Screening Of Alkaline Catalase Producing Strains, And The Enzyme's Purification And Characterization

A slightly halophilic and facultatively alkaliphilic strain F26 has been screened out from the soda lake located in the Hulunbeir area of Inner Mongolia Autonomous Region for its relatively high catalase activity. Its 16S rDNA sequence was closely related to the alkaliphilic Bacillus gibsonii (DSM 8722) and Bacillus horikoshii (DSM 8719) with similarity of 99 %. A five-isozyme system could be detected in the cells when strain F26 was cultivated under the condition of pH 9.8 and 5 % NaCl. Moreover, the expression of multiple catalases system was found to be growth-dependent, pH-dependent and NaCl-dependent. An alkaline catalase, isozyme 4, has been purified to homogeneity from strain F26, with a four–step procedure consisting of ammonium sulfate precipitation, ion exchange, gel filtration and hydrophobic interaction chromatography. The native catalase had a molecular mass of 140000 Da and showed the typical Soret band appearing at 408 nm. The pyridine hemochrome spectrum indicated the presence of protoheme IX as the prosthetic group. The apparent Km value for enzyme activity on H2O2 was calculated to be 32.5 mM. The isozyme was not reduced by dithionite but was strongly inhibited by cyanide, azide, hydroxylamine and 3-amino-1,2,4-triazole (the specific inhibitor of typical catalase). No peroxidase activity of this enzyme was detected when using dianisidine, DAB and phenylenediamine as electron donor. Moreover, the N-terminal sequence exhibited substantial similarity to the monofunctional catalase subgroup rather than catalase-peroxidase or Mn-catalase subgroup. Therefore, we conclude that isozyme 4 belongs to the typical catalases. On the other hand, the new purified enzyme displayed the unique properties of a sharp maximum activity at pH 11 and an uncommon structure of homodimer, which are both distinct from the common characteristics of the monofunctional catalase. To our knowledge, pH 11 is the most alkaline condition for optimum catalysis and enzyme stability among the catalases reported up to now and the structure of homodimer is also the first report among alkaline catalases. Another catalytic property, halo-alkali-stability with a half life of 90 h under the condition of pH 10.5 and 0.5M NaCl, might make the catalase possibly useful in the treatment of H2O2 - bleaching effluents. Besides, though isozyme 4 derived from a mesophile, it showed thermosensitive just like the psychrophilic catalase, so we describe it as a cold-adapted enzyme. A thermoalkaliphilic strain A6 was also screened out at the same time from the hot spring located in the Tengchong hot sea of Yunnan province. Its 16S rDNA sequence was not closely related to any other species. The most homologous one, called Bacillus horti, showed similarity of only 91 %, though strain A6 was also spore-formed. Comparison of the growth conditions between this two strains exhibited distinct difference in survival temperature range. Therefore, we conclude that strain A6 is a novel species at least, and it is quite possible that A6 also stands for a novel genus equal to Brevibacillus and Aneurinibacillus.