CN117004589A - Aminopeptidase mutant and application thereof - Google Patents

Abstract

The invention relates to an aminopeptidase mutant with improved heat stability, belonging to the technical fields of genetic engineering, enzyme engineering and food engineering. The aminopeptidase mutant is a polypeptide of SEQ ID NO:2, and the Q137P mutation is generated based on the wild aminopeptidase shown in the specification. The optimal reaction temperature of the aminopeptidase mutant Q137P is increased by 5 ℃, and the residual enzyme activity of the Q137P is still 29.5 percent after the Q137P is preserved for 30min at 60 ℃, which is increased by 40.5 percent compared with the original aminopeptidase. Is beneficial to widening the application of aminopeptidase under the high-temperature condition in the technical field of food processing.

Description

Aminopeptidase mutant and application thereof

Technical field:

the invention relates to an aminopeptidase mutant with improved heat stability, belonging to the technical fields of genetic engineering, enzyme engineering and food engineering.

The background technology is as follows:

aminopeptidases (APs for short, EC 3.4.11) are a class of exoproteases that selectively degrade polypeptide chains and protein N-terminal amino acid residues, releasing free amino acids. The aim of reducing the bitter taste can be achieved by catalyzing and degrading hydrophobic amino acid residues at the N end of the bitter peptide. Therefore, the aminopeptidase can significantly improve the hydrolysis degree of casein and soy protein hydrolysate and reduce bitterness. The aminopeptidase enzyme method has high debitterizing hydrolysis efficiency, mild conditions and easy operation, and is widely applied to the modern industries such as food and the like.

Aminopeptidases have great commercial utility, however few natural aminopeptidases are capable of catalyzing the reactions desired by people with high efficiency under conditions of convenient production, economical economy, etc. The existing aminopeptidase generally has the problems of low yield, poor enzyme activity, easy inactivation under high temperature conditions and the like. Aminopeptidases of high catalytic activity and stability are lacking in the fields of food processing and the like.

The invention comprises the following steps:

in view of the above problems, an object of the present invention is to provide an aminopeptidase mutant having improved enzymatic activity and stability. The invention obtains mutants with improved aminopeptidase stability through site-directed mutagenesis.

It is an object of the present invention to provide an aminopeptidase mutant which is defined in SEQ ID NO:2, and the amino acid sequence obtained by Q137P mutation is shown as SEQ ID NO:4 is shown in the figure;

it is a second object of the present invention to provide a gene encoding the aminopeptidase mutant;

it is a third object of the present invention to provide a recombinant vector or recombinant strain comprising the aminopeptidase mutant gene;

the expression vector used for the recombinant vector may be one of vectors known to those skilled in the art for producing proteins by gene recombination, such as expression vectors WB980, LY-3, etc.;

in one embodiment of the invention, the expression vector is pET28;

in a specific embodiment of the invention, the host cell used by the recombinant strain is E.coli BL21.

The fourth object of the present invention is the use of the recombinant vector or recombinant strain described above, in particular in the production of the aminopeptidase mutant according to the first aspect.

The fifth object of the present invention is the use of the aminopeptidase mutant according to claim one, in particular in catalyzing the degradation of proteins, for hydrolysing peptide bonds of proteins to produce polypeptides or amino acids; and the aminopeptidase mutant has improved stability compared with the wild-type.

The beneficial effects are that:

the invention uses site-directed mutagenesis technology to directionally evolve aminopeptidase from bacillus subtilis in vitro to obtain aminopeptidase mutant with improved enzyme activity and stability. The specific enzyme activity of Q137P is 4935U/g, and the specific enzyme activity of the wild aminopeptidase W168AP is 4253U/g. The optimal reaction temperature of Q137P is increased by 5 ℃, and after the heat preservation is carried out for 30min at 60 ℃, the residual enzyme activity is still 29.5%, which is increased by 40.5% compared with the original strain.

Description of the drawings:

fig. 1: aminopeptidase gene verification diagram.

Fig. 2: optimal temperature profile of WT and mutant Q137P.

Fig. 3: temperature stability profile of WT and mutant Q137P.

The specific embodiment is as follows:

the invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

The invention is defined as follows:

nomenclature of amino acids and DNA nucleic acid sequences

Using the accepted IUPAC nomenclature for amino acid residues, the three letter/single letter code format is used. The DNA nucleic acid sequence uses accepted IUPAC nomenclature.

In the present invention, 168AP represents the gene sequence of B.subtilis 168 source wild type aminopeptidase, as shown in SEQ ID NO:1 is shown in the specification; the coding gene sequence of the aminopeptidase mutant Q137P is shown as SEQ ID NO: 3.

W168AP represents the amino acid sequence of b.subtilis 168 derived wild type aminopeptidase as set forth in SEQ ID NO:2 is shown as follows:

MKKLLTVMTMAVLTAGTLLLPAQSVTPAAHAVQISNSERELPFKAKHAYSTI

SQLSEAIGPRIAGTAAEKKSALLIASSMRKLKLDVKVQRFNIPDRLEGTLSSAG

RDILLQAASGSAPTEEQGLTAPLYNAGLGYQKDFTADAKGKIALISRGDLTYY

EKAKNAEAAGAKAVIIYNNKESLVPMTPNLSGNKVGIPVVGIKKEDGEALTQ

QKEATLKLKAFTNQTSQNIIGIKKPKNIKHPDIVYVTAHYDSVPFSPGANDNG

SGTSVMLEMARVLKSVPSDKEIRFIAFGAEELGLLGSSHYVDHLSEKELKRSE

VNFNLDMVGTSWEKASELYVNTLDGQSNYVWESSRTAAEKIGFDSLSLTQG

GSSDHVPFHEAGIDSANFIWGDPETEEVEPWYHTPEDSIEHISKERLQQAGDLVTAAVYEAVKKEKKPKTIKKQMKAKASDIFEDIK。

the amino acid sequence of the aminopeptidase mutant Q137P is shown in SEQ ID NO:4, as follows: MKKLLTVMTMAVLTAGTLLLPAQSVTPAAHAVQISNSERELPFKAKHAYSTISQLSEAIGPRIAGTAAEKKSALLIASSMRKLKLDVKVQRFNIPDRLEGTLSSAGRDILLQAASGSAPTEEQGLTAPLYNAGLGYPKDFTADAKGKIALISRGDLTYYEKAKNAEAAGAKAVIIYNNKESLVPMTPNLSGNKVGIPVVGIKKEDGEALTQQKEATLKLKAFTNQTSQNIIGIKKPKNIKHPDIVYVTAHYDSVPFSPGANDNGSGTSVMLEMARVLKSVPSDKEIRFIAFGAEELGLLGSSHYVDHLSEKELKRSEVNFNLDMVGTSWEKASELYVNTLDGQSNYVWESSRTAAEKIGFDSLSLTQGGSSDHVPFHEAGIDSANFIWGDPETEEVEPWYHTPEDSIEHISKERLQQAGDLVTAAVYEAVKKEKKPKTIKKQMKAKASDIFEDIK.

The method for measuring the enzyme activity of the aminopeptidase comprises the following steps:

the enzyme activity measurement method of aminopeptidase refers to the LNA method, i.e., 1 enzyme activity unit (U/mL) is defined as the amount of enzyme required for hydrolyzing leucine paranitroaniline by 1mL of enzyme solution under a certain condition (the condition is 60 ℃ C., pH 9, unless otherwise specified) for 1 min. Three replicates were set for each sample and the results averaged.

(1) Measurement procedure

a. Centrifuging the fermentation broth 13000r/min for 2min to obtain supernatant as crude enzyme solution, and performing n-time dilution by using Tris-HCl buffer solution with pH of 9;

b. control group: taking 0.2mL of diluted enzyme solution, adding the diluted enzyme solution into an enzyme activity test tube containing 2.6mL of Tris-HCl buffer solution with pH of 9, preheating for 5min at 60 ℃, adding 1mL of 40% acetic acid solution, reacting for 10min, adding 0.20mL of 50mM LNA solution (0.1256 g of leucine paranitroaniline is dissolved to a constant volume of 10mL by using absolute ethyl alcohol), uniformly mixing, and standing for 10min;

c. experimental group: taking 0.2mL of diluted enzyme solution, adding the diluted enzyme solution into an enzyme activity test tube containing 2.6mL of Tris-HCl buffer solution with pH of 9, preheating for 5min at 60 ℃, adding 0.20mL of 50mM LNA solution into the test tube, reacting for 10min, adding 1mL of 40% acetic acid to terminate the reaction, uniformly mixing, and standing for 10min;

d. absorbance at 405nm was measured using a visible light spectrophotometer.

(2) Calculation formula

The enzyme activity was calculated according to the following formula:

wherein:

Δod—difference between absorbance of experimental and control;

n-dilution factor;

4-total volume of reactants;

0.2-the volume of enzyme solution added to the reaction system;

slope 0.0411 of K-p-nitroaniline standard curve;

1/10-reaction time 10 in 1 min.

Reference Y.Fundoian o-Hershcovitz, L.Rabinovitch, S.Shulami, V.Reiland, G.Shoham, and Y.Shoham (2005) The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase.FEMS Microbiol Lett,243,157-163.

The present invention provides a method for efficiently producing aminopeptidase by culturing a strain expressing aminopeptidase under suitable conditions and collecting the aminopeptidase from the culture.

According to a preferred embodiment of the invention, the proper condition is that the culture temperature is 35-37 ℃, the rotation speed is 200-220r/min, and the fermentation medium and the seed liquid medium are LB medium, and the composition is as follows:

LB medium: 5g/L yeast powder, 10g/L peptone, 10g/L sodium chloride and the balance of water;

the partial buffer solution involved in the enzyme purification process is as follows (water-based membrane suction filtration after all solutions are prepared, and water used for purifying protein is membrane-coated water):

Lysis Buffer：25mM Tris-HCl，0.5mM EDTA，10mM NaCl，1mM PMSF，pH 7.4；

wash Buffer:25mM Tris-HCl,0.1mM EDTA,1mM DTT,5mM imidazole, pH 7.4;

elution Buffer:25mM Tris-HCl,0.1mM EDTA,1mM DTT,150mM imidazole, pH 7.4.

Primer information according to the present invention and examples is shown in Table 1:

TABLE 1

Primer name	Sequence(s)
		137-F	CGGGATTGGGCTATCCTAAGGACTTTACCGCTGACGC
137-R	ATAGCCCAATCCCGCATTGTAAAGCGGG
		B.subtilis168AP-F	ATCGAATGAGCTTACAGGATCATGAAAAAGCTTTTGACTGTCAT
B.subtilis168AP-R	TTCTAATTACCCTCCCCCGGGTTATTTGATATCTTCAAAAATGTCAGATGC

The invention is further illustrated by the following detailed description.

Example 1: acquisition of aminopeptidase Gene

(1) Acquisition of aminopeptidase Gene

The aminopeptidase gene sequence (SEQ ID NO. 1) derived from B.subtilis 168 is obtained through NCBI database searching, and a primer is designed. The amplification was performed by PCR using the B.subtilis 168 genome as a template and the upstream and downstream primers Bacillus subtilis AP-R and Bacillus subtilis AP-F, and the amplification reaction system and reaction conditions were as follows in Table 2:

TABLE 2

The amplification procedure was set up as follows: pre-denaturation: 98 ℃ for 5min; denaturation: 98 ℃ for 10s; annealing: 55-65 ℃ for 5-15s; extension: 72 ℃ for 5s; reacting for 30 cycles; extension: 72℃for 10min.

And (3) carrying out agarose gel electrophoresis on the PCR product, wherein the size of an aminopeptidase gene electrophoresis band is about 1400bp (shown in figure 1), and recovering the PCR product by using a small amount of DNA recovery kit to obtain the aminopeptidase gene fragment 168AP.

(2) Construction of aminopeptidase plasmid

1) The plasmid was subjected to double digestion to obtain a linear vector, and vector pET-28a (+) was subjected to double digestion using Nde I and Xho I, and the digested system was as shown in Table 3.

TABLE 3 cleavage reaction System

After the preparation of the enzyme digestion system is completed according to the table 3, the reaction is carried out in a water bath kettle at 37 ℃ for 2 hours, and after the reaction is completed, agarose gel recovery is carried out on the reaction product, thus obtaining the pET-28a (+) linear vector.

2) Construction of recombinant plasmids

(1) A seamless cloning system was prepared according to Table 4, and the components were gently mixed after addition.

TABLE 4 seamless cloned ligation system

(2) The prepared sample was reacted at 50℃for 15min.

(3) After the reaction, the sample is placed on ice for cooling for a few seconds, and the ligation product pET28-168AP can be directly transferred into E.coli BL21 by scratching.

3) The ligation product was chemically transferred into E.coli BL21 as follows;

(1) Taking out the centrifuge tube containing E.coli BL21 competent cells from the refrigerator at-80 ℃, placing the centrifuge tube into ice for standing for about 5min, adding a connection product pET28-168AP after melting, gently mixing the mixture by a pipetting gun, and carrying out ice bath for 30min.

(2) The centrifuge tube was removed from the ice bath and rapidly transferred to a 42 ℃ water bath heat shock for 90s, followed by 2min of ice bath.

(3) In an ultra-clean bench, 750 μl of LB culture medium is added into each centrifuge tube, cultured in a shaker at 37deg.C 200 r/mm for more than 45min, plated, cultured at 37deg.C for 12h, and positive transformants are screened for verification, and the correct recombinant strain is named BL21/pET28-168AP.

(4) Construction of mutant plasmids

Taking plasmid pET28-168AP as a template, selecting 137-F and 137-R for Q137P site-directed mutagenesis, and the amplification reaction system is as follows:

the amplification procedure was set up as follows: pre-denaturation: 98 ℃ for 5min; denaturation: 98 ℃ for 10s; annealing: 55-65 ℃ for 5-15s; extension: 72 ℃ for 5s; reacting for 30 cycles; extension: 72℃for 10min.

Removing a plasmid template from the amplified product by using DpnI enzyme, carrying out self cyclization on the PCR product after template removal by using T4 ligase to obtain pET28-Q137P, carrying out P conversion on the newly constructed plasmid pET28-Q137P into large intestine competent cells, picking up transformants, carrying out colony PCR on each transformant, carrying out sequencing on the PCR product by using gold and other intelligent biotechnology limited company, and naming a mutant strain which contains Q137P and is correctly sequenced as BL21/pET28-Q137P.

Example 2: shake flask expression and enzymatic property determination of aminopeptidase and mutants thereof in e.coli BL21

1) Shaking and fermenting:

the genetically engineered strains BL21/pET28-168AP and BL21/pET28-Q137P are respectively streaked on LB solid plates containing kanamycin in three areas, cultured overnight, single colonies are picked up, inoculated into 5mL LB test tubes, cultured for 10 hours at the temperature of 37 ℃ at 200rpm, transferred into 50mL LB culture medium (250 mL shake flask) according to the inoculum size of 2%, cultured at the temperature of 37 ℃ at 200rpm until the bacterial density (OD ₆₀₀ ) IPTG was added to the mixture at 0.6 for induction and expression at 28℃for 12 hours.

2) Purification recovery of aminopeptidase protein

The method adopts a nickel ion affinity chromatography method to purify the protein, and comprises the following specific purification steps:

(1) The fermented broth was centrifuged at 8000rpm for 10min to collect the cells, which were resuspended in Lysis Buffer, and 1% lysozyme and 1% PMSF were added (after adding lysozyme, PMSF) in an amount of 20mL Lysis Buffer.

(2) The resuspended cells were magnetically stirred under ice bath conditions for 25min.

(3) Crushing the thalli for 25min by using an ultrasonic crusher, centrifuging at 10000rpm for 30min after crushing, and collecting supernatant after centrifugation, and then passing through the resin twice by using a Lysis Buffer.

(4) The supernatant was mixed with the resin and poured into a beaker and magnetic stirring was combined for 40min (rotational speed not exceeding 100 rpm).

(5) After the completion of the combination, the supernatant was poured into a resin tube, and the resin was allowed to flow out naturally, and the resin was precipitated.

(6) The residual resin in the beaker is blown and sucked by a gun head, the resin tube is filled, the Flow through is finished, the Wash Buffer (10 mL) with twice the column volume is used for washing off the impurity protein in the resin, and finally the target protein is eluted by 10mL Elution Buffer, and the Elutation Buffer is fully combined with the resin for 5min when the target protein is eluted, and then the target protein flows out, and the target protein which flows out is received.

(7) Selecting a 30KD replacement tube, fully flushing a filter membrane of the replacement tube by using membrane-passing water, adding the membrane-passing water, centrifuging for 20min by using a high-speed machine, pouring target protein into the replacement tube for concentration after cleaning, adding 10mL of 20mM phosphate buffer solution for replacement when the concentration is 1mL, repeating the operation when the replacement solution is 1mL, replacing twice, collecting enzyme liquid in a collection tube as purified enzyme liquid, and preserving at 4 ℃ for measuring enzyme properties.

And (3) detecting the enzyme activity of the aminopeptidase W168AP and the aminopeptidase Q137P after purification, and dividing the enzyme activity (U/mL) by the protein concentration (g/mL) to obtain specific enzyme activity (U/g), wherein the specific enzyme activity of the wild aminopeptidase W168AP is 4253U/g, and the specific enzyme activity of the aminopeptidase Q137P is 4935U/g.

3) Optimal reaction temperature and temperature stability of aminopeptidase mutant

Optimum reaction temperature:

the enzyme activities (pH 9) of the purified aminopeptidase W168AP and Q137P proteins are respectively measured at 30 ℃, 40 ℃, 50 ℃, 55 ℃,60 ℃, 65 ℃, 70 ℃, 75 ℃ and 80 ℃, and the highest enzyme activity at different measured reaction temperatures is 100 percent, and as can be seen from figure 2, the Q137P is obviously improved in the temperature range of 50-65 ℃, and the optimal reaction temperature is improved from 60 ℃ to 65 ℃.

Temperature resistance:

the purified aminopeptidase W168AP and Q137P proteins are respectively placed at 30 ℃, 40 ℃, 50 ℃, 55 ℃,60 ℃, 65 ℃, 70 ℃, 75 ℃ and 80 ℃ for 30min to determine the enzyme activity, the initial enzyme activity is taken as 100%, and according to the figure 3, the mutant Q137P still has 29.5% residual enzyme activity after heat preservation at 60 ℃ for 30min, the W168AP residual is 21%, and the Q137P is improved by 40.5% compared with the wild aminopeptidase.

Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited to the embodiments shown, but rather, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations in form and details can be made therein without departing from the spirit and principles of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims (8)

1. An aminopeptidase mutant, characterized in that the aminopeptidase mutant is characterized by an amino acid sequence set forth in SEQ ID NO:2, and the Q137P mutation is generated based on the wild aminopeptidase shown in the specification.

2. The aminopeptidase mutant of claim 1, wherein the aminopeptidase mutant has an amino acid sequence as set forth in SEQ ID NO: 4.

3. A gene encoding the aminopeptidase mutant of claim 1.

4. A recombinant vector or recombinant strain comprising the coding gene of claim 3.

5. The recombinant vector of claim 4, wherein the expression vector employed includes, but is not limited to WB980, LY-3, pET28.

6. The recombinant strain of claim 4, wherein the host cell employed is E.coli BL21.

7. Use of the recombinant vector or recombinant strain of claim 4 for the production of the aminopeptidase mutant of claim 1.

8. Use of the aminopeptidase mutant of claim 1 for catalyzing protein degradation.