Orthophosphate Anion Enhances the Stability and Activity of Endoxylanase from Bacillus sp. Min Tae Park, Myung Seop Lee, Ja Young Choi, Sun Chang Kim, Gyun Min Lee Department of Biological Sciences, Korea Advanced Institute of Science and Technology 373-1, Kusong-Dong, Yusong-Gu, Taejon 305-701, Korea; fax: 82-42-869-2610; e-mail: gmlee@mail.kaist.ac.kr Received 24 February 2000; accepted 28 September 2000 Abstract: Endoxylanase, for which the optimum tem- perature is 60°C (optimum pH 7), is labile to heat. Be- cause the isoelectric point (pI) value of this xylanase is 10.6, the net charge of this enzyme is positive at pH 7. Thus, ions are likely to influence its enzyme structure and the thermal stability of endoxylanase may improve. Among the various ions tested, orthophosphate anion (HPO 4 2- ) was found to significantly improve not only the stability but the activity of xylanase. When K 2 HPO 4 con- centration was increased from 50 mM to 1.2 M, the T m value of xylanase was increased from 60.0°C to 74.5°C. The affinity of xylanase on xylan also increased along with K 2 HPO 4 concentration. Thus, the xylanase activity at 0.6 M K 2 HPO 4 was 2.3-fold higher than that at 50 mM K 2 HPO 4 , and 120.2-fold higher than that in 40 mM MOPS buffer. This enhanced activity in the presence of K 2 HPO 4 probably takes place because the orthophosphate anion affects the binding and catalytic residues of endox- ylanase. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 434–440, 2001. Keywords: activity; endoxylanase; orthophosphate an- ion; stability INTRODUCTION Endo--1,4-D-xylanase (E.C. 3.2.1.8) is a xylan-degrading enzyme that cleaves xylan in a random manner. Xylanases are used in the bleaching of pulps for paper manufacturing (Ragauskas et al., 1994). The prebleaching of pulp with xylanases can lower the utilization of toxic chemicals used for the traditional bleaching process, such as chlorine and hypochlorite. The thermostability of xylanase is a critical factor for successful bio-bleaching, because this process is carried out at high temperatures (Ra ¨ttö et al., 1994). Xylanases are also applied to improve the product qualities of human food and animal feed (Coughlan and Hazlewood, 1993). In particular, it is known that enzyme hydrolysates, composed mainly of low-member oligomers, such as xylo- biose and xylotriose, selectively promote the growth of in- testinal bifidobacteria, which are beneficial for maintenance of healthy microflora. Thus, it is desirable to increase the binding affinity of xylanase on xylan. Protein stability can be increased by several methods, such as random and rational mutations, chemical modifica- tion, and use of additives (O’Fagain, 1995). Salts, one of these additives, influence protein stability directly by pref- erential binding to the folded or unfolded protein, and/or indirectly by changing the properties of the solvent water (Timasheff, 1992). A remarkable increase in the conforma- tional stability of RNase T 1 in the presence of salts was shown to result primarily from the preferential binding of salt ions to the folded protein at ion binding sites (Pace and Grimsley, 1988). Salts also influence protein activity significantly. The ac- tivity of N 5 ,N 10 -methylenetetrahydromethanopterin reduc- tase was found to be stimulated over 100-fold by sulfate and phosphate (Ma et al., 1991). The activity and conforma- tional stability of HIV-1 protease were both enhanced under high-salt conditions (Szeltner and Polga ´r, 1996). On the other hand, -isopropylmalate isomerase was found to be inhibited by a high concentration of (NH 4 ) 2 SO 4 , although its thermal stability was markedly enhanced (Bigelis and Umbarger, 1976). However, the mechanisms underlying the effects of salts on enzyme stability and activity are poorly understood. Endo--1,4-D-xylanase, originating from Bacillus sp., has a high isoelectric point (pI) value of 10.6 (Lee et al., 1997). Thus, the endoxylanase structure may be influenced by ions, resulting in significant changes in its stability and activity without chemical or genetic manipulations. In this study, we investigate the effects of various ions on the sta- bility and activity of this endoxylanase. MATERIALS AND METHODS Enzyme Preparation The source of endoxylanase is Bacillus sp. isolated from ordinary farm compost (Lee et al., 1997). The gene coding for this endoxylanase was overexpressed in B. subtilis Correspondence to: G. M. Lee Contract grant sponsor: Ministry of Science and Technology, Korea © 2001 John Wiley & Sons, Inc.