CN116656655A - Aminopeptidase and coding gene thereof - Google Patents
Aminopeptidase and coding gene thereof Download PDFInfo
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- CN116656655A CN116656655A CN202310225690.XA CN202310225690A CN116656655A CN 116656655 A CN116656655 A CN 116656655A CN 202310225690 A CN202310225690 A CN 202310225690A CN 116656655 A CN116656655 A CN 116656655A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/485—Exopeptidases (3.4.11-3.4.19)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/11—Aminopeptidases (3.4.11)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application discloses aminopeptidase and a coding gene thereof, wherein the aminopeptidase is an aminopeptidase sequence derived from Bacillus licheniformis YN, and belongs to the field of food enzyme engineering. According to the application, the electrophoretically pure aminopeptidase is obtained by purifying Bacillus licheniformis YN fermentation liquor, and the aminopeptidase has catalytic activity on substrates leucine-p-nitroaniline, methionine-4-nitroaniline, L-proline-4-nitroaniline, L-lysine-4-nitroaniline, L-arginine-4-nitroaniline, L-valine-4-nitroaniline and L-phenylalanine-4-nitroaniline, wherein the substrates with highest catalytic activity are proline, leucine and lysine in sequence, and the specific enzyme activity reaches 1125U/mg when Leu-pNA is used as the substrate. The enzyme sequence is analyzed by MALDI-TOF-MS, and the primer is designed to clone the aminopeptidase gene to obtain the nucleic acid and protein sequence of the enzyme, thereby laying a foundation for the industrial application of aminopeptidase with high catalytic efficiency.
Description
Technical Field
The application belongs to the technical field of food enzyme engineering, and particularly relates to aminopeptidase and a coding gene thereof.
Background
Aminopeptidases (APs for short, ec 3.4.11) are a class of exoproteases that selectively cleave amino acid residues from the N-terminus of proteins and polypeptides to produce free amino acids. In some proteolysis, the polypeptide chain of single or multiple hydrophobic amino acids contained in the peptide chain has a small part of the hydrophobic amino acids carried by the carboxyl terminal exposed, and is called as bitter peptide, and the bitter taste becomes a main obstacle for the application of soybean protein hydrolysate or polypeptide in the food field.
At present, the protein debittering mainly comprises a physical method, a chemical method and a biological method. The enzyme method biological debittering has the advantages of green, environmental protection, controllable process and the like, and is widely applied to protein debittering, wherein aminopeptidase is one of the most commonly used debittering enzymes, and the aminopeptidase can relieve the bitter taste of the protein by hydrolyzing the N-terminal hydrophobic amino acid residue of the peptide. Aminopeptidase has the purpose of reducing the bitter taste of protein hydrolysates such as casein and soy protein, and is widely applied to industries such as food processing.
The existing aminopeptidase generally has the problems of low yield, poor enzyme activity and the like. Aminopeptidases having high catalytic activity are lacking in the fields of food processing and the like. Therefore, the preparation of aminopeptidase with high catalytic performance is of significance for high value-added processing of food proteins.
Disclosure of Invention
The application aims to: in order to overcome the defects in the prior art, the application provides aminopeptidase and a coding gene thereof, in particular to aminopeptidase BlynAPs with high catalytic activity derived from bacillus licheniformis YN (Bacillus licheniformis YN), which has higher specific enzyme activity for Leu-pNA.
The technical scheme is as follows: in order to achieve the above purpose, the application adopts the following technical scheme:
the application relates to aminopeptidase and a coding gene thereof, wherein the aminopeptidase is an aminopeptidase sequence derived from Bacillus licheniformis YN, and belongs to the field of food enzyme engineering. According to the application, the electrophoretically pure aminopeptidase is obtained by purifying Bacillus licheniformis YN fermentation liquor, and the aminopeptidase has catalytic activity on substrates leucine-paranitroaniline, methionine-4-nitroaniline, L-proline-4-nitroaniline, L-lysine-4-nitroaniline, L-arginine-4-nitroaniline, L-valine-4-nitroaniline and L-phenylalanine-4-nitroaniline, wherein the substrates with highest catalytic activity are proline, leucine and lysine in sequence, and when Leu-pNA is used as a substrate, the specific enzyme activity reaches 1125U/mg. The enzyme sequence is analyzed by MALDI-TOF-MS, and the primer is designed to clone the aminopeptidase gene to obtain the nucleic acid and protein sequence of the enzyme, thereby laying a foundation for the industrial application of aminopeptidase with high catalytic efficiency.
It is a first object of the present application to provide an aminopeptidase whose amino acid sequence is as follows (a) or (b):
(a) An amino acid sequence as shown in SEQ ID NO. 1;
(b) Aminopeptidase derived from SEQ ID NO.1 by substitution and/or deletion and/or addition of one or several amino acid residues of the amino acid sequence of SEQ ID NO. 1.
Alternatively, as an embodiment of the present application, the specific enzyme activity of the aminopeptidase is 1125U/mg when leucine-p-nitroaniline Leu-pNA is used as a substrate.
Alternatively, as an embodiment of the present application, the specific enzyme activity of the aminopeptidase is 1395U/mg when proline-4-nitroaniline Pro-PNA is used as a substrate.
Alternatively, as an embodiment of the present application, when lysine-4-nitroaniline Lys-pNA is used as a substrate, the specific enzyme activity of the aminopeptidase is 911U/mg.
Alternatively, as an embodiment of the present application, the aminopeptidase is derived from bacillus licheniformis YN (Bacillus licheniformis YN). The strain is obtained by applicant self-screening and is named by self-numbering.
It is a second object of the present application to provide a nucleotide sequence which encodes an aminopeptidase as described above.
Alternatively, as an embodiment of the present application, the nucleotide sequence is a DNA molecule as described in any one of the following 1) to 3):
1) A DNA molecule with a coding region shown as SEQ ID NO. 2;
2) A DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in 1) and which encodes an aminopeptidase-related protein;
3) A DNA molecule having more than 90% homology with the DNA sequence defined in 1) and encoding a protein related to aminopeptidase.
Alternatively, as an embodiment of the present application, the stringent conditions may be hybridization and washing of a membrane with a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65℃in a DNA or RNA hybridization experiment.
The third object of the present application is to provide a method for preparing aminopeptidase as described above, comprising the steps of:
(1) Cloning the nucleotide sequence of claim 3 or 4 into an expression vector, and transferring the expression vector into an expression cell to obtain a cell containing the recombinant vector;
(2) Culturing the cell containing the recombinant vector obtained in the step (1), and separating and purifying the cell from the culture by IPTG induction to obtain the aminopeptidase.
Alternatively, as an embodiment of the present application, the electrophoretically pure aminopeptidase is obtained from Bacillus licheniformis YN fermentation broth having a high yield of enzyme activity by separation and purification.
It is a fourth object of the present application to provide a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the above-described gene.
Alternatively, as an embodiment of the present application, the recombinant expression vector includes any one of pMD-19T vector, pXMJ19 vector, pET series vector and pPICZ series vector.
Alternatively, as an embodiment of the present application, the host of the transgenic cell includes any one of E.coli, corynebacterium, bacillus, saccharomyces, or filamentous fungi.
A fifth object of the present application is to provide the use of an aminopeptidase as described above in the pharmaceutical, food or cosmetic field, said use being at least one of the following (c 1) to (c 3):
(c1) As aminopeptidases;
(c2) Preparing a functional oligopeptide;
(c3) Preparing the taste peptide.
Alternatively, as an embodiment of the present application, the method for preparing the taste peptide of (c 2) functional oligopeptide or (c 3) comprises the steps of: the aminopeptidase is added into a hydrolysis reaction system containing protein substrates, and the enzyme hydrolysis reaction is carried out for 2 to 6 hours at the temperature of 25 to 65 ℃ to obtain the corresponding functional oligopeptide including the taste peptide.
Alternatively, as an embodiment of the present application, the temperature of the enzymatic hydrolysis reaction is preferably 30 to 60 ℃, especially 30 to 40 ℃.
Alternatively, as an embodiment of the present application, the protein substrate may include any one or more of leucine-p-nitroaniline, methionine-4-nitroaniline, L-proline-4-nitroaniline, L-lysine-4-nitroaniline, L-arginine-4-nitroaniline, L-valine-4-nitroaniline, and L-phenylalanine-4-nitroaniline.
It is a sixth object of the present application to provide a functional oligopeptide or a taste peptide, prepared according to the use. Or specifically provides a taste peptide, which is prepared according to the application.
It is a seventh object of the present application to provide a recombinant microorganism obtained by introducing the above-mentioned gene into a microorganism of interest and overexpressing the gene.
An eighth object of the present application is to provide the use of said recombinant microorganism for the production of a taste peptide.
It is a ninth object of the present application to provide a kit for producing a taste peptide, said kit comprising said recombinant microorganism.
The tenth object of the present application is to provide a recombinant bacterium obtained by silencing a gene encoding the aminopeptidase in the genome of a target bacterium.
The beneficial effects are that: the aminopeptidase with high catalytic activity and the coding gene thereof provided by the application are characterized in that the aminopeptidase with high enzyme activity yield is obtained from Bacillus licheniformis YN fermentation broth through separation and purification, the enzyme sequence is analyzed by MALDI-TOF-MS, and the aminopeptidase gene is cloned by designing a primer, so that the nucleic acid and protein sequence of the aminopeptidase are obtained. According to measurement, the aminopeptidase has catalytic activity on substrates leucine-p-nitroaniline, methionine-4-nitroaniline, L-proline-4-nitroaniline, L-lysine-4-nitroaniline, L-arginine-4-nitroaniline and L-valine-4-nitroaniline, wherein the substrates with highest catalytic activity are proline, leucine and lysine in sequence, when Leu-pNA is taken as the substrate, the specific enzyme activity of the electrophoresis pure enzyme is 1125U/mg, when proline-4-nitroaniline Pro-PNA is taken as the substrate, the specific enzyme activity of the aminopeptidase is 1395U/mg, and when lysine-4-nitroaniline Lys-pNA is taken as the substrate, the specific enzyme activity of the aminopeptidase is 911U/mg.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of aminopeptidase purification provided by the examples of the present application; wherein lane M is a protein Marker; the other lanes are proteins in different purification steps.
FIG. 2 shows the results of a substrate specificity study of aminopeptidases of the present application.
FIG. 3 shows the results of enzyme activity studies at various temperatures of aminopeptidases of the examples of the present application.
FIG. 4 shows the results of temperature stability studies of aminopeptidases of the present application.
Detailed Description
The application will be further described with reference to the drawings and examples. The application will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are illustrative of the present application and should not be construed as limiting the application described in detail in the claims.
Example 1 obtaining of aminopeptidase Gene sequence
According to the embodiment of the application, the electrophoretically pure aminopeptidase is obtained from Bacillus licheniformis YN fermentation broth with high enzyme activity yield through separation and purification, the enzyme sequence is analyzed by MALDI-TOF-MS, and the primer is designed to clone the aminopeptidase gene, so that the nucleic acid and protein sequence of the aminopeptidase are obtained.
The specific process is as follows:
the inventors have obtained a Bacillus licheniformis strain by self-screening and named Bacillus licheniformis YN (B.lichenifermis YN). The bacillus licheniformis YN (B.lichenifermis YN) is inoculated in a fermentation culture medium (1.2% wheat protein, 0.5% fructose and 0.5% yeast powder) with an inoculum size of 2%, and then is placed at 39 ℃ for culturing for 42 hours by a 200-rotation shaking table, so that fermentation liquor with aminopeptidase activity reaching 5000U/mL is obtained.
Centrifuging the fermentation liquor at 8000rpm and 4 ℃ for 10min to remove thalli, and obtaining a supernatant crude enzyme solution. Slowly adding ammonium sulfate into the crude enzyme solution under ice bath, stirring while adding until saturation is 65%, and standing in a refrigerator at 4deg.C overnight after ammonium sulfate is completely dissolved.
The above mixture was centrifuged at 8000rpm at 4℃for 10min, and the precipitate was collected and redissolved in 50mM Tris-HCl buffer solution pH 8.5. Further purification was performed by passing through a 0.22 μm filter.
(1) Anion exchange chromatography linear elution
Hitrap Q HP column (5 ml) was connected to AKTA protein purification system. Solution A was 50mM Tris-HCl, pH8.5, and solution B was 50mM Tris-HCl, pH8.5, containing 1M NaCl. 10mL of the sample passing through the 0.22 mu m filter membrane is injected by using the solution A pre-balanced filler, and the solution B is subjected to gradient elution, wherein the flow rate is 5.0mL/min. Removing impurities from 30% B, collecting target protein by a 40% B branch pipe, detecting at 280nm wavelength, and measuring aminopeptidase activity.
SDS-PAGE analysis of samples from different protein collection tubes was performed and found to yield electrophoretically pure, non-heterogeneous, active aminopeptidases as shown in FIG. 1.
(2) Aminopeptidase protein bands were then excised on a PAGE gel and digested with trypsin. And analyzing the released short peptide sequence by mass spectrometry MALDI-TOF-MS to obtain partial amino acid sequence of the aminopeptidase. Extracting B.lichenifermis YN genome DNA, and designing corresponding primers by combining partial amino acid sequences obtained by mass spectrum MALDI-TOF-MS determination, wherein the primer sequences are as follows:
F:5’-ATGAGCGACTTCCGGTC-3’；
R：5’-TTAGGACCCGGGGCCGTCGAGCAGG-3’。
PCR amplification is carried out by taking B.lichenifermis YN genome DNA as a template, a target fragment obtained by amplification is connected to a pMD-19T vector, and is transformed into escherichia coli JM109, and after resistance screening and PCR verification, plasmids are extracted and sent to Shanghai students for sequencing. Sequencing results show that the nucleotide sequence is shown as SEQ ID NO.2, the nucleotide sequence is named BlynAPs, and the enzyme is identified as aminopeptidase; the amino acid sequence coded by the nucleotide sequence is shown as SEQ ID NO. 1.
Example 2 enzymatic Property analysis of aminopeptidases
1. Enzyme Activity measurement
The enzyme activity of the purified aminopeptidase was determined using Leucine-p-nitroaniline (L-Leucine-p-nitroaniline, leu-pNA) as a substrate.
The reaction system is as follows: 50mmol/L of Tirs-HCl (pH 8.5) buffer solution and a proper amount of enzyme solution react for 10min at 50 ℃ by taking 10mmol/L of Leu-pNA as a substrate, and 40% acetic acid stops the reaction, and an enzyme-labeled instrument is used for detecting the absorbance at 405 nm. A blank was also placed and the enzyme was inactivated with 40% acetic acid. The specific method is shown in Table 1.
Table 1 sample and blank treatment modes
Definition of aminopeptidase enzyme activity: the amount of enzyme required to hydrolyze Leu-pNA to 1. Mu.g pNA per minute at pH8.5 and a temperature of 50℃is 1 enzyme activity unit, indicated as U.
The enzyme activity calculation formula of the aminopeptidase is as follows:
U＝Y×20×138.12×N/10 (1)
wherein U represents enzyme activity, and the unit is U/mL; y is the concentration of pNA calculated from the standard curve in mmol/L; n is the dilution multiple of the enzyme solution; 20 is the dilution multiple of the total reaction system to the sample enzyme solution; 138.12 is the relative molecular mass of p-nitroaniline; 10 is a reaction time of 10min.
In this example, n=500 and y=0.25 in the sample group were substituted into formula (1), and the enzyme activity was calculated as 34530U/mL.
2. Protein content determination:
the protein content of the samples was determined by the Bradford method.
3. The calculation formula of specific enzyme activity:
specific enzyme activity (U/mg) =enzyme activity (U)/protein amount (mg) (2)
And (3) performing enzyme activity and protein content measurement on the electrophoresis pure aminopeptidase obtained by purifying the B.lichenifermis YN fermentation supernatant. The result shows that the protein amount is 30.69mg/mL, and the specific enzyme activity is 1125U/mg after substituting the protein amount into the formula (2).
4. Substrate specificity Studies of enzymes
The measurement system and method were as shown in Table 1 above, with Leucine-p-nitroaniline (L-Leucine-p-nitroaniline, leu-pNA), L-methionine-4-nitroaniline (L-Methionine pnitroanilide, L-Met-PNA), L-proline-4-nitroaniline (L-Proline pnitroanilide trifluoroacetate salt, pro-PNA), L-Lysine-4-nitroaniline (L-Lysine p-nitroanilide dihydrobromide, lys-PNA), L-Arginine-4-nitroaniline (L-Arginine p-nitroanilide dihydrochloride, arg-PNA), L-valine-4-nitroaniline, L-phenylalanine-4-nitroaniline as a control, respectively.
As a result, as shown in FIG. 2, aminopeptidase has catalytic activity on the above substrates, wherein the substrate having the highest catalytic activity is proline (1395U/mg, 124%), followed by leucine (1125U/mg, 100%) and lysine (911U/mg, 81%).
5. Investigation of the optimal reaction temperature of the enzyme
The water bath temperatures in Table 1 were controlled at 30, 40, 50, 60, 70, 80, 90℃respectively, and the other steps were unchanged, and the enzyme activities at different temperatures were measured. The aminopeptidase activity was found to be highest at 70℃and reached 1475U/mg. The relative enzyme activities at different temperatures are calculated by taking the highest enzyme activity of aminopeptidase at 70 ℃ as a control, and the result is shown in figure 3, wherein the optimal enzyme activity temperature of aminopeptidase is 60-70 ℃, and the relative enzyme activities are maintained above 60% when the enzyme activity of aminopeptidase is 30-90 ℃.
The temperature in the method for measuring the enzyme activity in Table 1 is a temperature range commonly used in the literature, and specifically, the optimum temperature of different enzymes, and also factors such as stability of the enzyme during measurement at high temperature need to be considered, so that the temperature selected does not necessarily coincide with the enzyme activity measurement temperature selected at the initial stage of the study.
6. Temperature stability study of enzymes
The purified enzyme solution with the concentration of 100mg/mL is incubated at 30, 40, 50, 60, 70, 80 and 90 ℃ for 1h, then cooled on ice for 5min, and the residual enzyme activity is measured, and an unheated and incubated sample with an ice bath is used as a control.
As a result, as shown in FIG. 4, the enzyme activity of aminopeptidase gradually decreased with increasing incubation temperature at 30 to 90℃to show that incubation temperature has an important effect on the enzyme activity stability of aminopeptidase, wherein the appropriate incubation temperature is 30 to 60℃and can be suitably adjusted to 25 to 65℃so that the relative enzyme activity at this time is maintained at 85% or more.
EXAMPLE 3 use of aminopeptidase
The aminopeptidase prepared in example 1 is applied to the fields of pharmacy, food or cosmetics, and can be used as aminopeptidase, and the application fields of the aminopeptidase belong to the protection scope of the application. The application particularly suggests that it may be used in particular for the preparation of functional oligopeptides, such as for the preparation of taste peptides.
The method for preparing the functional oligopeptide such as the taste peptide by utilizing the aminopeptidase comprises the following steps: the aminopeptidase prepared in the example 1 is added into a hydrolysis reaction system containing a protein substrate, and the enzyme hydrolysis reaction is carried out for 2 to 6 hours at the temperature of 25 to 65 ℃ to obtain the corresponding taste peptide or functional oligopeptide. In the embodiment, the enzyme hydrolysis reaction is carried out for 3 hours at 50 ℃ to obtain the corresponding functional oligopeptide, namely the taste peptide, which comprises the following specific steps:
(1) Protease enzymolysis: preparing a soybean protein isolate solution of 100mg/mL, regulating pH=9 by using a NaOH (1 mol/L) solution, adding alkaline protease with enzyme ratio of 1000U/g to a substrate, reacting for 3 hours at 50 ℃, inactivating enzyme in a water bath of 95 ℃ for 15 minutes after the reaction is finished, and rapidly cooling to room temperature, wherein the product is used as a blank control group;
(2) Aminopeptidase enzymolysis: and (3) continuously adding aminopeptidase with enzyme ratio of 2000U/g to the substrate into the blank control group obtained in the step (1), reacting for 3 hours at 50 ℃, and finally thoroughly inactivating enzyme in a boiling water bath for 15 minutes, and cooling to room temperature, wherein the product is used as an experimental group.
(3) And (3) centrifuging: and centrifuging enzymolysis solutions of the blank control group and the experimental group to obtain supernatant, filtering the supernatant to obtain two groups of soybean oligopeptide, and detecting the bitter value of the soybean oligopeptide.
The bitterness of the sample is detected by using an electronic tongue technology, the bitterness of the blank group oligopeptide obtained by enzymolysis of soybean protein isolate by alkaline protease is 9.89, the bitterness of the experimental group oligopeptide obtained by enzymolysis of alkaline protease and aminopeptidase is 5.36, the bitterness is reduced by 45.8%, and the bitterness is obviously improved.
Similarly, it can be deduced from the nature of the gene sequence that the DNA molecule which hybridizes with the DNA sequence of the DNA molecule whose coding region is shown as SEQ ID NO.2 under stringent conditions and which codes for an aminopeptidase-related protein, or the DNA molecule which has more than 90% homology with its DNA sequence and which codes for an aminopeptidase-related protein, has the same functional properties as the above-mentioned nucleotides shown as SEQ ID NO.2, and these nucleotides can be used for the preparation of aminopeptidases. In addition, the gene can be further prepared into a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium, wherein the recombinant expression vector comprises any one of a pMD-19T vector, a pXMJ19 vector, a pET series vector and a pPICZ series vector, and the host of the transgenic cell comprises any one of escherichia coli, corynebacterium, bacillus, saccharomycetes or filamentous fungi. Furthermore, the above-mentioned gene or recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium can be further used for preparing a recombinant microorganism, such as a recombinant microorganism obtained by introducing the above-mentioned gene into a microorganism of interest and overexpressing the gene, which recombinant microorganism is used for producing functional oligopeptide, including a kit for producing taste peptide, which kit includes the recombinant microorganism. The gene encoding the aminopeptidase in the genome of the target bacterium can be silenced to obtain a recombinant bacterium.
It should be noted that the aminopeptidase obtained by the present application further includes aminopeptidases derived from SEQ ID NO.1 by substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence of SEQ ID NO.1, and all have the same functional characteristics as the amino acid sequence shown as SEQ ID NO. 1.
The foregoing is only a preferred embodiment of the application, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present application, and such modifications and adaptations are intended to be comprehended within the scope of the application.
Claims (10)
1. An aminopeptidase, wherein the amino acid sequence of the aminopeptidase is (a) or (b) as follows:
(a) An amino acid sequence as shown in SEQ ID NO. 1;
(b) Aminopeptidase derived from SEQ ID NO.1 by substitution and/or deletion and/or addition of one or several amino acid residues of the amino acid sequence of SEQ ID NO. 1.
2. The aminopeptidase of claim 1, wherein the aminopeptidase has a specific activity of 1125U/mg when Leu-pNA is used as a substrate, or a specific activity of 1395U/mg when proline-4-nitroaniline Pro-pNA is used as a substrate, or a specific activity of 911U/mg when lysine-4-nitroaniline Lys-pNA is used as a substrate.
3. A nucleotide sequence encoding the aminopeptidase of claim 1.
4. A nucleotide sequence according to claim 3, characterized in that the nucleotide sequence is a DNA molecule according to any one of the following 1) to 3):
1) A DNA molecule with a coding region shown as SEQ ID NO. 2;
2) A DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in 1) and which encodes an aminopeptidase-related protein;
3) A DNA molecule having more than 90% homology with the DNA sequence defined in 1) and encoding a protein related to aminopeptidase.
5. The method for producing aminopeptidase according to claim 1 or 2, characterized by comprising the steps of:
(1) Cloning the nucleotide sequence of claim 3 or 4 into an expression vector, and transferring the expression vector into an expression cell to obtain a cell containing the recombinant vector;
(2) Culturing the cell containing the recombinant vector obtained in the step (1), and separating and purifying the cell from the culture by IPTG induction to obtain the aminopeptidase.
6. A recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium comprising the gene of claim 3 or 4, characterized in that the recombinant expression vector comprises any one of a pMD-19T vector, a pXMJ19 vector, a pET-series vector and a pPICZ-series vector; the host of the transgenic cell comprises any one of escherichia coli, corynebacterium, bacillus, saccharomycete or filamentous fungi.
7. Use of an aminopeptidase according to claim 1 or 2 in the pharmaceutical, food or cosmetic field, characterized in that the use is at least one of the following (c 1) to (c 3):
(c1) As aminopeptidases;
(c2) Preparing a functional oligopeptide;
(c3) Preparing the taste peptide.
8. The use according to claim 7, wherein the method of (c 2) preparing a functional oligopeptide or (c 3) preparing a taste peptide comprises the steps of: the aminopeptidase is added into a hydrolysis reaction system containing protein substrates, and the enzyme hydrolysis reaction is carried out for 2 to 6 hours at the temperature of 25 to 65 ℃ to obtain the corresponding functional oligopeptide or flavor peptide.
9. A taste peptide prepared according to the use of claim 7 or 8.
10. A recombinant microorganism obtained by introducing the gene according to claim 3 or 4 into a microorganism of interest and overexpressing the gene.
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