WO2021129353A1 - 一种透明质酸水解酶及其编码序列和利用其制备寡聚透明质酸盐的方法 - Google Patents

一种透明质酸水解酶及其编码序列和利用其制备寡聚透明质酸盐的方法 Download PDF

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WO2021129353A1
WO2021129353A1 PCT/CN2020/133978 CN2020133978W WO2021129353A1 WO 2021129353 A1 WO2021129353 A1 WO 2021129353A1 CN 2020133978 W CN2020133978 W CN 2020133978W WO 2021129353 A1 WO2021129353 A1 WO 2021129353A1
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hyaluronic acid
nucleotide
host cell
amino acid
hydrolase
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French (fr)
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李斌
余允东
徐飞
祝俊
邵凡涛
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江苏诚信药业有限公司
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase

Definitions

  • This application belongs to the technical field of genetic engineering and enzyme engineering, and in particular relates to a hyaluronic acid hydrolase and its coding sequence and a method for preparing oligomeric hyaluronate using the same.
  • Hyaluronic acid (HyaluroIlic acid, referred to as HA), also known as hyaluronic acid, is a macromolecular mucopolysaccharide, a branchless polymer glycosaminoglycan composed of repeating units of N-acetylglucosamine and D-glucuronic acid disaccharides. Sugars, N-acetylglucosamine and D-glucuronic acid are repeatedly and alternately connected by ⁇ -1,3 and ⁇ -1,4 glycosidic bonds. The two monosaccharides in the molecule are composed of equal molar ratios, and the molecular formula is (C 14 H 21 NO 11 ) n . Its structural formula is:
  • Hyaluronic acid exists in the interstitium of animal tissues or the capsule of some bacteria. It is widely used in medicine, cosmetics, food and other fields. Its molecular weight is generally 1 ⁇ 10 5 -10 7 Da. Studies have shown that molecular weight has a great influence on the activity of hyaluronic acid, and hyaluronic acid of different molecular weights even show diametrically opposite activities.
  • Oligomeric hyaluronic acid is hyaluronic acid with a molecular weight of less than 10,000 Da. It has an angiogenic effect in the body, promotes the proliferation of endothelial cells in vitro, and promotes wound healing.
  • the hydroxyl group, carboxyl group and other polar groups in the oligomeric hyaluronic acid molecule can form hydrogen bonds with water molecules to bind a large amount of water, and the water retention effect is obvious. Therefore, the oligomeric hyaluronic acid can be used for sun protection, anti-aging, and moisturizing. In cosmetics.
  • the methods of hyaluronic acid degradation mainly include physical degradation, chemical degradation, and biodegradation.
  • Physical degradation methods include heating, ultrasonic degradation, ultraviolet, mechanical shear degradation, ⁇ -ray radiation degradation, 60 Co radiation and microwave degradation.
  • the physical degradation method does not require chemical reagents, does not pollute the environment, the post-treatment process is simple, and the relative molecular mass distribution range of the obtained product is narrow, and the thermal stability is good. However, this method has a long degradation time, which is not conducive to large-scale production and reduces costs.
  • the chemical degradation method is mostly used.
  • Chemical degradation methods mainly include alkaline hydrolysis, acid hydrolysis and oxidative degradation.
  • the chemical degradation method can control the relative molecular weight of the product by controlling the amount of chemical reagents added and the reaction time.
  • the degradation time is short and the production cost is reduced.
  • its disadvantage is that it requires more severe reaction conditions (such as higher acid-base concentration, etc.) ) In order to achieve the greatest degree of degradation.
  • the glycosidic bond on the sugar chain is broken, but the structure of the residues of the monosaccharides (glucuronic acid and acetylglucosamine) is also destroyed.
  • the acetyl group is hydrolyzed, and the six-membered ring of the monosaccharide is broken.
  • the infrared spectrum is inconsistent with the European Pharmacopoeia standard spectrum), which has a certain impact on the biological activity of the prepared oligomeric hyaluronic acid.
  • the oligomeric hyaluronic acid prepared by the chemical degradation method is also easy to brown, and the production process will pollute the environment.
  • CN101429255A discloses a method for preparing low-molecular-weight sodium hyaluronate, that is, sodium hyaluronate solid powder is subjected to acid-catalyzed degradation in an organic solvent containing acid (such as sulfuric acid, hydrochloric acid, etc.) with an organic solvent concentration of 70-100%. Preparation of low molecular weight sodium hyaluronate with a molecular weight of 5000-900000 Da. However, this method needs to remove the organic solvent, and the process is cumbersome.
  • an organic solvent containing acid such as sulfuric acid, hydrochloric acid, etc.
  • the reaction conditions of enzymatic hydrolysis are mild, without strong acid and alkali, the prepared oligomeric hyaluronic acid will not brown, and will not cause environmental pollution, and the disaccharide structure of oligomeric hyaluronic acid prepared by enzymatic hydrolysis is complete, infrared
  • the map is consistent with the European Pharmacopoeia, so enzymatic hydrolysis is most suitable for preparing oligomeric hyaluronic acid.
  • the existing hyaluronidase has poor activity and high cost, and is not suitable for industrial production.
  • This application provides a hyaluronic acid hydrolase and its coding sequence and a method for preparing oligomeric hyaluronate using the same.
  • the hyaluronic acid hydrolase has relatively high activity, and when the hydrolase is used to prepare oligomeric hyaluronic acid, the operation is simple and the efficiency is high.
  • the present application provides a hyaluronic acid hydrolase, the hyaluronic acid hydrolase having any one of the amino acid sequences shown in (I), (II) or (III):
  • the above-mentioned amino acid sequences all have the activity of hyaluronic acid hydrolase, and the hyaluronic acid hydrolase provided in the present application has high activity, can efficiently hydrolyze hyaluronic acid, and hydrolyze it into oligomeric hyaluronic acid with lower molecular weight. , And the content of the product is higher. The efficiency of bioenzymatic preparation of oligomeric hyaluronic acid is greatly improved, which is beneficial to industrial production.
  • the hyaluronic acid hydrolase provided by the present application can also be applied to the fields of medical treatment and beauty.
  • amino acid sequence shown in SEQ ID NO. 1 is derived from Bacillus lentus, and includes 1233 amino acids with a molecular weight of 138.14 KDa.
  • the hyaluronic acid hydrolase has the amino acid sequence shown in SEQ ID NO. 97.5%, 98%, 98.5%, 99%, 99.5%, etc.) homologous amino acid sequences.
  • sequence of SEQ ID NO.1 is:
  • the present application provides a nucleotide encoding the hyaluronic acid hydrolase of the first aspect, the nucleotide having one of the nucleotide sequences shown in (I), (II) or (III) anyone:
  • the hyaluronic acid hydrolase has a nucleotide sequence that is greater than or equal to 90% (for example, 90%, 91%, 92%, 93%, 94%) with the nucleotide sequence shown in SEQ ID NO. %, 95%, 96%, 97%, 98%, or 99%, etc.) homologous nucleotide sequences.
  • sequence of SEQ ID NO. 2 is:
  • the present application provides a vector containing at least one copy of the nucleotide as described in the second aspect.
  • the obtaining of the vector is a conventional technical means in the art, as long as the method for obtaining the vector with the nucleotide sequence is feasible, there is no special limitation here, and those skilled in the art can follow Need to choose a suitable carrier preparation method.
  • the present application provides a recombinant host cell comprising the vector as described in the third aspect and/or the nucleotide encoding the hyaluronic acid hydrolase as described in the first aspect.
  • the host cell is a eukaryotic cell and/or a prokaryotic cell.
  • the eukaryotic cell includes any one or a combination of two or more of yeast cells, mammalian cells, insect cells or plant cells, preferably Pichia pastoris.
  • the prokaryote includes Escherichia coli and/or Bacillus subtilis.
  • the present application provides a method for preparing the hyaluronic acid hydrolase as described in the first aspect, including the following steps:
  • the host cell comprises a nucleotide encoding the hyaluronic acid hydrolase of the first aspect
  • the hyaluronic acid hydrolase expressed by the host cell is collected.
  • the method for preparing recombinant host cells is conventional technical means in the art.
  • the host cell is a eukaryotic cell
  • methods such as electroporation, DNA transfection, and microscopic injection can be used; when the host cell is a prokaryotic cell, the method can be used.
  • electroporation and other methods to prepare are conventional technical means in the art.
  • the collected hyaluronic acid hydrolase can be prepared into freeze-dried powder, tablet or liquid for use.
  • the present application also provides a method for synthesizing oligomeric hyaluronate using the hyaluronic acid hydrolase as described in the first aspect, which includes the following steps:
  • the mass concentration of hyaluronic acid and/or hyaluronic acid salt in step (1) is 1-20%, for example, it can be 1%, 2%, 5%, 8%, 10%, 12%, 14%, 16%, 18% or 20%, etc.
  • the temperature of the enzymatic hydrolysis is 30-50°C, for example, 30°C, 32°C, 35°C, 38°C, 40°C, 42°C, 45°C or 50°C, etc.
  • the pH is 4-9, for example It can be 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 8.5, or 9, etc.
  • the temperature of the inactivation is 60-100°C, for example, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C, etc.
  • 100min for example, may be 10min, 15min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, etc.
  • the inorganic salt is a soluble inorganic salt
  • the inorganic salt is a sodium salt, preferably sodium chloride.
  • the volume ratio of the alcohol and/or ketone to the filtrate is (3-10):1, for example, it can be 3:1, 3.5:1, 4:1, 5:1, 6:1, 7: 1, 8:1, 9:1 or 10:1, etc.
  • the hyaluronic acid hydrolase provided by this application has high enzymatic activity, and its enzymatic activity can reach 8.0 ⁇ 10 6 IU/mg after testing.
  • the use of the enzyme hyaluronic acid to generate oligomeric hyaluronic acid has high efficiency and high efficiency, and solves the shortcoming of low activity of hyaluronic acid hydrolase used in the prior art.
  • the method for preparing oligomeric hyaluronic acid provided in this application has simple operation, mild conditions, no damage to the product structure, no environmental pollution, and low cost of the hydrolyzed hyaluronidase derived from fermentation, which is suitable for large-scale industrial production. Compared with obtaining hyaluronic acid from animal sources, its production cost is greatly reduced, which is conducive to industrial production.
  • Figure 1 is the proton nuclear magnetic resonance spectrum of the oligomeric sodium hyaluronate prepared in Example 3.
  • LB liquid medium Lia-Bertani Medium
  • YPD liquid medium Yeast Extract Peptone Dextrose Medium
  • YPD solid medium yeast powder 10g, peptone 20g, glucose 20g, agar powder 20g, add water to make the volume to 1L;
  • BMGY liquid medium Bacted Glycerol-complex Medium
  • yeast powder 10g peptone 10g
  • YNB yeast Nitrogen Base without Amino Acid
  • glycerol 10g glycerol 10g
  • biotin 0.004g phosphoric acid Adjust the pH to 6.0 with salt buffer (0.1M), and add water to make the volume to 1L.
  • MD Minimal Dextrase Medium solid medium
  • YNB 13.4g biotin 0.4mg
  • glucose 20g agar powder 20g, add water to make the volume to 1L
  • agar powder 20g add water to make the volume to 1L
  • the formula of the fermentation medium is: 14.9 g of magnesium sulfate heptahydrate, 18.2 g of potassium sulfate, 0.93 g of calcium sulfate dihydrate, 40 g of glycerol, 26.7 mL of 85% phosphoric acid, and 4.13 g of potassium hydroxide. The volume is adjusted to 1L with water.
  • restriction enzymes EcoR I, Not I and Sac I used were purchased from New England Biolabs, and the restriction enzymes were used in accordance with the instructions for use of the restriction enzymes.
  • This example is used to prepare genetically engineered strains containing the nucleotide sequence of hyaluronic acid hydrolase.
  • Restriction endonuclease Sac I was used to linearize the recombinant plasmid.
  • Pichia pastoris GS115 Pichia pastoris GS115, purchased from invitrogen
  • YPD medium for activation
  • the activated GS115 was inserted into 50mL YPD medium at 0.5% inoculum and cultured to logarithm at 30°C.
  • the cells obtained by centrifugation were washed twice with 20mL sterile water, and then washed twice with 20mL sterile 1M sorbitol, and 1mL 1M sorbitol solution was added to resuspend the cells to obtain GS115 competent cells;
  • Lyticase purchased from sigma
  • Example 2 the recombinant engineering strain prepared in Example 1 was used to prepare hyaluronic acid hydrolase.
  • Example 2 Streak the recombinant engineered bacteria prepared in Example 1 on a YPD plate, culture it upside down at 30°C; pick a single colony with a diameter of 1mm on the plate and inoculate it into 50mL YPD liquid medium, culture it with shaking at 200rpm for 24h at 30°C.
  • OD 600 reaches 5
  • 10% of the inoculum amount is inoculated into 300 mL YPD liquid medium.
  • the shake flask used is a 1L Erlenmeyer flask.
  • the culture is shaken on a shaker at 30° C. and 200 rpm. When the OD 600 reaches 12, the culture is stopped.
  • the fermentation medium After the fermentation medium is configured per liter of material, it is poured into a 30L fermentor and sterilized at 121°C for 30 minutes; after cooling, the temperature is controlled at 30°C, and the pH value is adjusted to 5.0 with ammonia water. Inoculate the grown seed liquid into the tank with an inoculum amount of 5%. Adjust the speed and ventilation according to the dissolved oxygen, and control the dissolved oxygen above 30%;
  • the hyaluronidase precipitate was dissolved in phosphate buffer solution (pH 6.0, 50mmol/L), dialyzed overnight to remove residual ammonium sulfate, and finally passed through a 1 ⁇ 10 4 Da ultrafiltration membrane to remove small molecular impurities, and the purified transparent Plasmidase.
  • the hyaluronidase activity in the fermentation broth determined by the Chinese Pharmacopoeia method was 1.0 ⁇ 10 5 IU/mL, and the purified hyaluronidase activity was 8.0 ⁇ 10 6 IU/mg.
  • This embodiment provides a method for preparing oligomeric sodium hyaluronate.
  • the temperature Raise to 65°C, maintain for 30min, add 1kg NaCl, filter the enzymatic hydrolysate with 0.45 ⁇ m nylon filter membrane, then filter through membrane, precipitate with 500L ethanol to obtain sodium hyaluronate precipitate, then use ethanol to dehydrate the precipitate, then vacuum dry Obtain oligomeric sodium hyaluronate.
  • the oligomeric hyaluronic acid is 0.89kg of white particles.
  • the absorbance of glucuronic acid and the content of glucuronic acid at 530nm are measured by a spectrophotometer.
  • the content of hyaluronic acid is calculated to be 97.5% and the molecular weight is 9800Da. , PH 6.8.
  • This embodiment provides a method for preparing oligomeric sodium hyaluronate.
  • the enzymatic hydrolysate is filtered, and then filtered through a membrane and precipitated with 500L of ethanol to obtain sodium hyaluronate precipitate.
  • the precipitate is dehydrated with ethanol and then vacuum dried to obtain oligomeric sodium hyaluronate.
  • the oligomeric hyaluronic acid is 0.87kg of white particles.
  • the absorbance value of glucuronic acid and the content of glucuronic acid at 530nm are measured by a spectrophotometer.
  • the content of hyaluronic acid is calculated to be 96.5% and the molecular weight is 5100Da. , PH 6.9.
  • This embodiment provides a method for preparing oligomeric sodium hyaluronate.
  • the enzymatic hydrolysate is filtered, and then filtered through a membrane and precipitated with 1000 L of ethanol to obtain sodium hyaluronate precipitate, which is dehydrated with ethanol, and then dried in vacuum to obtain oligomeric sodium hyaluronate.
  • the oligomeric hyaluronic acid is 0.92kg of white particles.
  • the absorbance of glucuronic acid and the content of glucuronic acid at 530nm are measured by a spectrophotometer, and the content of hyaluronic acid is calculated to be 95.8% and the molecular weight is 1200Da , PH 7.1.
  • the hyaluronidase provided by this application is expressed in Pichia pastoris, its activity in the fermentation broth is 1.0 ⁇ 10 5 IU/mL, and the purified hyaluronidase activity is 8.0 ⁇ 10 6 IU/mg, when it is expressed in other hosts (such as Escherichia coli), its enzymatic activity is more excellent; therefore, using the hydrolyzed hyaluronidase provided in this application to produce oligomeric sodium hyaluronate is simple and efficient High, suitable for large-scale industrial production.

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Abstract

一种透明质酸水解酶及其编码序列和利用其制备寡聚透明质酸盐的方法。带有编码透明质酸水解酶的核苷酸序列可以通过转导、转化、结合转移的方法转入工程菌中，通过调节所述编码基因的表达，高效表达透明质酸水解酶，为寡聚透明质酸的生产提供有效途径。使用所述透明质酸水解酶水解透明质酸，操作简单，条件温和，效率较高，得到的寡聚透明质酸的纯度较高，同时发酵来源的透明质酸水解酶成本较低，适合大规模的工业化生产。

Description

一种透明质酸水解酶及其编码序列和利用其制备寡聚透明质酸盐的方法 技术领域
本申请属于基因工程和酶工程技术领域，尤其涉及一种透明质酸水解酶及其编码序列和利用其制备寡聚透明质酸盐的方法。
背景技术
透明质酸(HyaluroIlic acid，简称HA)又名玻璃酸，是一种大分子粘多糖，由N-乙酰氨基葡糖和D-葡糖醛酸双糖重复单元构成的无分支高分子糖胺聚糖，N-乙酰氨基葡糖和D-葡糖醛酸通过β-1,3和β-1,4糖苷键反复交替连接，分子中两种单糖按等摩尔比组成，分子式为(C 14H 21NO 11) n。其结构式为：
Figure PCTCN2020133978-appb-000001
透明质酸存在于动物组织间质或某些细菌的荚膜中，广泛用于医药、化妆品、食品等领域，分子量一般为1×10 5-10 7Da。研究表明，分子量对透明质酸的活性有很大影响，不同分子量的透明质酸甚至表现出截然相反的活性。
寡聚透明质酸是分子量小于10000Da的透明质酸，其在体内具有促血管生成作用，在体外可促进内皮细胞的增生，促进创伤愈合。此外，寡聚透明质酸分子中的羟基、羧基和其他极性基团可与水分子形成氢键而结合大量的水分，保水效果明显，因此寡聚透明质酸可用于防晒、抗衰老、保湿化妆品中。
目前，透明质酸降解的方法主要有物理降解、化学降解、生物降解三大类。 物理降解法包括加热、超声波降解、紫外线、机械剪切力降解、γ-射线辐射降解、 60Co射线和微波降解等。物理降解法不需要化学试剂，不污染环境，后处理过程简单，且得到产品的相对分子质量分布范围窄，热稳定性好，但是此法降解时间较长，不利于大规模生产，为降低成本，则多使用化学降解法。化学降解法主要有碱水解、酸水解和氧化降解。
化学降解法通过控制化学试剂的加入量和反应时间即可控制产品的相对分子质量，降解时间短，降低了生产成本，但是其缺点在于需要较剧烈的反应条件(如较高的酸碱浓度等)才能达到最大程度的降解。此时，不但糖链上的糖苷键断裂，单糖(葡糖醛酸和乙酰氨基葡糖)残基的结构也遭到破坏，如乙酰基被水解掉，单糖六元环断裂等(表现在红外图谱上是与欧洲药典标准图谱不一致)，对制得的寡聚透明质酸的生物学活性产生一定影响。化学降解法制备的寡聚透明质酸还容易褐变，生产过程会污染环境。
CN101429255A公开了一种低分子透明质酸钠的制备方法，即透明质酸钠固体粉末在含有酸(如硫酸、盐酸等)的有机溶剂中，有机溶剂浓度70-100％，进行酸催化降解，制备分子量5000-900000Da的低分子透明质酸钠。但该方法需要脱除有机溶剂，工艺繁琐。
生物降解中酶解法反应条件温和，不用强酸强碱，制备的寡聚透明质酸不会发生褐变，不会造成环境污染，并且酶解法制备的寡聚透明质酸的双糖结构完整，红外图谱与欧洲药典一致，因此酶解法最适合制备寡聚透明质酸。但是，现有的透明质酸酶活性较差，成本较高，不适用于工业化生产。
因此，需要提供一种活性较高、能够高效水解透明质酸的透明质酸水解酶来制备寡聚透明质酸。
发明内容
本申请提供了一种透明质酸水解酶及其编码序列和利用其制备寡聚透明质酸盐的方法。所述透明质酸水解酶活性较高，利用该水解酶制备寡聚透明质酸时操作简单，效率较高。
第一方面，本申请提供一种透明质酸水解酶，所述透明质酸水解酶具有(I)、(II)或(III)所示的氨基酸序列中的任意一个：
(I)如SEQ ID NO.1所示的氨基酸序列；
(II)与SEQ ID NO.1所示的氨基酸序列具有≥90％同源性的氨基酸序列；
(III)SEQ ID NO.1所示的氨基酸序列经修饰、取代、缺失或添加至少一个氨基酸获得的氨基酸序列。
以上所述的氨基酸序列均具有透明质酸水解酶的活性，且本申请提供的透明质酸水解酶活性较高，能够高效水解透明质酸，将其水解为分子量较低的寡聚透明质酸，且产物的含量较高。对生物酶法制备寡聚透明质酸的效率有较大的提升，有利于工业化生产。同时，本申请提供的透明质酸水解酶还能够应用于医疗、美容等领域。
本申请中，所述SEQ ID NO.1所示的氨基酸序列来自迟缓芽孢杆菌(Bacillus lentus)，包括1233个氨基酸，其分子量为138.14KDa。
作为本申请的优选技术方案，所述透明质酸水解酶具有与SEQ ID NO.1所示的氨基酸序列具有≥95％(例如可以是95％、95.5％、96％、96.5％、97％、97.5％、98％、98.5％、99％或99.5％等)同源性的氨基酸序列。
其中，SEQ ID NO.1的序列为：
Figure PCTCN2020133978-appb-000002
Figure PCTCN2020133978-appb-000003
第二方面，本申请提供编码第一方面所述的透明质酸水解酶的核苷酸，所述核苷酸具有(I)、(II)或(III)所示的核苷酸序列中的任意一个：
(I)编码第一方面所述的透明质酸水解酶的核苷酸序列；
(II)如SEQ ID NO.2所示的核苷酸序列；
(III)与SEQ ID NO.2所示的核苷酸序列具有≥85％同源性的核苷酸序列。
作为本申请的优选技术方案，所述透明质酸水解酶具有与SEQ ID NO.2所示的核苷酸序列具有≥90％(例如可以是90％、91％、92％、93％、94％、95％、96％、97％、98％或99％等)同源性的核苷酸序列。
其中，SEQ ID NO.2的序列为：
Figure PCTCN2020133978-appb-000004
Figure PCTCN2020133978-appb-000005
Figure PCTCN2020133978-appb-000006
Figure PCTCN2020133978-appb-000007
第三方面，本申请提供一种载体，所述载体含有至少一个拷贝的如第二方面所述核苷酸。
本申请中，所述载体的获得为本领域的常规技术手段，只要能够获得带有所述核苷酸序列的载体的方法都是可行的，在此不做特殊限定，本领域技术人员可以根据需要选择合适的载体制备方法。
第四方面，本申请提供一种重组的宿主细胞，包含如第三方面所述的载体和/或编码第一方面所述的透明质酸水解酶的核苷酸。
根据本申请，所述宿主细胞为真核细胞和/或原核细胞。
优选地，所述真核细胞包括酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞中的任意一种或两种以上的组合，优选为毕赤酵母。
优选地，所述原核生物包括大肠杆菌和/或枯草芽孢杆菌。
第五方面，本申请提供一种制备如第一方面所述的透明质酸水解酶的方法， 包括如下步骤：
制备重组的宿主细胞，其中所述宿主细胞包含编码第一方面所述的透明质酸水解酶的核苷酸；
培养所述宿主细胞；以及
收集所述宿主细胞表达的透明质酸水解酶。
本申请中，制备重组宿主细胞的方法为本领域的常规技术手段，例如当宿主细胞是真核细胞，可用电穿孔、DNA转染、显微镜注射等方法；当宿主细胞是原核细胞时，可用电穿孔等方法进行制备。
本申请中可以将收集得到的透明质酸水解酶制备成冻干粉、片剂或液体进行使用。
第六方面，本申请还提供一种利用如第一方面所述的透明质酸水解酶合成寡聚透明质酸盐的方法，包括如下步骤：
(1)配制含透明质酸和/或透明质酸盐的溶液，将使用第六方面所述的方法制备得到的透明质酸水解酶与所述溶液混合，酶解后得到酶解液；以及
(2)将所述酶解液灭活，加入无机盐，搅拌至完全溶解，过滤，向滤液中加入醇和/或酮，析出所述寡聚透明质酸盐。
根据本申请，步骤(1)所述透明质酸和/或透明质酸盐的质量浓度为1-20％，例如可以是1％、2％、5％、8％、10％、12％、14％、16％、18％或20％等。
优选地，所述酶解的温度为30-50℃，例如可以是30℃、32℃、35℃、38℃、40℃、42℃、45℃或50℃等，pH为4-9，例如可以是4、4.5、5、5.5、6、6.5、7、8、8.5或9等。
优选地，所述灭活的温度为60-100℃，例如可以是60℃、65℃、70℃、75℃、80℃、85℃、90℃、95℃或100℃等；时间为10-100min，例如可以是10min、 15min、20min、30min、40min、50min、60min、70min、80min、90min或100min等。
本申请中，所述无机盐为易溶性无机盐，所述无机盐为钠盐，优选为氯化钠。
优选地，所述醇和/或酮与所述滤液的体积比为(3-10):1，例如可以是3:1、3.5:1、4:1、5:1、6:1、7:1、8:1、9:1或10:1等。
本申请所述的数值范围不仅包括上述例举的点值，还包括没有例举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本申请不再穷尽列举所述范围包括的具体点值。
与现有技术相比，本申请的有益效果为：
(1)本申请提供的透明质酸水解酶酶活高，经检测其酶活可达8.0×10 6IU/mg。采用该酶透明质酸生成寡聚透明质酸，高效率较高，解决了现有技术中使用的透明质酸水解酶活性低的缺点。
(2)本申请中提供的制备寡聚透明质酸的方法操作简单，条件温和，对产品结构无破坏，无环境污染，而且发酵来源的水解透明质酸酶成本低、适合大规模工业化生产，相比于从动物来源获取透明质酸，其生产成本大大降低，利于工业化生产。
附图说明
图1为实施例3制备的寡聚透明质酸钠的核磁共振氢谱。
图2为实施例3制备的寡聚透明质酸钠的核磁共振碳谱。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本申请的技术方案，但下述的实例仅仅是本申请的简易例子，并不代表或限制本申请的权利保护范围， 本申请的保护范围以权利要求书为准。
以下实施例中LB液体培养基(Luria-Bertani Medium)的配方为：酵母粉5g，蛋白胨10g，氯化钠10g，调节pH到7.0，加水定容至1L；
YPD液体培养基(Yeast Extract Peptone Dextrose Medium)的配方为：酵母粉10g，蛋白胨20g，葡萄糖20g，加水定容至1L；
YPD固体培养基(YPD平板)的配方为：酵母粉10g，蛋白胨20g，葡萄糖20g，琼脂粉20g，加水定容至1L；
BMGY液体培养基(Buffered Glycerol-complex Medium)的配方为：酵母粉10g，蛋白胨10g，YNB(无氨基酵母氮源，Yeast Nitrogen Base without Amino Acid)13.4g，甘油10g，生物素0.004g，用磷酸盐缓冲液(0.1M)调节pH至6.0，加水定容至1L。
MD(Minimal Dextrase Medium)固体培养基的配方为：YNB 13.4g、生物素0.4mg、葡萄糖20g，琼脂粉20g，加水定容至1L；
发酵培养基的配方为：七水硫酸镁14.9g、硫酸钾18.2g、二水硫酸钙0.93g、甘油40g、85％磷酸26.7mL、氢氧化钾4.13g，加水定容至1L。
以下实施例中，所用限制性内切酶EcoR Ⅰ、Not Ⅰ和Sac Ⅰ购自New England Biolabs公司，使用时按照限制性内切酶使用说明书进行操作。
实施例1
本实施例用于制备含有透明质酸水解酶核苷酸序列的基因工程菌株。
1.将全基因合成的含有序列SEQ ID NO.1(由常州基宇生物科技有限公司合成)的序列经限制性内切酶EcoR Ⅰ和Not Ⅰ酶切后重组到酵母表达载体pPIC9k(购自invitrogen公司)，并转化到E.coli Top10感受态(购自北京全式金生物技术有限公司)中；
将E.coli Top10置于LB液体培养基中，37℃、160rpm转振荡培养过夜，提取重组质粒；
利用限制性内切酶Sac Ⅰ将重组质粒线性化。
2.将线性化重组质粒导入毕赤酵母
将Pichia Pastoris GS115(巴斯德毕赤酵母GS115，购自invitrogen公司)单菌落挑入YPD培养基中活化，活化的GS115按0.5％的接种量接入50mL YPD培养基中30℃培养至对数期，离心获得的菌体用20mL无菌水洗2次，再用20mL无菌1M山梨醇洗2次，加入1mL 1M山梨醇溶液重悬菌体获得GS115感受态细胞；
将上一步中Sac Ⅰ线性化的重组质粒片段加入到80μL GS115感受态细胞中冰浴5分钟，电转化后加入800μL山梨醇将细胞移至1.5mL无菌离心管中，25℃温育2小时后，离心涂MD平板，30℃培养至长出菌落后，划线分离出单菌落；
将单菌落挑入无菌水中加入适量溶壁酶(Lyticase，购自sigma公司)后37℃温育1小时消化细胞壁，取部分消化产物加入PCR体系挑选阳性克隆，所述阳性克隆即为含有所述透明质酸水解酶核苷酸序列的重组工程菌株。
将阳性克隆挑入YPD液体培养基后转接入BMGY液体培养基中，培养至OD 600为1.0时接入1％甲醇诱导，诱导72小时，每24小时补加一次甲醇用于诱导表达所述透明质酸水解酶。
实施例2
本实施例使用实施例1制备得到的重组工程菌株制备透明质酸水解酶。
取实施例1制备得到的重组工程菌在YPD平板上划线，30℃，倒置培养过夜；平板上挑取直径1mm的单菌落接种至50mL YPD液体培养基中，30℃， 200rpm振荡培养24h，OD 600达到5，以10％的接种量接种到300mL YPD液体培养基中，所用摇瓶为容量1L三角瓶，30℃、200rpm摇床振荡培养，当OD 600达到12时停止培养。
将发酵培养基按每升料配置好后，倒入30L发酵罐，121℃灭菌30min；降温后控制温度30℃，使用氨水调节pH值至5.0。将长好的种子液接种进罐，接种量5％。根据溶氧调节转速和通气，控制溶氧量在30％以上；
培养24小时，待溶氧量突变上升，此时湿重约为140g/L，开始补料50％甘油(w/v)，补料速度为每小时在每升发酵液中补充15mL甘油，补料时控制溶氧保持在30％以上；待菌种湿重长至180g/L左右，停止补加甘油，以7.2mL/L发酵液/小时流速流加100％甲醇并保持，诱导10小时后，pH值调节为6.0，诱导24小时，pH调节为7.0，补料速度保持不变，根据溶氧调节转速和通气，保持溶氧在30％以上。诱导96小时菌体湿重为340g/L，放罐，离心，收集上清液。
将上清液中加入硫酸铵，使其浓度为15％，过滤除去产生的沉淀，然后缓缓加入硫酸铵，使其浓度达到50％为止，得到的沉淀即为透明质酸酶，将得到的透明质酸酶沉淀溶于磷酸缓冲溶液(pH 6.0，50mmol/L)中，透析过夜，以去除残留的硫酸铵，最后经过1×10 4Da的超滤膜除去小分子杂质，得到纯化后透明质酸酶。采用中国药典方法测定发酵液中透明质酸酶活力为1.0×10 5IU/mL，纯化后的透明质酸酶活力为8.0×10 6IU/mg。
实施例3
本实施例提供一种寡聚透明质酸钠的制备方法。
向100L不锈钢溶解罐中加入100L纯化水，边搅拌边向该溶解罐中加入分子量为1.2×10 6Da的透明质酸钠1kg，待完全溶解后，用氢氧化钠调节PH至 6.0，并升温至45℃，加入实施例2中的制备得到的透明质酸水解酶1L，使用乌氏粘度计，采用特性粘度法测酶解溶解液中产物分子量，当酶解至分子量小于10000Da时，将温度升高至65℃，维持30min，加入1kg NaCl，用0.45μm的尼龙滤膜过滤酶解液，然后经过膜过滤、使用500L乙醇沉淀得到透明酸钠沉淀，再将沉淀使用乙醇脱水，然后真空干燥即得寡聚透明质酸钠。
该寡聚透明质酸为白色颗粒0.89kg，采用咔唑法，通过分光光度计测定530nm处葡萄糖醛酸的吸光度值与葡萄糖醛酸含量，计算出透明质酸的含量为97.5％，分子量为9800Da，pH 6.8。
本实施例制备的寡聚透明质酸钠的核磁共振图谱，其中图1为核磁共振氢谱，D-葡萄糖醛酸 1H NMR(300MHz,D 2O+NaOD)δ/ppm：4.46-4.469(1H,H-1)，3.437(1H,H-2)，3.552-3.580(1H,H-3)，3.692-3.742(1H,H-4)，3.692-3.742(1H,H-5)；N-乙酰氨基葡萄糖 1H NMR(300MHz,D 2O+NaOD)δ/ppm：4.520(1H,H-1)，3.692-3.742(1H,H-2)，3.692-3.742(1H,H-3)，3.437(1H,H-4)，3.437(1H,H-5)，3.692-3.742(1H,H-6proR)，3.937-3.974(1H,H-6proS)，1.962(1H,H-Me)；
图2为核磁共振碳谱，D-葡萄糖醛酸 13C NMR(300MHz,D 2O+NaOD)δ/ppm：104.460(C-1)，73.246(C-2)，76.443(C-3)，82.708(C-4)，80.026(C-5)，176.815(C＝O)，N-乙酰氨基葡萄糖 13C NMR(300MHz,D 2O+NaOD)δ/ppm：103.534(C-1)，56.857(C-2)，83.432(C-3)，72.696(C-4)，79.470(C-5)，63.941(C-6)，25.130(C-Me)，177.353(C＝O)。
综合核磁共振氢谱和碳谱的图谱数据可得，该结果与理论寡聚透明质酸钠的图谱基本吻合，证明所得产物为寡聚透明质酸钠。
实施例4
本实施例提供一种寡聚透明质酸钠的制备方法。
向100L不锈钢溶解罐中加入100L纯化水，边搅拌边向该溶解罐中加入分子量为1.2×10 6Da的透明质酸钠1kg，待完全溶解后，用氢氧化钠调节pH至6.0，并升温至45℃，加入实施例2中的制备得到的透明质酸水解酶1L，酶解至分子量至5000Da时，将温度升高至65℃，维持30min，加入1kg NaCl，用0.45μm的尼龙滤膜过滤酶解液，然后经过膜过滤、使用500L乙醇沉淀得到透明酸钠沉淀，该沉淀使用乙醇脱水，然后真空干燥即得寡聚透明质酸钠。
该寡聚透明质酸为白色颗粒0.87kg，采用咔唑法，通过分光光度计测定530nm处葡萄糖醛酸的吸光度值与葡萄糖醛酸含量，计算出透明质酸的含量为96.5％，分子量为5100Da，pH 6.9。
实施例5
本实施例提供一种寡聚透明质酸钠的制备方法。
向100L不锈钢溶解罐中加入100L纯化水，边搅拌边向该溶解罐中加入分子量为1.2×10 6Da的透明质酸钠1kg，待完全溶解后，用氢氧化钠调节pH至6.0，并升温至45℃，加入实施例2中的制备得到的透明质酸水解酶1L，酶解至分子量至1000Da时，将温度升高至65℃，维持30min，加入1kg NaCl，用0.45μm的尼龙滤膜过滤酶解液，然后经过膜过滤、使用1000L乙醇沉淀得到透明酸钠沉淀，该沉淀使用乙醇脱水，然后真空干燥即得寡聚透明质酸钠。
该寡聚透明质酸为白色颗粒0.92kg，采用咔唑法，通过分光光度计测定530nm处葡萄糖醛酸的吸光度值与葡萄糖醛酸含量，计算出透明质酸的含量为95.8％，分子量为1200Da，pH 7.1。
综上所述，本申请提供的透明质酸水解酶在毕赤酵母中表达之后，其在发酵液中的活力为1.0×10 5IU/mL，纯化后的透明质酸酶活力为8.0×10 6IU/mg，当 其在其他宿主(例如大肠杆菌)中表达时，其酶活力更加优异；因此，使用本申请提供的水解透明质酸酶生产寡聚透明质酸钠，操作简单，效率较高，适合大规模的工业化生产。
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 一种透明质酸水解酶，其具有(I)、(II)或(III)所示的氨基酸序列中的任意一个：
    (I)如SEQ ID NO.1所示的氨基酸序列；
    (II)与SEQ ID NO.1所示的氨基酸序列具有≥90％同源性的氨基酸序列；
    (III)SEQ ID NO.1所示的氨基酸序列经修饰、取代、缺失或添加至少一个氨基酸获得的氨基酸序列。
  2. 根据权利要求1所述的透明质酸水解酶，其中，所述透明质酸水解酶具有与SEQ ID NO.1所示的氨基酸序列具有≥95％同源性的氨基酸序列。
  3. 一种编码权利要求1或2所述的透明质酸水解酶的核苷酸，其具有(I)、(II)或(III)所示的核苷酸序列中的任意一个：
    (I)编码权利要求1或2所述的透明质酸水解酶的核苷酸序列；
    (II)如SEQ ID NO.2所示的核苷酸序列；
    (III)与SEQ ID NO.2所示的核苷酸序列具有≥85％同源性的核苷酸序列。
  4. 根据权利要求3所述的核苷酸，其中，所述核苷酸具有与SEQ ID NO.2所示的核苷酸序列具有≥90％同源性的核苷酸序列。
  5. 一种载体，其含有至少一个拷贝的如权利要求3或4所述的核苷酸。
  6. 一种重组的宿主细胞，其包含如权利要求5所述的载体和/或编码权利要求1或2所述的透明质酸水解酶的核苷酸。
  7. 根据权利要求6所述的宿主细胞，其中，所述宿主细胞为真核细胞和/或原核细胞。
  8. 根据权利要求7所述的宿主细胞，其中，所述真核细胞包括酵母细胞、哺乳动物细胞、昆虫细胞或植物细胞中的任意一种或两种以上的组合，优选为毕赤酵母。
  9. 根据权利要求7所述的宿主细胞，其中，所述原核生物包括大肠杆菌和/或枯草芽孢杆菌。
  10. 一种制备如权利要求1或2所述的透明质酸水解酶的方法，其包括如下步骤：
    制备重组的宿主细胞，其中所述宿主细胞包含编码权利要求1或2所述的透明质酸水解酶的核苷酸；
    培养所述宿主细胞；以及
    收集所述宿主细胞表达的透明质酸水解酶。
  11. 一种利用如权利要求1或2所述的透明质酸水解酶合成寡聚透明质酸盐的方法，其包括如下步骤：
    (1)配制含透明质酸和/或透明质酸盐的溶液，将所述透明质酸水解酶与所述溶液混合，酶解后得到酶解液；以及
    (2)将所述酶解液灭活，加入无机盐，搅拌至完全溶解，过滤，向滤液中加入醇和/或酮，析出所述寡聚透明质酸盐。
  12. 根据权利要求11所述的方法，其中，步骤(1)所述溶液中透明质酸和/或透明质酸盐的质量浓度为1-20％。
  13. 根据权利要求11所述的方法，其中，步骤(1)所述酶解的温度为30-50℃，pH为4-9。
  14. 根据权利要求11所述的方法，其中，步骤(2)所述灭活的温度为60-100℃，时间为10-100min。
  15. 根据权利要求11所述的方法，其中，步骤(2)所述无机盐为钠盐，优选为氯化钠；
    优选地，步骤(2)所述醇和/或酮与所述滤液的体积比为(3-10):1。
PCT/CN2020/133978 2019-12-27 2020-12-04 一种透明质酸水解酶及其编码序列和利用其制备寡聚透明质酸盐的方法 WO2021129353A1 (zh)

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