PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE XYLANASE FROM SPOROTHRIX CARNIS

Abstract

Xylanase plays an important role in the degradation of lignocellulose and is an important industrial enzyme used in biofuel, animal feed, pharmaceutical, and wood processing industries. Thermostability of enzymes has been the focus of attention, due to long shelf-life at high temperatures. Xylanase was produced by growing S. carnis in a xylan-based basal medium. The enzyme was purified to homogeneity using ammonium sulfate precipitation, ion exchange chromatography and gel filtration. The final purification was 4.05-fold with a recovery of 26.64% and a specific activity of 52.87 U/mg. The molecular weight of the enzyme was 46.87 kDa using sodium dodecyl polyacrylamide gel electrophoresis.The optimum pH and temperature for the purified xylanase were 10.0 and 60oC respectively. The enzyme was found to be highly thermostable with half-lives of 11, 7, 6, 5, and 3 hours at 50oC, 60oC, 70oC, 80oC and 90oC, respectively. About 35.71% of xylanase activity was retained after an hour at pH 9.0. The enzyme exhibited bifunctionality in its specificity,with the ability to degrade both beechwood xylan and two cellulosic substrates, avicel and carboxymethyl cellulose.The activity of xylanase was enhanced in the presence of Mg2+, Mn2+, Ca2+, PO4 3- and CH2(COO)2 2- and was inhibited in the presence of Cu2+, Hg2+, EDTA, Cl-, and CO3 2-.Activity was inhibited by urea, sodium dodecyl sulphate and iodoacetic acid, but was activated by 5.5’-dithiobis-(2-nitrobenzoic acid). Km values of 0.36, 0.76, and 0.60 mg/ml were obtained when beechwood xylan, avicel, and carboxymethyl cellulose respectively, were used as substrates. Vmax values in the same order were 222.22, 140.85 and 23.81 μmol min-1 mg-1. The unique characteristics of xylanase from Sporothrix carnis such as stability atpH and temperature extremes, bifunctionality, and remarkable kinetic parameters make it suitable for use in lignocellulosic bioconversion processes.