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CN-121991935-A - Neutral protease mutant and application thereof in repairing amino acid fermentation wastewater

CN121991935ACN 121991935 ACN121991935 ACN 121991935ACN-121991935-A

Abstract

The invention belongs to the technical field of biology, and discloses a neutral protease mutant, wherein the amino acid sequence of the neutral protease mutant is mutated on the basis of the amino acid sequence of the neutral protease shown as SEQ ID NO. 1, wherein (1) amino acid alanine at position 50 is replaced by aspartic acid, (2) valine at position 171 is replaced by leucine, or (3) amino acid alanine at position 50 is replaced by aspartic acid, and valine at position 171 is replaced by leucine. Compared with the wild type, the neutral protease mutant has the advantages of improved enzyme activity, lower optimal pH value and improved stability, and is suitable for repairing amino acid wastewater with lower pH value.

Inventors

  • Shi fulong
  • ZHU XIANDA
  • Ma Lanjiang
  • WANG SHIWEI
  • ZHENG JIAJIA
  • WANG JIANWEN
  • MA HONGJUAN
  • LV BAOLONG

Assignees

  • 宝鸡阜丰生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260304

Claims (10)

  1. 1. A neutral protease mutant is characterized in that the amino acid sequence of the neutral protease mutant is mutated by (1) substituting aspartic acid for alanine at position 50, (2) substituting leucine for valine at position 171, or (3) substituting aspartic acid for alanine at position 50 and leucine for valine at position 171 in the neutral protease amino acid sequence shown in SEQ ID NO. 1.
  2. 2. The neutral protease mutant according to claim 1, wherein the amino acid sequence of the neutral protease mutant is shown in SEQ ID NO. 3.
  3. 3. The neutral protease mutant according to claim 1, wherein the amino acid sequence of the neutral protease mutant is shown in SEQ ID NO. 4.
  4. 4. The neutral protease mutant according to claim 1, wherein the amino acid sequence of the neutral protease mutant is shown in SEQ ID NO. 5.
  5. 5. A gene encoding the neutral protease mutant according to any one of claims 2 to 4.
  6. 6. A gene expression cassette comprising a gene encoding the neutral protease mutant according to any one of claims 2 to 4.
  7. 7. A recombinant expression vector comprising the gene of claim 5 or the gene expression cassette of claim 6.
  8. 8. A host cell expressing the neutral protease mutant of any one of claims 2-4 or carrying the gene expression cassette of claim 6 or carrying the recombinant expression vector of claim 7.
  9. 9. The recombinant cell of claim 8, wherein the recombinant cell is a recombinant yeast cell.
  10. 10. Use of the neutral protease mutant according to any one of claims 1 to 4 for the remediation of amino acid fermentation wastewater.

Description

Neutral protease mutant and application thereof in repairing amino acid fermentation wastewater Technical Field The invention belongs to the technical field of biology, and particularly relates to a neutral protease mutant and application thereof in repairing amino acid fermentation wastewater. Background The main pollutants of the amino acid fermentation wastewater are residual amino acids, proteins, polypeptides, saccharides, organic acids and a small amount of nitrogen and phosphorus, and the wastewater generally has the characteristics of high COD, high ammonia nitrogen and good biodegradability. The residual protein content in the fermentation wastewater is large, and the wastewater treatment difficulty is increased. The neutral protease can directly catalyze the hydrolysis of proteins and polypeptides which are not completely extracted in the wastewater into micromolecular peptides and amino acids, reduces COD and Total Organic Carbon (TOC) of the wastewater, can be directly added, and is suitable for the pretreatment of amino acid fermentation wastewater such as glutamic acid, lysine and threonine. Chinese patent No. 104860494B uses complex enzyme preparation containing neutral proteinase to repair sewage, hydrolyzes high molecular weight organic matter into small molecular organic matter, improves the utilization rate of microorganism, and finally achieves the purpose of sludge reduction. Chinese patent No. 1814755B discloses a high temperature neutral proteinase which is separated from bacillus licheniformis, the full length of the proteinase amino acid sequence is 377 amino acids, the proteinase has better thermal stability, the optimal reaction pH7.0-7.2 and the optimal reaction temperature of 65 ℃ can be inhibited by EDTA and pMSF. However, the existing neutral protease has unstable activity in complex sewage environments (such as high temperature or non-neutral pH), has poor enzymolysis effect, and limits the application range. Mutation of neutral proteases may result in alteration of beneficial properties. The current neutral protease mutation construction technology comprises site-directed mutagenesis, traditional mutagenesis and other methods. Site-directed mutagenesis is based on enzyme crystal structure, homology modeling, engineering of active centers, substrate binding pockets or stability-related amino acids by PCR targeting. Traditional mutagenesis is to obtain mutants by mutagenesis of strains by physical, chemical or/and plasma (ARTP) or the like, screening of high-yield mutants and isolation and purification. Disclosure of Invention In order to further improve the biological performance of neutral protease, the invention provides a neutral protease mutant and application thereof in repairing amino acid fermentation wastewater. The invention is realized by the following scheme. A neutral protease mutant has an amino acid sequence which is mutated in the neutral protease amino acid sequence shown in SEQ ID NO. 1 by (1) substitution of alanine for aspartic acid at amino acid position 50, (2) substitution of valine for leucine at amino acid position 171, or (3) substitution of alanine for aspartic acid at amino acid position 50 and valine for leucine at amino acid position 171. The invention also relates to a gene for encoding the neutral protease mutant. The invention also relates to a gene expression cassette and a recombinant expression vector containing the neutral protease mutant, and a host cell containing the gene expression cassette and the recombinant expression vector. Preferably, the recombinant cell is a recombinant yeast cell. The invention also relates to a method for preparing the neutral protease mutant according to claim 1, comprising culturing the recombinant cell according to claim 4. The invention also relates to the use of neutral protease mutants, preferably in amino acid fermentation wastewater. The neutral protease mutant has the advantages of improved enzyme activity, strong pH and temperature adaptability, low pH resistance, good high temperature resistance and the like compared with a wild type, can react in a wide pH value and temperature range, and is beneficial to improving the flexibility of an amino acid fermentation wastewater treatment process. The neutral protease mutant can still maintain 70-80% of enzyme activity at pH5, and NP-M3 can still maintain more than 40% of enzyme activity at pH3, so that the pH application range is obviously improved compared with that of a wild type, and the neutral protease mutant is suitable for repairing amino acid fermentation wastewater with pH of 3-5. The optimal temperature of the wild type neutral protease and the mutant neutral protease is 65 ℃, and the mutant neutral protease has enzyme activity in the range of 35-75 ℃, and the mutant neutral protease maintains higher enzyme activity when deviating from the optimal temperature. Drawings FIG. 1 relative enzyme activities of neutral proteases at different pH condit