EXTRACTION, PURIFICATION AND CHARACTERIZATION OF XYLANASE FROM BACCILUS LICHENIFORMIS LR1C USING CASSAVA(MANIHOT ESCULANTA)PEELS AS SUBSTRATE

Abstract

Xylanases are glycosidases (O-glycoside hydrolases, EC 3.2.1.8) which catalyze the endohydrolysis of 1,4-β-D-xylosidic linkages in xylan. Which have great application in various industries such as textile industry. This study was aimed at extracting,purifying and characterizing xylanase from Bacillus licheniformis LR1C isolated from water squeezed out of soaked dried powdery cassava peels.The isolated Bacteria were identified using standard microbiological techniques and screened on xylan-agar plates for xylanase production.Assay of xylanase activity and protein determination were determined by dinitrosalicylic acid and Bradford methods respectively. The purification of xylanasewas carriedout according to a procedure that consisted of (NH4)2SO4 fractionation, ion exchange chromatographyon DEAE Sephadex A-50 and gel-filtration on Sephadex G-100 respectively. The subunit molecular weight of the purified xylanase was estimated bydodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).The physicochemical propertiestemperature and pH optimal,thermostability, pH stability and enzymes kinetics were studied for the purified xylanase using standard methods. Xylanase activity,inhibition pattern and Ki of inhibitors on purified xylanase were investigated. The highest bacterial population with the value 6.6×108 CFU/mLwas recorded for the sample coded 2 bcollected from factory b while the least occurred in sample with code 3a (0.8×108 CFU/mL) from factory a. The bacterial isolates were tentatively identified and classified into genera Bacillus, Lactobacillus, Micrococcus, Streptococcus andCorynebacteria.The identities of the best three xylanase-producing bacterial strainswith code 1b-7, 1b-7(c) and 5a-9(a) were further authenticated as Bacillus licheniformis, B. cereus and B. cereusby 16SrRNAgene sequence analysis.Out of the entire bacteria screened, B. licheniformis LR1C had the best xylanase activity; it was therefore selected for further studies. The ion exchange chromatography gave 10.00 fold purification with 11.68% recovery and gel filtration on Sephadex G-100 gave 19.43 fold purification with 7.92% recovery for xylanase from B. licheniformis LR1C cultivated in an enzyme production with cassava peels. The subunit and native molecular weight of the purified xylanase were 51kDa and 200,KDa respectively. The optimum enzyme activity occurred at 70 oC and pH 7.0. The xylanase activity was stable at 40°C+for 30 mins and a pH of 5. The metallic ion (Cu 2+, Pb 2+ Na+ and Hg+) strongly inhibited the activity of enzyme while (Mg 2+, Mn 2+, K +, Na + and Cu 2+)stimulated the activity of the enzyme. The apparent Km and Vmax of purified xylanase were19.28mg/ml and 23.23μmol/min/mlrespectively. Purified xylanase from B. licheniformis LR1C had a greater affinity for its normal substrate xylan. The kinetic inhibition studies was investigated with the use of the PbCl2, HgCl2, and homoterocarpin given different inhibition patterns with Ki values of 11.0, 35.0 and 5.0 mM respectively. The enzyme posseses potentials for application in industries and has established the utilization of cassava peels as a novel substitute to the commercial substrate known to be expensive in the production of xylanase.