| 摘要: |
| 采用Tris-HCl缓冲液(50mmol/L,pH7.5)抽提、Q-sepharose F.F.阴离子交换层析、Sephacryl S-300凝胶过滤层析、SDS-PAGE等方法,进行中华管鞭虾虾头蛋白酶的分离纯化、酶学性质及其动力学特性的研究。结果表明,中华管鞭虾虾头蛋白酶粗提物经层析后,得到聚丙烯酰胺凝胶电泳纯的一酶组分。该蛋白酶的比活力从359.39U/mg增加到8277.83U/mg,提高了23.03倍,回收率达37.69%,SDS-PAGE显示该蛋白酶只含一条谱带,相对分子量为24.50kDa。以偶氮酪蛋白作为底物,该酶的米氏常数Km值为0.28mg/ml,最适温度为55℃,最适pH为7.5;蛋白酶在60℃以下比较稳定,放置1h后活性仍保持在60%以上,而在60℃以上时蛋白酶活性急剧下降,80℃放置1h后,酶活性只残留21.32%。10mmol/L Cu2+、Zn2+和EDTA对该蛋白酶有较强的抑制作用,抑制率分别约为97%、96%和55%,10mmol/LMg2+能显著促进蛋白酶活性,而10mmol/L Fe2+、Ba2+、Ca2+对蛋白酶活性的影响不大。推测中华管鞭虾蛋白酶可能为一种金属蛋白酶。 |
| 关键词: 中华管鞭虾, 蛋白酶, 纯化, 生化特性 |
| DOI： |
| 分类号: |
| 基金项目:国家科技部十五攻关项目,2001BA501A-25号 |
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| PURIFICATION AND BIOCHEMICAL PROPERTIES OF PROTEASE IN SOLENOCERA CRASSICORNIS |
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YANG Wen-Ge1,2, XUE Chang-Hu2, XU Da-Lun1, HE Xiong3, PAN Yun-Di1, CHEN Shi-Guo2
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1.Faculty of Life Science and Biotechnology, Ningbo University, Ningbo;2.Division of Life Science and Technology, Ocean University of China, Qingdao;3.Zhejiang Pharmaceutical College, Ningbo
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| Abstract: |
| Solenocera crassicornis is an important shrimp species in fishery resource and wide ly distributed in the East China Sea. S. crassicornis are usually frozen for worldwide trade. How ever, the shrimp head that takes 40% of the body weight is often discarded during the process. In order to fully utilize the discarded, a purification procedure for protease from S. crassicornis heads was developed. To our best knowledge, it is the first trial to study the protease in detail from S. crassicornis to abstract and use the marine proteases.
The procedures of extracting and purifying protease from S. crassicornis heads include three steps: preparing crude protease with Tris-HCl buffer solution (50mmol/L, pH7.5),ion-exchanging with Q-sepharose F.F., and gel-filtering with sephacry lS-300HR. The enzymology nature and dynamics characteristics were studied with biochemical and electrophoretic techniques.
The enzyme was purified to an electrophoretically homogenous state, reaching 23.03-fold purification with a yield of 37.69%. The abstracted protease showed a single band in its relative molecular weight at 24.5kD a based on sodium dodecyl sulphate-polycrylamide gel electrophoresis (SDS-PAGE). Calculated from Lineweaver-Burk plot of 1/v versus 1/[S], the apparent Km value of the enzyme was 0.28mg/ml. Using azocasein as the substrate, the optimum temperature was 55°C and the optimum pH of the enzyme reaction was 7.5. This protease shows a good thermal stability below 60°C. More than 60% of protease activity still remained after incubation at 60°C for 1h. But the enzyme became inactivated rapidly when temperature rose above 60°C, and only 21.32% of protease activity remained at 80°C for 1h.
Effects of some metal ions and EDTA on the protease activity were also investigated. The results indicated that the protease activity could be inhibited by 10mmol/L of Cu2+, Zn2+ and EDTA, with their inhibitory ratios at 97%, 96% and 55%, respectively. 10mmol/L of Mg2+ could obviously increase the enzyme activity, while 10mmol/L of Ca2+, Ba2+ and Fe2+ had no significant effect on the pro tease activity. It is speculated that the protease from S. crassicornis should be a type of metalloenzyme. |
| Key words: Solenocera crassicornis, Protease, Purification, Biochemical properties |
