High-temperature-resistant high-enzyme-activity broad-spectrum aminopeptidase mutant and application thereof

Abstract

The application belongs to the field of microbial genetic engineering and protein engineering, and particularly relates to a high-temperature-resistant high-enzyme-activity broad-spectrum aminopeptidase mutant and application. Q80A Compared with the protein before mutation, the protease has 30% higher leucine aminopeptidase enzyme activity and 60% higher phenylalanine aminopeptidase enzyme activity under the condition of 50 DEG C, and the production and application of aminopeptidase are promoted.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and protein engineering technology, specifically relating to high-temperature resistant, high-enzyme-activity, broad-spectrum aminopeptidase mutants and their applications. Background Technology

[0002] Bitterness resulting from the exposure of hydrophobic amino acids in protein hydrolysates is a key bottleneck restricting their application in food. Aminopeptidase (AP), as an important class of exonucleases, can specifically catalyze the hydrolysis of N-terminal residues of proteins or peptides, and is widely used in peptide debittering and the preparation of bioactive peptides. It is also a commonly used debittering enzyme in food processing. However, currently, there is a scarcity of aminopeptidases with debittering potential; most are leucine aminopeptidases, which have limited substrate specificity and cannot meet practical debittering needs. Furthermore, most aminopeptidases have poor thermostability, with optimal temperatures concentrated in the 35–40℃ range, making it impossible to synergize with endonucleases under high industrial conditions, severely restricting their industrial application. This patent has screened a broad-spectrum aminopeptidase mutant that still exhibits high catalytic activity at high temperatures, showing promising prospects for industrial application.

[0003] This application describes the assay of aminopeptidase activity using the method described by Qin et al. A synthetic amide chromogenic substrate with a specific amino acid at its N-terminus was selected. Under specific temperature and pH conditions, aminopeptidase hydrolyzes the peptide chain from the N-terminus of the substrate, releasing a p-nitroaniline (pNA) chromogenic group (colorless before release, yellow after release). The OD of the solution was measured using a microplate reader. 405 The absorbance value is used to calculate the enzyme activity of aminopeptidase. The advantages of this method are high substrate specificity (only aminopeptidase can hydrolyze it, without interference from other enzymes); and rapid detection, allowing real-time monitoring of absorbance changes. Summary of the Invention

[0004] The purpose of this invention is to provide a high-temperature resistant, high-enzyme-activity, broad-spectrum aminopeptidase mutant protein, the amino acid sequence of which is shown in SEQ ID NO.3.

[0005] Another object of the present invention is to provide the use of a high-temperature resistant, high-enzyme-activity, broad-spectrum aminopeptidase mutant protein in the hydrolysis of leucine and / or phenylalanine.

[0006] To achieve the above objectives, the present invention adopts the following technical measures: The applicant, by mining a broad-spectrum aminopeptidase protein sequence from Bacillus licheniformis DW2 (as shown in SEQ ID NO.1), modified the aminopeptidase. By mutating glutamine at position 80 to alanine, the leucine activity of the aminopeptidase at 50°C was increased by 30%, and the phenylalanine activity by 60%, resulting in a significant increase in enzyme activity. The amino acid sequence of the obtained heat-resistant, high-activity broad-spectrum aminopeptidase mutant protein is shown in SEQ ID NO.3, and one of the genes encoding it is shown in SEQ ID NO.4.

[0007] The scope of protection of this invention also includes: The fusion protein obtained by fusing the mutant protein shown in SEQ ID NO.3 with a protein purification tag.

[0008] The gene encoding the mutant or fusion protein shown in SEQ ID NO.3.

[0009] Expression cassettes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells containing the above-mentioned coding genes.

[0010] The recombinant microorganism mentioned above is recombinant Bacillus licheniformis.

[0011] The mutant protein shown in SEQ ID NO.3, the above-mentioned fusion protein, the encoding gene of the mutant or fusion protein described in SEQ ID NO.3, and the application of expression cassettes, recombinant vectors, recombinant microorganisms or ex vivo recombinant cells having the above-mentioned encoding gene in the preparation of aminopeptidase.

[0012] The use of the protein shown in SEQ ID NO.3, the above-mentioned fusion protein, the encoding gene, the expression cassette having the above-mentioned encoding gene, the recombinant vector, the recombinant microorganism or the ex vivo recombinant cell in the hydrolysis of leucine and / or phenylalanine.

[0013] The use of the protein shown in SEQ ID NO.3, the above-mentioned fusion protein, the encoding gene, the expression cassette having the above-mentioned encoding gene, the recombinant vector, the recombinant microorganism or the recombinant cell in vitro in the preparation of debittering agents.

[0014] A method for increasing aminopeptidase activity includes mutating the 80th amino acid of the protein shown in SEQ ID NO.1 from glutamine to alanine.

[0015] A method for preparing a thermostable, broad-spectrum aminopeptidase includes introducing an expression vector expressing the mutant protein or fusion protein into Bacillus licheniformis for fermentation.

[0016] The preferred Bacillus licheniformis mentioned above is Bacillus licheniformis DW2.

[0017] The coding gene described above is preferably the one shown in SEQ ID NO.4.

[0018] Compared with the prior art, the present invention has the following advantages: This invention obtains a mutant by site-directed mutagenesis of aminopeptidase derived from Bacillus licheniformis DW2, which increases the activity of leucine aminopeptidase by 30% and the activity of phenylalanine aminopeptidase by 60% at 50°C, thus promoting the production and application of aminopeptidase. Detailed Implementation

[0019] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0020] Example 1:

[0021] Construction of aminopeptidase mutant expression strains derived from Bacillus licheniformis: 1) The aminopeptidase gene (shown in SEQ ID NO.2) from Bacillus licheniformis DW2 (CN116334120B) was ligated into the expression vector pHY-300 to construct the recombinant plasmid pHY-300-ywaD. The recombinant plasmid was transformed into Bacillus licheniformis DW2 to obtain recombinant Bacillus licheniformis expressing wild-type YwaD.

[0022] 2) Using the plasmid pHY-300-ywaD expressing the wild-type YwaD gene as a template, primers Q80A-F and Q80A-R were used to amplify the DNA fragment via PCR. The PCR product was purified, and the DNA fragment was recombinantly circularized using Winzyme's HI-Efficience CloningMix. The recombinant DNA was then transformed into *E. coli* DH5α competent cells. The cells were plated on Tet-resistant plates and incubated at 37°C. Colony PCR was then performed to verify the transformants. If the sequencing results of the target fragment matched the design using primers amp-F and amp-R, the mutant vector was successfully constructed.

[0023] Recombinant plasmids of the mutants were extracted using a plasmid extraction kit and prepared for use. The recombinant vector was transformed into Bacillus licheniformis DW2 competent cells. The bacterial cells were plated on culture plates containing Tet resistance for screening and incubated at 37°C. Colony PCR was performed to verify the transformants using primers amp-F and amp-R. If the target fragment size was correct, the strain was identified as engineered strain pHY-300-ywaD. Q80A Construction successful, expressing mutant protein ywaD Q80A That is, the 80th amino acid of the protein shown in SEQ ID NO.1 is mutated from Q to A, resulting in ywaD. Q80A The protein sequence is shown in SEQ ID NO.3. The proteins expressed by the recombinant bacteria in Table 1 are also named according to this rule.

[0024] The applicant also prepared other mutant proteins as control groups. The construction process of recombinant strains expressing these mutant proteins can be found in the recombinant Bacillus licheniformis pHY-300-ywaD strain. Q80A The corresponding primers used in the construction of the recombinant strains are shown in Table 1. As shown in Table 1, the control recombinant strains include pHY-300-ywaD. K57R pHY-300-ywaD A23I pHY-300-ywaD S243W pHY-300-ywaD L246T pHY-300-ywaD G25R pHY-300-ywaD Q54W pHY-300-ywaD R250Q pHY-300-ywaD H29I pHY-300-ywaD Q80Y .

[0025] Table 1 Primers used for site-directed mutagenesis .

[0026] Example 2:

[0027] Assay of the activity of aminopeptidase expressed by recombinant Bacillus licheniformis prepared in Example 1: Wild-type YwaD recombinant Bacillus licheniformis and different mutant YwaD recombinant Bacillus licheniformis prepared in Example 1 were streaked onto LB agar plates (containing 20 μg / mL Tet) and cultured overnight at 37°C to obtain single colonies. Single colonies were then picked and activated in 5 mL of liquid LB medium containing Tet, and cultured with shaking at 37°C for 12 h. 1 mL of the activated bacterial culture was then inoculated into 30 mL of liquid LB medium containing Tet and cultured overnight at 37°C with shaking. The overnight culture seed culture was inoculated into 30 mL of TB medium at a 1% inoculation rate and fermented at 37°C and 230 rpm for 48 h. The supernatant was collected by centrifugation.

[0028] The TB culture medium formulation includes: 12 g / L peptone, 24 g / L yeast extract, 5 g / L glycerol, 16.43 g / L dipotassium hydrogen phosphate, 2.31 g / L potassium dihydrogen phosphate, and natural pH.

[0029] The aminopeptidase assay was performed according to the method of Qin et al. The reaction system consisted of 50 mmol / L Tris-HCl (pH 8.0) buffer and an appropriate amount of enzyme solution, using 20 mmol / L Leu-pNA or Phe-pNA as the substrate. The reaction was carried out at 50℃ for 10 min, and the absorbance at 405 nm was measured using a microplate reader. Enzyme activity was defined as the amount of enzyme required to release 1 μg of pNA per minute at pH 8.0 and 50℃, which is defined as one enzyme activity unit (U). The enzyme activity results are shown in Table 2. Table 2. Activities of aminopeptidases expressed by different recombinant Bacillus licheniformis prepared in Example 1. .

[0030] As shown in the table above, the enzyme activity of wild-type YwaD is defined as 100% at 50℃. The enzyme activity of YwaD mutant Q80A is significantly improved. At 50℃, the relative enzyme activity of leucine is 130.71%, an increase of 30.71%; and the relative enzyme activity of phenylalanine is 160.88%, an increase of 60.88%.

[0031] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A synthetically produced aminopeptidase mutant protein, wherein the amino acid sequence of the mutant protein is shown in SEQ ID NO.

3.

2. The fusion protein obtained by fusing the mutant protein of claim 1 with a protein purification tag.

3. The gene encoding the mutant or fusion protein of claim 1.

4. An expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the gene encoding as described in claim 3.

5. The mutant protein of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, and the use of the expression cassette, recombinant vector, recombinant microorganism or ex vivo recombinant cell having the encoding gene of claim 3 in the preparation of aminopeptidase.

6. The mutant protein of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, and the use of an expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the encoding gene of claim 3 in the hydrolysis of leucine and / or phenylalanine.

7. The mutant protein of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, and the use of the expression cassette, recombinant vector, recombinant microorganism or ex vivo recombinant cell having the encoding gene of claim 3 in the preparation of debittering agents.

8. A method for increasing aminopeptidase activity, comprising mutating the 80th amino acid of the protein shown in SEQ ID NO.1 from glutamine to alanine.

9. A method for preparing a thermostable broad-spectrum aminopeptidase, comprising introducing an expression vector expressing the mutant protein of claim 1 or the fusion protein of claim 2 into Bacillus licheniformis for fermentation.

10. The method according to claim 9, wherein the Bacillus licheniformis is Bacillus licheniformis DW2.