Isolation and Purification of Heterotetrameric Catalase from a Desiccation Tolerant Cyanobacterium Lyngbya arboricola

The desiccation tolerant cyanobacterium Lyngbya arboricola, isolated from bark surfaces of Mangifera indica, possessed up to four stable isoforms of catalase in addition to other antioxidative enzymes, for several years under a dry state. Purification of the two most persistent isoforms of catalase (Cat) has been undertaken by employing acetone precipitation, ethanol: chloroform treatment, gel filtration and ion exchange chromatography. The two isoforms of catalase remained almost unchanged on varying matric and osmotic hydration levels of mats of the cyanobacterium. The purification procedures resulted in a 1.3 % yield of purified single isoform (0.22 mg mL-1 protein) with 709 Units mg-1 specific activity and a purity index of 0.83. Five millimolar of dithiothreitol (DTT) was observed to be pertinent in maintaining the optimum redox state of the enzyme. The purification procedures additionally facilitated the simultaneous elimination and procurement of phycoerythrins (PE) and mycosporine-like amino acids (MAA). Each purified isoform gave a single band (~45kDa) upon SDS-PAGE and denaturing urea isoelectric focusing (IEF) depicted the presence of 2 subunits each of CatA and CatB. The monoisotopic mass and pI value of CatA and CatB as revealed by LC-MS analysis and internal amino acid sequencing was 78.96, 5.89 and 80.77, 5.92, respectively, showing resemblance with CatA of Erysiphe graminis subs. hordei and CatB of Ajellomyces capsulata. The heterotetrameric monofunctional catalase (~320 kDa), due to its stability in the form of resistance to ethanol: chloroform, its thermoalkaliphilic nature and the presence of innumerable hydrophobic amino acid residues (~40%), thus exhibited its potential for biotechnological applications.