Neutral protease mutant and application thereof in repairing amino acid fermentation wastewater

Abstract

The invention belongs to the technical field of biology, and discloses a neutral protease mutant, the amino acid sequence of which is mutated on the basis of the amino acid sequence of neutral protease as shown in SEQ ID NO: 1: (1) substitution of 50th amino acid alanine into aspartic acid, (2) substitution of 171st valine into leucine, or (3) substitution of 50th amino acid alanine into aspartic acid, or (4) substitution of 171st valine into leucine, or (5) substitution of 171st valine into leucine, or (6) substitution of 171st valine into leucine, or (7) substitution of 171st valine into leucine. And valine at the 171st site is replaced by leucine. Compared with a wild type, the neutral protease mutant disclosed by the invention has the advantages that the enzyme activity is improved, the optimum pH is lower, the stability is improved, and the neutral protease mutant is suitable for repairing amino acid wastewater with lower pH.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to neutral protease mutants and their application in the remediation of amino acid fermentation wastewater. Background Technology

[0002] The main pollutants in amino acid fermentation wastewater are residual amino acids, proteins, polypeptides, sugars, organic acids, and small amounts of nitrogen and phosphorus. This type of wastewater typically exhibits high COD, high ammonia nitrogen, and good biodegradability. The high residual protein content in fermentation wastewater increases the difficulty of wastewater treatment.

[0003] Neutral proteases can directly catalyze the hydrolysis of incompletely extracted proteins and peptides in wastewater into small molecule peptides and amino acids, reducing COD and total organic carbon (TOC) in wastewater. They can be added directly and are suitable for the pretreatment of fermentation wastewater containing amino acids such as glutamic acid, lysine, and threonine.

[0004] Chinese invention patent CN104860494B uses a complex enzyme preparation containing neutral protease to remediate wastewater, hydrolyzing high molecular weight organic matter into smaller organic molecules, improving the utilization efficiency of microorganisms, and ultimately achieving sludge reduction. Chinese invention patent CN1814755B isolates a high-temperature neutral protease from Bacillus licheniformis. This protease has a full-length amino acid sequence of 377 amino acids, exhibits good thermal stability, and has an optimal reaction pH of 7.0-7.2 and an optimal reaction temperature of 65℃. It can be inhibited by EDTA and pMSF. However, existing neutral proteases exhibit unstable activity and poor enzymatic hydrolysis in complex wastewater environments (such as high temperatures or non-neutral pH), limiting their application scope.

[0005] Mutating neutral proteases can produce beneficial performance changes. Current techniques for constructing neutral proteases through mutation include site-directed mutagenesis and conventional mutagenesis. Site-directed mutagenesis is based on enzyme crystal structure and homology modeling, using PCR to target and modify the active site, substrate binding pocket, or stability-related amino acids. Conventional mutagenesis involves mutagenizing strains using physical, chemical, and / or plasma-induced mutation methods (ARTP), screening for high-yielding mutants, and then isolating and purifying the mutants. Summary of the Invention

[0006] In order to further improve the biological performance of neutral protease, this invention provides a neutral protease mutant and its application in the remediation of amino acid fermentation wastewater.

[0007] The present invention is achieved through the following scheme.

[0008] A neutral protease mutant having its amino acid sequence modified as shown in SEQ ID NO:1 by the following mutations: (1) replacing the 50th amino acid alanine with aspartic acid, (2) replacing the 171st amino acid valine with leucine, or (3) replacing the 50th amino acid alanine with aspartic acid and replacing the 171st amino acid valine with leucine.

[0009] This invention also relates to genes encoding the aforementioned neutral protease mutants.

[0010] The present invention also relates to a gene expression cassette encoding the above-described neutral protease mutant, a recombinant expression vector, and a host cell containing the above-described gene expression cassette and recombinant expression vector. Preferably, the recombinant cell is a recombinant yeast cell.

[0011] The present invention also relates to a method for preparing the neutral protease mutant of claim 1, comprising culturing the recombinant cells of claim 4.

[0012] This invention also relates to the application of neutral protease mutants, preferably in amino acid fermentation wastewater.

[0013] The beneficial effects achieved by this invention mainly include, but are not limited to, the following aspects: Compared with the wild type, the neutral protease mutant of this invention exhibits improved enzyme activity, strong pH and temperature adaptability, and good tolerance to low pH and high temperatures. It can react within a wide pH and temperature range, which is beneficial for improving the flexibility of amino acid fermentation wastewater treatment processes. The neutral protease mutant of this invention can still maintain 70-80% enzyme activity at pH 5, and NP-M3 can still maintain more than 40% enzyme activity at pH 3. The pH application range is significantly improved compared with the wild type, making it suitable for remediating amino acid fermentation wastewater with a pH of 3-5. The optimal temperature for both the wild type and the mutant neutral protease is 65℃, and they exhibit enzyme activity in the range of 35-75℃. The mutant neutral protease maintains higher enzyme activity when deviating from the optimal temperature. Attached Figure Description

[0014] Figure 1 Relative enzyme activity of neutral proteases under different pH conditions.

[0015] Figure 2 Relative enzyme activity of neutral protease after treatment at pH 5 for 180 min and 360 min. Detailed Implementation

[0016] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0018] Example 1 The wild-type enzyme gene sequence used in this invention has been disclosed in document CN1814755A.

[0019] The structure of the neutral protease with high homology was obtained using the RCSB PDB and NCBI databases. Homology modeling was performed using the SWISS-MODEL website, and the sequence was compared with the wild-type sequence to screen for two mutation sites that might affect activity. Single-mutant and double-mutant neutral protease mutants were then designed. First, GenScript Biotech synthesized the mutant NP-M3 with two mutation sites. Then, using the NP-M3 gene as a template, primers for the mutation sites were designed, and the mutants NP-M1 and NP-M2 with a single mutation site were amplified using overlap extension PCR. The amino acid sequence of the wild-type neutral protease is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2. The amino acid sequences of mutants NP-M1, NP-M2, and NP-M3 are shown in SEQ ID NO: 3-5, respectively. Compared to the wild-type enzyme, mutant NP-M3 has the following mutations: Ala alanine A (gca) at position 50 is mutated to aspartic acid D (gac); Val valine V (gtc) at position 171 is mutated to Leu leucine L (ctc); mutant NP-M1 has the same mutation: Ala alanine A (gca) at position 50 is mutated to aspartic acid D (gac); and mutant NP-M2 has the same mutation: Val valine V (gtc) at position 171 is mutated to Leu leucine L (ctc). The corresponding nucleotide sequence changes are shown in parentheses.

[0020] Expression vector construction: The gene fragments of the neutral protease wild-type and mutant were digested with EcoRI and PstI and ligated into the pGBKT7 vector digested with the same enzyme to construct yeast recombinant expression vectors pGBKT7-WT, pGBKT7-NP-M1, pGBKT7-NP-M2, and pGBKT7-NP-M3 capable of expressing secreting neutral protease. These vectors were then transformed into Saccharomyces cerevisiae AH109 to express the target protein.

[0021] Preparation of competent cells: AH109 yeast strain was inoculated into YPAD medium and cultured at 30℃ and 200rpm in a shaker until OD600=0.6. The cells were collected, washed twice with sterile water, and then resuspended in 100mM LiAc solution to prepare competent yeast cells.

[0022] Plasmid transformation: The recombinant plasmid was mixed with salmon sperm DNA and added to competent yeast cells. The mixture was gently stirred, and a PEG / LiAc mixture (50% PEG3350, 1M LiAc, and 10×TE Buffer in a volume ratio of 8:1:1) was added. The cells were incubated at 30°C for 30 min, followed by heat shock treatment at 42°C for 15 min, and then rapidly cooled on ice for 5 min. The cells were collected by centrifugation, resuspended in sterile water, and plated on SD auxotrophic plates. The plates were incubated upside down at 30°C for 3-5 days until single colonies appeared.

[0023] Screening for auxotrophic culture: Single colonies from SD / -Trp auxotrophic culture plates were transferred to new auxotrophic plates and incubated at 30°C for 3 days. Colony growth was then observed. Colonies that could grow on auxotrophic culture media were preliminarily identified as positive clones.

[0024] Fermentation expression: The selected engineered bacteria were inoculated into SD / -Trp liquid medium and cultured at 30℃ and 200rpm in a constant temperature shaker until the OD600 of the bacterial solution reached 0.8, at which point the yeast was in the logarithmic growth phase. Then, the culture was inoculated into fresh SD / -Trp liquid medium at a 10% inoculation ratio and cultured at 30℃ and 200rpm for another 48 hours. After culturing, the culture was stopped, and the supernatant was collected by centrifugation at 6000rpm for 3 minutes. The supernatant was then concentrated through an ultrafiltration membrane with a molecular weight cutoff of 20kDa to obtain a crude enzyme solution. The crude enzyme solution was then detected by SDS-PAGE electrophoresis. A significant band was found at the 27kDa position, which was consistent with the expected molecular weight of the protease, indicating that the neutral protease was successfully expressed.

[0025] The enzyme activity of the crude enzyme solution was determined by the quantitative Folin-Ciocalteu method. The neutral protease of the target molecular weight was further purified by precipitation with saturated ammonium sulfate, ion exchange chromatography, and gel filtration chromatography. The enzyme activity of the purified neutral protease was then determined by the quantitative Folin-Ciocalteu method. Each experiment was repeated three times, and the average value was calculated. The enzyme solution temperature was set at 55℃, the standard operating temperature for neutral protease. The enzyme activities of wild-type and mutant strains are shown in Table 1 below.

[0026] Table 1

[0027] As shown in Table 1 above, the enzyme activities of mutants NP-M2 and NP-M3 are increased to a certain extent compared with wild-type, while the enzyme activity of mutant NP-M1 is not significantly different from that of wild-type, with no obvious increase.

[0028] Example 2 Enzymatic Properties Analysis Optimal pH: Using the purified enzyme protein from Example 1 as the research object, the activities of wild-type and mutant neutral proteases were detected under different pH conditions to explore the optimal pH value and pH stability. Enzyme activity was tested at 55°C within the pH range of 3.0 to 9.0. Three parallel samples were set up for each pH value, and their average values ​​were calculated. The relative enzyme activity was used as a reference for the optimal enzyme activity. The final results are shown below. Figure 1 As shown in the figure. The experimental results show that the optimal pH for both wild-type and mutant neutral proteases is 7.0. However, NP-M1, NP-M2 and NP-M3 can still maintain 70-80% enzyme activity at pH 5, and NP-M3 can still maintain more than 40% enzyme activity at pH 3. The pH application range is significantly improved, making it suitable for the remediation of amino acid fermentation wastewater with a pH of 3-5.

[0029] Enzyme tolerance was studied by investigating the residual enzyme activity after treatment at pH 5 for 180 min and 360 min. Figure 2 As shown, the results indicate that the neutral protease mutant is more stable under acidic conditions at pH 5 compared to the wild type.

[0030] Optimal temperature: The temperature adaptability of the neutral protease was further studied within the range of 35℃ to 75℃. The experiment was conducted at pH 7.0, with temperature gradients set at 5℃ intervals. At each temperature value, three parallel samples were set up, and the relative enzyme activity of each sample was measured and the average value was calculated. The final results are shown in Table 2.

[0031] Table 2

[0032] The experimental results showed that the optimal temperature for both wild-type and mutant neutral proteases was 65℃, and the enzyme activity was within the range of 35-75℃. The mutant neutral protease maintained higher enzyme activity when deviating from the optimal temperature.

[0033] Example 3 Application of neutral proteases in the degradation of macromolecular proteins in amino acid fermentation wastewater.

[0034] After centrifuging to collect bacterial proteins, amino acid fermentation wastewater will retain a certain concentration of protein. Simulated amino acid fermentation wastewater was prepared with a pH of 5.5, a protein content of 20 g / L, and an average molecular weight of 83,000 Da. At pH 5.5 and 50°C, 1.5% of the purified neutral protease prepared in Example 1 was added, and the hydrolysis time was 3 hours. After hydrolysis, the proportion of low molecular weight peptides (100-2000 Da) was measured, and the results are shown in Table 3.

[0035] Table 3

[0036] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the disclosed technical content without departing from the scope of the technical solution of the present invention, resulting in equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of the technical solution of the present invention.

Claims

1. A neutral protease mutant, characterized in that, The amino acid sequence of the neutral mutant is made by the following mutations in the amino acid sequence of the neutral protease as shown in SEQ ID NO:1: (1) the 50th amino acid alanine is replaced with aspartic acid, (2) the 171st amino acid valine is replaced with leucine, or (3) the 50th amino acid alanine is replaced with aspartic acid and the 171st amino acid valine is replaced with leucine.

2. The neutral protease mutant according to claim 1, characterized in that, The amino acid sequence of the neutral protease mutant is shown in SEQ ID NO:

3.

3. The neutral protease mutant according to claim 1, characterized in that, The amino acid sequence of the neutral protease mutant is shown in SEQ ID NO:

4.

4. The neutral protease mutant according to claim 1, characterized in that, The amino acid sequence of the neutral protease mutant is shown in SEQ ID NO:

5.

5. A gene encoding a neutral protease mutant according to any one of claims 2-4.

6. A gene expression cassette containing a gene encoding a mutant of the neutral protease as described in any one of claims 2-4.

7. A recombinant expression vector containing the gene of claim 5 or the gene expression cassette of claim 6.

8. A host cell expressing any one of the neutral protease mutants of claims 2-4, or carrying the gene expression cassette of claim 6, or carrying the recombinant expression vector of claim 7.

9. The recombinant cell according to claim 8, characterized in that, The recombinant cells are recombinant yeast cells.

10. The application of the neutral protease mutant according to any one of claims 1-4 in the remediation of amino acid fermentation wastewater.

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