Purifi cation and Molecular Cloning of Catalase from Rhizobium radiobacter Strain 2-1

Hydrogen peroxide is widely used for chemical treatment in various industries particularly in the textile, paper, pulp, food and dairy industries as a more environmentally-friendly alternative to chlorine that forms toxic and carcinogenic chemicals called dioxins as by-products7,9,33). However, the rapid formation of reactive oxygen species (ROS) from hydrogen peroxide, including hydroxyl radicals is also a threat6,10,25). In light of the growing concern over environmental problems, hydrogen peroxide should be decomposed before disposal. Currently, hydrogen peroxide is either diluted with large quantities of water, which is expensive and wasteful, or treated with agents such as sodium bisulphite or hydrosulphite, which leaves salt in the water5). Alternatively, the application of catalases has been suggested. These enzymes play an important role in reducing the formation of the highly reactive hydroxyl radical that arises from H2O2 degradation via the Fenton reaction13). Catalases are assigned to three phylogenetically distinct groups: two groups comprise heme catalases and one group comprises nonheme catalases8,22). Generally, a few thermostable versions of a monofunctional catalase32), catalase peroxidases4,12,32,34) and Mn catalases2,17) have been described. True catalases are usually homo tetramers, with a subunit size of approximately 60 kDa. Each subunit contains one heme group20,21). Since the hydrogen peroxide bleaching step occurs at elevated temperatures and pH (>60°C and pH 9), commercially available catalases that are optimally active at 20–50°C and at neutral pH. The pretreatment was recurred the temperature and pH prior to their use23,27). From this, thermostable catalases hold a great potential for application in the various diff erent industries due to the increasing trend to develop environmentally friendly technologies. The availability of catalase that can function at high temperature and pH would be attractive for the above mentioned applications. However, many of the reported catalases exhibit low thermal stability, several are rapidly inactivated in the presence of hydrogen peroxide, and most show low activity and stability at elevated temperature and pH, making them unsuitable for industrial applications30). Nakayama et al. isolated strain 2-1 that can produce high-activity catalase from industrial waste. From phylogenetic analysis, strain 2-1 was identified as Rhizobium radiobacter, formally Agrobacterium tumefaciens35,36) (Nakayama et al., unpublished data). In this study, we describe the purifi cation of catalase from Rhizobium radiobacter strain 2-1. The catalase showed extremely high activity and exceptional stability at elevated temperatures and pHs compared with many other reported catalases. Furthermore, we have determined its N-terminal amino acid sequence and cloned the catalase encoding gene. The alignment result of cloned gene showed no homology with those of other Purifi cation and Molecular Cloning of Catalase from Rhizobium radiobacter Strain 2-1