JP2006204260A - Hyaluronic acid synthase and its gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a hyaluronic acid synthase having a high expression efficiency in a host and having reduced occurrence of environmental pollution and a gene encoding the enzyme. <P>SOLUTION: The hyaluronic acid synthase comprises (a) a DNA composed of a specific base sequence or its complementary strand, (b) a DNA that is hybridized with the DNA of (a) under a stringent condition and has a hyaluronic acid synthase activity, (c) a DNA that has 96.8% or above homology to the DNA of (a) and encodes a peptide having a hyaluronic acid synthase activity or (d) a DNA that encodes a polypeptide in which one or several amino acids are deleted, substituted, added or inserted in a specific amino acid sequence and which has a hyaluronic acid synthase activity. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a hyaluronic acid synthase and a hyaluronic acid synthase gene.

  Hyaluronic acid (hereinafter also referred to as “HA”) is a high-molecular-weight glucosaminoglycan, and a disaccharide unit (GlcAβ1-3GlcNAc: GlcA is glucuronic acid) in which glucuronic acid is linked to N-acetylglucosamine with β1,3 bonds. GlcNAc represents N-acetylglucosamine), and is a linear polysaccharide composed of repeating β1,4 bonds. HA is known to exist widely as an extracellular matrix component of various tissues such as vitreous, skin, and ligaments in vertebrates. HA works with other extracellular matrix components to form a three-dimensional structure, functions not only as a structure for maintaining water retention and flexibility of tissues, but also interacts with cell surface receptors together with HA binding proteins. Regulates cell behavior such as cell adhesion, migration, and differentiation, and is also involved in phenomena such as morphogenesis, wound healing, cancer invasion, and metastasis.

  The biosynthesis of hyaluronic acid was conducted in the 1950s and 1960s by cell-free experiments using streptococci. Two types of sugars, UDP-glucuronic acid and UDP-N-acetylglucosamine, were used to elongate the hyaluronic acid chain. The activity of hyaluronic acid synthase (hereinafter also referred to as “HAS”) localized in the streptococcal cell membrane was shown by using nucleotides (see Non-Patent Document 1). It has been difficult to solubilize and purify the enzyme as a stable active form for many years, but in 1993, the hyaluronic acid synthase gene (hasA) of Streptococcus pyogenes was isolated. Since then (see Non-Patent Document 2), active recombinant HAS has been obtained, and research on various properties of HAS has progressed.

  Thereafter, a hyaluronic acid synthase (hereinafter also referred to as “HAS”) gene was isolated from a mammal which is a eukaryote. First, HAS1 (Non-Patent Documents 3 to 4, Patent Documents 1 to 3) is isolated, and then HAS2 (Non-Patent Documents 5 to 6 and Patent Document 4) and HAS3 (Non-Patent Documents 7 and 4) are successively used. Isolated on. These are highly homologous and are thought to form a family. In addition to these, the HAS gene was also cloned from Streptococcus equisimilis (Non-patent Document 8, Patent Document 5) and Pasteurella multicida (Non-patent Document 9, Patent Document 6). Furthermore, among the known genes, those having high homology with the above-mentioned HAS exist, and A98R of PBCV-1 virus that infects Xenopus DG42 (Non-patent Document 10) or chlorella-like green algae (Non-patent Document 11). ) Has been reported to have HAS activity.

A98R has been reported to have different metal requirements than other prokaryotic and eukaryotic HAS and require manganese ions for HAS activity.
JP-A-9-224673 WO97 / 38113 WO97 / 40174 WO98 / 00551 WO99 / 23227 WO99 / 51265 Markovitz, M. et al., J. Biol. Chem. 234, 2343, (1959) DeAngelis, PL et al., J. Biol. Chem., 268, 19181 (1993) Itano, N. et al., J. Biol. Chem., 271, 9875 (1996) Itano, N. et al., Biochem. Biophys. Res. Commun., 222, 816 (1996) Watanabe, K. et al., J. Biol. Chem., 271, 22945 (1996) Spicer, AP et al., J. Biol. Chem., 271, 23400 (1996) Spicer, AP et al., J. Biol. Chem., 272, 8957 (1997) Kumari, K. et al., J. Biol. Chem., 272, 32539 (1997) DeAngelis, PL et al., J. Biol. Chem., 273, 8454 (1998) DeAngelis, PL et al., J. Biol. Chem., 271, 23657 (1996) DeAngelis, PL et al., Science, 278, 1800 (1997)

  An object of the present invention is to provide a hyaluronic acid synthase capable of producing hyaluronic acid under conditions with high expression efficiency in a host and reduced occurrence of environmental pollution, and a gene encoding the enzyme.

The present invention provides the following hyaluronic acid synthase and a gene encoding the enzyme.
1. Hyaluronic acid synthase gene comprising the following DNA (a), (b), (c) or (d):
(A) DNA comprising the base sequence represented by SEQ ID NO: 1 or its complementary strand;
(B) a DNA that hybridizes with the DNA of (a) above under stringent conditions and encodes a protein having hyaluronic acid synthase activity;
(C) DNA encoding a polypeptide having a homology of more than 96.8% with the DNA of (a) and having hyaluronic acid synthase activity;
(D) DNA encoding a polypeptide having one or several amino acids deleted, substituted, added or inserted in the amino acid sequence of SEQ ID NO: 5 and having hyaluronic acid synthesis activity.
2. Hyaluronic acid synthase gene comprising the following DNA (a), (b), (c) or (d):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 2 or its complementary strand;
(B) a DNA that hybridizes with the DNA of (a) above under stringent conditions and encodes a protein having hyaluronic acid synthase activity;
(C) DNA encoding a polypeptide having a homology of more than 96.5% with the DNA of (a) and having hyaluronic acid synthase activity;
(D) DNA encoding a polypeptide having one or several amino acids deleted, substituted, added or inserted in the amino acid sequence of SEQ ID NO: 5 and having hyaluronic acid synthesis activity.
3. Hyaluronic acid synthase comprising the following polypeptides (A) to (C):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 5,
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, added, deleted or inserted in the amino acid sequence of (A), and having hyaluronic acid synthase activity;
(C) a polypeptide having a homology of more than 97.8% with the amino acid sequence of (A) and having a hyaluronic acid synthase activity; Hyaluronic acid synthase comprising the following polypeptides (A) to (C):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6,
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, added, deleted or inserted in the amino acid sequence of (A), and having hyaluronic acid synthase activity;
(C) a polypeptide having a homology of more than 97.8% with the amino acid sequence of (A) and having hyaluronic acid synthase activity

According to the present invention, hyaluronic acid synthase that can be highly expressed in a host such as yeast and does not require metals that cause environmental pollution such as Mn ²⁺ ions and a gene encoding the enzyme can be provided.

  Hyaluronic acid synthase is an enzyme that synthesizes hyaluronic acid having a polymer structure composed of a repeating structure of glucuronic acid and glucosamine using UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates.

  The hyaluronic acid synthase of the present invention described in SEQ ID NO: 5 or 6 is a hyaluronic acid synthase derived from the Chlorella virus CVHI1 strain and CVKA1 strain. The chlorella viruses CVHI1 and CVKA1 are chlorella viruses of a different strain from the chlorella virus PBCV-1 isolated from Paramecium.

  The hyaluronic acid synthase of the present invention is magnesium ion-requiring and has high activity in the presence of magnesium ions. On the other hand, it is described in DeAngelis et al, 1997, Science, 278, 1800-1803 that a known hyaluronic acid synthase (A98R) derived from chlorella virus PBCV-1 is manganese ion-requiring. Manganese ions, unlike magnesium ions, have a negative impact on the environment and require waste liquid treatment.

  The hyaluronic acid synthase of the present invention is superior to A98R, which is a known hyaluronic acid synthase, in that it requires magnesium ions without adverse effects on the environment. In addition, although the hyaluronic acid synthase of this invention expresses high activity by magnesium ion, you may have activity lower than presence in magnesium presence in the presence of manganese ion.

  One or several (usually 9 or less, preferably 7 or less, more preferably 5 or less, more preferably 3 or less) amino acids of hyaluronic acid synthase have been deleted, substituted, added or inserted, and hyaluronic acid Polypeptides having synthetic activity, as well as polypeptides having homology of more than 97.8% (preferably more than 98%, more preferably more than 99%) and hyaluronic acid synthesis activity are preferably magnesium ion auxotrophic Is a polypeptide that is part of hyaluronic acid synthase and has hyaluronic acid synthesizing activity, and is a deletion, addition, insertion or substitution of hyaluronic acid synthase and has hyaluronic acid synthesizing activity Polypeptides, including polypeptides having greater than 97.8% homology and having hyaluronic acid synthesis activity . A polypeptide that is a part of hyaluronic acid synthase and has hyaluronic acid synthesizing activity includes an amino acid site that is essential for exhibiting hyaluronic acid synthesizing activity in the hyaluronic acid synthase as described above, and is not essential A part of the polypeptide is deleted and has a hyaluronic acid synthesis activity.

  In the DNA encoding the hyaluronic acid synthase or the amino acid sequence of the hyaluronic acid synthase in the present invention, one or several (usually 9 or less, preferably 7 or less, more preferably 5 or less, more preferably 3 or less) The DNA encoding the polypeptide having the amino acid deletion, substitution, addition or insertion and having hyaluronic acid synthesizing activity is not particularly limited as long as it encodes the hyaluronic acid synthase or polypeptide. Those that differ in sequence due to the degeneracy of.

  In one embodiment of the present invention, the hyaluronic acid synthase gene of the present invention has a nucleotide sequence of SEQ ID NO: 1 of more than 96.8%, preferably more than 97%, more preferably more than 98%, even more preferably 99%. It is a DNA encoding a polypeptide having super homology and having hyaluronic acid synthase activity.

  In another embodiment of the present invention, the hyaluronic acid synthase gene of the present invention has a nucleotide sequence of SEQ ID NO: 2 and more than 96.5%, preferably more than 97%, more preferably more than 98%, still more preferably DNA encoding a polypeptide having a homology of more than 99% and having hyaluronic acid synthase activity.

  In the present specification, “stringent conditions” means that only a base sequence encoding a polypeptide having hyaluronic acid synthase activity equivalent to a specific hyaluronic acid synthase hybridizes with the specific sequence (so-called specific hybrid). A base sequence encoding a polypeptide that does not have equivalent activity means a condition that does not form a hybrid (so-called non-specific hybrid) with the specific sequence. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, it hybridizes at 42 ° C. in 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0,2 M NaH 2 PO 4, 20 mM EDTA · 2Na, pH 7.4). Further, the condition of washing at 42 ° C. with 0.5 × SSC is an example of the stringent condition of the present invention, but is not limited thereto.

The stringent conditions are preferably highly stringent conditions. The highly stringent conditions are not particularly limited as long as the gene encoding A98R does not hybridize. For example, washing is performed at 60 ° C. at a salt concentration corresponding to 0.1 × SSC and 0.1% SDS. It is a condition that is called.
The hyaluronic acid synthase of the present invention is obtained by transforming a host with an expression vector inserted so that the DNA encoding the hyaluronic acid synthase is transcribed and translated in a form having the above properties. It can be obtained by culturing the transformant in an appropriate medium and collecting it from the obtained culture.
Examples of host cells include microbial cells such as E. coli, yeast, and bacteria, animal cells such as insect cells, COS-1 cells, and CHO cells, and plant cells such as Arabidopsis cells.

  Various vectors can be used depending on the host to be transformed. For example, pMAL-p2, pUC19 etc. in E. coli, pYES, pYC, pYI, pYL, pYEUra3TM etc. in yeast, pBLUE Bac4 etc. in insect cells, pSVK3, pFLAG-CMV-2, pcDNA3.1 in COS-1 cells. , PRc / CMV2, etc. In Arabidopsis cells, pBI and the like can be mentioned.

  Known promoters can be widely used for each host. For example, constitutive expression promoters such as PMA1 promoter, ADH1 promoter, GAL1 promoter, PGK promoter, PHO5 promoter, GAPDH promoter, etc. can be used in yeast.

  By inserting the gene encoding the hyaluronic acid synthase of the present invention into an appropriate vector such as pYEUra3TM, a target hyaluronic acid synthase expression vector can be obtained.

  Preparation of a transformant by introducing an expression vector into a host cell can be performed by a method commonly used in the field of genetic engineering. Examples include lipofection method, DEAE-dextran method, calcium chloride method, protoplast method, competent method, electroporation method and the like.

  The hyaluronic acid synthase of the present invention can be obtained by culturing the transformant obtained by the above method in an appropriate medium and collecting it from the resulting culture.

  In one embodiment of the present invention, the hyaluronic acid synthase of the present invention is obtained by crushing and centrifuging the transformant cell body (for example, yeast) obtained by enzymatic treatment, ultrasonic crushing, or the like. A cell-free extract can be obtained, and the cell-free extract can be obtained as a membrane fraction by ultracentrifugation.

  Further, from the obtained membrane fraction, it is solubilized using a surfactant such as dichitokinin or CHAPS (3-[(3-cholamidopropyl) dimethylamino) -1-propanesulphonic acid) and purified by a conventional purification method. You can also. Examples of purification methods include salting out with ammonium sulfate and sodium sulfate, centrifugation, dialysis, ultrafiltration, ion exchange chromatography, adsorption chromatography, hydrophobic chromatography, gel filtration chromatography, affinity chromatography, electrophoresis, etc. And can be purified by appropriately combining them.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated still more concretely, this invention is not limited to these.

Example 1 Method for Isolating Chlorella Virus-Derived Hyaluronic Acid Synthase Gene PCR primers were prepared in order to isolate the chlorella virus hyaluronic acid synthase gene (cvHAS) by PCR. Primers are already disclosed in Chlorella virus genome sequence information (Kutish et al, 1996, Virology, 223, 303-317) (Li et al, 1995, Virology, 212, 134-150) (Li et al, 1997 , Virology, 237, 360-377) (Lu et al, 1995, Virology, 206, 339-352) (Lu et al, 1996, Virology, 216, 102-123) and identification information of hyaluronic acid synthesis gene from chlorella virus (Graves et al, 1999, virology, 257, 15-23) (DeAngelis et al, 1997, Science, 278, 1800-1803) designed and added restriction enzyme sites necessary for introduction into expression vectors What was made was produced. The restriction enzyme site added a SacI site to the 3 ′ primer.
5 'primer (SEQ ID NO: 3)
5'- ATG GGT AAA AAT ATA ATC ATA ATG GTT TCG-3 '
3 'primer (SEQ ID NO: 4)
5 '-AAT TGA GCT CTC ACA CAG ACT GAG CAT TGG TAG −3'
The genomic DNA of the chlorella virus CVHI1 strain and CVKA1 strain was used as a PCR template. PCR was performed using a reaction program of 25 cycles of 94 ° C. for 2 minutes (94 ° C. for 15 seconds, 55 ° C. for 30 seconds, 68 ° C. for 2 minutes) using KOD-plus- (Toyobo) as the DNA polymerase. The obtained PCR fragment was inserted into the SmaI site and SacI site of the multicloning site of the expression vector pLTex321s. The DNA sequences of the CVHI1 strain-derived hyaluronic acid synthase gene cvHAS-HI (SEQ ID NO: 1) and the CVKA1 strain-derived hyaluronic acid synthase gene cvHAS-KA (SEQ ID NO: 2) were confirmed by a DNA sequencer.

Example 2 Method for Expression and Extraction of cvHAS in Yeast
Yeast (BY4743) containing cvHAS-HI or cvHAS-KA was cultured at 30 ° C. overnight. 1 ml of this preculture was added to 100 mL of SD medium (0.67% Yeast nitrogen base w / o AA, 2% glucose, 0.02% adenine sulfate, 0.02% L-tryptophan, 0.02% L-histidine, 0.03% lysine monohydrochloride, 0.03% L-methionine and -0.03% L-leucine were inoculated) and cultured at 30 ° C. for 18 hours. Thereafter, in order to induce the expression of recombinant cvHAS, the cells were cultured at a low temperature (10 ° C.) for 48 hours. After completion of the culture, the cells are collected by centrifuging the culture, and 30 ml of buffer (50 mM phosphate buffer (pH 7.0), 5% glycerol, 1 mM EDTA, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride) After suspending in (containing PMSF) and 1 mM dithiothreitol, the cells were disrupted by a French press. The disrupted solution is centrifuged (12,000 rpm, 20 minutes), and then the supernatant is ultracentrifuged (x100,000 g, 1 hour) to extract the crude membrane fraction, and then with 200 μl of buffer (same as above). Resuspended to obtain a crude cvHAS extract.

Example 3 Measurement of hyaluronic acid synthase activity Hyaluronic acid synthase activity was determined by synthesizing hyaluronic acid in vitro using a cvHAS crude extract produced in yeast and measuring the concentration of hyaluronic acid in the reaction mixture. expressed. In vitro hyaluronic acid synthesis consisted of 50 mM Tris buffer (pH 7.0), 20 mM MgCl2, 0.1 mM EGTA, 10 mM 2-ME, 0.05% BSA, 1 mM UDP-GlcA, 1 mM UDP-GlcNAc, and 10 μl of cvHAS crude extract The reaction solution was adjusted to a total volume of 100 μl and reacted at 37 ° C. for 20 hours. After the reaction, the reaction was completed by heating at 90 ° C. for 3 minutes. After the reaction solution was centrifuged, the supernatant was used for measurement of hyaluronic acid.

  For the measurement of hyaluronic acid, a hyaluronic acid plate “Chugai” (Fujirebio), a reagent for measuring hyaluronic acid by hyaluronic acid binding protein, was used. The hyaluronic acid plate “Chugai” is a sandwich-bound hyaluronic acid quantification method using hyaluronic acid binding protein. After the hyaluronic acid in the specimen is bound to the hyaluronic acid binding protein immobilized on the microtiter plate, the enzyme-labeled hyaluronic acid binding protein is bound to form a sandwich. Subsequently, when tetramethylbenzidine (TMB) and hydrogen peroxide are added, TMB is oxidized and colored by the action of the peroxidase of the labeling enzyme. After adding the reaction stop solution, the hyaluronic acid concentration of the sample can be evaluated by measuring absorbance (A450) with a plate reader at a wavelength of 450 nm and a control wavelength of 620 nm. A standard curve was prepared using the standard hyaluronic acid solution attached to this kit, and the concentration of hyaluronic acid synthesized in the reaction solution was calculated from the absorbance of the reaction solution before and after the above reaction.

  The protein concentration of the crude extract was measured using Bio-Rad protein assay reagent (BioRad) using BSA as a standard protein.

  Hyaluronic acid synthase activity is calculated by calculating the amount of hyaluronic acid synthesized per hour (ng-HA / h / mg-protein) by 1 mg of protein contained in the crude hyaluronic acid extract. 1 represents HAS activity.

  In Table 1, a crude extract derived from yeast transformed with a vector containing no hyaluronic acid synthase was designated as mock. Crude extracts derived from yeast transformed with a vector containing hyaluronic acid synthase derived from the chlorella virus CVHI1 and CVKA1 strains were expressed as cvHAS-HI and cvHAS-KA, respectively.

As shown in the results of Table 1, it was confirmed that the isolated gene has hyaluronic acid synthase activity.
Example 4 Optimum temperature of cvHAS Using the cvHAS crude extract whose hyaluronic acid synthase activity was confirmed in Example 3, the optimum temperature of cvHAS was examined. For in vitro hyaluronic acid synthesis, 50 mM Tris buffer (pH 7.0), 20 mM MgCl2, 0.1 mM EGTA, 10 mM 2-ME, 0.05% BSA, 1 mM UDP-GlcA, 1 mM UDP-GlcNAc and 10 μl of cvHAS crude extract were used. The reaction solution was adjusted to a total volume of 100 μl and reacted at a temperature range of 10 to 42 ° C. for 1 hour. The hyaluronic acid synthase activity of the cvHAS crude extract was measured by the method shown in Example 3, and the relative activity at each temperature was expressed. The results are shown in FIG. As shown in the results of FIG. 1, it was confirmed that the optimum temperature of cvHAS-HI was around 37 ° C.
Example 5 Optimal pH of cvHAS
Using the cvHAS crude extract whose hyaluronic acid synthase activity was confirmed in Example 3, the optimum pH of cvHAS was examined. In vitro hyaluronic acid synthesis consisted of 20 mM MgCl2, 0.1 mM EGTA, 10 mM 2-ME, 0.05% BSA, 1 mM UDP-GlcA, 1 mM UDP-GlcNAc, 10 μl of cv HAS crude extract and 50 mM phosphate buffer (pH 5.5). -7.5), and the reaction solution adjusted to a total volume of 100 μl was reacted at 37 ° C. for 1 hour. The hyaluronic acid synthase activity of the cvHAS crude extract was measured by the method shown in Example 3, and the relative activity at each pH was expressed. The result is shown in FIG. As shown in the results of FIG. 2, it was confirmed that the optimum pH of cvHAS-HI was around 7.0.
Example 6 Metal ion requirement of cvHAS Using the cvHAS crude extract whose hyaluronic acid synthase activity was confirmed in Example 3, the metal ion requirement of cvHAS was examined. In vitro hyaluronic acid synthesis consists of 50 mM Tris buffer (pH 7.0), 20 mM MgCl2, or MnCl2, 0.1 mM EGTA, 20 mM 2-ME, 0.05% BSA, 1 mM UDP-GlcA, 1 mM UDP-GlcNAc, and 10 μl cv HAS The reaction solution containing the crude extract and adjusted to a total volume of 100 μl was reacted at 37 ° C. for 1 hour and 2 hours. After the reaction, the reaction was completed by heating at 90 ° C. for 3 minutes. The hyaluronic acid synthase activity of the cvHAS crude extract was measured by the method shown in Example 3 to show the activity in the presence of each metal ion. The results are shown in FIG. As shown in the results of FIG. 3, cvHAS has a high metal requirement for Mg2 + ions, and it was confirmed that the enzyme activity was 10% or less in the presence of Mn2 + ions compared to the presence of Mg2 + ions.

The relative activity of hyaluronic acid synthase at each temperature is shown. The relative activity at each pH of hyaluronic acid synthase is shown. The hyaluronic acid synthase activity in the presence of each metal ion is shown.

Claims (4)

Hyaluronic acid synthase gene comprising the following DNA (a), (b), (c) or (d):
(A) DNA comprising the base sequence represented by SEQ ID NO: 1 or its complementary strand;
(B) a DNA that hybridizes with the DNA of (a) above under stringent conditions and encodes a protein having hyaluronic acid synthase activity;
(C) DNA encoding a polypeptide having a homology of more than 96.8% with the DNA of (a) and having hyaluronic acid synthase activity;
(D) DNA encoding a polypeptide having one or several amino acids deleted, substituted, added or inserted in the amino acid sequence of SEQ ID NO: 5 and having hyaluronic acid synthesis activity. Hyaluronic acid synthase gene comprising the following DNA (a), (b), (c) or (d):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 2 or its complementary strand;
(B) a DNA that hybridizes with the DNA of (a) above under stringent conditions and encodes a protein having hyaluronic acid synthase activity;
(C) DNA encoding a polypeptide having a homology of more than 96.5% with the DNA of (a) and having hyaluronic acid synthase activity;
(D) DNA encoding a polypeptide having one or several amino acids deleted, substituted, added or inserted in the amino acid sequence of SEQ ID NO: 5 and having hyaluronic acid synthesis activity. Hyaluronic acid synthase comprising the following polypeptides (A) to (C):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 5,
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, added, deleted or inserted in the amino acid sequence of (A), and having hyaluronic acid synthase activity;
(C) A polypeptide having a homology of more than 97.8% with the amino acid sequence of (A) and having a hyaluronic acid synthase activity. Hyaluronic acid synthase comprising the following polypeptides (A) to (C):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6,
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, added, deleted or inserted in the amino acid sequence of (A), and having hyaluronic acid synthase activity;
(C) A polypeptide having a homology of more than 97.8% with the amino acid sequence of (A) and having a hyaluronic acid synthase activity.

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