阅读说明：本技术 一种微生物源氨基肽酶及其制备与应用 (Microbial aminopeptidase and preparation and application thereof ) 是由 程昌勇 宋厚辉 孙静 杭奕 于 2019-10-08 设计创作，主要内容包括：本发明涉及基因工程和生物酶技术,旨在提供一种微生物源氨基肽酶及其制备与应用。该氨基肽酶的氨基酸序列如SEQ ID NO.1所示,在食品工业和生物制药行业作为蛋白质或多肽的水解酶应用。本发明采用基因工程表达进行实现,克服了天然菌中氨基肽酶含量少的缺点,可以批量发酵；可溶性表达形式避免了其他微生物源蛋白酶为包涵体的缺点。该蛋白酶广泛存在于不同物种的生物体中；具有食品风味改善,生物活性肽制备和神经性化学毒剂特异性清除等功能。其物理特征特征为可溶性形式,且保留天然活性,因而具备广泛应用场景。(The invention relates to genetic engineering and biological enzyme technology, and aims to provide a microbial aminopeptidase and preparation and application thereof. The amino acid sequence of the aminopeptidase is shown as SEQ ID NO.1, and the aminopeptidase can be used as a hydrolase of protein or polypeptide in the food industry and the biopharmaceutical industry. The invention is realized by adopting gene engineering expression, overcomes the defect of low aminopeptidase content in natural bacteria, and can be fermented in batch; the soluble expression form avoids the disadvantage that other proteases of microbial origin are inclusion bodies. The protease is widely present in organisms of different species; has the functions of improving the flavor of food, preparing bioactive peptide, specifically eliminating nerve chemical toxic agent, etc. The physical characteristics of the protein are soluble forms, and the natural activity is retained, so that the protein has a wide application scene.)

1. An aminopeptidase derived from a microorganism, wherein the amino acid sequence of the aminopeptidase is represented by SEQ ID NO. 1.

2. Use of the aminopeptidase of claim 1 as a hydrolase for proteins or polypeptides in the food industry and biopharmaceutical industry.

3. The method for producing aminopeptidase of claim 1, wherein a PCR amplification reaction is carried out using a genomic DNA of a wild-type strain of Listeria monocytogenes as a template to obtain a target fragment containing an aminopeptidase target gene and a cleavage site of the aminopeptidase target gene; carrying out double enzyme digestion on a target gene and a vector by using restriction enzymes Kpn I and BamH I, and then connecting the target gene into an expression vector pET30a (+); transforming the ligation product into competent cells E.coli DH5 alpha, and culturing on Kana/LB solid agar medium; putting the positive cloning recombinant bacterium plasmid into a competent cell E.coli Rosetta, taking a monoclonal colony for amplification culture, and separating and purifying to obtain a recombinant protein, namely the microbial aminopeptidase, through ultrasonic disruption, imidazole elution and nickel ion resin column affinity chromatography.

4. The method according to claim 3, characterized in that it comprises in particular the steps of:

(1) amplifying the genome DNA of a Listeria wild strain by using a specific primer, and designing enzyme cutting sites of Kpn I and BamH I on the primer while amplifying, wherein the sequences of the specific primer are respectively as follows:

an upstream primer: 5'-CGGGGTACCATGACAGTATTTAGTGAAAAGTTAGAAAAGTATGC-3', respectively; a downstream primer: 5'-CGCGGATCCTTAGAACGCCCAGTCGCCTTTA-3', respectively;

the PCR amplification system is as follows: KOD-plus-Neo 1. mu.L, 10 XPCR Buffer for KOD-plus-Neo 5. mu.L, MgSO₄mu.L, dNTPs 5. mu.L, upstream primer 1. mu.L, downstream primer 1. mu.L, DNA template 2. mu.L, plus ddH₂O to 50 μ L; the PCR amplification conditions were: pre-denaturation at 94 ℃ for 30s, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 68 ℃ for 1min for 30s, circulation for 25-35 times, and extension at 68 ℃ for 8-12 min;

(2) respectively carrying out double digestion on pET30a (+) plasmid and amplification product by using restriction enzymes Kpn I and BamH I, recovering the target product after electrophoresis, and then carrying out overnight connection at 16 ℃ by using T4 DNA ligase to connect the target gene with the vector; transforming the ligation product into a competent cell E.coli DH5 alpha, coating the competent cell E.coli DH5 alpha on a Kana/LB agar solid culture medium, and culturing overnight at 37 ℃ to obtain a recombinant strain; culturing in Kana/LB agar solid culture medium with kanamycin concentration of 50mg/L and agar concentration of 1.5%;

(3) selecting the recombinant strain to be monoclonal to 5mL Kana/LB agar liquid culture medium, and staying overnight at 37 ℃; extracting plasmid after culturing, and sequencing;

(4) transforming the plasmid with correct sequencing into E.coli Rosetta, and culturing overnight; then, the recombinant strain is selected to be monoclonal to 500mL Kana/LB culture medium, and cultured at 37 ℃ and 150 r/min; culturing to OD_600nmWhen the concentration is 0.6, adding IPTG with the final concentration of 0.6mM, and inducing for 8 hours at 30 ℃ at the speed of 100-300 r/min to obtain protein induction expression bacterial liquid; centrifuging the bacterium liquid 3700r/min for 15min, discarding the supernatant, and collecting the precipitate; then adding 30mL of 50mM PBS, and using a cell ultrasonic crusher to crack cells; centrifuging at 3700r/min for 15min to collect supernatant, combining the supernatant with nickel column at 4 deg.C for 4h, and purifying with column; the heteroprotein was eluted using 30mL, 50mM imidazole and thenEluting the target protein by using 6-8mL of 400mM imidazole to finally obtain the purified active protein, namely the microbial aminopeptidase.

Technical Field

The invention belongs to gene engineering and biological enzyme technology, and particularly relates to preparation and application of microbial aminopeptidase.

Background

Aminopeptidases (APs for short, EC 3.4.11) are exoproteases that selectively cleave amino acid residues from the N-terminus of proteins and polypeptides to produce free amino acids, and are widely found in organisms of various species, including mammals, plants, microorganisms, and the like. Because of the variety of aminopeptidases, which modify the N-terminal residues of various proteins, the proteases have wide applications in the food industry and biopharmaceuticals. For example, the method can be used for removing the bitter taste of protein hydrolysate, the protein hydrolysate hydrolyzed by protease usually has the bitter taste, and the bitter taste can be removed by further hydrolyzing the protein hydrolysate by aminopeptidase; the deep hydrolysis of the protein, the compound use of aminopeptidase and protease, can greatly improve the degree of hydrolysis of the protein, in the preparation process of soy sauce brewing, cheese production and other protein hydrolysis products, can improve the utilization ratio of the protein, save the cost; the preparation of bioactive polypeptide can be carried out by controlling the enzymolysis of protein, the bioactive polypeptide is mostly cell growth factor, the content of the bioactive polypeptide in the body is extremely low, but the bioactivity is extremely high, and the bioactive polypeptide has the functions of health care and beauty treatment. Because of its high production and application value, the research on aminopeptidase has been more and more focused.

Currently, commercially available aminopeptidases are mainly produced by extraction from animals and plants and microbiological methods. The extraction of aminopeptidase from animals and plants is generally mainly used for characterization of enzymology properties and diagnosis of related diseases; in the latter case, although there are many kinds of aminopeptidase-producing microorganisms, most of the strains are not suitable for mass production of aminopeptidase due to low enzyme-producing ability or potential safety hazard.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects in the prior art and provides a microbial aminopeptidase and preparation and application thereof.

In order to solve the technical problem, the solution of the invention is as follows:

provides a microbial aminopeptidase, the amino acid sequence of the aminopeptidase is shown in SEQ ID NO. 1.

The invention further provides the use of said aminopeptidase in the food industry and in the biopharmaceutical industry as a hydrolase for proteins or polypeptides.

The invention also provides a preparation method of the protease, which is characterized in that genome DNA of a wild Listeria monocytogenes strain is used as a template to carry out PCR amplification reaction to obtain a target fragment containing an aminopeptidase target gene and containing a restriction enzyme cutting site; carrying out double enzyme digestion on a target gene and a vector by using restriction enzymes Kpn I and BamH I, and then connecting the target gene into an expression vector pET30a (+); transforming the ligation product into competent cells E.coli DH5 alpha, and culturing on Kana/LB solid agar medium; putting the positive cloning recombinant bacterium plasmid into a competent cell E.coli Rosetta, taking a monoclonal colony for amplification culture, and separating and purifying to obtain a recombinant protein, namely the microbial aminopeptidase, through ultrasonic disruption, imidazole elution and nickel ion resin column affinity chromatography.

In the invention, the preparation method specifically comprises the following steps:

(1) amplifying the genome DNA of a Listeria wild strain by using a specific primer, and designing enzyme cutting sites of Kpn I and BamH I on the primer while amplifying, wherein the sequences of the specific primer are respectively as follows:

an upstream primer: 5'-CGGGGTACCATGACAGTATTTAGTGAAAAGTTAGAAAAGTATGC-3', respectively;

a downstream primer: 5'-CGCGGATCCTTAGAACGCCCAGTCGCCTTTA-3', respectively;

the PCR amplification system used was: KOD-plus-Neo 1. mu.L, 10 XPCR Buffer for KOD-plus-Neo 5. mu.L, MgSO₄mu.L, dNTPs 5. mu.L, upstream primer 1. mu.L, downstream primer 1. mu.L, DNA template 2. mu.L, plus ddH₂O to 50 μ L; the PCR amplification conditions were: pre-denaturation at 94 ℃ for 30s, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 68 ℃ for 1min for 30s, circulation for 25-35 times, and extension at 68 ℃ for 8-12 min;

(2) respectively carrying out double digestion on pET30a (+) plasmid and amplification product by using restriction enzymes Kpn I and BamH I, recovering the target product after electrophoresis, and then carrying out overnight connection at 16 ℃ by using T4 DNA ligase to connect the target gene with the vector; transforming the ligation product into a competent cell E.coli DH5 alpha, coating the competent cell E.coli DH5 alpha on a Kana/LB agar solid culture medium, and culturing overnight at 37 ℃ to obtain a recombinant strain; culturing in Kana/LB agar solid culture medium with kanamycin concentration of 50mg/L and agar concentration of 1.5%;

(3) selecting the recombinant strain to be monoclonal to 5mL Kana/LB agar liquid culture medium, and staying overnight at 37 ℃; extracting plasmid after culturing, and sequencing;

(4) transforming the plasmid with correct sequencing into E.coli Rosetta, and culturing overnight; then, the recombinant strain is selected to be monoclonal to 500mL Kana/LB culture medium, and cultured at 37 ℃ and 150 r/min; culturing to OD_{600 nm}When the concentration is 0.6, adding IPTG with the final concentration of 0.6mM, and inducing for 8 hours at 30 ℃ at the speed of 100-300 r/min to obtain protein induction expression bacterial liquid; centrifuging the bacterium liquid 3700r/min for 15min, discarding the supernatant, and collecting the precipitate; then adding 30mL of 50mM PBS, and using a cell ultrasonic crusher to crack cells; centrifuging at 3700r/min for 15min to collect supernatant, combining the supernatant with nickel column at 4 deg.C for 4h, and purifying with column; eluting the hybrid protein by using 30mL of 50mM imidazole, and eluting the target protein by using 6-8mL of 400mM imidazole to finally obtain the purified active protein, namely the microbial aminopeptidase.

Compared with the prior art, the invention has the beneficial effects that:

1. the aminopeptidase in the invention selects mature commercial engineering bacteria escherichia coli Rosetta for expression, and the bacteria have the characteristics of safety, stable expression and easy culture; the preparation technology of the microbial aminopeptidase is realized by adopting genetic engineering expression, overcomes the defect of low aminopeptidase content in natural bacteria, and can realize batch fermentation; meanwhile, the aminopeptidase in the invention is in a soluble expression form, so that the defect that other microbial protease is inclusion body is avoided. The aminopeptidase of the present invention was tested in a number of repeated experiments to confirm that it had aminopeptidase activity.

2. The microbial aminopeptidase of the present invention belongs to the aminopeptidase M29 family II, and is derived from Listeria monocytogenes. The protease is an exoprotease which can selectively cut off amino acid residues from the N end of proteins and polypeptides to generate free amino acids, and is widely present in organisms of different species, including mammals, plants, microorganisms and the like. Aminopeptidases are diverse in kind and can modify the N-terminal residues of a wide variety of proteins. On one hand, in bacteria and cells, the protease plays a role in participating in modification and maturation of target proteins, antigen presentation of immune cells and detection indexes of liver and kidney injury; on the other hand, in industrial applications, the aminopeptidase has functions of food flavor improvement, preparation of bioactive peptides, specific elimination of nerve chemical poisons, and the like.

3. The recombinant strain constructed by the method can efficiently and rapidly express active protein, the target protein is high in yield, the protein has high aminopeptidase activity, better technical support is provided for industrial production, food processing and medical health care of the protease, and the recombinant strain has higher research and potential application values.

4. The physical characteristics of the microbial aminopeptidase are characterized by a soluble form, and natural activity is retained, so that the microbial aminopeptidase has a wide application scene.

Drawings

Fig. 1 shows the induced expression and purification of recombinant protein in e.

In the figure, M is a protein Marker, and 1, 2, 3, 4 and 5 are purified recombinant proteins.

Detailed Description

The Listeria monocytogenes wild strain refers to an EGD-e strain, and the strain is a standard strain purchased from ATCC.

The main reagents are as follows: LB medium, agarose H, agar, imidazole, ampicillin, kanamycin, IPTG were purchased from Shanghai Biotech, BHI medium was purchased from Oxoid, UK, KOD-plus-Neo (PCR kit) was purchased from TOYOBO, PCR product purification/recovery kit was purchased from Shanghai Rier maple Biotech, Inc., restriction endonuclease was purchased from NEB, T4 ligase was purchased from Takara, plasmid extraction kit was purchased from Tiangen Biochemical, Inc.

The main apparatus is as follows: shaking table (HZ-9211K), vortex oscillator (ZEALWAY GI541), multifunctional enzyme labeling instrument (BioTek SynergyTM H1), gradient PCR instrument (Eppendorf), gel imaging system (UVP), metal bath (Thermo), biological safety cabinet (BSC-II), protein electrophoresis instrument (BIORAD)

The technical solution of the present invention is further explained by the following embodiments with reference to the attached drawings, but the scope of the present invention is not limited in any way by the embodiments.

1. Construction of recombinant expression bacteria

(1) Specific primers (SEQ ID NO.2 and SEQ ID NO.3) are adopted to amplify the genome DNA of a Listeria wild strain (such as EGDe standard strain of ATCC or other Listeria standard strains), and Kpn I and BamH I enzyme cutting sites are designed on the primers during amplification, wherein the amplification primers are as follows:

SEQ ID NO.2：

upstream primer 5'-CGGGGTACCATGACAGTATTTAGTGAAAAGTTAGAAAAGTATGC-3'

SEQ ID NO.3：

Downstream primer 5'-CGCGGATCCTTAGAACGCCCAGTCGCCTTTA-3'

The PCR amplification system is as follows: KOD-plus-Neo 1. mu.L, 10 XPCR Buffer for KOD-plus-Neo 5. mu.L, MgSO₄mu.L, dNTPs 5. mu.L, upstream primer 1. mu.L, downstream primer 1. mu.L, DNA template 2. mu.L, plus ddH₂O to 50. mu.L. The PCR amplification conditions were: pre-denaturation at 94 ℃ for 30s, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 68 ℃ for 1min for 30s, circulation for 25-35 times, and extension at 68 ℃ for 8-12 min.

(2) The plasmid pET30a (+) and the amplification product are respectively cut by restriction enzymes Kpn I and BamH I, and the target product is recovered after 1% electrophoresis. The gene of interest was then ligated to the vector using T4 DNA ligase overnight at 16 ℃. Finally, the ligation product is transformed into a competent cell E.coli DH5 alpha, coated on a Kana/LB agar solid culture medium, kept stand at 37 ℃ and cultured overnight to obtain a recombinant strain; the culture was carried out in Kana/LB agar solid medium, kanamycin concentration was 50mg/L, and agar concentration was 1.5%.

(3) The recombinant strain is selected to be monoclonal to 5mL Kana/LB liquid culture medium, cultured overnight at 37 ℃ at 150r/min, and then identified by methods such as PCR, double enzyme digestion plasmid, sequencing and the like.

2. Expression and purification of recombinant proteins

And (3) transforming the positive plasmid with correct sequencing into an escherichia coli competent cell E. The recombinant strain is selected to be monoclonally cultured in 5mL liquid Kana/LB culture medium for overnight culture, and then 1mL bacterial liquid is taken to be transferred into 500mL Kana/LB culture medium for fermentation culture at 37 ℃ and 150 r/min. Culturing to OD_600nmWhen the concentration is 0.6, adding IPTG with the final concentration of 0.6mM, and inducing for 8 hours at 30 ℃ at the speed of 100-300 r/min to obtain the protein induction expression bacterial liquid. Centrifuging the bacterial solution 3700r/min for 15min, discarding the supernatant, collecting the precipitate, adding 30mL of 50mM PBS, lysing the cells with a cell ultrasonication instrument, centrifuging 3700r/min for 15min, collecting the supernatant, combining the supernatant with a nickel column at 4 ℃, 4h, and purifying with a column. The purified active protein (i.e., the microbial aminopeptidase of the present invention) is finally obtained by eluting the hybrid protein with 30mL of 50mM imidazole and eluting the target protein with 6-8mL of 400mM imidazole.

The initial LB culture medium comprises, by mass, 0.9-1.6% of tryptone, 0.8-1.4% of sodium chloride, 0.5-0.7% of yeast extract, and the balance of deionized water.

The purity of the active protein was checked by SDS-PAGE.

The amino acid sequence of the aminopeptidase was determined as follows: (1-410aa)

The format of the triple codon of the amino acid sequence is shown as SEQ ID NO. 1.

3. Protease activity assay

The exoenzyme activity of the aminopeptidase is performed by a method of hydrolyzing amino acid p-nitroaniline.

The specific method comprises the following steps: in a 96-well plate, 200. mu.l of a reaction system was added with 25mM Tris-HCl buffer, 0.5. mu.M purified aminopeptidase and 1mM amino acid p-nitroaniline, respectively, and OD was measured with a microplate reader_405nmDetecting absorbance value, setting 3 parallel experiments and enzyme labeling for each reactionThe reading interval of the instrument is 1h, and the reading is continuously carried out for 12 h. Through repeated determination, the aminopeptidase in the invention respectively takes Leu-pNA, Arg-pNA and Lys-pNA as substrates, and utilizes the Mie's equation to obtain the enzyme activity reaction efficiency parameter k_cat/K_mThe values are 3.149X 10 respectively⁵min^-1M^-1、1.536×10⁵min^-1M^-1And 1.035X 10⁴min^-1M^-1. This shows that the aminopeptidase of the present invention has strong aminopeptidase activity and wide substrate selectivity, and can recognize and cut various N-terminal residues of protein or polypeptide, so as to hydrolyze and modify protein, and has the highest leucine residue hydrolyzing efficiency.

In the production of hydrolyzed proteins or polypeptides actually used in the food industry and biopharmaceutical industry, the aminopeptidase of microbial origin of the present invention should be present in liquid form.

Sequence listing

<110> Zhejiang agriculture and forestry university

<120> microbial aminopeptidase, and preparation and application thereof

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Pro Val Asp Ala Ala Pro Leu Val Arg Leu Ile Ser Lys Tyr Ala Phe

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Glu Val Gly Ala Glu Asp Val Ile Met Asp Trp Arg Asp Glu Glu Leu

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Gly Ala Leu Arg Tyr Lys Asn Ala Pro Leu Arg Val Phe Glu Ser Ala

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Pro Val His Arg Val Ala Glu Lys Thr Glu Leu Ala Lys Glu Gly Ala

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Cys Phe Ile Ser Ile Thr Ser Glu Asp Pro Asp Leu Leu Asn Gly Val

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Asp Ser Asn Lys Ile Ala Thr Phe Gln Lys Thr Met Gly Gly Ala Met

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Ser Glu Phe Arg Glu Leu Met Gln Ala Asn Val Val Ser Trp Thr Val

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Val Ala Ala Ala Ser Gln Gly Trp Ala Ala Lys Val Phe Pro Asp Leu

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Thr Pro Ala Glu Gln Met Glu Thr Leu Trp Glu Ala Ile Phe Glu Thr

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Thr Arg Ile Asn Thr Glu Asn Pro Val Glu Thr Trp Lys Asn His Asp

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Gln Thr Leu Ala Ala Lys Ala Glu Ser Leu Asn Glu Lys Gln Phe Thr

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Ser Leu His Tyr Thr Ala Pro Gly Thr Asp Leu Thr Ile Gly Leu Pro

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Glu Phe Met Ala Asn Met Pro Thr Glu Glu Val Phe Cys Cys Ala Asp

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Lys Leu Lys Val Glu Gly Tyr Val Ser Ser Thr Lys Pro Leu Ser Tyr

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Ala Gly Asn Ile Ile Asp Asp Phe Lys Ile Thr Phe Glu Lys Gly Arg

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Ile Val Gly Val Glu Ala Ala Ser Gly Glu Glu Ile Leu Lys Asp Leu

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Ile Ala Thr Asp Glu Gly Ser His Tyr Leu Gly Glu Val Ala Leu Val

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Pro Asp Pro Ser Pro Ile Ser Gln Ser Gly Ile Leu Phe Tyr Asn Thr

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Ser Ser Glu Met Asp Ile Asp Gly Val Thr Glu Ser Gly Glu Val Val

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Pro Val Phe Arg Lys Gly Asp Trp Ala Phe

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