CN85103913A - 胶原酶制备工艺 - Google Patents
胶原酶制备工艺 Download PDFInfo
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- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims abstract description 3
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- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 238000013016 damping Methods 0.000 claims description 5
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- 235000013372 meat Nutrition 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
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- 238000000746 purification Methods 0.000 claims description 4
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 claims description 3
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- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- MKUXAQIIEYXACX-UHFFFAOYSA-N aciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 2
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- 102000035195 Peptidases Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 1
- 241000607632 Vibrio alginolyticus chemovar iophagus Species 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
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- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明属于酶工程或生物工程的技术领域。胶原酶是一种在生理pH及温度条件下水解天然胶原酶的水解蛋白酶，我们将组织梭菌Cl·histolyticum64008经改进的培养基、发酵液静止发酵得到毒性低的胶原酶，将发酵液经过无菌过滤，硫酸铵沉淀、脱盐、冻干获得胶原酶粗品，再经DEAE柱层析及Sephadex G-25脱盐，冻干后可得精品，该胶原酶是细胞分离的重要工具酶，其油膏制剂用于治疗清疮脱痂，其注射用于治疗椎脊盘突出症
Description
本发明属酶工程或生物工程技术领域。
胶原酶（Collagenase）是一种在生理PH及温度条件下水解天然胶原蛋白酶，胶原酶不仅是细胞分离的重要工具酶，而且是外用清疮脱痂，局部注射治疗椎脊盘突出症的良好药物。
1927年Ssadilow首先使用了“Collagenase”的名称，1938年Grassmenn第一次证明胶原酶的存在，它能作用于天然胶原分子的特异性螺旋体结构部份，通过对各种菌产生胶原酶的研究，认为只有溶组织梭状芽孢杆菌（Clostridum histolyticum）与介毒无色杆菌（Achro-mobacter iophagus）较有价值，在胶原酶的制备方面，美国哥伦比亚大学I.mandl博士提出的发酵配方提取方法及检定方法至今仍为各国研究工作者采用。五十年代提纯胶原酶的方法有四类：（1）硫酸铵沉淀法（2）有机溶剂沉淀分离法，（3）电泳法（4）吸附洗脱法。1959年，Grant.N.H.Alburn，H.z报导使用DEAE离子交换层析精制胶原酶的方法，七十年代至今采用的方法主要有两类：（一）利用各种交换材料及分子筛进一步分离提纯胶原酶（二）用亲和层析提纯胶原酶。
原工艺路线长，发酵液配方复杂。
本发明为了提高胶原酶的质量，改进了发酵液配方，改进了提取精制方法，提高收率。
本发明制备工艺为：
1.菌种Cl.histolyticum 64008。
2.培养基配方：酵母膏0.2-1%，酵母粉0.2-1%，明胶1%-3%，肉胨2%-6%。
3.发酵液培养温度：37℃-42℃，周期为40-45小时，静止发酵。
4.发酵液经无菌过滤后，在10℃以下，加硫酸铵沉淀胶原酶，使浓度达到45-55%（重量/体积）所获沉淀溶于PH7-8，Tris-Ca++缓冲液在低温与氮气加压下经超滤膜脱盐，冷冻干燥获胶原酶粗品。
5.胶原酶粗品经以PH5-6，HAC-NaAC缓冲液平衡之DEAE纤维素柱层析，下柱液再经PH6.0-7.5电导水平衡的SephadexG-25柱，获得溶液经冰冷干燥即为胶原酶精制品，活力达到800-1000单位/毫克蛋白质。
实施例1、
菌种组织梭菌Cl.histolyticum64008
一级种液：酵母膏0.2%-1.0%，牛肉膏1%-2%，肉胨2%-6%，明胶1%-3%;二级种液：酵母膏0.2%-1.0%肉胨2%-6%，明胶1%-3%;接种量3%-5%，培养温度37℃-42℃，周期24小时，发酵液：酵母膏0.2%-1%，酵母粉0.2%-1%，明胶1%-3%，肉胨2%-6%，PH7-8，温度37℃-42℃，周期40-45小时，静止发酵，发酵液效价在400单位/毫克，毒性安全剂量小白鼠尾静脉注射50单位/只。发酵液经无菌过滤，泵压至盐水冷却锅，在低温下加入硫酸铵使达50%浓度（重量/体积），再加入1%氯化钙，放置过夜，收集硫酸铵沉淀，溶解于0.05克分子/Tris-Ca++缓冲液，离心除杂质收集粗酶液，以超滤膜脱盐，无菌过滤，冷冻干燥，得胶原酶粗品，较发酵液的比活力提高5-10倍，酶活性收率为50%。
例2：
以PH5-6的醋酸缓冲液平衡DEAE-Cellulose装柱3.6×50厘米柱，进样量控制在30万-50万单位，粗酶溶于醋酸缓冲液，上柱，收集胶原酶部份，再上Sephadex G-25柱（3.6×50厘米），以NaCL，NaoH调节水使电导达40-70微欧-1，收集胶原酶部份，冷冰干燥，所获得胶原酶精品纯度可达800-1000单位/毫克蛋白质，每1200胶原酶单位中，蛋白酶含量低于10单位。
按本工艺制得的胶原酶，产品质量符合要求，制备的胶原酶试剂可供各研究单位分离细胞用，制备药理规格的外用油膏制剂可用于治疗清疮脱痂，制备的注射剂可用于治疗椎脊盘突出症。
Claims (1)
1、一种利用组织梭菌Cl，histolgticum64008经培养发酵后再经DEAE纤维素及分子筛分离提纯胶原酶的工艺，本发明的特征在于将菌种经组份为酵母膏0.2-1%，酵母粉0.2-1%，明胶1-3%，肉胨2-6%的培养基在温度为37-42℃，周期40-45小时静止发酵后，无菌过滤，在10℃以下加硫酸铵沉淀胶原酶，使浓度达到45-55%(重量/体积)，所获沉淀溶于PH7-8Tris-Ca++缓冲液在低温与氮气加压下经招滤膜脱盐，冷冻干燥获胶原酶粗品，然后经以PH5-6，HAC-NaAc缓冲液平衡之DEAE纤维素柱层析，下柱液再经PH6.0-7.5电导水平衡的SephadexG-25粒，得到溶液经冷冻干燥即为胶原酶精品。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 85103913 CN85103913A (zh) | 1985-06-19 | 1985-06-19 | 胶原酶制备工艺 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 85103913 CN85103913A (zh) | 1985-06-19 | 1985-06-19 | 胶原酶制备工艺 |
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| Publication Number | Publication Date |
|---|---|
| CN85103913A true CN85103913A (zh) | 1986-12-24 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 85103913 Pending CN85103913A (zh) | 1985-06-19 | 1985-06-19 | 胶原酶制备工艺 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1058750C (zh) * | 1992-03-03 | 2000-11-22 | 山西医科大学 | 一种从双胸蚓中提取胶原酶的方法 |
| CN101870965A (zh) * | 2010-06-01 | 2010-10-27 | 青岛康地恩生物科技有限公司 | 一种高酶活速溶酶制剂的生产方法 |
| CN103320361A (zh) * | 2013-06-27 | 2013-09-25 | 山东大学 | 一株产酸的梭菌及其在造纸废水处理中的应用 |
| CN101534920B (zh) * | 2006-11-01 | 2013-10-16 | 比奥根艾迪克Ma公司 | 通过低ph和二价阳离子分离生物大分子的方法 |
| CN104508127A (zh) * | 2012-04-18 | 2015-04-08 | 菲迪亚制药股份公司 | 用于产生和纯化来自溶藻弧菌(Vibrio alginolyticus)的胶原酶的新方法 |
-
1985
- 1985-06-19 CN CN 85103913 patent/CN85103913A/zh active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1058750C (zh) * | 1992-03-03 | 2000-11-22 | 山西医科大学 | 一种从双胸蚓中提取胶原酶的方法 |
| CN101534920B (zh) * | 2006-11-01 | 2013-10-16 | 比奥根艾迪克Ma公司 | 通过低ph和二价阳离子分离生物大分子的方法 |
| CN101870965A (zh) * | 2010-06-01 | 2010-10-27 | 青岛康地恩生物科技有限公司 | 一种高酶活速溶酶制剂的生产方法 |
| CN104508127A (zh) * | 2012-04-18 | 2015-04-08 | 菲迪亚制药股份公司 | 用于产生和纯化来自溶藻弧菌(Vibrio alginolyticus)的胶原酶的新方法 |
| CN103320361A (zh) * | 2013-06-27 | 2013-09-25 | 山东大学 | 一株产酸的梭菌及其在造纸废水处理中的应用 |
| CN103320361B (zh) * | 2013-06-27 | 2015-05-13 | 山东大学 | 一株产酸的梭菌及其在造纸废水处理中的应用 |
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