Protein & Peptide Letters, 2013, 20, 0000-0000 1 0929-8665/13 $58.00+.00 © 2013 Bentham Science Publishers Crystal Structure and Fluorescence Analysis of Alkaline Thermostable Xylanase from Bacillus sp. (NCL 87-6-10) L. Satyanarayan 1 , Sushama M. Gaikwad, H. Balkrishnan and C.G. Suresh* Division of Biochemical Sciences, National Chemical Laboratory, Pune - 411008, India; Present address: 1 Department of Chemistry, University of Florida, Gainesville, Florida-32611, USA Abstract: Structural information deduced from the new crystal form of xylanase from Bacillus sp (NCL 87-6-10) (AT- BXYL-C) helped us to identify the active site and interpret the stability of the enzyme. The analysis of the tetragonal crys- tal structure of ATBXYL-C with a bound and cleaved xylotriose revealed the two glutamic acid residues in the structure that could act as nucleophile (Glu94) and base (Glu184) in the enzyme activity and also the tryptophan residues interact- ing with the substrate. The cleavage of xylotriose in the crystal showed xylobiose to be the major product. Intrinsic fluo- rescence of the enzyme showed the presence of tryptophans in partially exposed to the solvent at the active site and sur- face tryptophans in electropositive environment. The titration experiments with xylobiose and xylotriose revealed slightly enhanced preference for longer chain X3 compared with X2. The crystal structure also account for some of the factors, such as increased number of ionic interactions and additional interactions at the N-terminus, which contributed to in- creased alkalophilicity and thermostability of the enzyme. Keywords: Alkaline thermophile Bacillus xylanase (ATBXYL-C), active site residues, fluorescence studies, three-dimensional structure. INTRODUCTION Xylanases are microbial enzymes that depolymerise po- lymeric xylan strands into monomeric pentosan sugars which are then consumed by bacteria and fungi as primary carbon source. The potential of the enzyme for application in paper industry has resulted in considerable research interest in cel- lulose free xylanases [1]. High pH and temperature em- ployed in pulping operations has triggered intensive search for alkalophilic and thermostable xylanases. A xylanase from Bacillus sp. (NCL 87-6-10) that was reported from our labo- ratory exhibited stability both at high pH and temperature. The enzyme is active up to pH 10 and 70  C [2]. The poten- tial application of the enzyme in paper and pulp industry has been reported [3]. Following the previous reports, the en- zyme will be referred as alkaline thermophilic Bacillus xy- lanase-C (ATBXYL-C) in subsequent discussion [2-4]. Studies on the structure-function relationship have al- ways been one of the central issues in the investigations of biological macromolecules. Attempts to correlate structural changes in proteins to alterations in their biological activities have been hitherto largely confined to the side-chain func- tional groups. The utility of intrinsic fluorescence studies is greatly extended by its use in conjunction with X-ray crystal- lographic studies, which mutually supplement each other. Despite the biotechnological importance of xylanases isolated from extremophilic organisms, very few reports characterizing the structure-function relationship of these enzymes are documented. In the present report we describe *Address correspondence to this author Division of Biochemical Sciences, National Chemical Laboratory, Pune- 411008. India; Tel: +91 20 25902236; Fax: +91 20 25902648; E-mail:cg.suresh@ncl.res.in the structural studies and fluorescence behaviour of AT- BXYL-C. Microenvironment of the active site Trp residue and the binding affinity of two xylo-oligo saccharides have been determined by fluorescence titration experiments. The results from fluorescence studies are analyzed with reference to the crystal structure of ATBXYL-C complexed with hy- drolysed xylotriose. MATERIALS AND METHODS Enzyme Production The alkalophilic thermophilic Bacillus (NCL 87-6-10) was isolated from soil samples of Calicut in South India rich in decomposing coconut detritus. Fermentation of the organ- ism was carried out in wheat bran-yeast extract medium con- taining wheat bran (3% w/v), tryptone (1 to 3% w/v) and tween-80 (0.5 to 1%). The maximum xylanase activity ob- tained was 170 IU/ml after 48 hours of growth [2, 3]. Enzyme Purification The culture filtrate (500ml) was brought to 60% ammo- nium sulphate saturation; the precipitate was collected by centrifugation at 10,000 x g for 30 min, dissolved in mini- mum volume of 20 mM potassium phosphate buffer pH 7.0 and extensively dialyzed against the same buffer. The dia- lyzed enzyme solution was concentrated to ~5ml using ultra filtration over 10,000 Da cut-off membrane. The solution was loaded onto a CM-sephadex column (28x4cm) pre- equilibrated with 20 mM potassium phosphate buffer, pH 7.0. The bound enzyme was eluted with 0-0.8 M KCl gradi- ent. The fractions showing xylanase activity were pooled and concentrated using Amicon ultra filtration cell. The purity of