南极大磷虾（Euphausia superba）胰蛋白酶样酶分离纯化、酶学性质探索及其生物学活性研究
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摘要
南极大磷虾隶属于节肢动物门甲壳纲,是南极生态系统的关键物种,资源丰富,具有巨大的开发和利用潜力。国外研究表明,南极大磷虾体内含有多种蛋白酶,能清除机体坏死组织和细胞、治疗角膜碱烧伤和消除牙斑等,具有广泛的医学用途,而其体内蛋白酶活性40%来源于胰蛋白酶。因此,对其胰蛋白酶样酶进行分离纯化,酶学性质探索和生物学活性研究,具有一定的理论和应用价值。
本文采用20-70%饱和度硫酸铵分级沉淀、Sephacryl S100凝胶过滤柱层析和DEAE Sepharose-F.F离子交换柱层析,纯化得到一种胰蛋白酶TLEⅠ,分子量大约在30.0KDa,纯化倍数为3.19。
胰蛋白酶酶学性质研究发现:酶促反应的速率受pH、温度影响。在37°C,以BApNA为底物的反应体系中,在较广泛的pH范围(3.0~11.0)内,酶具有活性,最适宜反应pH在7.0~9.0之间;在pH 8.0,以BApNA为底物的反应体系中,酶在较广泛的温度范围(4-80°C)内具有活性,反应最适宜温度为30°C~50°C。Mg~(2+)、Ca~(2+)(0~0.5 mmol/L)对胰蛋白酶有一定的激活作用。Fe~(3+)、Cu~(2+)、Zn~(2+)、丙三醇和丙酮对胰蛋白酶有一定的抑制作用。Mn~(2+)、乙醇和二甲基亚砜在低浓度时对酶起激活作用,高浓度下表现为抑制作用。
建立兔机械创伤模型,以提取的粗酶、纯化酶、木瓜蛋白酶和生理盐水分别涂抹伤口,以肉眼观察和伤口愈合面积为指标对比各组实验结果。结果显示,粗酶和纯化酶都有很好的效果,粗酶更佳。
分别采用番泻叶、大黄、5-羟色胺(5-HT)建立小鼠腹泻模型1h后,以生理盐水为阴性对照,思密达为阳性对照,分别灌胃不同剂量(20mg.kg~(-1)和40mg.kg~(-1))的南极磷虾蛋白酶制剂,统计6h内小鼠腹泻发生率和腹泻累积次数。结果显示,南极磷虾蛋白酶可显著降低番泻叶引起的小鼠腹泻次数,降低大黄,5-羟色胺引起的小鼠腹泻次数。
采用右旋葡聚糖硫酸钠(DSS)诱导小鼠溃疡性结肠炎(UC)模型,以生理盐水为阴性对照,柳氮磺胺吡啶为阳性对照,分别灌胃不同剂量(20mg.kg~(-1)和40mg.kg~(-1))的南极磷虾蛋白酶制剂,通过肉眼及实体观察,EGF的测定,组织切片观察对比各组实验结果。结果显示:灌胃南极磷虾蛋白酶的小鼠在饮用DSS诱导UC后,能够保持良好的精神状态,少有便血现象,并能抑制小鼠体重的减轻;同时,南极磷虾蛋白酶能够明显提高UC小鼠血液中EGF表达水平,从组织切片结果来看,南极磷虾蛋白酶也能降低溃疡症状的发生,其作用效果在一定程度上与其浓度成正比。
Euphausia superba which belongs to Arthropoda Crustacea, is the key species to the Antarctic ecological system. It is rich in resources and has great potential for the development and use. Foreign studies show that the Antarctic krill proteinases is very effective in eliminating necrotic tissues and cells, treatment of corneal alkali burn and eliminating plaque etc. It has great medical purposes and 40% of protease activity comes from trypsin. Accordingly the purification of Euphausia pacifica’s trypsin-like enzyme, the enzymatic characteristics study and the research on its biological activities has significant theoretic and practical importance.
In this paper, The separation and purification of trypsin-like enzymes was conducted by saturated-precipitation method using 20-70% ammonium sulfate, Sephacryl S100 gel chromatography and DEAE Sepharose-F.F ion exchange chromatography. One component(TLEⅠ) was obtained and its molecular weight was about 30.0KDa. purification multiple was 3.19.
The purified trypsin-like enzymes were active in extremely wide pH range of 3.0~11.0 and the optimum values were between pH 7.0 and 9.0 at 37°C with BApNA as subtrate. And they were active in temperature range of 4~80°C, extremely wide, with optimum values between 30 and 50°C at pH 8.0, BApNA as subtrate. With metal ion, Mg~(2+)、Ca~(2+) (0~0.5 mmol/L) had activation on trypsin-like enzymes, Fe~(3+)、Cu~(2+)、Zn~(2+)、Glycerine and Acetone had inhabitation on trypsin-like enzymes. Mn~(2+)、Ethanol and Dimethyl sulphoxide(DMSO),activate trypsin-like enzymes, when their concentration is low, but they inhibit trypsin-like enzymes significantly in high concentration. the rabbit model of mechanical trauma was established with the crude enzyme extracted and pure enzymes compared with papain and saline to clean the wound. The result showed that crude enzyme and the pure enzyme had a very good result, crude enzyme was better.
Diarrhea was induced in mice by cathartics (senna, rhubarb or 5-hydroxytryptamine(5-HT)). After 1h, the mouse were given by Antarctic krill proteinases at different doses(20mg.kg~(-1)& 40mg.kg~(-1))while normal saline was negative control and smecta was positive control. The ratio of diarrhea and the accumulated times of diarrhea of mice were measured within six hours. The result showed that the Antarctic krill proteinases could decrease the times of diarrhea of mice induced by senna significantly, decrease the times of diarrhea of mice induced by rhubarb and 5-HT.
Dextran sodium sulfate (DSS) was used to induce UC in mice and mice treated with Antarctic krill proteinases at different doses(20mg.kg~(-1)& 40mg.kg~(-1))while normal saline was negative control and Sulfasalazine was positive control. The results of each group were compared by visual observation, EGF determination and histological observation. The result showed that the Antarctic krill proteinases could inhibite the reduction of mice weight, keep them in good spirits and reduce the phenomenon of blood in the stool. At the same time, the Antarctic krill proteinases could significantly improve the expression level of EGF in UC mice. It could also reduce the occurrence of ulcer symptoms, and the effect is proportional to its concentration.
本文采用20-70%饱和度硫酸铵分级沉淀、Sephacryl S100凝胶过滤柱层析和DEAE Sepharose-F.F离子交换柱层析,纯化得到一种胰蛋白酶TLEⅠ,分子量大约在30.0KDa,纯化倍数为3.19。
胰蛋白酶酶学性质研究发现:酶促反应的速率受pH、温度影响。在37°C,以BApNA为底物的反应体系中,在较广泛的pH范围(3.0~11.0)内,酶具有活性,最适宜反应pH在7.0~9.0之间;在pH 8.0,以BApNA为底物的反应体系中,酶在较广泛的温度范围(4-80°C)内具有活性,反应最适宜温度为30°C~50°C。Mg~(2+)、Ca~(2+)(0~0.5 mmol/L)对胰蛋白酶有一定的激活作用。Fe~(3+)、Cu~(2+)、Zn~(2+)、丙三醇和丙酮对胰蛋白酶有一定的抑制作用。Mn~(2+)、乙醇和二甲基亚砜在低浓度时对酶起激活作用,高浓度下表现为抑制作用。
建立兔机械创伤模型,以提取的粗酶、纯化酶、木瓜蛋白酶和生理盐水分别涂抹伤口,以肉眼观察和伤口愈合面积为指标对比各组实验结果。结果显示,粗酶和纯化酶都有很好的效果,粗酶更佳。
分别采用番泻叶、大黄、5-羟色胺(5-HT)建立小鼠腹泻模型1h后,以生理盐水为阴性对照,思密达为阳性对照,分别灌胃不同剂量(20mg.kg~(-1)和40mg.kg~(-1))的南极磷虾蛋白酶制剂,统计6h内小鼠腹泻发生率和腹泻累积次数。结果显示,南极磷虾蛋白酶可显著降低番泻叶引起的小鼠腹泻次数,降低大黄,5-羟色胺引起的小鼠腹泻次数。
采用右旋葡聚糖硫酸钠(DSS)诱导小鼠溃疡性结肠炎(UC)模型,以生理盐水为阴性对照,柳氮磺胺吡啶为阳性对照,分别灌胃不同剂量(20mg.kg~(-1)和40mg.kg~(-1))的南极磷虾蛋白酶制剂,通过肉眼及实体观察,EGF的测定,组织切片观察对比各组实验结果。结果显示:灌胃南极磷虾蛋白酶的小鼠在饮用DSS诱导UC后,能够保持良好的精神状态,少有便血现象,并能抑制小鼠体重的减轻;同时,南极磷虾蛋白酶能够明显提高UC小鼠血液中EGF表达水平,从组织切片结果来看,南极磷虾蛋白酶也能降低溃疡症状的发生,其作用效果在一定程度上与其浓度成正比。
Euphausia superba which belongs to Arthropoda Crustacea, is the key species to the Antarctic ecological system. It is rich in resources and has great potential for the development and use. Foreign studies show that the Antarctic krill proteinases is very effective in eliminating necrotic tissues and cells, treatment of corneal alkali burn and eliminating plaque etc. It has great medical purposes and 40% of protease activity comes from trypsin. Accordingly the purification of Euphausia pacifica’s trypsin-like enzyme, the enzymatic characteristics study and the research on its biological activities has significant theoretic and practical importance.
In this paper, The separation and purification of trypsin-like enzymes was conducted by saturated-precipitation method using 20-70% ammonium sulfate, Sephacryl S100 gel chromatography and DEAE Sepharose-F.F ion exchange chromatography. One component(TLEⅠ) was obtained and its molecular weight was about 30.0KDa. purification multiple was 3.19.
The purified trypsin-like enzymes were active in extremely wide pH range of 3.0~11.0 and the optimum values were between pH 7.0 and 9.0 at 37°C with BApNA as subtrate. And they were active in temperature range of 4~80°C, extremely wide, with optimum values between 30 and 50°C at pH 8.0, BApNA as subtrate. With metal ion, Mg~(2+)、Ca~(2+) (0~0.5 mmol/L) had activation on trypsin-like enzymes, Fe~(3+)、Cu~(2+)、Zn~(2+)、Glycerine and Acetone had inhabitation on trypsin-like enzymes. Mn~(2+)、Ethanol and Dimethyl sulphoxide(DMSO),activate trypsin-like enzymes, when their concentration is low, but they inhibit trypsin-like enzymes significantly in high concentration. the rabbit model of mechanical trauma was established with the crude enzyme extracted and pure enzymes compared with papain and saline to clean the wound. The result showed that crude enzyme and the pure enzyme had a very good result, crude enzyme was better.
Diarrhea was induced in mice by cathartics (senna, rhubarb or 5-hydroxytryptamine(5-HT)). After 1h, the mouse were given by Antarctic krill proteinases at different doses(20mg.kg~(-1)& 40mg.kg~(-1))while normal saline was negative control and smecta was positive control. The ratio of diarrhea and the accumulated times of diarrhea of mice were measured within six hours. The result showed that the Antarctic krill proteinases could decrease the times of diarrhea of mice induced by senna significantly, decrease the times of diarrhea of mice induced by rhubarb and 5-HT.
Dextran sodium sulfate (DSS) was used to induce UC in mice and mice treated with Antarctic krill proteinases at different doses(20mg.kg~(-1)& 40mg.kg~(-1))while normal saline was negative control and Sulfasalazine was positive control. The results of each group were compared by visual observation, EGF determination and histological observation. The result showed that the Antarctic krill proteinases could inhibite the reduction of mice weight, keep them in good spirits and reduce the phenomenon of blood in the stool. At the same time, the Antarctic krill proteinases could significantly improve the expression level of EGF in UC mice. It could also reduce the occurrence of ulcer symptoms, and the effect is proportional to its concentration.
引文
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