Indian Journal of Experimental Biology Vol. 53, June 2015, pp. 356-363 Production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching Leya Thomas*, Raveendran Sindhu, Parameswaran Binod & Ashok Pandey Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram-695 019, Kerala, India Received 21 November 2014; revised 03 December 2014 Here, we described the production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching. Various process parameters affecting xylanase production by B. pumilus were optimized by adopting a Plackett-Burman design (PBD) as well as Response surface methodology (RSM). These statistical methods aid in improving the enzyme yield by analysing the individual crucial components of the medium. Maximum production was obtained with 4% yeast extract, 0.08% magnesium sulphate, 30 h of inoculum age, incubation temperature of 33.5 °C and pH 9.0. Under optimized conditions, the xylanase activity was 372 IU/ml. Media engineering improved a 5-fold increase in the enzyme production. Scanning electron microscopy (SEM) showed significant changes on the surface of xylanase treated pulps as a result of xylan hydrolysis. Increased roughness of paper carton fibres was apparent in scanning electron micrograph due to opening of the micro fibrils present on the surface by xylanase action. The untreated pulp did not show any such change. These results demonstrated that the B. pumilus MTCC 5015 xylanase was effective in bio-bleaching of paper carton. Keywords: Paper carton, Response surface methodology (RSM), Xylan Xylanases (EC 3.2.1.8) catalyse the hydrolysis of xylan, which is the second most abundant polysaccharide component in the plant cell walls. Xylans are polysaccharide made from units of xylose. The xylanolytic enzyme system carrying out the xylan hydrolysis is usually composed of a repertoire of hydrolytic enzymes: β-1,4-endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-glucuronidase, acetyl xylan esterase, and phenolic acid (ferulic and p-coumaric acid) esterase and act cooperatively to convert xylan into its constituent sugars. Microbial xylanases are preferred catalysts for xylan hydrolysis due to their high specificity, mild reaction conditions and negligible substrate loss and by-product generation 1 . Xylanases are produced by several bacteria, algae, protozoa, fungi, gastropods and arthropods 2 . On the basis of their amino acid sequences and structural homologies, endo-1,4- xylanases have been mainly grouped in families 10 and 11 of the glycoside hydrolases, although other families including families 5, 7, 8, and 43 also contain some xylanolytic enzymes 3,4 . Xylanases have a wide range of industrial applications including bulk chemicals, bio-bleaching of wood pulps, food additives, laundry detergents and fabric care compositions 1,5 . Application of these enzymes in paper industries for pulp treatment, and in improving the effectiveness of conventional bleaching chemicals has increased interest in them. The cost- effective production of xylanase can partly be achieved by using cheaply available agro-industrial residues such as wheat bran 6,7 . Solid-state fermentation (SSF) using various lignocellulosic substrates has been reported previously 8,9 and has been regarded as the best option with advantages, such as higher productivity as well as lower operational and capital costs. Several Bacillus spp. produce high levels of extracellular xylanase 10 . Several reports are available for xylanase production by Bacillus pumilus, including B. pumilus ASH 3 and B. pumilus SV-205 4 . Xylanase production is known to be affected by various nutritional and physiological factors, such as carbon source, nitrogen source, inoculum size and age, pH of the media, additives and incubation temperature 11 . Hence, optimization of various process parameters is of prime importance for the cost effective production of enzyme. —————— *Correspondence: Phone: 91 471 2515279; Fax: 91 471 2491712 E-mail: layam31@gmail.com