Purification and characterization of an extracellular mutanase from Trichoderma harzianum

Abstract

Trichoderma harzianum (CCM F-470) was found to produce large amounts of extracellular mutanase (0.33 U ml−1, 1.85 U mg protein−1) when grown aerobically on the optimized mutan medium in fermenter culture with an automatic pH control. The mutanase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The enzyme was recovered with a 94-fold increase in specific activity and a yield of 73%. The molecular weight of the purified enzyme proved to be 67 kDa, as estimated by SDS-PAGE, and to be 274 kDa, as determined by size-exclusion HPLC. These results indicate that the native mutanase is probably a tetramer protein. The isoelectric point was at 7.11, and the carbohydrate content in the purified enzyme was 4.42%. The pH and temperature optima were 5.5 and 40°C, respectively. The enzyme remained stable over a pH range of 4.5-6.0 and up to 35° for 1 h. The values of Km and Vmax under standard assay conditions were 1.2 × 10−3 g ml−1 and 8.48 × 10−2 U mg−1, respectively.