Purification and characterization of a β-mannanase from Trichoderma harzianum strain T4

An extracellular β-mannanase was isolated from crude extract samples of the mesophilic fungus Trichoderma harzianum strain T4 when grown on wheat bran as the carbon source. The induction profile showed that the β-mannanase activity was detected from the sixth day of cultivation period. The enzyme was purified to apparent homogeneity by ultrafiltration, gel filtration and ion-exchange chromatography procedures. The purified β-mannanase (Man I) had molecular masses of 32.5 and 36.5 kDa, as determined by SDS-PAGE and gel filtration chromatography, respectively. It was most active at 55 °C and pH 3.0 and showed thermal stability with half-lives approx. of 5 h at 55 °C and 4 h at 60 °C and at 70 °C, Man I showed half-life of 1 h. Man I showed Km and Vmax values of 1.3 mg/ml and 3.6 IU/ml with β-galactomannan as the substrate, respectively. The purified enzyme was not active against cellulose and xylan. Man I was highly activated by β-mercaptoethanol, cysteine, 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) and dithiothreitol (DTT). On the other hand, N-bromosuccinimide (NBS) was a strong inhibitor of Man I activity. The thermal stability of the purified Man I make this enzyme attractive for use in industrial applications.