The production, purification and characterization of endo-1,4-ß-mannanase from newly isolated strains from Scopulariopsis candida

English: Mannan polysaccharides occur in hemicellulose fraction of the plant cell walls. The hydrolysis of these polymers involves the action of enzymes such as β-mannanase, β-mannosidase and α- galactosidase which are produced by both fungi and bacteria. The current study reports on the production of β-mannanase, β-mannosidase and α-galactosidase by newly isolated Scopulariopsis candida strains LMK004 and LMK008. The effect of medium composition and carbon source on growth and enzyme production was evaluated in a liquid culture. A combination of Vogel’s medium and locust bean gum was found to stimulate growth and increase β-mannanase production. Optimal β-mannanase production of 7800 nkat/g biomass for LMK004 and 13300 nkat/g biomass for LMK008 was achieved in media containing 10% NaCl, 1X Vogel’s medium, 1% yeast extract and 1% locust bean gum. Both strains secreted trace amounts (less than 1 nkat/ml) of β-mannosidase and α-galactosidase indicating that these enzymes may be retained intracellularly. Native-PAGE and SDS-PAGE were used together with the zymogram to assess purity and to estimate the molecular weight of the proteins. The molecular weight of LMK004 β-mannanase was estimated to be ≈41 kDa whereas that of LMK008 β-mannanase could not be determined due to excessive loss of protein material during dialysis. The β-mannanase from LMK004 was most active at pH 5 and 50 °C, and retained ≥ 80% of its activity at pH 5 – 6.5 after 24 hrs of incubation at 4 °C. In contrast, the LMK008 β- mannanase retained ≥ 60% activity between pH 6 – 7. Both enzymes remained stable for 3 hrs at temperature between 30 °C and 40 °C, and showed loss of activity at higher temperatures. The two enzymes displayed different degrees of halotolerance. The LMK008 β-mannanase tolerated high NaCl concentrations with 60% activity remaining after incubation for 2 hrs at 20% NaCl, whereas the LMK004 β-mannanase was only active between 0% - 10% NaCl. It is clear from the current study that the two strains of S. candida produce distinct β-mannanases which may be useful candidates in low water activity reactions.