Hess J M, Kelly R M
Department of Chemical Engineering, North Carolina State University, Box 7905, Stinson Road, Raleigh, North Carolina 27695-7905, USA.
Biotechnol Bioeng. 1999 Mar 5;62(5):509-17. doi: 10.1002/(sici)1097-0290(19990305)62:5<509::aid-bit2>3.0.co;2-7.
The inactivation behavior of the xylose isomerase from Thermotoga neapolitana (TN5068 XI) was examined for both the soluble and immobilized enzyme. Polymolecular events were involved in the deactivation of the soluble enzyme. Inactivation was biphasic at 95 degrees C, pH 7.0 and 7.9, the second phase was concentration-dependent. The enzyme was most stable at low enzyme concentrations, however, the second phase of inactivation was 3- to 30-fold slower than the initial phase. Both phases of inactivation were more rapid at pH 7.9, relative to 7.0. Differential scanning calorimetry of the TN5068 XI revealed two distinct thermal transitions at 99 degrees and 109 degrees C. The relative magnitude of the second transition was dramatically reduced at pH 7.9 relative to pH 7.0. Approximately 24% and 11% activity were recoverable after the first transition at pH 7.0 and 7.9, respectively. When the TN5068 XI was immobilized by covalent attachment to glass beads, inactivation was monophasic with a rate corresponding to the initial phase of inactivation for the soluble enzyme. The immobilized enzyme inactivation rate corresponded closely to the rate of ammonia release, presumably from deamidation of labile asparagine and/or glutamine residues. A second, slower inactivation phase suggests the presence of an unfolding intermediate, which was not observed for the immobilized enzyme. The concentration dependence of the second phase of inactivation suggests that polymolecular events were involved. Formation of a reversible polymolecular aggregate capable of protecting the soluble enzyme from irreversible deactivation appears to be responsible for the second phase of inactivation seen for the soluble enzyme. Whether this characteristic is common to other hyperthermophilic enzymes remains to be seen.
对来自嗜热栖热菌（TN5068 XI）的木糖异构酶在可溶性和固定化酶状态下的失活行为进行了研究。可溶性酶的失活涉及多分子事件。在95℃、pH 7.0和7.9条件下，失活呈双相，第二相呈浓度依赖性。该酶在低酶浓度下最稳定，然而，失活的第二相比初始相慢3至30倍。相对于pH 7.0，在pH 7.9时两个失活阶段都更快。对TN5068 XI进行差示扫描量热法分析，发现在99℃和109℃有两个明显的热转变。相对于pH 7.0，在pH 7.9时第二个转变的相对幅度显著降低。在pH 7.0和7.9时，第一次转变后分别约有24%和11%的活性可恢复。当TN5068 XI通过共价连接固定在玻璃珠上时，失活呈单相，速率与可溶性酶失活的初始相相对应。固定化酶的失活速率与氨释放速率密切相关，氨释放可能源于不稳定的天冬酰胺和/或谷氨酰胺残基的脱酰胺作用。第二个较慢的失活阶段表明存在一种未折叠中间体，而固定化酶未观察到这种中间体。失活第二相的浓度依赖性表明涉及多分子事件。可溶性酶失活的第二相似乎是由于形成了一种能够保护可溶性酶免于不可逆失活的可逆多分子聚集体。这种特性是否为其他嗜热酶所共有还有待观察。
