CN110205315B

CN110205315B - Nitrilase XiNit2, and coding gene and application thereof

Info

Publication number: CN110205315B
Application number: CN201910461834.5A
Authority: CN
Inventors: 刘建国; 刘德健; 苏石晶; 贾庆珠; 谭雯斐; 陈京生; 郗丽君; 窦珂; 王明
Original assignee: China University of Petroleum East China
Current assignee: China University of Petroleum East China
Priority date: 2019-05-30
Filing date: 2019-05-30
Publication date: 2021-01-01
Anticipated expiration: 2039-05-30
Also published as: CN110205315A

Abstract

The invention discloses nitrilase XiNit2, and a coding gene and application thereof. The protein provided by the invention is derived from aromatic octylate (Xinfangfangia sp.), is nitrilase, is named as XiNit2 protein, and is a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table; protein consisting of an amino acid sequence shown in a sequence 3 in a sequence table. The invention also protects the application of the XiNit2 protein as nitrilase. The invention also protects a preparation for degrading nitrile compounds, and the active ingredient of the preparation is XiNit2 protein. The invention has great application prospect in the fields of relevant chemical industry, environmental pollution treatment and the like.

Description

Nitrilase XiNit2, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to nitrilase XiNit2, and a coding gene and application thereof.

Background

In the chemical field, nitrile compounds are not only widely used as raw materials in fine chemicals, but also as intermediates in many chemical products, such as nitriles in the production of amines, amino compounds, carboxylic acids and heterocyclic compounds, but also as highly toxic pollutants, which pose serious threats to the health of animals, plants and humans themselves. The chemical method for processing nitrile compounds needs to be carried out under strong acid (strong base), high pressure and high temperature conditions, and a large amount of salt is generated, so that the later separation is difficult, and residues can continuously cause harm. The biological method for degrading nitrile compounds has the advantages of mild reaction conditions and low cost, and the key point is that the nitrile compounds can be degraded into water and carbon dioxide through the own metabolic function, so that the real green degradation is achieved.

Nitrilase (Nitrilase, EC 3.5.5.1) is an important industrial enzyme belonging to Nitrilase superfamily, which not only can degrade nitrile pollutants, but also can convert cheap nitrile compounds into intermediates of products with high added values, such as industries, foods, medicines 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. However, according to the current research reports, nitrilases generally have the problems of poor thermal stability, narrow substrate spectrum, weak tolerance and the like, and the nitrilase only derived from Pyrococcus sp. Therefore, it is a hot point of research to find nitrilase with better quality.

Disclosure of Invention

The invention aims to provide nitrilase XiNit2, and a coding gene and application thereof.

The protein provided by the invention is derived from aromatic sinense (Xinfangfangia. sp), is nitrilase, is named as XiNit2 protein, and is (a1) or (a2) or (a3) or (a4) or (a5) or (a6) or (a 7):

(a1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(a2) a protein consisting of an amino acid sequence shown in a sequence 3 in a sequence table;

(a3) a fusion protein comprising (a 1);

(a4) a fusion protein obtained by linking a tag-containing short peptide to the terminus of (a 1);

(a5) a fusion protein obtained by attaching a tag to the terminus of (a 1);

(a6) and (a1), or (a2), or (a3), or (a4), or (a5) is subjected to substitution and/or deletion of one or more amino acid residues and/or addition of a protein which has a nitrilase function and is derived from the nitrilase.

(a7) A protein derived from an aromatic octanoate bacterium, having a homology of 99% or more with (a1), and having a nitrilase function.

The labels are specifically shown in table 1.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG
	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL
			HA
	9	YPYDVPDYA

The coding gene of the XiNit2 protein also belongs to the protection scope of the invention.

The gene is a DNA molecule as described in any one of the following 1) to 4):

1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

2) the coding region is DNA molecule shown as 1-846 bit nucleotide in sequence 4 in the sequence table;

3) a DNA molecule which hybridizes with the DNA sequence defined in 1) or 2) under strict conditions and codes a protein with the function of nitrilase;

4) derived from aromatic sincalides, has more than 90 percent of homology with the DNA sequence limited by 1) and encodes a DNA molecule with a protein with a nitrilase function.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

The recombinant expression vector, the expression cassette or the recombinant microorganism containing the gene all belong to the protection scope of the invention.

The invention also protects the application of the XiNit2 protein as nitrilase. When the XiNit2 protein is used as the nitrilase, the temperature is 30-70 ℃ and the pH is 3-11. When the XiNit2 protein was used as the nitrilase, the temperature used was 60 ℃ and the pH used was 7.

The invention also protects the application of the XiNit2 protein in degrading nitrile compounds. When the XiNit2 protein is used for degrading nitrile compounds, the adopted temperature is 30-80 ℃, and the adopted pH value is 3-11. When the XiNit2 protein is used for degrading nitrile compounds, the temperature is 60 ℃, and the pH is 7.

The invention also protects the application of the XiNit2 protein in preparing nitrilase.

The invention also protects a preparation for degrading nitrile compounds, and the active ingredient of the preparation is XiNit2 protein.

Any of the above nitrile compounds is an aliphatic nitrile, an aromatic nitrile, or an acyl nitrile.

The nitrilase XiNit2 provided by the invention has high enzyme activity, wide substrate, wide reaction temperature and wide reaction pH, and has high tolerance to metal ions, metal ion chelating agents, reducing agents, organic solvents and surfactants.

The invention has great application prospect in the fields of relevant chemical industry, environmental pollution treatment and the like.

Drawings

FIG. 1 is a photograph of strain DLY 26.

FIG. 2 is a phylogenetic tree.

FIG. 3 is a bacterial growth curve.

FIG. 4 is a chromatogram of gas chromatography detection.

FIG. 5 shows the acrylonitrile degradation rate at each time point of the shaking culture.

FIG. 6 is Superdex^TM20010/60 chromatogram for separating and purifying by gel chromatography column.

FIG. 7 is an electrophoretogram of XiNit1 protein solution.

FIG. 8 shows the relative enzyme activity results when the optimum pH was measured.

FIG. 9 shows the relative enzyme activity results when pH stability was measured.

FIG. 10 shows the relative enzyme activity results when the optimal reaction temperature was measured.

FIG. 11 shows relative enzyme activity results when temperature stability was measured.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 3 isolation, identification and preservation of Arisaema sinense DLY26

First, separate

About 1ml of an activated sludge sample (obtained from a petrochemical oil refining and chemical wastewater treatment system) was sequentially diluted with sterile distilled water to 10-fold, 20-fold, 50-fold, 100-fold and 1000-fold volumes. And coating 100 mul of the diluted sample on the surface of a solid culture medium by using a coating plate method, culturing at 30 ℃, and observing the growth condition of surface colonies every day. Colonies with different colors and morphologies are picked, purified and cultured by a three-region streaking method until single colonies with the same morphologies, sizes and other characteristics grow on the surface of the culture medium.

II, identification

The strain obtained by purification was inoculated on a TSA plate, cultured at 30 ℃ for 24 hours, and then the morphology, size, and the like of the cells were observed using a transmission electron microscope (see FIG. 1 for a photograph of the strain DLY 26). Single colonies were picked from the surface of the TSA plate cultured for 24 hours, and gram-stained according to a standard method, and the gram-stained bacteria were observed using an inverted fluorescence microscope. Preparing a semi-solid culture medium TSB, and observing the motility and the aerobic condition of bacteria by adopting a puncture inoculation method.

Based on liquid medium TSB: preparing culture medium systems with different NaCl concentrations (NaCl gradient is 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0g/100mL), placing in a shaking table at 30 ℃ after inoculation for shaking culture for 3-5d, and detecting the growth condition of bacteria at OD600 by using a UV-2450 spectrophotometer to determine the optimal salinity growth range; preparing liquid culture media with different pH values (pH gradient set to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0), inoculating, placing in a shaking table at 30 ℃ for shaking culture for 3-5d, and taking an non-inoculated culture medium with the same culture conditions as a blank control to determine the optimal growth pH range; the temperature gradient is set to 4 ℃, 20 ℃, 28 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃ and 50 ℃ to determine the optimal growth temperature range.

On the basis of a liquid carbon source culture medium, different carbohydrates with different concentrations are added to be used as a unique carbon source, and the assimilation of bacteria is detected.

The nitrate reducing ability, nitrite reducing ability, denitrification ability, and the presence or absence of lipase and catalase of the bacteria were determined by referring to the methods in the manual for identifying common bacteria systems. Voges-Proskauer test (VP test) of bacteria using the Haibo gram-negative bacteria identification System (Qingdao, China), detection of hydrogen sulfide (H) of bacteria₂S) production, Urease (URE), Gelatin (GEL) hydrolysis. It can also be used to evaluate the presence or absence of other physiological and biochemical features, such as Arginine (ADH), Sorbitol (SOR), Sucrose (SAC), etc., to test for the presence of arginase, sorbitol dehydrogenase, sucrase.

In X.soli ZQBW^T、R.megalophilus JA194^TAnd Fal. halotolerans JA744^TAs a reference strain. The results for strain DLY26 and the reference strain are shown in tables 2 and 3 and 4.

TABLE 2

Biochemical characteristics	DLY26	ZQBW^T	JA194^T	JA744^T
					Temperature range	4-45	26-35	4-45	4-40
Optimum temperature	30	30	30	30
					pH range	5-11	6-12	3-11	3-11
Optimum pH	7.0	7.0	7.0	7.0
					Salinity Range (%)	0-5.0	0-5.0	0-5.0	0-5.0
Optimum salinity (%)	1.0	0	0	0
					Cell size (μm)	1.2-2.0*,0.4-0.6	1.5-2.6*,0.7-0.9	1.2-1.0*,1.5-2.0	2.0-5.0*,1.0-1.2
Flagellum	-	-	-	+
					Cell shape	Rod	Rod	Oval Rod	Oval Rod
Movement property	-	-	-	+
					GC content (%)	63.9	67.0	66.67	74-76

TABLE 3

TABLE 4

ONPG

ADH

LDH

ODC

CIT

H₂S

URE

IND

VP

GEL

RHA

MEL

AMY

OX

DLY26

+

W

+

-

+

DLY30

+

-

+

W

+

-

+

-

+

JA194^T

-

+

W

+

-

+

JA744^T

+

W

+

-

+

Note: ONPG: beta-galactosidase; ADH: arginine; LDH: lysine; ODC: ornithine; and (3) CIT: a citrate salt; h₂S: hydrogen sulfide production test; URE: urease; IND: indole; GEL: gelatin; RHA: rhamnose; MEL: melibiose; AMY: amygdalin; OX: an oxidase enzyme.

The bacterial genomic DNA extraction kit is used for extracting the genomic DNA of the strain DLY26, and 16S rDNA amplification is carried out by adopting universal primers 27F and 1492R. The amplified product was analyzed for product bands by agarose gel electrophoresis to obtain target bands and purified using Tiangen DNA purification kit DP 209. The purified product was ligated into the vector pMD19-T and the vector of interest was transformed into E.coli DH 5. alpha. using the pMD19-T cloning kit. The recombinant strain is sent to Qingdao Okagaku Biotechnology limited company for sequencing to obtain a 16S rRNA sequence. The sequence of 16S rRNA of each relevant gene was searched using GenBank databases and phylogenetic trees of the strains were constructed using MEGA version 7.0. Phylogenetic analysis of bacteria was performed using the nearest neighbor ligation (NJ). Based on 1000 replicates, the topology of the phylogenetic tree was determined using Bootstrap analysis. See fig. 2.

The above identification results showed that the strain DLY26 belongs to the family Rhodobacteriaceae (Rhodobacteraceae), Arisaema (Xinfangfangia).

III, preservation

Aromatic sinense (Xinfangfangia sp.) DLY26 has been preserved in the China center for type culture Collection (address: China, Wuhan university; zip code: 430072) in 2019 at 1 month and 2 days, and the preservation number is CCTCC No: m2019001.

Example 2 degradation of Acrylonitrile by Arisaema sinense DLY26

Na₂HPO₄/NaH₂PO₄The formula of the buffer solution is as follows: disodium hydrogen phosphate 14.2g, sodium dihydrogen phosphate 12.2g, dissolved in 800mL ddH₂O, using ddH₂And adding O to the volume of 1L, and adjusting the pH value to 7.0.

The formula of the activation medium is as follows: tryptone Soy Broth Medium (TSB)30g, in ddH₂Dissolving O, diluting to 1L, and adjusting pH to 7.0.

Basic culture medium: disodium hydrogen phosphate 2.0g, potassium dihydrogen phosphate 1.0g, yeast powder 0.5g, magnesium sulfate 0.2g, ferrous sulfate 0.03g, dissolved in 800mL Na₂HPO₄/NaH₂PO₄Buffer, then add glycerol 3.25mL and mix well, then add Na₂HPO₄/NaH₂PO₄Buffer volume to 1L.

Firstly, preparing seed liquid

Mixing the materialsInoculating the strain DLY26 into an activation culture medium, and performing shaking culture at 30 ℃ and 150rpm to obtain a seed solution. OD of seed liquid_600nmThe value is 0.6-0.8.

Secondly, preparing a cell dry weight standard curve

1. And (3) inoculating the seed solution prepared in the step one to an activation culture medium according to the inoculation amount of 10%, and then carrying out shaking culture at 30 ℃ and 150rpm for 48 hours.

2. After completion of step 1, 40mL of sample was taken and OD adjusted with activation medium_600nmValue, obtaining OD_600nmThe values of the respective bacterial liquids were different.

3. And (3) taking the bacterial liquid obtained in the step (2), centrifuging at 8000rpm for 15min, and collecting bacterial precipitates.

4. And (3) taking the thallus precipitate obtained in the step (3), fully washing the thallus precipitate by using normal saline, drying the thallus precipitate at the temperature of 60 ℃, and measuring the dry weight of the thallus precipitate.

5. Preparation of OD_600nmStandard curve of values versus biomass (dry cell weight). Standard curve equation: y is 2.895 x-0.036; r²0.9988; x is OD_600nmThe value y is the dry weight of the cells (unit: g/L).

Thirdly, making a bacterial growth curve and detecting the degradation rate of acrylonitrile

1. Inoculating the seed liquid prepared in the first step into a basal culture medium containing 10mM acrylonitrile (as a unique nitrogen source) according to the inoculation amount of 10%, performing shaking culture at 30 ℃ and 150rpm, and detecting OD every 12h_600nmThe value is obtained. The results show that OD was obtained after 48 hours of shaking culture_600nmThe value reaches a maximum value.

2. Taking the culture system of the step 1 after shaking culture for 48h, centrifuging at 8000rpm for 15min, collecting thallus precipitate, and washing for 2-3 times (the washing method is Na₂HPO₄/NaH₂PO₄Suspending the cells in buffer, centrifuging at 8000rpm for 15min, collecting the cells), and adding Na₂HPO₄/NaH₂PO₄Suspending the cells in a buffer to obtain OD_600nmBacterial suspension with value of 0.43.

3. 100mL of a basal medium containing 20mM acrylonitrile was added with 5mL of the bacterial suspension prepared in step 2, followed by shaking culture at 150rpm at 30 ℃ for 104 hours. Sampling every 2h for 0-8h, sampling every 12h for 9-32h, and sampling every 24h for 33-104 h; sampling 5mL each time; acrylonitrile was added to the culture system after each sampling to make the concentration thereof in the system 20 mM.

4. Preparation of bacterial growth curves

Taking 3mL of the sample from step 3, and determining the OD_600nmValues were calculated for biomass (biomass in dry cell weight) per liter of culture system at each time point of shaking culture according to the standard curve of step two.

The results are shown in FIG. 3. During the cultivation, the biomass of the bacteria is increased and then decreased. The maximum value is reached at 4h of cultivation. After 4h of culture, the biomass of the thallus shows a tendency of reducing along with the prolonging of the culture time (the reason is that along with the gradual accumulation of acrylic acid which is an acrylonitrile product, the harm toxicity to bacteria is larger and larger, and the death rate of the bacteria is gradually larger than the growth rate).

5. Detecting the degradation rate of acrylonitrile

Taking 1mL of the sample from the step 3, centrifuging at 8000rpm for 2min, taking supernatant, adding 0.1mL of 2M hydrochloric acid aqueous solution into the supernatant to terminate the reaction, then taking 200 mu L of the supernatant, adding 800 mu L of ethyl acetate, uniformly mixing, standing for 30min, and collecting an ethyl acetate phase.

The acrylic acid content in the ethyl acetate phase was determined by gas chromatography. Relevant parameters of gas chromatography: the chromatographic column is a polar HP-FFAP capillary column (30m multiplied by 320 mu m multiplied by 0.25 mu m), the injection port temperature is 260 ℃, the column box temperature is 190 ℃, the FID detector temperature is 260 ℃, the hydrogen flow rate is 40mL/min, the air flow rate is 450mL/min, the nitrogen tail gas flow rate is 40mL/min, the sample injection amount is 0.2 mu L, and the split ratio is 50: 1.

And adopting ethyl acetate as a solvent to prepare acrylic acid standard solutions with various concentrations. The acrylic acid standard solution was checked by gas chromatography (parameters as above). A standard curve of peak area versus acrylic acid concentration was prepared. 2.9507X +6.9485, coefficient of correlation R²99.17%; y represents the peak area and X represents the acrylic acid concentration (in mmol/L).

Detecting ethyl acetate standard, acrylonitrile standard and acrylic acid standard by gas chromatography according to the above parameters, and obtaining chromatogram as shown in figure 4. The peak time of ethyl acetate is 1.387min, the peak time of acrylonitrile is 2.187min, and the peak time of acrylic acid is 4.522 min.

The acrylic acid production was calculated from the standard curve and the peak area, and the acrylonitrile degradation rate was further calculated at each time point of the shaking culture, and the results are shown in FIG. 5. The culture time is 0-32 hours, and the degradation rate of acrylonitrile is gradually improved. The culture time is 32-104 hours, the degradation rate of acrylonitrile is basically unchanged and is maintained at about 79.4 percent.

Example 3 preparation of nitrilase (XiNit2 protein)

Through a large number of sequence analyses, comparison and functional verification, a new protein is found from arylcinals DLY26 and is named as XiNit2 protein, which is shown as a sequence 1 in a sequence table. The gene of coding XiNit2 protein in aromatic sinense DLY26 is named as XiNit2 gene, and the coding frame is shown as sequence 2 in the sequence table.

Construction of recombinant plasmid

1. Taking genome DNA of arylate sincalis DLY26 as a template, adopting a primer pair consisting of DN2-F and DN2-R to carry out PCR amplification, and recovering a PCR amplification product.

DN2-F：5’-CACCGCCGGATTCTCGTCTCTT-3’；

DN2-R：5’-ATGATGTTGCGGCCGAGATGT-3’。

2. And (3) connecting the PCR amplification product obtained in the step (1) with a pDE1vector to obtain a recombinant plasmid pDE1-xinit 2.

The pDE1Vector (pDE1Vector) is a component of the pDE1 directed Expression Kit. pDE1 directive Expression Kit: beijing Optimus Hippocrate Biotechnology Ltd, catalog number TSV-E1.

Through sequencing, the recombinant plasmid pDE1-xinit2 has an open reading frame shown in a sequence 4 of a sequence table (wherein, the 1-846 nucleotide is the open reading frame), and expresses the fusion protein shown in a sequence 3 of the sequence table. In the sequence 3 of the sequence table, amino acid residues at positions 2-7 form a His6 label, and amino acid residues at positions 22-263 form a XiNit2 protein.

Secondly, preparing recombinant bacteria

The recombinant plasmid pDE1-xinit2 was introduced into E.coli BL21(DE3) to obtain recombinant bacteriacide.

The pDE1vector was introduced into E.coli BL21(DE3) to obtain recombinant strain B.

Third, expression of proteins

1. The recombinant strain was inoculated into a liquid LB medium containing 50. mu.g/mL kanamycin and shake-cultured at 37 ℃ and 150rpm for 12 hours to obtain a seed solution.

2.1 volume portion of the seed liquid was inoculated into 99 volume portions of liquid LB medium containing 50. mu.g/mL kanamycin, and cultured at 37 ℃ with shaking at 200rpm until OD_600nmThe value was about 0.6, at which time IPTG inducer was added so that the concentration in the system became 0.5mmol/L, followed by shaking culture at 200rpm at 25 ℃ for 6 hours (induction expression), followed by centrifugation at 8000 Xg for 10min at 4 ℃ to collect the cell pellet.

3. The precipitate obtained in step 2 was washed with sodium phosphate buffer (0.1M, pH7.2), suspended in sodium phosphate buffer (0.1M, pH7.2) and sonicated (sonication parameters: power 200W, 6s per 4s sonication, total time 40min), then centrifuged at 4 ℃ at 10000 Xg for 20min and the supernatant collected.

The recombinant bacterium A is subjected to the supernatant obtained in the step, and named as crude enzyme liquid A.

And (3) carrying out the steps on the recombinant bacterium B to obtain supernatant, and naming the supernatant as crude enzyme solution B.

Fourth, purifying the protein

And (4) filtering the crude enzyme liquid A obtained in the step three by using a microfiltration membrane with the pore diameter of 0.22 mu m, and collecting the filtrate. Taking the filtrate, and adopting Superdex^TM20010/60 separating and purifying with gel chromatographic column. Mixing Superdex^TM20010/60 the gel chromatography column was connected to a fast protein liquid phase system with 1 XPBS buffer (pH 8.0) as the mobile phase at a flow rate of 0.1 mL/min. And collecting the solution after passing the column with the retention volume of 19-21mL corresponding to the elution peak, namely the XiNit2 protein solution. The chromatogram is shown in FIG. 6.

The electrophoretogram of the XiNit2 protein solution is shown in FIG. 7, with only one protein band, and corresponds to the predicted molecular weight (about 25 kD).

EXAMPLE 4 enzymatic Properties of nitrilase (XiNit2 protein)

No ammonia water: boiling deionized water, and keeping boiling state for 15 min.

In this example, water refers to ammonia-free water.

Preparing a sodium phenolate solution: phenol 25g, dissolved in 800mL water, then 78mL 4M aqueous sodium hydroxide solution was added, then water to 1L.

Preparing a sodium nitrosoferricyanide solution: 1g of sodium nitroferricyanide was dissolved in 100mL of water to obtain a mother liquor, which was diluted to 100-fold volume with water before use to obtain a sodium nitroferricyanide solution.

Preparing a sodium hypochlorite solution: 19.4mL of a commercial 10% sodium hypochlorite stock solution was made up to 1L with water.

Preparing an ammonia standard solution: preparing different ammonia (NH) by using ammonium chloride and water as raw materials₃) Ammonia standard solution of concentration.

3-cyanopyridine solution: contains 500mM 3-cyanopyridine and the balance of water.

The reaction principle is as follows: nitrilase can degrade 1 molecule of 3-cyanopyridine into 1 molecule of nicotinic acid and 1 molecule of ammonia, so that the enzyme activity can be measured by measuring ammonia. The color rendering principle is as follows: under alkaline conditions, sodium nitrosoferricyanide catalyzes the reaction of ammonia with phenol and sodium hypochlorite to produce a blue soluble substance that has a maximum absorption at 630 nm.

Effect of pH on nitrilase Activity

1. Optimum pH

The XiNit2 protein solution prepared in example 3 was diluted to 2 volumes with buffer solution, and the diluted solution was used as a test solution.

The detection method comprises the following steps: taking a centrifuge tube, adding 1mL of test solution, 0.1mL of 3-cyanopyridine solution and 3.9mL of buffer solution, reacting for 1h at 30 ℃, then adding 0.5mL of 2M HCl aqueous solution to stop the reaction, and then sampling 1mL (called sampling solution) to perform subsequent color reaction; taking a 10mL centrifuge tube, sequentially adding 1mL of sampling solution, 2mL of sodium phenolate solution, 3mL of sodium nitroferricyanide solution and 3mL of sodium hypochlorite solution, then supplementing to 10mL with water, reacting at 37 ℃ for 15min, and then determining OD_630nmThe value is obtained.

The following buffers were used respectively: a citrate buffer solution of pH3.0, a citrate buffer solution of pH4.0, a citrate buffer solution of pH5.0, a citrate buffer solution of pH6.0, a phosphate buffer solution of pH7.0, a phosphate buffer solution of pH8.0, a carbonate buffer solution of pH9.0, a carbonate buffer solution of pH10.0, and a carbonate buffer solution of pH 11.0. The formulation of citrate buffer is shown in Table 5. The formulation of the phosphate buffer is shown in Table 6. The formulation of the carbonate buffer is shown in Table 7.

TABLE 5

pH	0.1M aqueous citric acid solution (mL)	0.1M aqueous sodium citrate solution (mL)
			3.0	37.2	2.8
4.0	26.2	13.8
			5.0	16.4	23.6
6.0	7.6	32.4

TABLE 6

pH	0.2M aqueous disodium hydrogen phosphate solution (mL)	0.2M sodium dihydrogen phosphate aqueous solution (mL)
			6.0	61.5	438.5
7.0	305	195
			8.0	473.5	26.5

TABLE 7

The optimal pH of the XiNit2 protein was 7. OD when buffer solution corresponding to optimum pH is used_630nmThe value was taken as 100%, and the relative value when each buffer was used was calculated as the relative enzyme activity. The results are shown in FIG. 8.

2. Stability of pH

The XiNit2 protein solution prepared in example 3 was diluted to 2 volumes with buffer and incubated at 4 ℃ for 12 h. At the time of incubation 0, the sample solution was designated as 0, and after incubation for 12 hours, the sample solution was designated as 12.

The detection method comprises the following steps: taking a centrifuge tube, adding 1mL of test solution, 0.1mL of 3-cyanopyridine solution and 3.9mL of sodium phosphate buffer solution (100mM, pH7.2), reacting for 1h at 30 ℃, then adding 0.5mL of 2M HCl aqueous solution to stop the reaction, and then sampling 1mL (called sampling solution) to perform subsequent color reaction; taking a 10mL centrifuge tube, sequentially adding 1mL of sampling solution, 2mL of sodium phenolate solution, 3mL of sodium nitroferricyanide solution and 3mL of sodium hypochlorite solution, then supplementing to 10mL with water, reacting at 37 ℃ for 15min, and then determining OD_630nmThe value is obtained.

OD of test solution 0_630nmThe value was defined as 100%, and the relative value of each sample solution 12 was calculated as the relative enzyme activity. The results are shown in FIG. 9. The XiNit2 protein has the most stable enzyme activity at pH7.

Second, influence of temperature on nitrilase Activity

1. Optimum reaction temperature

The XiNit2 protein solution prepared in example 3 was diluted to 2 volumes with sodium phosphate buffer (100mM, pH7.2) and used as a test solution.

The detection method comprises the following steps: taking a centrifuge tube, adding 1mL of test solution, 0.1mL of 3-cyanopyridine solution and 3.9mL of sodium phosphate buffer solution (100mM, pH7.2), reacting for 1h (the reaction temperature is respectively set to be 30, 40, 45, 50, 55, 60 and 70 ℃), then adding 0.5mL of 2M HCl aqueous solution to terminate the reaction, and then sampling 1mL (called as sampling solution) to carry out subsequent color reaction; taking a 10mL centrifuge tube, sequentially adding 1mL of sampling solution, 2mL of sodium phenolate solution, 3mL of sodium nitroferricyanide solution and 3mL of sodium hypochlorite solution, then supplementing to 10mL with water, reacting at 37 ℃ for 15min, and then determining OD_630nmThe value is obtained.

The optimum reaction temperature is 60 ℃. OD at optimum reaction temperature_630nmThe value was taken as 100%, and the relative values at various reaction temperatures were calculated as relative enzyme activities. The results are shown in FIG. 10.

2. Temperature stability

The XiNit2 protein solution prepared in example 3 was diluted to 2 volumes with sodium phosphate buffer (100mM, pH7.2), and then incubated (at 40, 50, 60, 70, 80 ℃ respectively; for 0, 30, 60, 90, 120min respectively) as a test solution.

To incubate the corresponding OD of the test solution at 0h_630nmThe values are taken as 100%, and the relative enzyme activities of the test solutions at other incubation times are calculated respectively. The results are shown in FIG. 11. The temperature at which the activity of the XiNit2 protease is stabilized is 60 ℃.

Influence of Metal ions and chemical reagents on nitrilase Activity

The XiNit2 protein solution prepared in example 3 was diluted to 2 volumes with sodium phosphate buffer (100mM, pH7.0) and used as a test solution.

The detection method comprises the following steps: taking a centrifuge tube, adding 1mL of test solution, 0.1mL of 3-cyanopyridine solution, 10 mu L of compound aqueous solution or 0.25mL of organic solvent, supplementing to 5mL with sodium phosphate buffer solution (100mM, pH7.0), reacting at 60 ℃ for 1h, then adding 0.5mL of 2M HCl aqueous solution to stop the reaction, and then sampling 1mL (called sampling solution) to perform subsequent color reaction; taking a 10mL centrifuge tube, sequentially adding 1mL of sampling solution, 2mL of sodium phenolate solution, 3mL of sodium nitroferricyanide solution and 3mL of sodium hypochlorite solution, then supplementing to 10mL with water, reacting at 37 ℃ for 15min, and then determining OD_630nmThe value is obtained.

With OD corresponding to OD obtained without addition of aqueous/organic compound solution_630nmThe values are taken as 100%, and the relative enzyme activities after adding various compound aqueous solutions/organic solvents are respectively calculated.

Some compounds are shown in Table 8. The concentration of the partial compound in a 5mL system is 1mM or 5mM respectively. The results are shown in Table 8.

Some compounds are shown in Table 9. The concentration of the partial compound in a 5mL system was 1 mM. The results are shown in Table 9.

The organic solvents are shown in Table 10. The results are shown in Table 10.

TABLE 8

TABLE 9

Compound (I)	Relative enzyme activity
		SDS (surfactant)	94(±3)％
EDTA (Metal ion chelating agent)	100(±1)％
		L-glutathione (reducing agent)	93(±1)％
L-cysteine (reducing agent)	92(±2)％

Watch 10

Compound (I)	Relative enzyme activity
		Isopropanol (I-propanol)	87(±5.5)％
Triton	94(±4.7)％
		Methanol	91(±4.1)％
Glycerol	91(±5.2)％
		N-hexane	99(±3.4)％
Ethanol	98(±3.2)％
		Methylene dichloride	100(±1.8)％
N-heptane	97(±1.8)％
		Ether (A)	100(±2.6)％
Ethyl acetate	90(±2.7)％
		DMSO	68(±3.5)％
Span-80	97(±3.8)％
		Acetone (II)	95(±4.4)％

Substrate specificity of nitrilase

The detection method comprises the following steps: taking a centrifuge tube, adding 1mL of test solution, substrate aqueous solution, supplementing to 5mL with sodium phosphate buffer solution (100mM, pH7.0), reacting at 60 ℃ for 1h, then adding 0.5mL of 2M HCl aqueous solution to stop the reaction, and then sampling 1mL (called as sampling solution) to perform subsequent color reaction; taking a 10mL centrifuge tube, sequentially adding 1mL of sampling solution, 2mL of sodium phenolate solution, 3mL of sodium nitroferricyanide solution and 3mL of sodium hypochlorite solution, then supplementing to 10mL with water, reacting at 37 ℃ for 15min, and then determining OD_630nmThe value is obtained.

OD corresponding to 3-cyanopyridine as substrate_630nmThe relative enzyme activities for the respective substrates were calculated as values of 100%.

The substrates are shown in Table 11, respectively. The concentration of the substrate in the 5mL system was 10 mM. The results are shown in Table 11.

Lauronitrile, 3-phenylpropanenitrile, 4-chlorobutyronitrile, 2-methylglutaronitrile, valeronitrile, acrylonitrile, iminodiacetonitrile, succinonitrile, adiponitrile and sebaconitrile belong to the aliphatic nitriles. Benzonitrile, 3-cyanopyridine and 3-cyanothiophene belong to the class of aromatic nitriles. 1, 2-phenylethanedionitrile belongs to the group of acylnitriles.

TABLE 11

Substrate	Relative enzyme activity
		Lauronitrile (Dodecanielite)	113 (soil 2.5)%
3-Phenylpropionitrile (3-Phenylpropionitrile)	112 (soil 2.2)%
		4-Chlorobutyronitrile (4-Chlorobutyronitrile)	111 (soil 2.1)%
2-methylglutaronitrile (2-Methylglutarontrile)	113 (soil 2.5)%
		Valeronitrile (Pentaneniile)	112 (soil 2.2)%
Acrylonitrile (Acrylonitril)	113 (soil 2.5)%
		Iminodiacetonitrile (Iminodiacetonitril)	113 (soil 2.5)%
Succinonitrile (succinonitril)	113 (soil 2.5)%
		Adiponitrile (Dicyanobutane)	113 (soil 2.5)%
Benzonitrile (Benzonitile)	100 (soil 2.0)%
		3-cyanothiophene (Thiophene-3-carbonitile)	113 (soil 2.5)%
1, 2-Phenylethanedinitrile (1, 2-Phenylenediacetonitriles)	113 (soil 2.5)%
		Decanedinitile (Decanedinile)	113 (soil 2.5)%

Fifth, determination of enzyme activity

Enzyme activity (1U) is defined as: 1. mu. mol. L is formed every 1min^-1The amount of enzyme required for ammonia.

The detection method comprises the following steps: taking a centrifuge tube, adding 1mL of test solution, 0.1mL of 3-cyanopyridine solution and 3.9mL of sodium phosphate buffer solution (100mM, pH7.0), reacting for 1h at 60 ℃, then adding 0.5mL of 2M HCl aqueous solution to stop the reaction, and then sampling 1mL (called sampling solution) to perform subsequent color reaction; taking a 10mL centrifuge tube, sequentially adding 1mL of sampling solution, 2mL of sodium phenolate solution, 3mL of sodium nitroferricyanide solution and 3mL of sodium hypochlorite solution, then supplementing to 10mL with water, reacting at 37 ℃ for 15min, and then determining OD_630nmThe value is obtained.

Drawing a standard curve: adding 1mL of ammonia standard solution, 2mL of sodium phenolate solution, 3mL of sodium nitroferricyanide solution and 3mL of sodium hypochlorite solution into a centrifuge tube, supplementing the mixture to 10mL with water, reacting at 37 ℃ for 15min, and measuring OD_630nmThe value is obtained. The standard curve regression equation is: y 0.1024x +0.0265, R²0.9953; y is OD_630nmThe value, x, is the ammonia concentration (in. mu.g/mL).

According to OD_630nmAnd (4) detecting the ammonia content in the test solution by using a value and standard curve regression equation so as to calculate the enzyme activity.

The crude enzyme solution A prepared in example 3 was used as a test solution, and the enzyme activity was 25.5U/mL.

The crude enzyme solution B prepared in example 3 was used as a test solution, and the enzyme activity was 0U/mL.

The enzyme activity per unit volume of the test solution was measured by using the XiNit2 protein solution prepared in example 3 as the test solution. The protein concentration in the XiNit2 protein solution prepared in example 3 was measured. And dividing the enzyme activity of the test solution in unit volume by the protein content of the test solution in unit volume to obtain the specific activity of the protein, wherein the value is 231U/mg.

Sequence listing

<110> China university of Petroleum (east China)

<120> nitrilase XiNit1, and coding gene and application thereof

<130> XI1234567

<141> 2019-05-30

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 241

<212> PRT

<213> Arcarbon sinense (Xinfangfangia sp.)

<400> 1

Met Gln Leu Gln Ala Ala Ala Ala Ala Gly Ala Lys Leu Leu Leu Val

1 5 10 15

Pro Glu Leu Phe Leu Pro Gly Tyr Asn Arg Pro Asp Leu His Ala Glu

20 25 30

Leu Ala Gln Pro Ile Asp Gly Ala Trp Ile Thr Arg Leu Arg Gln Met

35 40 45

Ala Ala Gln Ala Gly Cys Gly Ile Cys Leu Gly Trp Ala Glu Arg Asp

50 55 60

Gly Ala Ala Val Tyr Asn Ala Ala Thr Thr Ile Gly Pro Asp Gly Ala

65 70 75 80

Ile Leu Gly His Tyr Arg Lys Ile Gln Leu Tyr Gly Pro Met Glu Arg

85 90 95

Ala Ser Phe Ala Arg Gly Asp Cys Leu Ala Pro Ser Phe Arg Leu Gly

100 105 110

Leu Arg Val Ala Met Leu Ile Cys Tyr Asp Ile Glu Phe Pro Gly His

115 120 125

Ala Ala Ala Leu Ala Ala Gly Gly Val Asp Leu Ile Leu Val Pro Thr

130 135 140

Ala Asn Pro Ala Gly Phe Asp Tyr Val Gln Glu Arg Leu Val Tyr Ala

145 150 155 160

Arg Ala His Glu Asn Asp Leu Val Val Ala Tyr Ala Asn Leu Val Gly

165 170 175

Pro Glu Gly Asp Val Thr Phe Gly Gly Arg Ser Val Ile Ala Gly Pro

180 185 190

Asp Gly Ala Pro Leu Ala Thr Ala Gly Ala Leu Gly Glu Ala Leu Leu

195 200 205

Ile Val Asp Leu Ala Ala Ala Ala Arg Val Pro Gln Asp Leu Arg Ser

210 215 220

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

225 230 235 240

Ile

<210> 2

<211> 729

<212> DNA

<213> Arcarbon sinense (Xinfangfangia sp.)

<400> 2

atgcagttgc aggcagctgc ggcggcgggg gcgaaactgc tgctggtgcc cgaactgttc 60

ctgccgggct ataatcgccc cgatctgcat gccgaactgg cccaaccgat cgacggcgcc 120

tggatcaccc ggctgcgcca gatggcggcg caggccggct gcggcatctg cctgggctgg 180

gccgagcggg acggtgccgc ggtctataat gcggcaacga cgatcgggcc ggatggggcg 240

atcctgggcc attaccgcaa gatccagctt tacggcccga tggagcgggc aagctttgcc 300

cgcggcgatt gtctggcgcc cagcttccgg ctggaggggc tgcgggtggc gatgctgatc 360

tgctatgaca tcgaatttcc cggccatgcg gcggcgctgg cggcgggggg tgtcgatctg 420

atcctggtgc caaccgcaaa tcccgccggt ttcgactatg tgcaagagcg gctggtctat 480

gcccgcgcgc atgaaaacga ccttgtcgtc gcctatgcca atctggtcgg gccggaaggc 540

gatgtgacct tcggcggccg gtcggtcatt gccgggccgg atggcgcgcc gctggccacg 600

gcgggggcct tgggcgaggc cctgctgatc gtggatctgg ccgcggcggc gcgggtgccg 660

caggatctgc gctcggccca gcggcaggaa taccgcgccg cccggccggg agggtcggcg 720

cagatctga 729

<210> 3

<211> 263

<212> PRT

<213> Artificial sequence

<400> 3

Met His His His His His His Arg Gly Ser Pro Phe Thr His Arg Arg

1 5 10 15

Ile Leu Val Ser Leu Met Gln Leu Gln Ala Ala Ala Ala Ala Gly Ala

20 25 30

Lys Leu Leu Leu Val Pro Glu Leu Phe Leu Pro Gly Tyr Asn Arg Pro

35 40 45

Asp Leu His Ala Glu Leu Ala Gln Pro Ile Asp Gly Ala Trp Ile Thr

50 55 60

Arg Leu Arg Gln Met Ala Ala Gln Ala Gly Cys Gly Ile Cys Leu Gly

65 70 75 80

Trp Ala Glu Arg Asp Gly Ala Ala Val Tyr Asn Ala Ala Thr Thr Ile

85 90 95

Gly Pro Asp Gly Ala Ile Leu Gly His Tyr Arg Lys Ile Gln Leu Tyr

100 105 110

Gly Pro Met Glu Arg Ala Ser Phe Ala Arg Gly Asp Cys Leu Ala Pro

115 120 125

Ser Phe Arg Leu Glu Gly Leu Arg Val Ala Met Leu Ile Cys Tyr Asp

130 135 140

Ile Glu Phe Pro Gly His Ala Ala Ala Leu Ala Ala Gly Gly Val Asp

145 150 155 160

Leu Ile Leu Val Pro Thr Ala Asn Pro Ala Gly Phe Asp Tyr Val Gln

165 170 175

Glu Arg Leu Val Tyr Ala Arg Ala His Glu Asn Asp Leu Val Val Ala

180 185 190

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

195 200 205

Ser Val Ile Ala Gly Pro Asp Gly Ala Pro Leu Ala Thr Ala Gly Ala

210 215 220

Leu Gly Glu Ala Leu Leu Ile Val Asp Leu Ala Ala Ala Ala Arg Val

225 230 235 240

Pro Gln Asp Leu Arg Ser Ala Gln Arg Gln Glu Tyr Arg Ala Ala Arg

245 250 255

Pro Gly Gly Ser Ala Gln Ile

260

<210> 4

<211> 888

<212> DNA

<213> Artificial sequence

<400> 4

atgcatcatc atcatcatca ccgcggatcg cccttcaccc accgccggat tctcgtctct 60

ttgatgcagt tgcaggcagc tgcggcggcg ggggcgaaac tgctgctggt gcccgaactg 120

ttcctgccgg gctataatcg ccccgatctg catgccgaac tggcccaacc gatcgacggc 180

gcctggatca cccggctgcg ccagatggcg gcgcaggccg gctgcggcat ctgcctgggc 240

tgggccgagc gggacggtgc cgcggtctat aatgcggcaa cgacgatcgg gccggatggg 300

gcgatcctgg gccattaccg caagatccag ctttacggcc cgatggagcg ggcaagcttt 360

gcccgcggcg attgtctggc gcccagcttc cggctggagg ggctgcgggt ggcgatgctg 420

atctgctatg acatcgaatt tcccggccat gcggcggcgc tggcggcggg gggtgtcgat 480

ctgatcctgg tgccaaccgc aaatcccgcc ggtttcgact atgtgcaaga gcggctggtc 540

tatgcccgcg cgcatgaaaa cgaccttgtc gtcgcctatg ccaatctggt cgggccggaa 600

ggcgatgtga ccttcggcgg ccggtcggtc attgccgggc cggatggcgc gccgctggcc 660

acggcggggg ccttgggcga ggccctgctg atcgtggatc tggccgcggc ggcgcgggtg 720

ccgcaggatc tgcgctcggc ccagcggcag gaataccgcg ccgcccggcc gggagggtcg 780

gcgcagatct gaccgcagac ggccgcaggc aaccgtccgg cggccgcttt cagccggccg 840

ggatcaggtg gcggtccagc tcggtcgaca tctcggccgc aacatcat 888

Claims

1. A protein which is (a1) or (a2) or (a5) as follows:

(a5) and (a) a fusion protein obtained by ligating a tag to the terminus of (a 1).

2. A gene encoding the protein according to claim 1.

3. The gene of claim 2, wherein: the gene is a DNA molecule as described in any one of the following 1) or 2) or 3) or 4):

2) DNA molecule shown in sequence 2 in the sequence table;

3) the coding region is shown as the 64 th-792 th nucleotide in the sequence 4 in the sequence table;

4) DNA molecule shown in sequence 4 in the sequence table.

4. A recombinant expression vector, expression cassette or recombinant microorganism comprising the gene of claim 2 or 3.

5. Use of the protein of claim 1 as a nitrilase.

6. The use of claim 5, wherein: the protein of claim 1, which is used at a temperature of 30-70 ℃ and a pH of 3-11.

7. The use of claim 6, wherein: when the protein of claim 1 is used, the temperature is 60 ℃ and the pH is 7.

8. Use of the protein of claim 1 for degrading nitrile compounds.

9. The use of claim 8, wherein: the use of the protein of claim 1 for the degradation of nitriles at a temperature of 30-80 ℃ and a pH of 3-11.

10. The use of claim 9, wherein: the use of the protein of claim 1 for the degradation of nitriles at a temperature of 60 ℃ and a pH of 7.

11. Use of the protein of claim 1 for the preparation of a nitrilase.

12. A preparation for degrading nitrile compounds, which contains the protein according to claim 1 as an active ingredient.