimal reaction conditions, the enzyme activity of the purified recombinant nitrilase is 0.78 mu mol/(mg. min).

EXAMPLE 5 degradation of nitriles by recombinant nitrilases

First, experiment method

A purified nitrilase solution was obtained in accordance with the method of example 4, and then degradation of other nitriles was examined by using the purified nitrilase solution, and the relative activity of the nitrilase was calculated by performing degradation at 45 ℃ and pH 8.0.

Second, experimental results

Table 1:

as shown in Table 1, the nitrilase (Nit3) has stronger hydrolysis capacities to acetonitrile, adiponitrile and butenenitrile, respectively 132%, 120% and 107%, and relative enzyme activity to acetonitrile of 89% under the same hydrolysis conditions relative to the hydrolysis efficiency of the substrate which is n-valeronitrile, and the nitrilase Nit3 has a very wide substrate spectrum and has higher hydrolase activity to various nitrile compounds.

Sequence listing

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Met Ala Gln Ala Ile Ile Gly Ala Asp Gly Glu Leu Val Ala Thr Arg

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Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Val Phe Gly Glu Gly

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Asp Gly Ser Asp Leu Gln Val His Glu Leu Pro Ile Gly Arg Val Gly

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Ala Leu Asn Cys Trp Glu His Phe Gln Pro Leu Thr Lys Tyr Ala Met

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gccgacgtgg ttgccttccc ggagacgttc atcccggggt atccgtggtg gatttggctg 180

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ctgaagcagc aactgttgct gcccggcgga ggtttcgcac ggatctacgg tccggatggc 780

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Claims

1. The amino acid sequence is shown as SEQ ID NO: 1, the application of the protein shown in the specification in catalyzing the hydrolysis of nitrile is characterized in that one of phenylacetonitrile, adiponitrile, butenenitrile and n-valeronitrile is used as a substrate, the hydrolysis temperature is 45 ℃, and the hydrolysis pH is 8.0.

2. The application of a recombinant vector in preparation of n-pentanoic acid by using n-valeronitrile as a substrate for nitrile hydrolysis is characterized in that the recombinant vector contains a nucleotide sequence shown as SEQ ID NO: 2, the hydrolysis temperature is 45 ℃, and the hydrolysis pH is 8.0.

3. Use of an engineered bacterium carrying the recombinant vector of claim 2 in the preparation of n-pentanoic acid by hydrolysis of nitrile using n-valeronitrile as a substrate at a hydrolysis temperature of 45 ℃ and a hydrolysis pH of 8.0.