OPTIMIZATION, PURIFICATION AND CHARACTERIZATION OF INDUCED XYLANASE PRODUCED FROM KOLANUT (COLA NITIDA, VENT) HUSK BY BACILLUS MEGATERIUM

Abstract

Xylanolytic enzymes brings about the hydrolysis of complex biomolecules into simpler monomers. This study deals with production of xylanase enzyme from the bacterium Bacillus megaterium KPWS using kolanut husk as substrate. The bacterium was screened positive for xylanase activity qualitatively on xylan nutrient agar and quantitatively under submerged fermentation technique. Nutritional and cultural parameters were optimized using one factor at a time technique. The extracellular xylanase produced from optimized B. megaterium KPWS culture was partially purified via ammonium sulphate precipitation (60%); ion exchange chromatography and gel filtration. The molecular weight of purified xylanase was determined through Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was characterized accordingly. The different conditions optimized for maximum production of xylanase included substrate concentration (1% xylan plus 1.5% kolanut husk), nitrogen source (yeast extract plus peptone), carbon source (sucrose), incubation period (24 h), pH (5), temperature (35oC) and inoculum size (1%). The obtained xylanase showed 4-fold purification with specific activity of 653.72 μmol/ml/mg and a molecular weight of 21 kDa as observed on SDS-PAGE. The optimum pH and temperature of the purified enzyme was 5.0 and 60oC respectively retaining more than 50% of its activity over a broad range of temperature (30oC-60oC) and pH (3.0-8.0). The enzyme showed Km of 29.50 mg/ml and Vmax of 1250 μmol/min/ml with birch wood xylan with specificity for xylan as substrate over others. The enzyme was inhibited by Cu2+, Pb2+, Fe3+ and Hg2+ and stimulated by K+, Ca2+, Mg2+, Na+ and Mn2+ metal ions. Also, chemical agent such as ethylenediamine tetra acetate (EDTA), sodium dodecyl sulphate (SDS) and urea stunted the enzyme activity in that order, while β-mercaptoethanol improved the activity of xylanase produced. The xylanase produced therein is moderately thermostable, acidic in nature which could be of industrial importance due to the cheap substrate source.