Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus
Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus
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DOI：
10.1016/j.procbio.2009.08.014
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发表时间：
2010-01-01
影响因子：
4.4
通讯作者：
Guisan, Jose M.
中科院分区：
文献类型：
--
作者：
Bolivar, Juan M.;Rocha-Martin, Javier;Guisan, Jose M.
首先，将固定在氰化物溴化琼脂糖珠(CNBR)上的酶(在固定化过程中没有涉及所有的酶亚基)与醛-葡聚糖进行了交联。该制剂不再释放酶亚基，在pH 4和25℃下变得完全稳定。然后，比较了许多不同的酶制剂(固定酶固定在CNBr上、该衍生物进一步与醛-葡聚糖交联、固定化在高活性氨基-环氧基载体上、固定化在含有少量氨基和多个环氧基的载体上、固定化在pH 7的乙氧基-琼脂糖珠上、该制剂进一步在pH 10孵育，最后在pH 10直接固定化在该载体上的酶)在pH 4和高温下的稳定性，其中解离和扭曲在酶失活中起到了相关的作用。最稳定的制剂是在pH 7下制备的，在pH 10下孵育，然后用氨基和环氧基载体固定GDH，第三种是在pH 10的乙二醛-琼脂糖固定化的酶。当不是所有的酶亚基都被固定时，活性根本不能恢复。在其他衍生物中，只有乙氧基制剂(最惰性的载体和那些预计会有更强烈的多点共价结合的载体)在水介质中重新孵育时具有显著的再活化作用。对复活条件进行了优化。在pH为7的条件下固定化，再在pH为10的条件下孵育，酶活恢复到100%。(C)爱思唯尔有限公司出版的2009年。
First, the enzyme immobilized on cyanide bromide agarose beads (CNBr) (that did not involve all enzyme subunits in the immobilization) has been crosslinked with aldehyde-dextran. This preparation did not any longer release enzyme subunits and become fully stable at pH 4 and 25 degrees C.Then, the stabilities of many different enzyme preparations (enzyme immobilized on CNBr, that derivative further crosslinked with aldehyde-dextran, enzyme immobilized on highly activated amino-epoxy supports, GDH immobilized on supports having a few animo groups and many epoxy groups, GDH immobilized on glyoxyl-agarose beads at pH 7, and that preparation further incubated at pH 10, and finally the enzyme immobilized on this support directly at pH 10) were compared at pH 4 and high temperatures, conditions where both dissociation and distortion play a relevant role in the enzyme inactivation. The most stable preparation was that prepared at pH 7 and incubated at pH 10, followed by GDH immobilized on amino and epoxy supports and the third one was the enzyme immobilized on glyoxyl-agarose at pH 10.The incubation of all enzyme preparations in saturated guanidine solutions produced the full inactivation of all enzyme preparations. When not all enzyme subunits were immobilized, activity was not recovered at all. Among the other derivatives, only glyoxyl preparations (the most inert supports and those where a more intense multipoint covalent attachment were expected) gave significant reactivation when re-incubated in aqueous medium. After optimization of the reactivation conditions. the enzyme immobilized at pH 7 and later incubated at pH 10 recovered 100% of the enzyme activity. (C) 2009 Published by Elsevier Ltd.