WO2020199521A1 - 一种硫酸软骨素酶的筛选，鉴定及优化表达 - Google Patents

一种硫酸软骨素酶的筛选，鉴定及优化表达 Download PDF

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WO2020199521A1
WO2020199521A1 PCT/CN2019/106448 CN2019106448W WO2020199521A1 WO 2020199521 A1 WO2020199521 A1 WO 2020199521A1 CN 2019106448 W CN2019106448 W CN 2019106448W WO 2020199521 A1 WO2020199521 A1 WO 2020199521A1
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chondroitin sulfate
sulfate
enzyme
chondroitin
chondrosulfatase
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张昊宁
陈松
汤传根
王璟
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南京汉欣医药科技有限公司
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Priority to US17/601,080 priority Critical patent/US11667901B2/en
Priority to EP19922714.1A priority patent/EP3933037A4/en
Publication of WO2020199521A1 publication Critical patent/WO2020199521A1/zh

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    • C12Y301/06004—N-Acetylgalactosamine-6-sulfatase (3.1.6.4)

Definitions

  • the invention relates to the screening, identification and optimized expression of chondroitin sulfate, belonging to the technical field of bioengineering.
  • Chondrosulphatase is a lyase that can degrade glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and hyaluronic acid into unsaturated disaccharides and oligosaccharides.
  • ChSase is divided into ChSase ABC, ChSase AC, ChSase B and ChSase C according to the different substrates. Chondroitin sulfate has important application value in biochemical and pharmaceutical fields.
  • chondroitin sulfate In basic research, it can be used as a tool enzyme for the quality research of chondroitin sulfate and the efficient preparation of chondroitin sulfate oligosaccharides with multiple biological activities;
  • chondroitin sulfate In medicine, chondroitin sulfate is used as a medicinal enzyme, which can degrade mucus in cystic fibrosis sites, promote the regeneration of nerve axes, reduce the symptoms of lumbar disc herniation, anti-tumor and enhance the adhesion between chondrocytes and cartilage And many other medicinal functions.
  • Chondroitin sulfate is mainly derived from microorganisms, and most of them are lyases.
  • Flavobacterium heparinum, Proteus pengii, Aeromonas sobria, Proteus vulgaris, etc. can produce chondroitin sulfate lyase. However, most of the chondroitin sulfate lyase derived from microorganisms is an intracellular enzyme with low enzyme activity. Tao Ke et al.
  • ChSase derived from Proteus was heterologously expressed in E. coli.
  • glyceraldehyde-3-phosphate dehydrogenase was co-expressed, but the enzyme activity after purification was reduced by 3.1 times. , So it is not conducive to industrial amplification.
  • the invention collects soil samples, sewage or silt from coasts, rivers, farmer's markets, slaughterhouses, canteens, etc., to screen, and identifies a chondroitin sulfate enzyme. Take an appropriate amount of soil sample, extract it with physiological saline, after enrichment and culture, apply gradient dilution to screen the plate, compare and select the colony with the largest ratio of the transparent circle to the strain diameter, inoculate the colony into the liquid fermentation medium, cultivate and extract the bacteria The group genome was subjected to metagenomic sequencing, and the sequence with the highest possibility of chondroitin sulfate was analyzed and screened.
  • the sequence was then artificially synthesized and further identified and optimized for expression in Escherichia coli and Bacillus subtilis.
  • the expression vector used was plasmid pBRSFDuet-1, pHT01, pHT43-His, pMA5 or pWB980.
  • the present invention screened and identified a chondroitin sulfate enzyme from the natural world, and its amino acid sequence was 85% similar to the known chondroitin sulfate enzyme reported by NCBI (National Center of Biotechnology Information). Then it was found in E. coli and Bacillus subtilis. Optimized expression in Bacillus to achieve efficient biosynthesis of chondroitin sulfate with high enzymatic activity, with the highest enzymatic activity of 11976.5U/L.
  • the chondroitin sulfate enzyme collects soil samples from coasts, rivers, farmer’s markets, slaughterhouses, canteens, etc., and screens sewage or sludge.
  • the amino acid sequence identified is the same as that of NCBI.
  • the highest similarity of chondroitin sulfate reported is 85%.
  • the chondroitin sulfate is optimized for expression in the engineered strains of Escherichia coli or Bacillus subtilis, including Escherichia coli MG1655, Escherichia coli DH5 ⁇ , Escherichia coli W3110, Escherichia coli BL21, and Bacillus subtilis 168 , Bacillus subtilis WB600, Bacillus subtilis WB800 and other hosts.
  • the engineered strain is further preferably Bacillus subtilis, because the strain is more safe and has a wider application range.
  • the expression vector of the chondroitin sulfate gene is plasmid pBRSFDuet-1 or pHT01.
  • the optimized expression of the chondroitinase gene includes codon optimization, ribosome binding site RBS optimization, promoter optimization, and the like.
  • the fermentation medium components are: yeast powder 12-18g/L, glucose 32-48g/L, potassium sulfate 3.2-4.8g/L, magnesium sulfate 1.8-2.2g/L L, phosphate buffer 40 ⁇ 60mM, FeCl 2 ⁇ 6H 2 O is 10.8 ⁇ 16.2mg/L; MnCl 2 ⁇ 4H 2 O is 0.8 ⁇ 1.2mg/L; ZnCl 2 is 1.36 ⁇ 2.04mg/L; CuCl 2 ⁇ 2H 2 O is 0.344 ⁇ 0.516mg/L; pH 5 ⁇ 9.
  • the engineered strain fermentation method is to inoculate the recombinant bacteria into the fermentation medium and ferment at 30-40°C for 20-80 h.
  • the method for determining the enzyme activity of the chondroitin sulfate enzyme is: sonicating the fermentation broth to disrupt the cells, centrifuging at 12000 rpm and 4°C for 20 minutes to take the supernatant as the crude enzyme solution; the crude enzyme solution is purified by MBP column (10mM maltose eluted). Add 40 microliters of appropriately diluted enzyme solution to 960 microliters of substrate solution (2g/L of C4S dissolved in 20mM of Tris-Hcl) based on the kinetics of enzyme activity determination method to take the initial reaction rate within 1 min.
  • Enzyme activity definition The amount of enzyme required for every 1 micromol of substrate consumed per unit time is defined as an enzyme activity unit.
  • the present invention also provides the application of the chondroitin sulfate in the preparation of products containing low molecular weight chondroitin sulfate in the fields of medicine, cosmetics and biology.
  • the present invention screens and identifies a chondroitin sulfate from the natural world, and its amino acid sequence is 85% similar to the chondroitin sulfate reported by NCBI.
  • Optimized expression in Bacillus subtilis and the expression vector is plasmid pBRSFDuet-1, pHT01, pHT43-His, pMA5 or pWB980, which realizes the high-efficiency biosynthesis of high-enzyme chondroitinase sulfate, and its highest enzyme activity is 11976.5U/L, Moreover, the whole process and post-treatment are relatively simpler, and it has potential and wide application value in preparing products containing low molecular weight chondroitin sulfate in the fields of medicine, cosmetics, and biology.
  • Figure 1 shows a schematic diagram of the construction of an E. coli vector optimized for expression of chondroitin sulfate
  • Figure 2 shows a schematic diagram of the construction of a Bacillus subtilis vector optimized for expression of chondroitin sulfate
  • Figure 3 shows the enzyme activity of optimized expression of chondroitin sulfate in Escherichia coli and Bacillus subtilis cultured in shake flasks for 50 hours.
  • SEQ ID NO. 1 sequence information is the amino acid sequence of a novel chondroitin sulphate enzyme identified by screening from nature in the present invention.
  • Example 1 Screening and identification of chondroitin sulfate
  • the selection medium (g/L) used was: chondroitin sulfate 4.5, ammonium chloride 2.5, sodium chloride 0.95, magnesium sulfate 1.0, dipotassium hydrogen phosphate 1.0, agar 20.0, bovine serum albumin 5.0, pH 7.0.
  • Example 2 Optimal expression of chondroitinase in Escherichia coli and Bacillus subtilis
  • CS(pBRSF)-F/CS(pBRSF)-R Use primers CS(pBRSF)-F/CS(pBRSF)-R to amplify CS DNA fragments by PCR (using artificially synthesized CS DNA as a template), and assemble the resulting CS DNA PCR product with the backbone pBRSFDuet-1 (use primer pBRSF- F/pBRSF-R amplification) to generate pBRSFDuet-1-CS recombinant plasmid.
  • CS(pHT)-F/CS(pHT)-R Use primers CS(pHT)-F/CS(pHT)-R to amplify CS DNA fragments by PCR (using synthetic CS DNA as a template), and assemble the resulting CS DNA PCR products with the backbone pHT01 (use primers pHT01-F/ pHT01-R amplification) to generate pHT01-CS recombinant plasmid.
  • the recombinant plasmid pBRSFDuet-1-CS was transformed into Escherichia coli MG1655, Escherichia coli DH5 ⁇ , Escherichia coli W3110, and Escherichia coli BL21, respectively, to obtain recombinant bacteria CSmg, CSdh, CSw300, and CSbl.
  • the recombinant plasmid pHT01-CS was transferred into Bacillus subtilis 168, Bacillus subtilis WB600, and Bacillus subtilis WB800, respectively, to obtain recombinant bacteria CS168, CS600, and CS800.
  • the primer information is as follows: 5’-3’ direction
  • IPTG IPTG inducer and corresponding antibiotics as needed (E. coli with kanamycin at a final concentration of 50ug/mL, Bacillus subtilis Add chloramphenicol with a final concentration of 25ug/mL to the bacilli, then place it at 220 rpm and 37°C for cultivation, and then perform enzyme activity determination after culturing for 50 hours.
  • the method for determining the enzyme activity of chondroitin sulfate is as follows: the fermentation broth is sonicated to disrupt the cells, and the supernatant is centrifuged at 12,000 rpm and 4°C for 20 minutes as the crude enzyme solution; the crude enzyme solution is purified by MBP column (eluted with 10 mM maltose).
  • Enzyme activity definition The amount of enzyme required for every 1 micromol of substrate consumed per unit time is defined as an enzyme activity unit.
  • the results of the enzyme activity determination are shown in Figure 3.
  • the recombinant bacteria can achieve high-enzyme activity chondroitin sulfate biosynthesis, and the chondroitin sulfate enzyme activity of CSdh is relatively high, reaching 11976.5 U/L.

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Abstract

一种硫酸软骨素酶的筛选，鉴定及优化表达，属于生物工程技术领域。从自然界中筛选鉴定出一种硫酸软骨素酶，其氨基酸序列与NCBI报道的硫酸软骨素酶最高相似度为85％，然后在大肠杆菌和枯草芽孢杆菌中优化表达，实现了高酶活的硫酸软骨素酶高效生物合成，其最高酶活为11976.5U/L，而且整个工艺和后处理相对更简单。在医药、化妆品、生物领域中制备含低分子量硫酸软骨素的产品中具有潜在而广泛的应用价值，为微生物系统高效发酵制备高酶活的硫酸软骨素酶奠定了基础，适合于工业化生产应用。

Description

一种硫酸软骨素酶的筛选，鉴定及优化表达 技术领域
本发明涉及一种硫酸软骨素酶的筛选，鉴定及优化表达，属于生物工程技术领域。
背景技术
硫酸软骨素酶(Chondrosulphatase，简称ChSase)是能够将硫酸软骨素、硫酸皮肤素和透明质酸等糖胺聚糖降解成不饱和二糖及寡糖的1种裂解酶。ChSase根据其作用底物的不同分为ChSase ABC、ChSase AC、ChSase B及ChSase C等。硫酸软骨素酶在生物化工及医药领域具有重要的应用价值，在基础研究中，它可以作为工具酶，用于硫酸软骨素质量研究和高效制备具有多种生物活性的硫酸软骨素低聚糖；在医药上，硫酸软骨素酶做为药用酶，具有降解囊性纤维变性部位的黏液、促进神经轴的再生、减轻腰椎间盘突出的症状、抗肿瘤和增强软骨细胞与软骨之间的黏附力等多种药用功能。硫酸软骨素酶主要来源于微生物，且多为裂解酶，肝素黄杆菌、彭氏变形杆菌和温和气单孢菌、普通变形杆菌等均可产硫酸软骨素裂解酶。然而大多数微生物来源的硫酸软骨素裂解酶为胞内酶且酶活力较低。陶科等人(硫酸软骨素裂解酶ABC产生菌的筛选及发酵工艺研究[J].中国抗生素杂志,2004,(29)3:138-140.)曾报道过利用一株彭氏变形杆菌发酵生产ChSase，该过程工艺较为复杂，而且在发酵后期，ChSase的酶活下降较为明显，整个过程的最高酶活仅为322.17U/L。苏昕等人(发酵法制备硫酸软骨素裂解酶及其酶分离纯化的研究[J].微生物学杂志,2005,(25)4:64-67.)报道了从鱼腹中筛选得到的一株温和气单孢菌，虽然其产酶能力较高，但是需要经过1步透析和4步柱层析的纯化工艺，过程较为复杂，成本较高，不利于工业放大。除了发酵来源微生物进行产酶，Li等人(Expression,purification and characterization of GAPDH-ChSase ABC I from Proteus vulgaris in Escherichia coli[J].Protein Expression and Purification,2016,(128):36-41.)也报道了将来源于变形杆菌的ChSase在大肠杆菌中进行异源表达，为提高ChSase的表达量，报道中共表达了甘油醛-3-磷酸脱氢酶，但是纯化后的酶活却降低了3.1倍，因此不利于工业放大。目前国内没有产品供应，几乎全部依靠进口，价格非常昂贵，限制了硫酸软骨素酶在制备低分子硫酸软骨素和医药方面的应用，因此，筛选高酶活的硫酸软骨素酶具有重要的意义。
发明内容
本发明从海岸边、河边、农贸市场、屠宰厂和食堂等处采集土样，污水或淤泥进行筛选，鉴定出一种硫酸软骨素酶。取适量土样，用生理盐水浸提，经富集培养后，梯度稀释涂筛选 平板，比较并挑选透明圈与菌株直径比值最大的菌落，将该菌落接种到液体发酵培养基中培养并提取菌群基因组进行宏基因组测序，分析并筛选出硫酸软骨素酶可能性最高的序列，然后人工合成该序列并在大肠杆菌和枯草芽孢杆菌中进一步鉴定与优化表达，所用表达载体为质粒pBRSFDuet-1、pHT01、pHT43-His、pMA5或pWB980。本发明从自然界中筛选鉴定出一种硫酸软骨素酶，其氨基酸序列与NCBI(全称National Center of Biotechnology Information)报道的已知硫酸软骨素酶最高相似度为85％，然后在大肠杆菌和枯草芽孢杆菌中优化表达，实现了高酶活的硫酸软骨素酶高效生物合成，其最高酶活为11976.5U/L。
在本发明的一种实施方式中，所述硫酸软骨素酶从海岸边、河边、农贸市场、屠宰厂和食堂等处采集土样，污水或淤泥进行筛选，鉴定得到的其氨基酸序列与NCBI报道的硫酸软骨素酶最高相似度为85％。
在本发明的一种实施方式中，所述硫酸软骨素酶在工程菌株大肠杆菌或枯草芽孢杆菌中优化表达，包括大肠杆菌MG1655，大肠杆菌DH5α，大肠杆菌W3110，大肠杆菌BL21，枯草芽孢杆菌168、枯草芽孢杆菌WB600、枯草芽孢杆菌WB800等宿主。
在本发明的一种实施方式中，所述工程菌株进一步优选为枯草芽孢杆菌，因为该菌种安全性更高，应用范围更广泛。
在本发明的一种实施方式中，所述硫酸软骨素酶基因的表达载体为质粒pBRSFDuet-1或pHT01。
在本发明的一种实施方式中，所述硫酸软骨素酶基因进行优化表达，包括密码子优化、核糖体结合位点RBS优化、启动子优化等。
在本发明的一种实施方式中，所述发酵培养基组分为：酵母粉12～18g/L，葡萄糖32～48g/L，硫酸钾3.2～4.8g/L，硫酸镁1.8～2.2g/L，磷酸盐缓冲液40～60mM，FeCl 2·6H 2O为10.8～16.2mg/L；MnCl 2·4H 2O为0.8～1.2mg/L；ZnCl 2为1.36～2.04mg/L；CuCl 2·2H 2O为0.344～0.516mg/L；pH 5～9。
在本发明的一种实施方式中，所述工程菌株发酵方法为将重组菌接种至发酵培养基，在30～40℃发酵20～80h。
在本发明的一种实施方式中，所述硫酸软骨素酶的酶活测定方法为：将发酵液超声破碎细胞，12000rpm 4℃离心20min取上清作为粗酶液；粗酶液经过MBP柱纯化(10mM的麦芽糖洗脱)。将40微升适当稀释的酶液加到960微升的底物溶液中(2g/L的C4S溶于20mM的Tris-Hcl)基于动力学的酶活测定方式取1min内的初始反应速率。酶活定义:单位时间内每消耗1微摩尔的底物所需要的酶量定义为一个酶活单位。
本发明还提供所述的硫酸软骨素酶在医药、化妆品、生物领域制备含低分子量硫酸软骨 素的产品中的应用。
本发明相对现有技术的有益效果如下所示：本发明从自然界中筛选鉴定出一种硫酸软骨素酶，其氨基酸序列与NCBI报道的硫酸软骨素酶最高相似度为85％，然后在大肠杆菌和枯草芽孢杆菌中优化表达，表达载体为质粒pBRSFDuet-1、pHT01、pHT43-His、pMA5或pWB980，实现了高酶活硫酸软骨素酶的高效生物合成，其最高酶活为11976.5U/L，而且整个工艺和后处理相对更简单，在医药、化妆品、生物领域制备含低分子量硫酸软骨素的产品中具有潜在而广泛的应用价值。
附图说明
图1所示为硫酸软骨素酶优化表达的大肠杆菌载体构建示意图；
图2所示为硫酸软骨素酶优化表达的枯草芽孢杆菌载体构建示意图；
图3所示为大肠杆菌和枯草芽孢杆菌中优化表达硫酸软骨素酶摇瓶上培养50h的酶活。
具体实施方式
实施例涉及的氨基酸序列信息：
SEQ ID NO.1序列信息为本发明从自然界中筛选鉴定出的新型硫酸软骨素酶氨基酸序列。
实施例1：硫酸软骨素酶的筛选与鉴定
从海岸边、河边、农贸市场、屠宰厂和食堂等处采集土样，污水或淤泥，取适量土样，用生理盐水浸提，经富集培养后，梯度稀释涂筛选平板，37℃培养3天后，挑选出有透明圈的菌落进行复筛。所用的筛选培养基(g/L)为：硫酸软骨素4.5，氯化铵2.5，氯化钠0.95，硫酸镁1.0，磷酸氢二钾1.0，琼脂20.0，牛血清白蛋白5.0，pH 7.0。在复筛平板中比较并挑选透明圈与菌株直径比值最大的菌落，将该菌落接种到液体发酵培养基中培养并提取菌群基因组进行宏基因组测序，分析并筛选出硫酸软骨素酶可能性最高的序列，将筛选出硫酸软骨素酶可能性最高的氨基酸序列与NCBI报道的硫酸软骨素酶进行对比，最终结果为该酶与NCBI报道的硫酸软骨素酶最高相似度为85％，然后人工合成该序列并在大肠杆菌和枯草芽孢杆菌中进一步鉴定，结果证明该酶确实是硫酸软骨素酶。
实施例2：在大肠杆菌和枯草芽孢杆菌中优化表达硫酸软骨素酶
表达系统的构建：
使用引物CS(pBRSF)-F/CS(pBRSF)-R通过PCR扩增CS DNA片段(使用人工合成CS  DNA为模板)，将所得的CS DNA PCR产物与骨架pBRSFDuet-1组装(使用引物pBRSF-F/pBRSF-R扩增)以产生pBRSFDuet-1-CS重组质粒。
使用引物CS(pHT)-F/CS(pHT)-R通过PCR扩增CS DNA片段(使用人工合成CS DNA为模板)，将所得的CS DNA PCR产物与骨架pHT01组装(使用引物pHT01-F/pHT01-R扩增)以产生pHT01-CS重组质粒。
重组菌的构建：
分别向大肠杆菌MG1655，大肠杆菌DH5α，大肠杆菌W3110，大肠杆菌BL21，转入重组质粒pBRSFDuet-1-CS，得到重组菌CSmg，CSdh，CSw300，CSbl。
分别向枯草芽孢杆菌168、枯草芽孢杆菌WB600、枯草芽孢杆菌WB800转入重组质粒pHT01-CS，得到重组菌CS168，CS600，CS800。
硫酸软骨素酶优化表达的大肠杆菌载体构建如图1所示；硫酸软骨素酶优化表达的枯草芽孢杆菌载体构建如图2所示。
引物信息如下：5’-3’方向
Figure PCTCN2019106448-appb-000001
分别挑取上述构建的7株重组菌株及对照菌(大肠杆菌转化pBRSFDuet-1空质粒，枯草芽孢杆菌转化pHT01空质粒)单克隆接种于5mL LB培养基，根据需要添加相应的抗生素(大肠杆菌加入终浓度为50ug/mL的卡那霉素(Kan)，枯草芽孢杆菌加入终浓度为25ug/mL的氯霉素)，置于200rpm 37℃培养10h，然后按10％的接种量转接于装液量为25mL的250mL三角摇瓶，培养基为发酵培养基，根据需要添加1.5mmol/L IPTG的诱导剂和相应的抗生素(大肠杆菌加入终浓度为50ug/mL的卡那霉素，枯草芽孢杆菌加入终浓度为25ug/mL的氯霉素)，然后置于220rpm 37℃培养，培养50h后进行酶活测定。硫酸软骨素酶的酶活测定方法为：将发酵液超声破碎细胞，12000rpm 4℃离心20min取上清作为粗酶液；粗酶液经过 MBP柱纯化(10mM的麦芽糖洗脱)。将40微升适当稀释的酶液加入960微升的底物溶液中(2g/L的C4S溶于20mM的Tris-HCl)基于动力学的酶活测定方式取1min内的初始反应速率。酶活定义:单位时间内每消耗1微摩尔的底物所需要的酶量定义为一个酶活单位。酶活测定结果如图3所示，重组菌都能实现高酶活硫酸软骨素酶的高效生物合成，其中CSdh的硫酸软骨素酶的酶活较高，达为11976.5U/L。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种硫酸软骨素酶，其特征在于，氨基酸序列如SEQ ID NO.1所示。
  2. 根据权利要求1所述的硫酸软骨素酶，其特征在于，所述硫酸软骨素酶从海岸边、河边、农贸市场、屠宰厂和食堂处的土样、污水或淤泥中筛选、鉴定得到的，其氨基酸序列与已知的硫酸软骨素酶最高相似度为85％。
  3. 根据权利要求2所述的硫酸软骨素酶，其特征在于，所述硫酸软骨素酶在工程菌株大肠杆菌或枯草芽孢杆菌中优化表达。
  4. 根据权利要求3所述的硫酸软骨素酶，其特征在于，所述硫酸软骨素酶在枯草芽孢杆菌中优化表达。
  5. 根据权利要求3所述的硫酸软骨素酶，其特征在于，所述硫酸软骨素酶基因的表达载体为质粒pBRSFDuet-1、pHT01、pHT43-His、pMA5或pWB980。
  6. 根据权利要求3所述的硫酸软骨素酶，其特征在于，所述硫酸软骨素酶基因进行优化表达，包括密码子优化、核糖体结合位点优化或启动子优化。
  7. 根据权利要求3所述的硫酸软骨素酶，其特征在于，发酵培养基组分为：酵母粉12～18g/L，葡萄糖32～48g/L，硫酸钾3.2～4.8g/L，硫酸镁1.8～2.2g/L，磷酸盐缓冲液40～60mM，FeCl 2·6H 2O为10.8～16.2mg/L；MnCl 2·4H 2O为0.8～1.2mg/L；ZnCl 2为1.36～2.04mg/L；CuCl 2·2H 2O为0.344～0.516mg/L；pH 5～9。
  8. 根据权利要求3所述的硫酸软骨素酶，其特征在于，所述工程菌株发酵方法为将重组菌接种至发酵培养基，在30～40℃发酵20～80h。
  9. 根据权利要求1所述的硫酸软骨素酶，其特征在于，所述硫酸软骨素酶的酶活测定方法为：将发酵液用超声破碎细胞，12000rpm 4℃离心20min取上清作为粗酶液；粗酶液经过MBP柱纯化，所述纯化使用10mM的麦芽糖洗脱；将40微升适当稀释的酶液加到960微升的底物溶液中，所述底物溶液为2g/L的C4S溶于20mM的Tris-HCl中，基于动力学的酶活测定方式取1min内的初始反应速率。
  10. 权利要求1所述的硫酸软骨素酶在医药、化妆品、生物领域制备含低分子量硫酸软骨素的产品中的应用。
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