Nitrite reductase mutants and uses thereof

Abstract

The present application relates to a kind of nitrite reductase mutant and its application, the amino acid sequence of the nitrite reductase mutant relative to wild-type nitrite reductase, with the substitution of amino acid in one or more positions corresponding to the 48th, 64th, 70th and 240th of SEQ ID NO:1.The nitrite reductase mutant provided by the present application has increased stability compared with wild-type nitrite reductase, and can be applied to degrade nitrite in sewage.The preparation method of the nitrite reductase mutant provided by the present application has simple process and mild conditions, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to nitrite reductase mutants and their applications. Background Technology

[0002] Nitrites are widely present in groundwater and surface water. The main causes of nitrite pollution in water bodies are the excessive use of chemical fertilizers and the improper disposal of animal feces, domestic sewage, and industrial wastewater. Studies have shown that drinking water containing nitrites can be toxic to the human body, and long-term consumption can lead to an increased incidence of cancer. Currently, the most common method for removing nitrites from wastewater is biological treatment. Compared with chemical treatment, biological methods have advantages such as lower cost and no secondary pollution.

[0003] Nitrite reductase (NIR) is an oxidoreductase extracted from plant cells that catalyzes the reduction of nitrite and is widely distributed in bacteria and archaea. Wild-type nitrite reductase exhibits low catalytic activity and poor stability for certain substrates. Existing technologies utilize protein engineering to mutate amino acids at specific positions based on the amino acid sequence of wild-type nitrite reductase, which has partially addressed the issue of poor stability. However, these nitrite reductase mutants still suffer from problems such as complex preparation processes and long fermentation times.

[0004] Therefore, there is a need for a nitrite reductase that simultaneously possesses the advantages of high enzyme activity, good stability, and simple and mild preparation methods. Summary of the Invention

[0005] The purpose of this invention is to provide a nitrite reductase mutant with high enzyme activity, good stability, and a simple preparation process.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a nitrite reductase mutant having an amino acid substitution at one or more positions corresponding to the 48th, 64th, 70th and 240th positions of SEQ ID NO:1.

[0008] In some embodiments, the nitrite reductase mutant possesses nitrite reductase activity. In some embodiments, the nitrite reductase mutant has increased stability compared to wild-type nitrite reductase; wherein the amino acid sequence of the wild-type nitrite reductase is shown in SEQ ID NO:1.

[0009] In some embodiments, the 48th amino acid of the nitrite reductase mutant can be substituted with serine, asparagine, glutamine, or threonine. In a preferred embodiment, the 48th amino acid of the nitrite reductase mutant is substituted with serine.

[0010] In some embodiments, the 64th amino acid of the nitrite reductase mutant can be substituted with glycine, alanine, valine, leucine, isoleucine, proline, or methionine. In a preferred embodiment, the 64th amino acid of the nitrite reductase mutant is substituted with glycine.

[0011] In some embodiments, the 70th amino acid of the nitrite reductase mutant can be substituted with glycine, alanine, valine, leucine, isoleucine, proline, or methionine. In a preferred embodiment, the 70th amino acid of the nitrite reductase mutant is substituted with alanine.

[0012] In some embodiments, position 240 of the nitrite reductase mutant may be substituted with serine, asparagine, glutamine, or threonine. In a preferred embodiment, position 240 of the nitrite reductase mutant is substituted with glutamine.

[0013] In some embodiments, the nitrite reductase mutant may have the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to it.

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned nitrite reductase mutant.

[0015] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the second aspect.

[0016] In some embodiments, the expression vector can be constructed by linking the above-mentioned 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.

[0017] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.

[0018] In some embodiments, the host cell may be a cell conventionally used to produce the aforementioned nitrite reductase mutant. In some embodiments, the host cell may be selected from fungal cells, bacterial cells, plant cells, insect cells, or mammalian cells. In some embodiments, the host cell may be selected from yeast cells, molds, or Escherichia coli.

[0019] In a fifth aspect, the present invention also provides a method for preparing the above-mentioned nitrite reductase mutant, comprising fermenting and culturing the above-mentioned host cells, collecting and preparing the above-mentioned nitrite reductase mutant.

[0020] In some embodiments, the method includes industrial-scale preparation of the above-mentioned nitrite reductase mutant under certain fermentation conditions in a production tank. In some embodiments, the fermentation conditions in the production tank are DO ≥ 30% and air flow rate 1:(1-2)vvm.

[0021] In a sixth aspect, the present invention provides the application of the nitrite reductase mutant disclosed herein in the degradation of nitrite.

[0022] In some embodiments, the nitrite reductase mutant is used for wastewater treatment.

[0023] In some embodiments, the nitrite reductase mutant can be used to degrade nitrite in wastewater. Attached Figure Description

[0024] Figure 1 The relative enzyme activity of nitrite reductase at different temperatures is shown.

[0025] Figure 2 The relative enzyme activity of nitrite reductase at different pH values ​​is shown. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0027] definition

[0028] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0029] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0030] Wild-type refers to the form found in nature. For example, naturally occurring or wild-type polypeptide or polynucleotide sequences are sequences that exist in organisms, can be isolated from natural sources, and have not been intentionally modified by human intervention.

[0031] Sequence identity, also known as sequence uniformity, refers to the "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences. This percentage represents the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are therefore not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0032] The expression “at least 85% sequence identity” as used in this document 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.

[0033] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing 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, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0034] The expression vector may contain the nucleic acid described in this invention, in a form suitable for expression in a host cell. This means that the expression vector includes one or more regulatory elements, selectable depending on the host cell for expression, which are operatively linked to the nucleic acid sequence to be expressed. Within the expression vector, "operative linking" is intended to link the target nucleotide sequence to the regulatory element in a manner that allows for nucleotide expression (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced).

[0035] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0036] Example 1: Establishment of Wild-Type Nitrite Reductase Genetically Engineered Bacteria

[0037] In this embodiment, based on the wild-type gene sequence of Pseudomonas sp. B21-044 nitrite reductase (GenBank: UVL19616.1) recorded in NCBI, the whole gene fragment (nucleotide sequence as shown in SEQ ID NO:2, and the encoded protein amino acid sequence as shown in SEQ ID NO:1) was artificially synthesized after sequence optimization. The gene was then inserted into the pET-28a plasmid by a gene synthesis company using BamHI and XhoI endonucleases to obtain a plasmid expressing wild-type nitrite reductase, named pET-WT. pET-WT was then transformed into Escherichia coli BL21(DE3) to establish a wild-type nitrite reductase genetically engineered bacterium.

[0038] Example 2: Construction of a nitrite reductase mutant library

[0039] In this embodiment, the protein reductase was engineered based on the pET-WT plasmid obtained in Example 1 using the error-prone PCR random mutation method.

[0040] Error-prone PCR amplification of the nitrite reductase gene: using a lower fidelity Taq polymerase, while utilizing Mn 2+ Alternative natural cofactor Mg 2+ Increase the probability of error.

[0041] 50 μL PCR system: 5×PCR Buffer (10 μL), dATP (2.5 mmol / L, 1 μL), dGTP (2.5 mmol / L, 1 μL), dCTP (2.5 mmol / L, 1 μL), dTTP (2.5 mmol / L, 1 μL), MgCl2 (5 mmol / L, 1 μL), MnCl2 (5 mmol / L, 1 μL), nitrite reductase template gene (1 μL), Taq DNA polymerase (5 U / μL, 0.5 μL), add sterile double-distilled water to 50 μL.

[0042] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 35 cycles of denaturation at 94℃ for 30 s, annealing at 50-65℃ for 40 s, and extension at 72℃ for 40 s; extension at 72℃ for 10 min; and cooling to 4℃.

[0043] The gene fragment amplified by error-prone PCR was ligated into the pET-28a vector. The ligated vector was then transformed into E. coli BL21(DE3) to construct a nitrite reductase gene mutant library.

[0044] Using Escherichia coli BL21(DE3) as the host and pET28a plasmid as the vector, an extended mutant nitrite reductase was expressed. High-activity mutant strains were screened using the following enzyme activity detection method.

[0045] The cell pellet was resuspended at 4°C in 100 mM citrate-sodium citrate buffer (pH 6.0) to a bacterial sludge concentration of 200 g / L. After sonication, cell debris was removed by centrifugation (13000 rpm, 30 min, 4°C). The clear supernatant of the lysate was collected to prepare the protein sample solution.

[0046] Enzyme activity assay: The enzyme catalytic reaction system was 270 μL. First, 125 μL of 0.1 mol / L pH 7.4 Tris-HCl buffer, 15 μL of 0.1 mol / L NaCl solution, 12.5 μL of 0.1 mol / L NaNO2 solution, and 7.5 μL of 0.1 mol / L methyl viologen solution were added to a 1.5 ml centrifuge tube, mixed well, and preheated at 30 °C for 5 min. Then, 70 μL of crude protein sample solution was added, and the mixture was preheated at 30 °C for 5 min. Next, 40 μL of 0.1 mol / L Na2S2O4 solution (Na2S2O4 dissolved in 0.1 mol / L NaHCO3 solution, freshly prepared) was added. At this point, the enzyme catalytic reaction was initiated. The mixture was placed in a 30 °C water bath and the timer was started. At 0 min and 30 min, 10 μL of the reaction solution was taken, 990 μL of water was added, and the mixture was shaken vigorously until the blue color completely disappeared, at which point the enzyme catalytic reaction was terminated.

[0047] Based on the enzyme activity assay results, highly active mutant strains were screened, and their genes were identified. The nucleotide sequence of the highly active mutant strain is shown in SEQ ID NO:4, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO:3. The screened highly active mutant was named the M1 mutant.

[0048] Example 3: Small-scale production of nitrite reductase mutant in shake flasks

[0049] In this embodiment, *E. coli* containing the M1 mutant gene plasmid pET-M1 was inoculated into 100 mL of LB medium containing kanamycin sulfate (100 μg / mL) (peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.2). *E. coli* was grown in a shaker at 37°C with shaking at 250 rpm for 16 hours. A 1:100 transfer ratio was then performed, with 1 mL of bacterial culture added to 100 mL of LB medium containing kanamycin sulfate. The culture was then incubated under the same conditions with shaking, and the absorbance at 600 nm was measured periodically to monitor bacterial growth density.

[0050] When the OD600 of the culture was 0.6 to 0.8, nitrite reductase gene expression was induced by adding isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 1 mM, followed by overnight incubation (10–16 hours). Cells were collected by centrifugation (10,000 rpm, 10 min, 4 °C), and the supernatant was discarded. The cell pellet was resuspended at 4 °C in 100 mM citrate-sodium citrate buffer (pH 6.0), resulting in a bacterial sludge concentration of 200 g / L. After sonication, cell debris was removed by centrifugation (13,000 rpm, 30 min, 4 °C). The clear supernatant of the lysate was collected to prepare the crude enzyme solution, which was stored at -20 °C.

[0051] Example 4: Fermentation production of nitrite reductase mutant

[0052] In this embodiment, *E. coli* containing the M1 mutant gene plasmid pET-M1 was inoculated into 120 mL LB medium containing kanamycin sulfate (100 mg / mL), peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.2. *E. coli* was grown overnight (10-16 hours) at 37°C with shaking at 250 rpm. The seed culture was added at a 2% inoculation rate to a 15 L fermenter containing 6 L of fermentation medium. The pH of the fermentation broth was maintained at 7.0-7.2 by adding ammonia. The fermentation temperature was 37°C, the stirring speed was 300-900 rpm, the dissolved oxygen was controlled at approximately 30%, and the air flow rate was 1:1-2 vvm. After 8 hours of culture, IPTG was added to a final concentration of 1 mmol / L to induce nitrite reductase expression. Fermentation continued for another 12-16 hours at 22°C. During fermentation, the culture was maintained by adding a feed solution containing 200 g / L glucose, 100 g / L yeast extract, and pH 7.2. After fermentation, the culture was homogenized and crushed directly using a high-pressure homogenizer. After centrifugation, filtration, and ultrafiltration concentration, a crude enzyme solution of the nitrite reductase mutant was prepared and stored at -20℃.

[0053] Example 5: Determination of nitrite reductase activity

[0054] In this embodiment, the crude enzyme solution of nitrite reductase mutant M1 prepared in Examples 3 and 4 was used to test enzyme activity.

[0055] Method for determining nitrite reductase activity: The enzyme-catalyzed reaction system is 270 μL. First, take 125 μL of 0.1 mol / L pH 7.4 Tris-HCl buffer, 15 μL of 0.1 mol / L NaCl solution, 12.5 μL of 0.1 mol / L NaNO2 solution, and 7.5 μL of 0.1 mol / L methyl viologen solution into a 1.5 ml centrifuge tube, mix well, and preheat at 30℃ for 5 min. Then add 70 μL of crude protein sample solution and preheat at 30℃ for 5 min. Add 40 μL of 0.1 mol / L Na2S2O4 solution (Na2S2O4 dissolved in 0.1 mol / L NaHCO3 solution, freshly prepared and used). At this point, the enzyme-catalyzed reaction is initiated. Place the tube in a 30℃ water bath and start timing. At 0 min and 30 min, take 10 μL of the reaction solution, add 990 μL of water, and shake vigorously until the blue color completely disappears, at which point the enzyme-catalyzed reaction is terminated.

[0056] Enzyme activity definition: The amount of enzyme required to degrade 1 ng NaNO2 per minute under the above conditions is defined as 1 enzyme activity unit; the change in sodium nitrite content in the sample was determined by the naphthylethylenediamine hydrochloride method.

[0057] Conclusion: The nitrite reductase mutant with the amino acid sequence shown in SEQ ID NO:3 had enzyme activities of 2300 U and 2800 U in small-scale production in shake flasks and fermentation tanks, respectively, both higher than the wild type's 200 U; compared with the wild type, it had a higher degradation effect on nitrite.

[0058] Example 6: Determination of the stability of nitrite reductase

[0059] The relative enzyme activity of nitrite reductase at different pH and temperature was determined with the enzyme activity measured at 30℃ and pH 7.0 as 100% enzyme activity.

[0060] The results are as follows Figure 1 and Figure 2 As shown, by Figure 1 It can be seen that when enzyme activity at 30℃ is taken as 100% enzyme activity, the relative enzyme activities measured at all temperatures are all below 100%, and the decreasing trend of enzyme activity intensifies with increasing temperature. Figure 2 It can be seen that when the pH is 7.5, its relative enzyme activity reaches 153.90%, while when the pH is 6.0, its relative enzyme activity is only 44.45%.

[0061] Therefore, the nitrite reductase with the amino acid sequence shown in SEQ ID NO:3 has high stability under conditions of 25-35℃ and pH 7.0-8.0. In particular, the optimal reaction temperature of the nitrite reductase is 30℃ and the optimal pH is 7.5.

[0062] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A nitrite reductase mutant, characterized in that, The amino acid sequence of the nitrite reductase mutant is shown in SEQ ID NO:

3.

2. The nitrite reductase mutant according to claim 1, characterized in that: The nitrite reductase mutant has nitrite reductase activity.

3. A nucleic acid molecule encoding a nitrite reductase mutant, characterized in that, The nucleic acid molecule encodes the nitrite reductase mutant as described in claim 1 or 2.

4. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. The host cell according to claim 5, characterized in that, The host cell is selected from fungal cells, bacterial cells, plant cells, insect cells, or mammalian cells.

7. The host cell according to claim 5, characterized in that, The host cell is selected from yeast cells, molds, or Escherichia coli.

8. The application of the nitrite reductase mutant according to claim 1 or 2 in the degradation of nitrite.

9. The application according to claim 8, characterized in that, The nitrite reductase mutant is used for wastewater treatment.

Images