Thermophilic laccase from xerophyte species Opuntia vulgaris

Two laccase temperature isoforms capable of oxidizing phenolic compounds to quinones were isolated and purified to homogeneity from the cladodes of the xerophyte species Opuntia vulgaris. These catalytically active proteins exhibit apparent molecular masses of 137 and 90kDa. Under reducing conditions, both isoforms yielded a subunit molecular mass of 43kDa, suggesting that the enzyme is a multimer of the 43kDa subunit. The 137kDa isoform when heated at 80°C for 3min generated three polypeptide bands on activity stained polyacrylamide gels exhibiting 137, 90 and 43kDa molecular forms. All isoforms of the enzyme exhibited an optimum pH of 10 when 2,6-dimethoxyphenol was used as a substrate. The optimum temperature of the 137kDa enzyme form was noted to be 80°C and that of the 90kDa enzyme form was 70°C. Denaturation kinetics of both the laccase isoforms carried out at their respective optimum temperatures for 30min exhibited enzyme activity in excess of their t _1/2 values throughout the assay period. The K_m for the 137kDa form was determined to be 2.2 ± 0.3mm and the V_max was 2.8 ± 0.2IU/mL. These high temperature stable laccase isoforms having alkaline pH optima can find significant industrial use.