Mechanisms of protein foldingand molecular breeding in halophilic enzymes

首先从角质盐杆菌（Halobacterium cutirubrum）中分离到核苷二磷酸激酶（NDK），它的稳定性和活性不需要高浓度的盐。这是第一个从极端嗜盐古菌中分离出的酶显示无盐特性的酶。通过ATP亲和柱一步纯化NDK，纯度为540倍。疏水色谱进一步纯化至均质。该酶在0 ~ 4 M NaCl条件下稳定，在2 M NaCl条件下活性最高。在0和4 M NaCl条件下，其活性最高可达70%。从角质瘤H染色体DNA中克隆并测序了ndk基因，其核苷酸序列已存入DDBJ，加入号为AB036344。我们在大肠杆菌中构建了该基因的表达载体。在大肠杆菌中表达的Ndkp定位于可溶性部分，但不具有酶活性。我们发现，这种表达酶的激活需要高盐浓度，一旦酶在高盐存在下被激活，它在脱盐后是稳定的。这一事实表明，Ndkp不需要高盐来维持稳定性和活性，但蛋白质折叠确实需要高盐。

We first isolated nucleoside diphsphate kinase (NDK) from Halobacterium cutirubrum which does not require high concentration of salts for its stability and activity. This is first one showing salt-free characteristics among enzymes isolated from extremely halophilic archaea. NDK was highly purified by one step ATP affinity column with 540-fold. It was further purified to homogeneity by hydrophobic chromatography. This enzyme was stable in the presence of 0〜4 M NaCl, and showed maximum activity at 2 M NaCl. It showed about 70 % of maximum activity at 0 and 4 M NaCl.The ndk gene was cloned and sequenced from H, cutirubrum chromosomal DNA and its nucleotide sequence has been deposited to DDBJ with accession number of AB036344.We have constructed the expression vector for this gene in E. coli. The expressed Ndkp in E. coli was localized in soluble fraction but did not have enzyme activity. We found that the high salt concentration was required for the activation of this expressed enzyme, and once enzyme was activated in the presence of high salt, it was stable after removal of salt. This fact indicated that Ndkp does not require high salt for stability and activity, but does require it for the protein folding.