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

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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.