Screening, Isolation And Characterization Of Laccase Enzymes From Namibian Termitomyces Schimperi And Kalaharituber Pfeilii

ABSTRACT

Few reports could be found on screening, isolation and characterisation of enzymes in local Fauna and Flora and little is known about laccase enzymes from Namibia origin hence there is a niche for enzyme studies in this area. This research has qualitatively screened Termitomyces schimperi and Kalaharituber pfeilii fruiting bodies for laccase enzymatic activity using α-naphtnol and 2, 2-azino-bis 3-ethylbenzthiazoline-6-sulphonic acid (ABTS). A clone of T. schimperi was also grown in the laboratory under controlled conditions. A purification protocol of laccase from K. pfeilii consisted of filtering the blended samples from the truffle’s outer layer, supernatant precipitation with ammonium sulphate at 80% saturation, ultrafiltration, size exclusion gel chromatography, and then anion exchange chromatography with DEAE Bio-Gel. For K. pfeilii, the final purification step resulted in a total activity (U) of 0.172, specific activity 15.317 U/mg, yield 23.6% and a purification fold of 880 was obtained. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed that K. pfeilii was homogenous according to the size with a band appearing at 60kDA. Following the same protocol, purification of laccases from T. schimperi fungal combs gave a total activity (U) of 0.0094 and specific activity of 3.901 (U/mg) the yield was 0.095 % while a 4 fold purification was achieved. The laccase from the solid state media of clones of T. schimperi in the laboratory was also purified with the same protocol. The final step which was an elution through DEAE ion exchange chromatography resulted in total activity (U) of 1.226 and specific activity of 47.406 (U/mg). The yield was 17% and a 25 folds purification of was achieved. The research further characterised the purified enzymes based on the optimum pH and temperature. A laccase from K. pfeilii has optimum temperature around 60°C and showed significant activity even at

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temperatures up to 80°C. The laccase from laboratory isolates of T. schimperi had optimum activity at 70°C and remained active even at 90°C.