Production of Alkaline Xylanase by an Alkalo-thermophilic Bacteria, Bacillus halodurans, MTCC 9512 isolated from Dung

An alkalo-thermophilic bacteria from dung has been isolated using Emerson medium in the agar plates. The bacteria has growth at the pH 10 and temperature 55°C. The bacteria was screened for the xylanase activity using Congo red dye followed by wash out by 1 mM sodium chloride. A clear zone around the colony in the replica plate was considered to have xylanase activity. The suspected colony in another replica plate was grown in Emerson broth and extracellular xylanase enzyme activity was analyzed by the colorimetric method using dinitro salicylic acid for estimation of reducing power. The morphological study of the bacteria was done after Gram stain and using 40x amplification in the phase contrast microscope. The isolated bacteria retained violet color after washing with acetone. Therefore, it is gram positive. Further characterization using various morphological, physiological and biochemical tests confirmed the bacteria as Bacillus halodurans and was given Accession number MTCC 9512 by IMTECH, Chandigarh. Growth conditions for the bacteria were optimized for maximum production of xylanase. The maximum amount of xylanase activity was found at the pH 9.5 and temperature 55°C. The growth of the bacteria and enzyme production were monitored up to 52 hours and it was found that the bacteria grew logarithmically up to 30 hours. Different carbon sources viz. xylan, sucrose, glucose, starch individually at 0.5% concentration were used in the Emerson growth medium. Maximum biomass growth was found with xylan whereas xylanase was maximally produced with glucose as carbon source. Therefore, glucose was considered to be the best inducer followed by xylan among the various carbon sources used. The enzyme was enriched by using 0–80% ammonium sulfate precipitation followed by desalting through Sephadex-G-25 gel filtration. The results indicated inhibitory nature of ammonium sulfate.