CN117946989A - Nitrite reductase mutant and application thereof - Google Patents
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Abstract
The invention relates to a nitrite reductase mutant and application thereof, wherein the amino acid sequence of the nitrite reductase mutant has amino acid substitution at one or more positions corresponding to 48 th, 64 th, 70 th and 240 th positions of SEQ ID NO. 1 relative to wild nitrite reductase. Compared with wild nitrite reductase, the nitrite reductase mutant provided by the invention has increased stability, and can be applied to degrading nitrite in sewage; the preparation method of the nitrite reductase mutant provided by the invention has the advantages of simple process, mild conditions and good application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a nitrite reductase mutant and application thereof.
Background
Nitrite is widely existing in underground water and surface water, and the main reasons for nitrite pollution in water bodies are excessive chemical fertilizer use, unreasonable disposal of animal manure, domestic sewage and industrial wastewater, and the like. Research shows that drinking water containing nitrite can produce toxic effect on human body, and long-term drinking can result in raising cancer incidence rate. The most common method for removing nitrite in wastewater at present adopts biological treatment, and compared with chemical treatment, the biological method has the advantages of low cost, no secondary pollution and the like.
Nitrite reductase (Nitrite reductase, NIR) is an oxidoreductase extracted from plant cells and catalyzes nitrite reduction, which is widely distributed in bacteria and archaea. The wild-type nitrite reductase has low catalytic activity on certain substrates and poor stability. In the prior art, amino acid mutation is carried out on a specific position on the basis of the amino acid sequence of the wild nitrite reductase by a protein engineering method, so that the problem of poor stability of the nitrite reductase is solved to a certain extent. However, these nitrite reductase mutants still have the problems of complex preparation process, long fermentation time and the like.
Therefore, a nitrite reductase which has the advantages of high enzyme activity, good stability, simple preparation method and process, mild condition and the like is needed.
Disclosure of Invention
The invention aims to provide a nitrite reductase mutant with high enzyme activity, good stability and simple preparation process.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, the invention provides a nitrite reductase mutant having an amino acid substitution at one or more of positions 48, 64, 70 and 240 corresponding to SEQ ID NO. 1.
In some embodiments, the nitrite reductase mutant has nitrite reductase activity. In some embodiments, the nitrite reductase mutant has increased stability as compared to a wild-type nitrite reductase; the amino acid sequence of the wild nitrite reductase is shown as SEQ ID NO. 1.
In some embodiments, amino acid 48 of the nitrite reductase mutant may be substituted with serine, asparagine, glutamine or threonine. In a preferred embodiment, the amino acid at position 48 of the nitrite reductase mutant is substituted with serine.
In some embodiments, the nitrite reductase mutant may be substituted with glycine, alanine, valine, leucine, isoleucine, proline, or methionine at amino acid 64. In a preferred embodiment, the amino acid at position 64 of the nitrite reductase mutant is substituted with glycine.
In some embodiments, the nitrite reductase mutant can be substituted with glycine, alanine, valine, leucine, isoleucine, proline, or methionine at amino acid 70. In a preferred embodiment, the nitrite reductase mutant has the amino acid at position 70 replaced with alanine.
In some embodiments, the nitrite reductase mutant may be substituted for serine, asparagine, glutamine or threonine at position 240. In a preferred embodiment, the nitrite reductase mutant is substituted with glutamine at position 240.
In some embodiments, the nitrite reductase mutant may have the amino acid sequence set forth in SEQ ID NO. 3, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.
In a second aspect, the invention provides a nucleic acid molecule encoding a nitrite reductase mutant as described above.
In a third aspect, the invention provides an expression vector comprising the nucleic acid molecule of the second aspect.
In some embodiments, the expression vector may be constructed by ligating the above-described nucleic acid molecules to various prokaryotic or eukaryotic expression vectors. In some embodiments, the prokaryotic or eukaryotic expression vector is selected from pGEX, pMAL or pET. In some embodiments, the prokaryotic expression vector is pET-28a.
In a fourth aspect, the invention provides a host cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.
In some embodiments, the host cell may be a cell conventionally used to produce the nitrite reductase mutants described above. In some embodiments, the host cell may be selected from a fungal cell, a bacterial cell, a plant cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is selected from the group consisting of a yeast cell, a mold, and E.coli.
In a fifth aspect, the present invention also provides a method for preparing the above nitrite reductase mutant, comprising fermenting the above host cell, collecting and preparing the above nitrite reductase mutant.
In some embodiments, the methods comprise industrially producing the above nitrite reductase mutants under certain tank fermentation conditions. In some embodiments, the fermentor fermentation conditions are DO 30% or greater and the air flow is 1:1-2 vvm.
In a sixth aspect, the invention provides the use of a nitrite reductase mutant of the disclosure for degrading nitrite.
In some embodiments, the nitrite reductase mutant is used for sewage treatment.
In some embodiments, the nitrite reductase mutant may be used to degrade nitrite in wastewater.
Drawings
FIG. 1 shows the relative enzyme activities of nitrite reductase at different temperatures.
FIG. 2 shows the relative enzymatic activities of nitrite reductase at different pH's.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.
Definition of the definition
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.
Wild type refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism, can be isolated from a natural source and is not intentionally modified by human manipulation.
Sequence identity, i.e., sequence identity, a "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since the gaps are not nucleotides or amino acids, the gaps present in the target sequence are not taken into account. Also, since the target sequence nucleotide or amino acid is counted, and the nucleotide or amino acid from the reference sequence is not counted, gaps in the reference sequence are not counted.
The expression "at least 85% sequence identity" as used herein may encompass at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.
Percent sequence identity can be calculated by the following procedure: determining the number of positions in which the same amino acid residue or nucleobase occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software that is readily available for online use and download. Suitable software programs are available from a variety of sources for alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is the bl2seq, which is part of the BLAST suite of programs available from the national center for Biotechnology information, BLAST website (BLAST. Ncbi. Lm. Nih. Gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to make a comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, needle, stretcher, water or Matcher, part of the EMBOSS suite of bioinformatics programs, and are also available from European Bioinformatics Institute (EBI) on www.ebi.ac.uk/Tools/psa.
An expression vector may comprise a nucleic acid according to the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the expression vector comprises one or more regulatory elements, selectable according to the host cell used for expression, which are operably linked to the nucleic acid sequence to be expressed. Within an expression vector, "operably linked" is intended to connect a nucleotide sequence of interest to a regulatory element in a manner that allows for expression of the nucleotide (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
Examples and figures are provided below to aid in the understanding of the invention. It is to be understood that these examples and drawings are for illustrative purposes only and are not to be construed as limiting the invention in any way. The actual scope of the invention is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the invention.
Example 1: establishment of wild nitrite reductase genetic engineering bacteria
In this example, according to the wild-type gene sequence GenBank: UVL19616.1 of Pseudomonas sp.B21-044 nitrite reductase recorded in NCBI, a whole gene fragment (the nucleotide sequence is shown as SEQ ID NO:2, the amino acid sequence of the encoded protein is shown as SEQ ID NO: 1) is artificially synthesized after sequence optimization, and the gene is inserted into pET-28a plasmid by a gene synthesis company through BamHI and XhoI endonucleases to obtain a plasmid expressing the wild-type nitrite reductase, named pET-WT, and the pET-WT is transferred into E.coli BL21 (DE 3) to establish a wild-type nitrite reductase genetic engineering bacterium.
Example 2: construction of a library of nitrite reductase mutants
In this example, nitrite reductase was protein engineered based on the pET-WT plasmid obtained in example 1 by using an error-prone PCR random mutation method.
Error-prone PCR amplification of nitrite reductase genes: adopts Taq polymerase with lower fidelity, and simultaneously utilizes Mn 2+ to replace a natural auxiliary factor Mg 2+ to increase error probability.
50. Mu.L of PCR system ：5×PCR Buffer(10μL),dATP(2.5mmol/L,1μL),dGTP(2.5mmol/L,1μL),dCTP(2.5mmol/L,1μL),dTTP(2.5mmol/L,1μL),MgCl2(5mmol/L,1μL),MnCl2(5mmol/L,1μL), nitrite reductase template gene (1. Mu.L), taq DNA polymerase (5U/. Mu.L, 0.5. Mu.L), and sterilized double distilled water was added to 50. Mu.L.
The PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 50-65℃for 40s and extension at 72℃for 40s for 35 cycles; the extension was continued at 72℃for 10min and cooled to 4 ℃.
The error-prone PCR amplified gene fragment was ligated into pET-28a vector. Transferring the connected vector into escherichia coli BL21 (DE 3) to establish a nitrite reductase gene mutation library.
The high-activity mutant strain is screened in high flux by using escherichia coli BL21 (DE 3) as a host, pET28a plasmid as a vector, expressing extended and mutated nitrite reductase and adopting the following enzyme activity detection method.
The cell pellet was resuspended at 4℃in 100mM citrate-sodium citrate buffer pH 6.0 at a bacterial sludge concentration of 200g/L. After ultrasonication, cell debris was removed by centrifugation (13000 rpm, 30min, 4 ℃). Collecting clear lysate supernatant to obtain protein sample solution.
And (3) enzyme activity detection: the enzyme catalytic reaction system is 270 mu L, 125 mu L of 0.1mol/L Tris-HCl buffer solution with pH of 7.4, 15 mu L of 0.1mol/L NaCl solution, 12.5 mu L of 0.1mol/L NaNO 2 solution and 7.5 mu L of 0.1mol/L methyl viologen solution are firstly taken and uniformly mixed in a 1.5ml centrifuge tube, and preheated at 30 ℃ for 5min; then 70 mu L of crude protein sample solution is added, and the mixture is preheated for 5min at 30 ℃; 40. Mu.L of 0.1mol/L Na 2S2O4 solution (Na 2S2O4 is dissolved in 0.1mol/L NaHCO 3 solution and is ready to use) was added, the enzyme-catalyzed reaction was started, placed in a water bath at 30℃and timed; 10 mu L of the reaction solution is taken at 0min and 30min respectively, 990 mu L of water is added, and the reaction is vigorously shaken until the blue color is completely removed, and the enzyme catalysis reaction is stopped.
And (3) screening high-activity mutant strains according to an enzyme activity detection result, and carrying out gene identification on the high-activity mutant strains, wherein the nucleotide sequence of the high-activity mutant strains is shown as SEQ ID NO. 4, and the amino acid sequence of protein coded by the high-activity mutant strains is shown as SEQ ID NO. 3. The high activity mutant selected was designated as M1 mutant.
Example 3: small-scale production of nitrite reductase mutants in shake flasks
In this example, E.coli containing the plasmid pET-M1 of the M1 mutant gene was inoculated into 100mL of LB medium containing kanamycin sulfate (100. Mu.g/mL) (peptone 10g/L, yeast extract 5g/L, naCl 10g/L, pH 7.2). Coli was grown in a shaker at 37℃with shaking at 250rpm for 16 hours. 1mL of the bacterial culture solution is placed in 100mL of LB culture medium containing kanamycin sulfate according to the proportion of 1:100 for transfer, and is subjected to shaking culture under the same conditions, and the absorbance value of the bacterial culture solution at 600nm is measured at regular time to monitor the bacterial growth density.
When the OD600 of the culture was 0.6 to 0.8, the expression of nitrite reductase gene was induced by adding isopropyl β -D-thiogalactoside (IPTG) to a final concentration of 1mM, followed by culturing for overnight (10 to 16 hours). Cells were collected by centrifugation (10000 rpm, 10min, 4 ℃) and the supernatant was discarded. The cell pellet was resuspended at 4℃in 100mM citrate-sodium citrate buffer pH6.0 at a bacterial sludge concentration of 200g/L. After ultrasonication, cell debris was removed by centrifugation (13000 rpm, 30min, 4 ℃). Clear lysate supernatant was collected to give crude enzyme solution which was stored at-20 ℃.
Example 4: fermentation production of nitrite reductase mutants
In this example, E.coli containing the plasmid pET-M1 of the M1 mutant gene was inoculated with 120mL of LB medium (peptone 10g/L, yeast extract 5g/L, naCl 10g/L, pH 7.2) containing kanamycin sulfate (100 mg/mL). Coli was grown overnight (10-16 hours) at 37℃in a shaker with shaking at 250 rpm. Adding the seed liquid into a 15L fermentation tank containing 6L fermentation medium according to an inoculum size of 2%, maintaining pH7.0-7.2 of the fermentation liquid by adding ammonia water, controlling dissolved oxygen to be about 30% in the fermentation process at a tank temperature of 37 ℃ and a stirring rotating speed of 300-900 rpm, controlling air flow to be 1:1-2 vvm, adding IPTG with a final concentration of 1mmol/L after culturing for 8 hours to induce the expression of nitrite reductase, and continuing to ferment for 12-16 hours at a tank temperature of 22 ℃. During the fermentation, the growth of the culture was maintained by adding a feed solution containing 200g/L glucose, 100g/L yeast extract, pH 7.2. After fermentation, the culture is directly homogenized and crushed by a high-pressure homogenizer, and is subjected to centrifugation, filtration and ultrafiltration concentration to prepare a nitrite reductase mutant crude enzyme solution which is stored at the temperature of minus 20 ℃.
Example 5: determination of nitrite reductase enzyme Activity
This example uses the crude enzyme solutions of nitrite reductase mutants M1 prepared in example 3 and example 4 for enzyme activity testing.
The method for measuring the activity of nitrite reductase comprises the following steps: the enzyme catalytic reaction system is 270 mu L, 125 mu L of 0.1mol/L Tris-HCl buffer solution with pH of 7.4, 15 mu L of 0.1mol/L NaCl solution, 12.5 mu L of 0.1mol/L NaNO 2 solution and 7.5 mu L of 0.1mol/L methyl viologen solution are firstly taken and uniformly mixed in a 1.5ml centrifuge tube, and preheated at 30 ℃ for 5min; then 70 mu L of crude protein sample solution is added, and the mixture is preheated for 5min at 30 ℃; 40. Mu.L of 0.1mol/L Na 2S2O4 solution (Na 2S2O4 is dissolved in 0.1mol/L NaHCO 3 solution and is ready to use) was added, the enzyme-catalyzed reaction was started, placed in a water bath at 30℃and timed; 10 mu L of the reaction solution is taken at 0min and 30min respectively, 990 mu L of water is added, and the reaction is vigorously shaken until the blue color is completely removed, and the enzyme catalysis reaction is stopped.
Definition of enzyme activity: the amount of enzyme required to degrade 1ng of NaNO 2 per minute under the above conditions is defined as 1 enzyme activity unit; and (3) measuring the content change of sodium nitrite in the sample by using a naphthalene ethylenediamine hydrochloride method.
Conclusion: the enzyme activities of the nitrite reductase mutant with the amino acid sequence shown as SEQ ID NO. 3, which are produced in a shake flask in a small-scale manner and produced by fermentation in a fermentation tank are 2300U and 2800U respectively, which are higher than that of a wild type strain of the nitrite reductase mutant with the amino acid sequence shown as SEQ ID NO. 3; has higher degradation effect on nitrite compared with wild nitrite reductase.
Example 6: determination of nitrite reductase stability
The relative enzyme activities of nitrite reductase at different pH and temperatures were detected with the enzyme activities measured at 30℃and pH7.0 as 100% enzyme activities, respectively.
As a result, as shown in FIGS. 1 and 2, it can be seen from FIG. 1 that the relative enzyme activity measured at each temperature was lower than 100% when the enzyme activity was 100% at 30℃and the decrease in enzyme activity was increased with the increase in temperature. As can be seen from FIG. 2, the relative enzyme activity reached 153.90% at pH 7.5, and only 44.45% at pH 6.0.
Therefore, the nitrite reductase with the amino acid sequence shown as SEQ ID NO. 3 has higher stability under the conditions of 25-35 ℃ and pH of 7.0-8.0, and particularly, the optimal reaction temperature of the nitrite reductase is 30 ℃ and the optimal pH is 7.5.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.
Claims (10)
1. A nitrite reductase mutant, characterized in that the mutant has an amino acid substitution at one or more of positions 48, 64, 70 and 240 corresponding to SEQ ID No. 1, relative to a wild type nitrite reductase.
2. The nitrite reductase mutant according to claim 1, characterized in that: the nitrite reductase mutant has nitrite reductase activity; preferably, the nitrite reductase mutant has increased stability compared to a wild type nitrite reductase.
3. The nitrite reductase mutant according to claim 1, characterized in that:
The nitrite reductase mutant has amino acid at position 48 replaced with serine, asparagine, glutamine or threonine; preferably, substituted with serine; and/or
The nitrite reductase mutant has amino acid at position 64 replaced with glycine, alanine, valine, leucine, isoleucine, proline or methionine; preferably, it is substituted with glycine; and/or
The nitrite reductase mutant has amino acid at position 70 replaced with glycine, alanine, valine, leucine, isoleucine, proline or methionine; preferably, substituted with alanine; and/or
The nitrite reductase mutant has amino acid at position 240 replaced with serine, asparagine, glutamine or threonine; preferably, it is substituted by glutamine.
4. The nitrite reductase mutant of claim 1, wherein the nitrite reductase mutant comprises an amino acid sequence having at least 85% sequence identity to SEQ ID No. 3.
5. A nucleic acid molecule encoding a nitrite reductase mutant according to any one of claims 1-4.
6. An expression vector comprising the nucleic acid molecule of claim 5.
7. A host cell comprising the nucleic acid molecule of claim 5 or the expression vector of claim 6.
8. The host cell of claim 7, wherein the host cell is selected from the group consisting of a fungal cell, a bacterial cell, a plant cell, an insect cell, and a mammalian cell; preferably, the host cell is selected from the group consisting of yeast cells, mold, E.coli.
9. Use of the nitrite reductase mutant of any one of claims 1-4 for degrading nitrite.
10. The use according to claim 9, characterized in that the nitrite reductase mutant is used for sewage treatment.
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