Purification and Characterization of a Novel Collagenase from Bacillus pumilus Col-J
Purification and Characterization of a Novel Collagenase from Bacillus pumilus Col-J
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短小芽孢杆菌 Col-J 中新型胶原酶的纯化和表征
DOI：
10.1007/s12010-009-8673-1
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Q Wu, C Li, CL Li, H Chen, SL L
中科院分区：
文献类型：
--
作者：
Q Wu, C Li, CL Li, H Chen, SL L
短小芽孢杆菌（Bacillus pumilus）Col-J胞外分泌的胶原酶经硫酸铵沉淀、SephadexG-100柱和SepharoseFastFlow柱层析纯化。纯化后的胶原酶比活力为87.33U/mg，回收率为7.00%。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测得胶原酶的相对分子质量为58.64 kDa。酶反应的最适温度为45 °C。在70 °C下孵育5分钟或在60 °C下孵育10分钟后，仍保留超过50%的原始活性。胶原酶在pH7.5时酶活最大，在pH6.5 -8.0范围内酶活稳定。Mn ~（2+）、Pb ~（2+）、乙二胺四乙酸、乙二醇四乙酸和β-巯基乙醇对胶原酶活性有强烈的抑制作用。Ca ~（2+）和Mg ~（2+）对酶活性有显著的促进作用。来自B的胶原酶pumilus Col-J对来自小牛皮肤的天然胶原蛋白显示出高度的特异性活性。该酶对胶原蛋白的Km和Vmax分别为0.79mg/mL和129.5U。
The collagenase, produced extracellular by Bacillus pumilus Col-J, was purified by ammonium sulfate precipitation followed by two gel filtrations, involving Sephadex G-100 column and Sepharose Fast Flow column. Purified collagenase has a 31.53-fold increase in specific activity of 87.33 U/mg and 7.00% recovery. The collagenase has a relative molecular weight of 58.64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal temperature for the enzyme reaction was 45 °C. More than 50% of the original activity still remained after 5 min of incubation at 70 °C or 10 min at 60 °C. The maximal enzyme activity of collagenase was obtained at pH 7.5, and it was stable over a pH range of 6.5–8.0. The collagenase activity was strongly inhibited by Mn2+, Pb2+, ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, and β-mercaptoethanol. However, Ca2+ and Mg2+ greatly increased its activity. The collagenase from B. pumilus Col-J showed highly specific activity towards the native collagen from calf skin. The Km and Vmax of the enzyme for collagen were 0.79 mg/mL and 129.5 U, respectively.
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影响因子：
--
作者：
Ivanko Ov;Varbanets' Ld
通讯作者：
Ivanko Ov;Varbanets' Ld
DOI：
10.5483/bmbrep.2002.35.6.576
发表时间：
2002-11
期刊：
Journal of biochemistry and molecular biology
影响因子：
--
作者：
P. Park;Sang-Hoon Lee;H. Byun;Soo-Hyun Kim;Se-Kwon Kim
通讯作者：
P. Park;Sang-Hoon Lee;H. Byun;Soo-Hyun Kim;Se-Kwon Kim
影响因子：
2.1
作者：
S. Miyoshi;Yuko Nitanda;K. Fujii;K. Kawahara;Tao Li;Yoko Maehara;T. Ramamurthy;Y. Takeda
通讯作者：
S. Miyoshi;Yuko Nitanda;K. Fujii;K. Kawahara;Tao Li;Yoko Maehara;T. Ramamurthy;Y. Takeda
影响因子：
4.2
作者：
L. Odell;J. Baumgartner;T. Xia;L. David
通讯作者：
L. Odell;J. Baumgartner;T. Xia;L. David
影响因子：
3.2
作者：
Jung, CM;Matsushita, O;Okabe, A
通讯作者：
Okabe, A