Construction of an Effective System for Expression of Manganese Catalase Gene from a Thermophilic Bacterium

1. 从日本温泉中分离的嗜热细菌Thermus sp.YS 8-13中克隆了一个新的过氧化氢锰酶基因，并测定了其核苷酸。909碱基开放阅读框编码302个氨基酸，推断分子量为33,303。在一个386碱基的长5'侧序列中，发现了一个类似于大肠杆菌的sd序列和一个启动子区域。一个102碱基的3'侧翼区域包含一个类似终止符的回文序列。推导出的氨基酸序列与植物乳杆菌锰过氧化氢酶的同源性总体为34%，但在某些可能形成含锰离子活性位点的区域同源性明显较高。锰过氧化氢酶基因在大肠杆菌中的表达将锰过氧化氢酶结构基因连接到高表达质粒pET体系中，并将质粒pETMNCAT引入大肠杆菌BL21。菌株BL21/pETMNCAT作为包涵体表达了大量36kDa的外源蛋白。由于表达蛋白的n端氨基酸序列与纯化的过氧化锰酶（Thermus sp.YS 8-13）相同，因此对该酶蛋白进行了增溶和重折叠。将包涵体用8M的氯化胍增溶后，在存在锰离子的情况下通过透析去除氯化胍，得到约一半的表达蛋白为可溶性活性过氧化锰酶。该亚基和天然酶的分子量、Vmax和K_M值均与真品Thermus sp.YS 8-13过氧化氢酶相同。

1. NUCLEOTIDE SEQUENCING OF A MANGANESE CATALASE GENEA novel manganese catalase gene from a thermophilic bacterium, Thermus sp.YS 8-13 isolated from a Japanese hot spring, was cloned and its nucleotide was determined. The 909-base open reading frame encoded 302 amino acids of deduced molecular weight 33,303. Within a 386-base long5'-flanking sequence, a SD-sequence and a promoter region analogous to that of E.coli were found. A 102-base 3'-flanking region contained a terminator-like palindrome sequence. The deduced amino acid sequence was 34% homologous overall to the sequence of Lactobacillus plantarum manganese catalase, but a significantly higher homology was found in certain regions which may form manganese ion containing active site.2. EXPRESSION OF MANGANESE CATALASE GENE IN E.COLIThe structural gene of manganese catalase was ligated into a high expression plasmid, pET system, and a resultant plasmid (pETMNCAT) was introduced into E.coli strain BL21. Strain BL21/pETMNCAT expressed significant amounts of a 36kDa foreign protein as inclusion bodies. As the N-terminus amino acid sequence of this expressed protein was identical to that of purified manganese catalase from Thermus sp.YS 8-13, solubilization and refolding of this enzyme protein was attempted. After solubilization of the inclusion body with 8M guanidium chloride, and removal of guanidium chloride by dialysis in the presence of manganese ion, about half of the expressed protein was obtained as a soluble and active manganese catalase. The molecular weights of the subunit and native enzyme, as well as the Vmax and K_M values were all identical to those of the authentic Thermus sp.YS 8-13 catalase.