JP5094461B2 - Gene encoding hyaluronic acid hydrolase - Google Patents

Description

  The present invention is a novel hyaluronic acid hydrolase derived from a microorganism and having an activity of hydrolyzing hyaluronic acid in an endo form, a gene encoding the enzyme, a method for producing the enzyme, and the enzyme The present invention relates to a method for producing low molecular weight hyaluronic acid.

  Hyaluronic acid has a repetitive structure of D-N-acetylglucosamine (hereinafter referred to as “GlcNAc”) and D-glucuronic acid (hereinafter referred to as “GlcUA”) [... GlcNAc (β-1,4) GlcUA (β -1,3) GlcNAc (β-1,4) GlcUA (β-1,3) ...], and is widely used in the fields of pharmaceuticals, foods and drinks, and cosmetics due to its high moisturizing function. ing.

In order to enhance the absorption of hyaluronic acid into the body, the use of low molecular weight hyaluronic acid is useful. Examples of the method for reducing the molecular weight include a method of treating with an alkali or an acid (for example, see Patent Document 1) and a method for enzymatic degradation (for example, see Patent Document 2). As a method for enzymatic degradation, there is known a method for reducing the molecular weight of hyaluronic acid with hyaluronidase. Known hyaluronidases are classified into the following three types according to the reaction mode.

Reaction mode 1: Hyaluronoglucosaminidase (EC 3.2.1.35)
The β-1,4 glycosidic bond connecting GlcNAc and GlcUA is hydrolyzed in an endo form. For example, mammals (human, bovine, etc.) are known from testis and serum, snake and bee venom (see, for example, Non-Patent Documents 1 and 2 and Patent Document 3).

Reaction mode 2: hyaluronoglucuronidase (EC 3.2.1.36)
The β-1,3 glycosidic bond connecting GlcUA and GlcNAc is hydrolyzed in an endo form. For example, the hill origin etc. (for example, refer patent document 4) are known.

Reaction mode 3: Hyaluronic acid lyase (EC 4.2.2.1)
The β-1,4 glycosidic bond connecting GlcNAc and GlcUA is decomposed in an endo form using β elimination reaction. For example, microorganisms such as Streptomyces or Streptococcus are known (for example, see Non-Patent Document 3).

However, the conventional hyaluronidase has the following problems. First, the hydrolyzable hyaluronoglucosaminidase (EC 3.2.1.35) of reaction mode 1 and the hydrolyzed hyaluronoglucuronidase (EC 3.2.1.36) of reaction mode 2 are derived from animals. However, it has been found that mass production of enzymes is difficult and expensive.
On the other hand, the reaction mode 3 hyaluronic acid lyase (EC 4.2.2.1) is derived from microorganisms and is advantageous from the viewpoint of production efficiency. However, the low molecular weight hyaluronic acid produced by this enzyme has 4,5-unsaturated uronic acid at the non-reducing end, and what is the non-reducing end structure (saturated) of hyaluronic acid present in the living body? Structurally different.
Considering the use of pharmaceuticals, foods and drinks, and cosmetics, it is preferable to use low molecular weight hyaluronic acid produced by hydrolysis. From such a technical background, it is hydrolyzed and hyaluronidase derived from microorganisms is strong. It was sought after.

JP 2006-265287 A JP 2006-271351 A JP 2006-119 A Special table 2003-502045 gazette “The Enzymes, 2nd edn” (USA), 1960, vol. 4, p. 447-460 “Biochemical and Biophysical Research Communications” (USA), 1997, Vol. 236, p. 10-15 “Biochimica et Biophysica Acta”, (Langoku), 1997, 1337, p. 217-226

  The problem to be solved by the present invention is to provide a microorganism-derived enzyme having the activity of hydrolyzing hyaluronic acid in an endo form and a method for producing low molecular weight hyaluronic acid hydrolyzed by the enzyme.

  Thus, as a result of intensive studies to solve the above problems, the present inventors have found hyaluronidase derived from microorganisms such as fungi. And it turned out that this enzyme is an enzyme which has the activity which hydrolyzes a hyaluronic acid by an endo type | mold novel as a microorganism origin. Furthermore, the gene encoding this enzyme and the method for producing this enzyme were clarified, and a method for producing low molecular weight hyaluronic acid using this enzyme was found, and the present invention was completed.

That is, the present invention provides the following inventions.
(1) A hyaluronic acid hydrolase derived from a microorganism and having an activity of hydrolyzing hyaluronic acid in an endo form.
(2) The hyaluronic acid hydrolase according to (1) above, wherein the amino acid sequence has a GEIDI or GEIDV motif.
(3) The hyaluronic acid hydrolase according to the above (1) or (2) having the following physicochemical properties (a) to (e):
(A) Action: hydrolyzes hyaluronic acid in an endo form to produce an even number of oligosaccharide units;
(B) Optimal pH: 2.5-3.5;
(C) Optimal temperature: 35-50 ° C;
(D) Thermal stability: 50 ° C. or lower;
And (e) Molecular weight: about 30,000 (SDS-PAGE method).
(4) Hyaluronic acid hydrolysis according to any one of (1) to (3) above, which is a protein comprising an amino acid sequence represented by the following (f), (g) or (h) Degradation enzyme:
(F) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4;
(G) a protein comprising an amino acid sequence in which one to a plurality of amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 3 or 4;
(H) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3 or 4.
(5) Production of hyaluronic acid hydrolase, wherein the enzyme is recovered from a culture of a microorganism having the ability to produce the hyaluronic acid hydrolase according to any one of (1) to (4) above Method.
(6) Gene consisting of the following DNA (i) , ( k) or (l):
(I) DNA comprising the base sequence represented by SEQ ID NO: 1 or 2;
(K) DNA encoding a protein having 80% or more identity with the DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 and having hyaluronic acid hydrolyzing activity;
(L) DNA encoding a protein having one or more DNAs deleted, substituted and / or added and having hyaluronic acid hydrolyzing activity in the DNA comprising the base sequence represented by SEQ ID NO: 1 or 2 .
(7) An enzyme preparation for reducing the molecular weight of hyaluronic acid, comprising the hyaluronic acid hydrolase according to any one of (1) to (4) above.
(8) A low molecular weight hyaluronic acid characterized in that the hyaluronic acid hydrolase according to any one of (1) to (4) above is allowed to act on hyaluronic acid to produce low molecular weight hyaluronic acid. Production method.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the low molecular weight hyaluronic acid hydrolyzed by the microorganism-derived enzyme which has the activity which hydrolyzes hyaluronic acid by an endo type, and this enzyme can be provided.

Hereinafter, the present invention will be described in detail.
First, the enzyme of the present invention (hereinafter referred to as “the present enzyme”) will be described. This enzyme is an enzyme that catalyzes a reaction of hydrolyzing hyaluronic acid in an endo form. The degradation site of hyaluronic acid is not particularly limited, and examples thereof include a β-1,4 glycosidic bond connecting GlcNAc and GlcUA.
This enzyme is not a lyase reaction that generates low molecular weight hyaluronic acid having 4,5-unsaturated uronic acid at the non-reducing end, but the same non-reducing end structure (saturated uronic acid) as hyaluronic acid existing in vivo It is particularly useful industrially because it catalyzes a hydrolysis reaction that produces a low molecular weight hyaluronic acid having a low molecular weight.

Confirmation of enzyme reaction and measurement of enzyme activity can be performed, for example, as follows.
Measuring the molecular weight of the product using an HPLC method (eg, described in Example 1, 5 “Analysis of low molecular weight hyaluronic acid”) indicates that endo-type hydrolysis is occurring.
In order to examine whether or not the β-1,4 glycosidic bond connecting GlcNAc and GlcUA has been degraded, the Morgan-Elson method, “The Journal of Biochemistry” (USA), 1955, 217, p. By referring to the procedure of 959-966, it may be confirmed that the reducing end of GlcNAc is generated.
In addition, in order to examine that this enzyme reaction is not a elimination reaction but a hydrolysis reaction, it is only necessary to confirm that there is no absorption at 232 nm characteristic of unsaturated uronic acid in the decomposition product (“glycan engineering”). And Pharmaceutical Development ", (Japan), Drug Side Effect Damage Relief and Research Promotion, edited by Yakuho Hokpo, December 1993, p.37).
In addition, these measuring methods show an example, and do not restrict | limit at all about the reagent of a measurement, a kind, density | concentration, and pH.

This enzyme is derived from a microorganism. It can be produced at low cost by ordinary culture, is easy to produce as compared with known animal-derived enzymes, and is industrially advantageous.
The microorganism here refers to a fungus [mushroom, fungus (mold), yeast] or a prokaryote (bacteria).
Examples of the fungi include filamentous fungi such as Penicillium genus (Aokabi), Aspergillus genus (Koji mold), Rhizopus genus, and yeasts such as Saccharomyces genus, Candida genus, and Debariomyces genus. Preferably, those derived from the genus Penicillium are used. Specifically, for example, Penicillium purpurogenum (Penicillium purpurogenum) and Penicillium funiculosum (Penicillium funiculosum), more specifically, Penicillium perprogenum IAM 13753 strain, IAM 13754 strain, Nimus 7095 strain, It is done.

  In addition, for example, in the SEQ ID NOs: 3 and 4, the present enzyme is characterized by 5 which is characteristic of a microorganism-derived polysaccharide hydrolase (EC 3.2.1.-) as shown at positions 112 to 116. It has an amino acid sequence motif (GEIDI or GEIDV) consisting of two amino acids (see “Applied Environmental Microbiology” (USA), 1998, Vol. 64, p. 385-391). These amino acid sequence motifs are not present in animal-derived hyaluronidase, indicating that the enzyme having these motifs is derived from a microorganism and the reaction is hydrolyzed. A hyaluronic acid degrading activity has not been found in a polysaccharide hydrolase derived from a microorganism having this motif, which has been conventionally known. This enzyme was found for the first time as an enzyme having hyaluronic acid hydrolyzing activity and having this motif.

Moreover, as one form of this enzyme, the hyaluronidase etc. which have the physicochemical property as described in the following (a)-(e), respectively, or those suitably combined are mentioned, for example.
(A) Action: Hyaluronic acid is hydrolyzed in an endo form to produce even-numbered oligosaccharide units.
(B) Optimal pH: 2.5 to 3.5
(C) Optimal temperature: 35-50 ° C
(D) Thermal stability: 50 ° C. or less (e) Molecular weight: about 30,000 (SDS-PAGE method)
Furthermore, examples of the present enzyme include a protein having an amino acid sequence represented by the following (f), (g), or (h).
(F) A protein comprising the amino acid sequence represented by SEQ ID NO: 3 or 4.
(G) A protein comprising an amino acid sequence in which one to a plurality of amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 3 or 4.
(H) A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 3 or 4.

Here, “one to a plurality of amino acids are deleted, substituted and / or added” means, for example, an arbitrary number of 1 to 30, preferably 1 to 20, and more preferably 1 to 10. Is deleted, substituted and / or added. In particular, a plurality of amino acids at the N-terminus of the enzyme may be removed by processing, so such consideration is necessary.
Moreover, in order to evaluate the homology of an amino acid sequence, various homology search programs and software are provided, and any of them may be used. In addition to the homology evaluation, preferably an identity evaluation is also used. The difference between homology and identity is that amino acids having the same properties (for example, glutamic acid and aspartic acid) are considered to be homologous, but are distinguished by identity. “Shows 80% or more homology” means that the homology or identity with the amino acid sequence represented by SEQ ID NO: 3 or 4 is, for example, 80% or more, preferably 90% or more, and most preferably 95%. That's it.

Next, a method for producing this enzyme will be described.
This enzyme-containing composition is produced, for example, by culturing the microorganisms exemplified above in a medium. As long as this enzyme-containing composition is obtained, the method for culturing microorganisms and the method for collecting enzyme compositions are not particularly limited. For example, the following methods can be employed.
Penicillium spp., Aspergillus spp., Aspergillus spp., Rhizopus spp., Etc., yeasts such as Saccharomyces spp., Candida spp. Incubate statically.
As the medium, for example, a synthetic medium, cooked cereal such as cooked rice can be used. The obtained culture can be used as it is as the enzyme-containing composition, but can also be extracted using a 0.2 M NaCl aqueous solution. Furthermore, the purity of the enzyme in the present enzyme-containing composition can be increased using ordinary enzyme purification methods. The present enzyme-containing composition thus obtained may be used as a preparation for reducing the molecular weight of hyaluronic acid.

Next, examples of the gene encoding the present enzyme (hereinafter referred to as “the present gene”) include genes comprising the following DNAs (i), (j), (k) or (l).
(I) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2.
(J) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2, and that encodes the protein having the above enzyme activity .
(K) DNA which shows a homology of 80% or more with the DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2, and encodes the protein having the above enzyme activity.
(L) A DNA comprising a nucleotide sequence represented by SEQ ID NO: 1 or 2, wherein one to a plurality of DNAs are deleted, substituted and / or added, and which encodes the protein having the enzyme activity.
These DNAs are highly likely to encode a polypeptide that brings about this enzyme activity, and transformants can be prepared and those having activity can be selected. In order to obtain a gene substantially identical to this gene, hybridization is performed under stringent conditions with a probe having a nucleotide sequence of SEQ ID NO: 1 or 2 or a complementary strand thereof, or a part thereof, Those encoding a polypeptide having this enzyme activity can be selected.

The stringent condition here is a condition in which only a specific hybrid is selectively formed and a signal is detected, but a non-specific hybrid is not formed. Such conditions vary slightly depending on the individual species, but can be easily determined by examining several salt concentrations or temperatures during hybridization and washing by conventional methods. As such conditions, for example, hybridization is performed overnight at 37 to 42 ° C. using DIG Easy Hyb reagent (manufactured by Roche Diagnostics). Washing is performed twice for 15 minutes using 0.5 × SSC and 0.1% SDS. The washing temperature is 45 ° C. or higher, preferably 52 ° C. or higher, more preferably 57 ° C. or higher. DNA that hybridizes under such conditions is highly likely to encode a protein having the present enzyme activity, but includes DNA having a mutation that loses activity. However, these can be easily removed by measuring the enzyme-producing ability of the transformant after transformation.
The homology evaluation of gene sequences can be performed in the same manner as the homology evaluation of amino acid sequences. In the present invention, “showing 80% or more homology” is, for example, 80% or more, preferably 90% or more, and most preferably 95% or more.

  As a method for obtaining this gene, a commonly used gene cloning method is used. For example, chromosomal DNA or mRNA can be extracted from microbial cells having the ability to produce the enzyme with reference to the method described in a conventional method, “Current Protocols in Molecular Biology”, WILEY Interscience, 1989. . Furthermore, cDNA can be synthesized using mRNA as a template. A library of chromosomal DNA or cDNA thus obtained is prepared. Then, based on the amino acid sequence of the enzyme, a suitable probe DNA is synthesized and used to screen from a chromosomal DNA or cDNA library, or the amino acid sequence or partial sequence information of the acquired gene is used. Based on the above, a suitable primer DNA is prepared, and DNA containing the gene fragment of interest is obtained by an appropriate polymerase chain reaction (PCR method) such as a degenerate PCR method, an inverse PCR method, a 5′-RACE method, or a 3′-RACE method. Can be amplified and ligated together to obtain DNA containing the full length gene. Furthermore, it is also possible to prepare an appropriate mixed base primer using information on the known gene sequence of similar enzyme, its peripheral sequence, and amino acid sequence, and amplify DNA containing the target gene fragment by PCR. it can.

  The gene obtained as described above was referred to as “The Operan”, p. 227, Cold Spring Harbor Laboratory, 1980, a vector DNA having a DNA sequence containing an expression region such as a promoter, an operator, and a ribosome binding site derived from an E. coli lactose operon or tryptophan operon. The recombinant DNA obtained by the insertion can be used to transduce, for example, Escherichia coli to obtain a high enzyme-producing strain.

  The vector DNA used may be any, for example, plasmid DNA or bacteriophage DNA. In addition to Escherichia coli, the resulting recombinant DNA is used to transform or transduce microorganisms such as other bacteria, yeast, filamentous fungi, actinomycetes, and animal cells, etc. Body or transductant can be obtained.

As long as this enzyme-containing composition is obtained, the method for culturing the transformant or transductant containing the recombinant DNA and the method for collecting the enzyme composition are not particularly limited. For example, the following methods can be employed.
The recombinant Escherichia coli is inoculated into an appropriate medium and cultured at 20 to 42 ° C. for 6 to 120 hours. The culture is preferably carried out by aeration and agitation deep culture, shaking culture, stationary culture or the like.
As the medium, for example, LB medium can be used. An appropriate amount of isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be appropriately added as an enzyme production-inducing substrate. After completion of the culture, the culture can be used as it is as the enzyme-containing composition.

  The cells are separated from the culture by, for example, operations such as filtration and centrifugation, and washed. It is preferable to recover the enzyme from the cells. In this case, the cells can be used as they are, but the cell walls using a cell wall lytic enzyme such as lysozyme, a method of destroying the cells using various disrupting means such as an ultrasonic crusher, a French press, or Dynamil. The enzyme can be recovered from the cells by a method for dissolving the enzyme, a method for extracting the enzyme from the cells using a surfactant such as Triton X-100, and the like.

  Furthermore, the enzyme content in the present enzyme-containing composition can be increased using ordinary enzyme purification methods. For example, an ammonium sulfate salting out method, an organic solvent precipitation method, an ion exchange chromatography method, a gel filtration chromatography method, a hydrophobic chromatography method and the like can be appropriately combined. The present enzyme-containing composition thus obtained can be used as an enzyme preparation for reducing the molecular weight of hyaluronic acid.

Low molecular weight hyaluronic acid can be produced as follows using this enzyme-containing composition.
The origin and average molecular weight of hyaluronic acid are not particularly limited, and those obtained from various animals (such as chicken caps) or microorganisms (such as Streptococcus) can be used. In addition, as the hyaluronic acid, various commercially available products (“Hyaluronic acid FCH” manufactured by Kibun Food Chemifa, “Hyaluronic sun HA-F” manufactured by Kewpie, etc.) can also be used.

  What is necessary is just to set suitably the conditions at the time of making this enzyme containing composition act on the said hyaluronic acid according to the kind of hyaluronic acid and hyaluronidase to be used. For example, when the enzyme-containing composition is allowed to act on hyaluronic acid having an average molecular weight of 1,800,000 to 2,200,000 derived from microorganisms, the enzyme-containing composition is added to a 0.1% hyaluronic acid solution, and if necessary The pH may be adjusted appropriately, and the reaction may be performed at 30 to 40 ° C. for about 1 to 72 hours.

  By the above reaction, hyaluronic acid having a reduced molecular weight is generated. A method for separating and purifying the low molecular weight hyaluronic acid from the reaction system after completion of the reaction is arbitrary. As the separation method, for example, a filtration method, a dialysis method, an extraction method, a gel filtration, a chromatography method using an ion exchange resin or silica gel, recrystallization, or the like can be appropriately used.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.

[Example 1]
(Confirmation of fungal hyaluronidase activity)
It was confirmed that Penicillium fungi produced hyaluronidase by the following method.
1. Test strains (4 types)
(1) Penicillium perprogenum IAM 13754 strain (2) Penicillium perprogenum IAM 13754 strain (3) Penicillium perprogenum IAM 7095 strain (4) Penicillium funiculosum IAM 13754 strain Preparation of hyaluronic acid Hyaluronic acid ("Hyaluronic acid FCH FCH200" manufactured by Kibun Food Chemifa Co., Ltd.) was dissolved in distilled water at a concentration of 0.1% to obtain a hyaluronic acid aqueous solution.
3. Preparation of Hyaluronidase-Containing Composition The test strain was inoculated into a cooked rice medium and statically cultured at 30 ° C. for 3 days. A 0.2 M NaCl aqueous solution was added to the culture, shaken for 1 hour, and a culture supernatant was prepared by centrifugation. This was used as a hyaluronidase-containing composition (hereinafter referred to as “the present enzyme-containing composition”). As a control, the cooked rice medium was allowed to stand in the same manner at 30 ° C. for 3 days, a 0.2 M NaCl aqueous solution was added, and the mixture was shaken for 1 hour, followed by centrifugation to obtain a centrifugal supernatant.
4). Hyaluronic acid low molecular weight treatment Hyaluronic acid aqueous solution was mixed with hyaluronidase-containing composition at an arbitrary ratio, and the mixture was allowed to stand at 37 ° C. for about 18 hours to carry out hyaluronic acid low molecular weight treatment.
5. Analysis of low molecular weight hyaluronic acid The average molecular weight of hyaluronic acid contained in the sample after the low molecular weight treatment was analyzed using a gel filtration column. The conditions for gel filtration are as follows.
Column: G4000SWXL (7.8 × 300 mm × 2, manufactured by Tosoh Corporation)
Eluent: 0.2M NaCl
-Flow velocity: 1 ml / min.
・ Detection: Absorbance (214nm)
・ Molecular weight reference material: Shodex STANDARD P-82

FIG. 1 shows the analysis result of a low molecular weight hyaluronic acid obtained by using a hyaluronidase-containing composition derived from Penicillium perprogenum IAM 13753 strain by a gel filtration column.
The upper part of FIG. 1 is a chart of hyaluronic acid before the molecular weight reduction treatment, and the lower part of FIG. The molecular weight peak of hyaluronic acid before the molecular weight reduction treatment was 1 million or more (the upper part of FIG. 1), but the molecular weight peak of the low molecular weight hyaluronic acid obtained after the treatment was about 10,000 (the lower part of FIG. 1). )Met.
For all the remaining three test strains, it was confirmed that the average molecular weight of hyaluronic acid contained in the sample after the molecular weight reduction treatment was smaller than the average molecular weight before the treatment, as in FIG. It was also confirmed that the average molecular weight of hyaluronic acid was reduced depending on the amount of enzyme composition added or the treatment time.

On the other hand, the above 3. For the centrifugal supernatant prepared as a control in Example 1, the average molecular weight of hyaluronic acid did not change before and after the molecular weight reduction treatment.
From the above, it was shown that all of the four test strains produced hyaluronidase that degrades hyaluronic acid in endo form, and that these enzyme-containing compositions can be used as low molecular weight preparations of hyaluronic acid. .

[Example 2]
(Production of the enzyme produced by Penicillium perprogenum IAM 13753)
A hyaluronidase-containing composition extracted from Penicillium perprogenum IAM 13753 strain was adsorbed on a Q-Sepharose FastFlow (GE Healthcare Biosciences) column (5 cm × 18 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.0). It was. After washing with 20 mM potassium phosphate buffer (pH 7.0), elution was carried out with the same buffer containing 0 to 1 M sodium chloride by the linear concentration gradient method, and fractions showing hyaluronidase activity were collected.

  Subsequently, ammonium sulfate was added and applied to a Toyopearl Butyl-650C (manufactured by Tosoh Corp.) column (2.5 cm × 15 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.0) containing 2M ammonium sulfate. After washing with 20 mM potassium phosphate buffer (pH 7.0) containing 2M ammonium sulfate, elution was carried out by the linear concentration gradient method using the same buffer containing 2 to 0M ammonium sulfate. The eluted active fraction was concentrated with Centriprep 10 (Amicon) and dialyzed against 20 mM potassium phosphate buffer (pH 7.0). The obtained enzyme was used in determining the physicochemical properties of the enzyme described in Example 3 below.

Example 3
(Physicochemical properties of this enzyme produced by Penicillium perprogenum IAM 13753)
As the physicochemical properties of the enzyme obtained in Example 2, the following (a) to (e) were examined.

(A) Action It was confirmed by the following method that this enzyme hydrolyzes the β-1,4 glycosidic bond connecting GlcNAc and GlcUA of hyaluronic acid in an endo form.
1. Analysis of low molecular weight hyaluronic acid by HPLC It was confirmed by the method described in No. 5 in Example 1 that this enzyme hydrolyzes hyaluronic acid in an endo form.
2. Confirmation of GlcNAc reducing end generation by Morgan-Elson method In order to determine whether the reducing end of the low molecular weight hyaluronic acid generated by this enzyme is GlcNAc or GlcUA, the amount of GlcNAc reducing end by the Morgan-Elson method is determined. went.
About the hyaluronic acid solution in which this enzyme is allowed to act, “The Journal of Biochemistry” (USA), 1955, Vol. 217, p. Measurements were performed according to the procedure described in 959-966.
As a result, it was confirmed that a GlcNAc reducing end was newly generated, and it was found that this enzyme decomposes a β-1,4 glycosidic bond connecting GlcNAc and GlcUA.
3. Absorbance analysis of low molecular weight hyaluronic acid Hyaluronic acid subjected to low molecular weight treatment using a hyaluronidase-containing composition derived from Penicillium perprogenum was separated by a gel filtration column. The absorbance spectrum of the fraction with an elution time (10 minutes) corresponding to a molecular weight of about 10,000 was measured using a multi-wavelength detector (MD2010plus manufactured by JASCO Corporation).
The resulting low molecular weight hyaluronic acid did not have absorption at 232 nm, which is characteristic of unsaturated uronic acid. Therefore, it was suggested that the molecular weight reduction reaction by the enzyme-containing composition is not a lyase reaction but a hydrolysis reaction.

(B) Optimal pH
The enzyme reaction was carried out at a temperature of 43 ° C. in the range of pH 2.5 to 6.0. The results are shown in FIG. Since this enzyme showed the highest activity at pH 3.0 and high activity at pH 2.5 to 3.5, the optimum pH of this enzyme was judged to be pH 2.5 to 3.5.

(C) Optimum temperature The activity of this enzyme was measured at various temperatures of 35 ° C or higher. The results are shown in FIG. The temperature range showing 50% or more of the activity in the vicinity of 43 ° C., which is the temperature showing the highest activity, was 35 to 50 ° C. From the above, it was determined that the optimum temperature range of this enzyme was 35-50 ° C.

(D) Thermostability The results of thermostability when treated at each temperature for 60 minutes are as shown in FIG. 4, and this enzyme was stable up to around 50 ° C.

(E) Molecular Weight The molecular weight was determined by SDS-PAGE [PAG Mini “Daiichi” 10/20 (Daiichi Chemical Co., Ltd.), BenchMark Protein Ladder (Invitrogen)]. The molecular weight of the subunit of this enzyme was about 30,000.

Example 4
(Cloning of the gene encoding this enzyme and recombinant expression in E. coli)
(1) Analysis of amino acid sequence of the enzyme The enzyme produced by Penicillium perprogenum IAM 13753 obtained in Example 2 was overnight in a 50 mM Tris-HCl buffer (pH 9.0) at 37 ° C. Then, the protein was treated with endoproteinase Asp-N (Takara Shuzo Co., Ltd.) and fragmented. The said fragment | piece was fractionated by the reverse phase HPLC using Capcell Pak C18 SG300 (made by Shiseido Co., Ltd.). The amino acid sequence of the obtained fragment peptide was analyzed by Edman method.

(2) Analysis of cDNA sequence encoding this enzyme Three kinds of bacterial cells obtained in Example 1-3 (Penicillium perprogenum IAM 13754 strain, Penicillium perprogenum IAM 13754 strain, Penicillium funiculosum IAM 13552 strain) were frozen in liquid nitrogen And then crushed. RNA was obtained from this crushed cell using ISOGEN (Nippon Gene).

  Based on the amino acid sequence obtained in (1) above, a PCR primer was prepared using a mixed base at the position where the codon was degenerated. Using three types of RNA as templates, RNA PCR Kit (AMV) Ver. DNA fragments amplified by 3.0 (Takara Shuzo Co., Ltd.) were isolated and their base sequences were determined. As a result, it was found that the obtained DNA fragment was a part of the gene encoding this enzyme (partial gene).

  Next, oligonucleotides necessary for 3'-RACE and 5'-RACE were designed based on the base sequences obtained by the above operations. RNA PCR Kit (AMV) Ver. 3.0 (manufactured by Takara Shuzo) and 5'-Full RACE Core Kit (manufactured by Takara Shuzo) were subjected to 3'-RACE and 5'-RACE, and the amplified DNA fragments were isolated and the nucleotide sequences were determined. . As a result, it was found that each DNA fragment is a part (partial gene) of the gene encoding this enzyme and has a start codon (ATG) or a stop codon.

By the above operation, the full length of the gene sequence encoding this enzyme was determined. The amino acid sequence was estimated based on the gene sequence information, and the N-terminal amino acid sequence obtained in (1) above was compared. As a result, the enzyme derived from Penicillium perprogenum IAM13753 (mature enzyme) It was found that part 5) was removed. Thereby, the gene sequence and amino acid sequence of this enzyme derived from Penicillium perprogenum IAM 13753 strain were estimated as SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
For Penicillium perprogenum IAM 13754 strain, the obtained gene sequence and the deduced amino acid sequence were completely identical with SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
Moreover, as for Penicillium funiculosum IAM13752 strain, as compared with the amino acid sequence of the enzyme derived from Penicillium perprogenum IAM13753, it was predicted that the N-terminal MKTSTLAAAVCQVFLGTRAVA (SEQ ID NO: 6) portion was removed. Thereby, the gene sequence and amino acid sequence of this enzyme derived from Penicillium funiculosum IAM 13752 were estimated as SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

The amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4 were examined for homology with various known hyaluronidases, but no significant homology was found. In addition, as shown in SEQ ID NO: 3 and SEQ ID NO: 4, the 5-amino acid sequence (GEIDI) characteristic of the microorganism-derived polysaccharide hydrolase (EC 3.2.1.-) is located at the 112th to 116th positions. It was found to have. An enzyme having such a motif sequence and having hyaluronidase activity has not been known so far.
Therefore, it was also shown from this result that this enzyme is a novel hyaluronic acid hydrolase derived from microorganisms.

(3) Cloning of full-length cDNA encoding this enzyme In order to obtain the full-length gene of the mature enzyme, a sequence in which an initiation codon (ATG) is added to the 5 ′ end of SEQ ID NO: 1 and SEQ ID NO: 2 is used. Considering a gene, an oligonucleotide containing a 5 ′ end or 3 ′ end (total 30 base oligonucleotide, 3 ′ end side complementary strand) was designed. For the Penicillium perprogenum IAM13753 strain, an EcoRV site was incorporated into the primer, and the product amplified by PCR was digested with EcoRV so that a coding region was obtained. That is, 5′-AGTGTGGGAGATACATCATGACAAGCTGCAA-3 ′ (30 mer) (SEQ ID NO: 7) and 5′-CTCCTTGAAACAGATCTCATTGATACAG-3 ′ (SEQ ID NO: 8) as an antisense primer were synthesized. For the Penicillium funiculosum IAM13752 strain, a coding region was obtained by incorporating a PvuII site into the primer and digesting the product amplified by PCR with PvuII. Specifically, 5′-GGGACAAGACAGCTGATGTACACGCTGGCAC-3 ′ (30 mer) (SEQ ID NO: 9) and 5′-TGATACATTCACAGCTGTCACTGGTACAA-3 ′ (SEQ ID NO: 10) were synthesized as antisense primers. RT-PCR was performed using the previously prepared RNA as a template and 3'-Full RACE Core Kit (Takara Shuzo). As a result of analyzing the base sequence of the obtained amplified DNA (about 900 bp), it completely matched with SEQ ID NO: 1 and SEQ ID NO: 2.

(4) Recombinant expression by E. coli of a gene encoding HAase A promoter derived from E. coli lactose operon or the like by treating 3'-RACE amplified DNA encoding HAase (about 900 bp) with a restriction enzyme (EcoRV or PvuII) , Inserted into a cloning site of pKK223-3 (manufactured by Amersham Pharmacia Biotech) having a DNA sequence containing an expression region such as an operator and a ribosome binding site (see The Operan, p. 227, Cold Spring Harbor Laboratory, 1980). Recombinant plasmid DNA pHAase was obtained.

  D. M.M. According to the method of Morrison (Methods in Enzymology, 68, p. 326-331, 1979), E. coli DH5α (manufactured by Takara Shuzo Co., Ltd.) was transformed with the recombinant plasmid DNA pHAase, E. E. coli DH5α (pHAase) was obtained. The recombinant plasmid pHAase was extracted from the cells using QIAGEN tip-100 (Qiagen) and purified to obtain a recombinant plasmid. The obtained E.I. E. coli DH5α (pHAase) was added to LB-IPTG-amp medium [bactotryptone 1% (w / v), yeast extract 0.5% (w / v), NaCl 0.5% (w / v), isopropyl- β-D-thiogalactopyranoside (1 mM) and ampicillin (50 μg / ml)] were cultured with shaking for 10 hours. As a result, the maximum HAase activity was confirmed in the cell disruption solution when the culture temperature was 25 ° C.

It is the figure which showed the analysis result by the gel filtration column of the low molecular-weight hyaluronic acid obtained using the hyaluronidase containing composition derived from Penicillium. FIG. 4 is a view showing the optimum pH of the present enzyme in Example 3. FIG. 4 is a view showing the optimum temperature of the present enzyme in Example 3. FIG. 4 is a view showing the thermal stability of the present enzyme in Example 3.

Claims (1)

A gene comprising the following DNA (i) , ( k) or (l):
(I) DNA comprising the base sequence represented by SEQ ID NO: 1 or 2;
(K) DNA encoding a protein having 80% or more identity with the DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 and having hyaluronic acid hydrolyzing activity;
(L) DNA encoding a protein having one or more DNAs deleted, substituted and / or added and having hyaluronic acid hydrolyzing activity in the DNA comprising the base sequence represented by SEQ ID NO: 1 or 2 .

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