KR100757278B1 - Hyperthermophilic Aminopeptidase P and Methods of Preparation Thereof - Google Patents

Abstract

The present invention relates to a highly thermophilic aminopeptidase P enzyme and a method for preparing the same, wherein Thermococcus sp. Novel highly thermophilic aminopeptidase P isolated from strain (KCTC 10859BP) and its functional equivalents, novel genes encoding them and methods for producing them. Proteolytic enzymes according to the invention are highly thermophilic.

Protease, Aminopeptidase P, Hyperthermia

Description

Hyperthermophilic Aminopeptidase P and Methods of Preparation Thereof

1 is Thermococcus sp. Dipeptidase from aminopeptidase P and P. horikoshii OT3 (PhOT3, gi: 14590819) from NA1 (TNA1), T. kodakarensis KOD1 (TkKOD1, gi: 57641390) and P. abyssi GE5 (PaGE5, gi: 14521082) Sequence comparison is shown. -Indicates a blank, the number on the right indicates the position of the last residue in the original sequence. The same residues between the four enzymes in the figure are indicated by an asterisk (*) and the conservative and semi-conservative substitutions by two points (:) and one point (.), Respectively. Putative active site residues that participate in metal ion coordination and proton exchange are shown in bold and underline, respectively.

2 shows the results of SDS-PAGE (12%) of purified enzymes. The molecular weight standards of M lanes are phosphorylase b (103 kDa), bovine serum albumin (77 kDa), ovalbumin (50 kDa), carbonic anhydrase; 34.3 kDa), soybean trypsin inhibitor (28.8 kDa) and lysozyme (20.7 kDa). The bands corresponding to the enzymes of the invention are indicated by arrows.

Figure 3 shows the effect of temperature (A) and pH (B, C) on the activity of aminopeptidase P (TNA1_APP). A: The sample temperature was increased from 40 to 100 ° C., and activity analysis was performed at standard conditions. B, C: sodium acetic acid (circle), pH 4-6; KMES (square), pH 5.5-7; MOPS (triangle), pH 6.57.5; HEPES (reverse triangle), pH 7-8 buffer solution (50 mM each) was used and hydrolysis of Lys (N ^ε -Abz) -Pro-Pro-pNA (transparency) and Met-Pro (filled) The activity analysis was carried out in the standard state.

4 shows heat inactivation of aminopeptidase P (TNA1_APP). The incubation time for the semilog curve of remaining activity is shown. APP (4 nM) was incubated in 50 mM sodium acetate buffer, 1.2 mM CoCl ₂ , at pH 5 at 80 ° C. or 90 ° C. At the time shown in the figure, a portion of the sample was taken and its activity was measured in the same buffer at 80 ° C using Met-Pro as substrate. The lines shown in the figure were obtained by linear regression analysis of the data. Small inset shows activation of enzyme by heat treatment.

5 shows the effect of metal ions on the activity of aminopeptidase P (TNA1_APP). Activity analysis was performed under standard conditions, but the concentration of divalent metal ions varied: Co ²⁺ (•), Mn ²⁺ (■), and Zn ²⁺ (▲).

6 shows the effect of increasing substrate concentration on aminopeptidase P (TNA1_APP) activity. Activity analysis was performed under standard conditions, but the concentration of Met-Pro varied.

Figure 7 shows a cleavage map of a recombinant plasmid having a recombinant aminopeptidase enzyme according to the present invention.

Recent advances in the study of genomes have produced enormous amounts of sequence information. With the general combination of conventional genetic engineering and genome research techniques, the genomic sequences of some highly thermophilic microorganisms are of interest because of their heat-resistant enzymes in the field of biotechnology, and many very thermostable enzymes have been developed for biotechnology purposes. have.

Aminopeptidase P

Aminopeptidase P (APP; X-Pro Aminopeptidase EC 3.4.11.9) is a peptidase that removes the N-terminal amino acid from a peptide whose second residue at the end is proline. Since enzymes with the specificity of APP were first purified from Escherichia coli , APP has been characterized from a wide range of biological sources, including the tissues of bacteria, nematodes and some mammals. Although the physiological role of APP in bacteria is not clear, mammalian APP is involved in the protein reorganization of collagen and the regulation of biologically active peptides such as substance P and bradykinin. APP from a significant number of lactococcal strains has been reported to contribute to the elimination of bitterness during the maturation of cheese by degrading peptides and involvement in releasing them into the cheese substrate. However, there are no reports on the properties of APP from archaea or thermophilic bacteria.

In order to facilitate the search for enzymes that are economically valuable and extremely stable to heat, and to find answers to the physiology of thermophilic archaea, especially at high temperatures, the present inventors have found that Thermococcus sp. NA1 (Bae et al) was isolated and deposited with the Biotechnology Center of Biotechnology (KCTC) on October 7, 2005, and was given a accession number of KCTC 10859BP. The entire genome sequence of the strain was determined to find many useful extremely thermally stable enzymes (Lee et al). Thermococcus sp. Analysis of genomic information of NA1 found aminopeptidase P gene belonging to thermostable aminopeptidase P, which was cloned, expressed using a recombinant vector, purified, and confirmed enzymatic activity. The present invention has been completed.

The present invention provides a novel highly thermophilic aminopeptidase P.

The present invention provides a method for producing a novel highly thermophilic aminopeptidase P.

The present invention provides a method for preparing aminopeptidase P. The manufacturing method is not limited thereto, but is preferably a manufacturing method by a genetic engineering method.

The present invention provides isolated DNA sequences encoding aminopeptidase P and recombinant vectors containing them.

In a first aspect, the present invention provides a nucleic acid sequence encoding aminopeptidase P that is stable at high temperature and an equivalent nucleic acid sequence to the sequence. The nucleic acid sequences are more specifically SEQ ID NO: 1.

"Equivalent nucleic acid sequences" include codon degenerate sequences of the aminopeptidase P enzyme sequences.

By "codon degenerate sequence" is meant a nucleic acid sequence that encodes a polypeptide of the same sequence as the naturally occurring aminopeptidase P enzyme disclosed herein but different from the naturally occurring sequence.

In a second aspect, the present invention provides aminopeptidase P. More specifically, aminopeptidase P represented by SEQ ID NO: 2 and functional equivalents thereof are provided.

“Functional equivalents” of the invention include amino acid sequence variants in which some or all of the aminopeptidase of SEQ ID NO: 2 is substituted, or a portion of the amino acid is deleted or added. Substitutions of amino acids are preferably conservative substitutions. Examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of amino acids is preferably located in portions not directly involved in the activity of aminopeptidase P.

In a third aspect, the present invention provides a recombinant vector comprising an isolated DNA fragment encoding the aminopeptidase P.

The term vector, as used herein, refers to a nucleic acid molecule capable of binding and transferring another nucleic acid thereto. The term expression vector includes plasmids, cosmids or phages capable of synthesizing proteins encoded by each recombinant gene carried by the vector. Preferred vectors are vectors capable of self-replicating and expressing the nucleic acid to which they are bound.

In a fourth aspect of the present invention, there is provided a cell transformed with a recombinant vector comprising an isolated DNA segment encoding the aminopeptidase P.

The term 'transformation' used in the present invention means that foreign DNA or RNA is absorbed into a cell and the genotype of the cell is changed.

Suitable transformed cells include, but are not limited to, prokaryotes, fungi, plants, animal cells, and the like. Most preferably, E. coli is used.

According to a fifth aspect of the present invention, there is provided a method for producing aminopeptidase P using the transformed cells or the Thermococcus sp.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are intended to illustrate the present invention, but the content of the present invention is not limited by the Examples.

Example 1 Primary Structure of Amino Peptidase P (APP) Gene and Expression of Recombinant Enzymes

(1) strains and growth conditions

Thermococcus sp. NA1 was isolated from deepwater hydrothermal vents in the area in the Park Manus region (3 ° 14 'S, 151 ° 42' E) in East Manus Basin. YPS medium was used for thermococcus sp. It was used to culture NA1, Thermococcus sp. Culture of NA1 and strain maintenance were done by standard methods. Thermococcus sp. To prepare NA1 seed cultures, YPS medium in 25 ml serum bottles was inoculated with a single colony formed on a pattagel plate and incubated at 90 ° C. for 20 hours. Seed culture was used to inoculate 700 ml of YPS medium in an anaerobic jar and incubated at 90 ° C. for 20 hours. The thermophilic archaea Thermococcus sp. NA1 (Bae et al) was deposited with the Biotechnology Center (KCTC) on October 7, 2005, and was assigned the accession number of KCTC 10859BP on October 20, 2005.

E. coli DH5α was used for plasmid propagation and nucleic acid sequencing. E. coli BL21-Codonplus (DE3) -RIL cells (stratazine, Lazola, Calif.) And plasmid pET-24a (+) (Novagen, Madison, Wisconsin) were used for gene expression. E. coli strains were incubated in Luria-Bertani medium at 37 ° C. and kanamycin was added to the medium to a final concentration of 50 μg / ml.

(2) DNA manipulation and sequencing

DNA manipulations were done in a standard manner as described by Samprock and Russell. Genomic DNA of Thermococcus sp. Was isolated by standard methods. Restriction enzymes and other modifying enzymes were purchased from Promega (Madison, Wisconsin). Small amounts of plasmid DNA from E. coli cells were made using the plasmid mini kit (Qiagen, Hilden, Germany). DNA sequencing was performed with an automated sequencer (AB3100) using the Big Die Terminator Kit (PE Applied Biosystems, Foster City, CA).

(3) Cloning and Expression of APP-Encoding Genes

Thermococcus sp . Flanked by Nde I and Xho I. The full length of the APP gene of NA1 includes genomic DNA and two primers (sense [5′-CGACCCGG CATAT GCGCCTCAACAAGCTCACTTCTCTG-3 ′; SEQ ID NO: 3] and antisense [5′-CTCCACAT CTCGA GCACGATTATCAGCTCCCTCGGTGCC-3 ′; SEQ ID NO: 4); The italic sequence in the sense primer is the Nde I site, and the italic sequence in the antisense primer is Xho I). The amplified sequence was digested with Nde I and Xho I and linked to Nde I / Xho I digested pET-24a (+). The ligand was transformed into E. coli DH5α. Candidates with the correct construct were selected by restriction enzyme digest and the DNA sequence of the clone was confirmed to have aminopeptidase P.

The resulting plasmid was transformed with E. coli BL21-CodonPlus (DE3) -RIL for expression. Overexpression of the APP gene was induced by addition of isopropyl-β- _D -thiogalactopyranoside (IPTG) to the midterm grade growth phase and incubation at 37 ° C. for 3 hours. Cells were obtained via centrifugation (6,000 × g for 20 min at 4 ° C.) and resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1M KCl and 10% glycerol. Cells were pulverized by ultrasound, centrifuged (20,000 xg for 30 min at 4 ° C.) and coenzyme samples were heat treated at 80 ° C. for 20 min. The resulting supernatant was treated on a column of TALON ^™ metal affinity resin (BD Bioscience Clontech, Palo Alto, Calif.) And 10 mM imidazole in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol. (Sigma, St. Louis, Missouri) and APP was eluted at 300 mM in buffer. The collected fractions were then buffer exchanged with 50 mM Tris-HCl (pH 8.0) buffer containing 10% glycerol using Centricon YM-10 (Millipore, Bedford, Mass.).

Protein concentration was determined from absorbance at 280 nm using a molar extinction coefficient of 33,710 M ⁻¹ cm ⁻¹ . The degree of purification of the protein was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis carried out by standard methods.

(4) Primary structure of APP gene and expression of recombinant enzyme

The inventors of the present invention are highly thermophilic archaea Thermococcus sp. Growing at a high temperature of 70-90 ° C. NA1 was isolated. By analyzing the genomic sequences, the inventors of the present invention have found an open reading frame (ORF) of the present invention consisting of 1,071 bp encoding a protein consisting of 356 amino acids and having a predicted molecular weight of 39,714 Da.

DNA sequence comparison analysis of T. kodakaraensis KOD1 (75% identity) (5), Pyrococcus abyssi GE5 (74% identity) putative APP gene and Pyrococcus horikoshii OT3 dipep peptidase claim (74% identity) with a protein of the invention of High similarity is shown (FIG. 1). Amino acid sequencing revealed that two aspartic acid residues, one histidine residue and two glutamic acid residues, all initially identified as active site residues involved in metal ion coordination in the crystal structure of E. coli APP, are perfectly conserved in TNA1_APP (Fig. 14).

Two other critical residues are also all conserved in high proportions; These two histidines are believed to play a role in exchanging protons with a solvent. Based on the above analysis, the open reading frame (ORF) is assumed to be APP, but since it is described that APP of all other archaea is estimated, it was necessary to reconfirm this fact by revealing the enzyme activity.

To confirm this, the APP gene was amplified using the PCR technique and the expressed enzyme was purified from the soluble cell extract by the method described above.

Analysis by SDS-PAGE shows that the 41-kDa protein of the present invention, which is the expected size of the fusion consisting of a 40-kDa APP protein and a 1-kDa peptide corresponding to LEH _6- (His-tag) at the C-terminus It is shown to be a major component of the purified sample (FIG. 2).

Example 2 Biochemical Characterization of Aminopeptidase P Enzyme

(1) enzyme analysis

APP-catalytic hydrolysis of Lys (N-Abz) -Pro-Pro-pNA (Bachem AG, Bubendorf, Switzerland) was observed by the release of Lys (N-Abz) -OH. This was observed with an emission wavelength of 417 nm and excitation wavelength of 320 nm at 80 ° C using an F-2000 fluorescence spectrometer (Hitachi, Tokyo, Japan). The reaction mixture (1 ml) consisted of 50 mM sodium acetate buffer (PH 5.0), 15 μM Lys (N-Abz) -Pro-Pro-pNA and 1.2 mM CoCl ₂ . The reaction was started by adding 2 μg APP. Assays based on the amount of proline released from the hydrolysis of Met-Pro (Bachem AG, Bubendorf) dipeptide were generally used. Proline concentrations were determined by modification of the colorimetric ninhydrin method by yalon and milnar. A ninhydrin agent was prepared by adding 3% (w / v) of ninhydrin to 60% (v / v) ice vinegar and 40% (v / v) phosphoric acid and incubating at 70 ° C for 30 minutes. APP assay mixture (300 μL) containing 50 mM sodium acetate buffer (pH 5.0), 4 mM Met-Pro, and 1.2 mM CoCl ₂ was incubated at 80 ° C. for 5 minutes. The reaction started with the addition of enzyme and the mixture was incubated at 80 ° C. for an additional 5 minutes. The reaction was terminated by the addition of glacial acetic acid (300 μl) and the ninhydrin agent (300 μl) was added. After heating at 80 ° C. for 10 minutes, the reaction was cooled in ice and the absorbance at 515 nm was measured. The amount of proline released was calculated using a molar extinction coefficient of 4,579 M ⁻¹ cm ⁻¹ for the ninhydrin-proline complex. One unit of APP is the amount of enzyme that releases 1 μmole of proline per minute under the assay conditions.

(2) Biochemical Characterization of APP Enzyme

The purified protein of the present invention was tested for activity against a typical APP substrate, which shows APP activity by hydrolyzing Lys (N ^ε -Abz) -Pro-Pro-pNA. APP is a protease that specifically removes N-terminal amino acids from a peptide whose second residue at the end is proline, although in many cases it is not possible to hydrolyze the dipeptide with proline at the C-terminal site.

In order to see if the recombinant enzyme of the present invention can hydrolyze the dipeptide and Lys (N ^ε -Abz) -Pro-Pro-pNA, the hydrolytic activity against Met-Pro was tested. The recombinant enzyme of the present invention was able to apparently hydrolyze Met-Pro dipeptides.

As shown in FIG. 3A, APP activity against Met-Pro was strongly promoted at high temperatures, showed an optimal activity temperature above 100 ° C., and was 10% less than the maximum activity observed at 40-50 ° C. . The same trend is also seen in experiments with Lys (N ^ε -Abz) -Pro-Pro-pNA. The effect of pH on APP activity was measured using various buffers in the pH range 4-8. Interestingly, the optimal pH of APP activity was shown differently at pH values 5 and 6.5-7 for Met-Pro (FIG. 3B) and Lys (N ^ε -Abz) -Pro-Pro-pNA (FIG. 3C), respectively.

Thermal stability was determined by incubating the enzymes of the present invention in 50 mM sodium acetate buffer, pH 5, 80 and 90 ° C. The APP was very stable to heat, with an enzymatic activity half-life of more than 100 minutes at 80 ° C. and more than 49 minutes at 90 ° C. Interestingly, APP maintained at 80 ° C was found to increase relative activity by 20% within 20 minutes (inset of Figure 4).

The presence of conserved amino acid residues for metal binding suggests that TNA1_APP may be affected by the addition of metal ions. As shown in FIG. 5, the addition of Co ²⁺ ions to the diafiltered enzyme of the present invention increased its enzymatic activity by 25-fold, while Mn ²⁺ and Zn ²⁺ increased their activity 5-fold and 4-fold, respectively. I was. These ions could not be replaced with other divalent cations (Ba ²⁺ , Ca ²⁺ , Cu ²⁺ , Fe ²⁺ , Mg ²⁺ and Ni ²⁺ ). The effects of Co ²⁺ , Mn ²⁺ and Zn ²⁺ concentrations on APP activity showed maximal activity at 3 mM CoCl ₂ , 20 mM MnCl ₂ and 0.4 mM ZnCl ₂ , showing very different responses. However, these cations caused inhibition when added above their optimum concentrations. Kinetics analysis was performed using Met-Pro, and rate constants such as K _m (0.96 mM), V _max (796 μmol min ^-1 mg ^-1 ) and k _cat (541 s ^-1 ) were obtained from the measured activity. Calculations were made (FIG. 6).

The metal-APP gene of the present invention was identified by sequencing, and the predicted function was reconfirmed through biochemical characterization of the recombinant enzyme expressed in E. coli . Database analysis shows that the amino acid sequence of TNA1_APP is closely related to two putative APP and one putative dipeptidase in genomic sequences of archaea T. kodakaraensis KOD1, P. abyssi GE5, and P. horikoshii OT3, respectively. .

It is also found in archaea Sulfolobus tokodaii str. 7 (39%) and E. coli O157 (26%) show similarity with APP and cytoplasmic APP of Drosophila melanogaster (17%), human (19%), rat (21%) and Caenorhabditis elegans (20%). In conclusion, the purified enzyme of the present invention exhibits APP activity as predicted by hydrolysis of Met-Pro and Lys (N ^ε -Abz) -Pro-Pro-pNA. All other APPs from mesophilic sources show optimal activity at temperatures up to 55 ° C.

Except for the heat stable thiol protease purified from the extracellular fraction of T. kodakaraensis KOD1, most of the proteases and peptidase isolated from Thermococcus spp. Showed increased activity at 70-85 ° C. at normal high temperatures. Given that, it is surprising that TNA1_APP exhibits an optimal temperature above 100 ° C. The fact that TNA1_APP is an extremely stable enzyme is evidenced by the fact that this enzyme has a t _1/2 of over 100 minutes at 80 ° C.

Furthermore, it appears that TNA1_APP is significantly activated by thermostable at high temperatures. Short-term heat-induced activations include P. horikoshii , Sulfolobus solfataricus or Thermococcus sp., Including acylamino acid-releasing enzymes, intracellular proteases, prolyl endopeptidase and carboxypeptidase. Also observed in other proteolytic enzymes purified from NA1. Although the mechanism has not been studied, it is presumed that heat-dependent morphological changes in the enzymes of the present invention increased its activity.

Guagliardi et al. Believe that the increase in activity by pre-heat treatment is characteristic of some enzymes from thermophilic biomass, and this reflects the activation of the low active state employed by these enzymes at normal temperatures.

The optimal pH of TNA1_APP is in the weakly acidic range (pH 5, 6.5-7) that distinguishes it from other APPs. Although membrane-bound APP isolated from bovine lung shows an optimal pH of 6.5 for bradykinin hydrolysis, most APPs show optimal activity at pH 7-9.

Furthermore, the optimal pH values differed in the hydrolysis of Met-Pro and Lys (N ^ε -Abz) -Pro-Pro-pNA.

Like enzymes from Lactococcus lactis and enzymes from other sources, Thermococcus sp. APP from NA1 is a metalloenzyme that is stimulated by Co ²⁺ and Mn ²⁺ .

Zn ²⁺ also showed a stimulatory effect, as opposed to Zn ²⁺ inhibition of APP in all mammals and C. elegans .

Proteases according to the invention are novel proteases that are highly thermophilic.

Aminopeptidase P is involved in the protein reorganization of collagen and the regulation of biologically active peptides such as substance P and bradykinin in mammals. APP from a large number of lactococcal strains is reported to contribute to the elimination of bitter taste during the maturation process of the cheese by decomposing peptides and releasing them into the cheese substrate.

references

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Claims (13)

A highly thermophilic aminopeptidase P enzyme having the amino acid sequence of SEQ ID NO.  A protein having the amino acid sequence of SEQ ID NO.  The gene of SEQ ID NO: 1.  A gene encoding the amino acid sequence of SEQ ID NO. A nucleic acid sequence encoding the highly thermophilic aminopeptidase P enzyme according to claim 1. The DNA nucleic acid sequence of claim 5 encoding the highly thermophilic aminopeptidase P enzyme of SEQ ID NO: 1. The nucleic acid sequence according to claim 5, wherein the DNA nucleic acid sequence encoding the highly thermophilic aminopeptidase P enzyme is equivalent to SEQ ID NO: 1 by codon degeneracy. delete delete Recombinant vector according to any one of claims 5 to 7, comprising a nucleic acid sequence. The recombinant vector according to claim 10, wherein said recombinant vector has a cleavage map as described in FIG. A cell transformed with the recombinant vector according to claim 10. Strain Thermococcus sp. Highly thermophilic aminopeptidase P enzyme production characterized by culturing NA1 (KCTC 10859BP) or the transformed cells according to claim 12, and separating the aminopeptidase P enzyme of SEQ ID NO: 2 from the cultured cells. Way.

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