Journal of Biotechnology 129 (2007) 717–722 Short communication Thermostabilization of Pichia stipitis xylitol dehydrogenase by mutation of structural zinc-binding loop Narayana Annaluru a , Seiya Watanabe a,b,c , Seung Pil Pack a,b , Ahmed Abu Saleh a , Tsutomu Kodaki a,b , Keisuke Makino a,b,d,∗ a Institute of Advanced Energy, Kyoto University, Gokasho, Uji 611-0011, Japan b CREST, JST (Japan Science and Technology Agency), Kyoto University, Gokasho, Uji 611-0011, Japan c Faculty of Engineering, Kyoto University, Kyotodaigaku-katsura, Saikyo-ku, Kyoto 615-8530, Japan d International Innovation Center, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-8501, Japan Received 23 December 2006; received in revised form 1 February 2007; accepted 5 February 2007 Abstract Xylitol dehydrogenase from Pichia stipitis (PsXDH) is one of the key enzymes for the bio-ethanol fermentation system from xylose. Previously, we constructed the C4 mutant (S96C/S99C/Y102C) with enhanced thermostability by introduction of structural zinc. In this study, for further improvement of PsXDH thermostability, we constructed the appropriate structural zinc-binding loop by comparison with other polyol dehydrogenase family members. A high thermostability of PsXDH was obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) showed a 10.8 ◦ C higher thermal transition temperature (T CD ) and 20.8 ◦ C higher half denaturation temperature (T 1/2 ) compared with wild-type. © 2007 Elsevier B.V. All rights reserved. Keywords: Thermostability; Xylitol dehydrogenase; Structural zinc; Site-directed mutagenesis 1. Introduction Improvement of protein thermostability is one of the major concerns of protein engineers since the protein structure has evolved and has been optimized for its function but not essen- tially for its stability. Several engineering approaches such as random mutagenesis, rational design, and the consensus approach using sequence databases and statistics have been employed to significantly modify the thermostability of a pro- tein (Lee and Vasmatzis, 1997; Van den Burg and Eijsink, 2002; Bornscheuer and Pohl, 2001; Lehmann and Wyss, 2001). Abbreviations: XDH, xylitol dehydrogenase; PsXDH, XDH from Pichia stipitis; MDR, medium-chain dehydrogenase/reductase; PDH, polyol dehy- drogenase; SDH, sorbitol dehydrogenase; CD, circular dichroism; T 1/2 , half denaturation temperature; T CD , thermal transition temperature; M. morganii, Morganella morganii; S. pombe, Schizosaccharomyces pombe; E. japonica, Eriobotrya japonica; H. jecorina, Hypocrea jecorina ∗ Corresponding author at: Institute of Advanced Energy, Kyoto University, Gokasho, Uji 611-0011, Japan. Tel.: +81 774 38 3517; fax: +81 774 38 3524. E-mail address: kmak@iae.kyoto-u.ac.jp (K. Makino). However, there is no standard or general procedure for protein thermostabilization, because the factors governing protein ther- mostability are not usually similar in all proteins (Pace et al., 1996; Vieille and Zeikus, 2001). Xylitol dehydrogenase from Pichia stipitis (PsXDH) plays an important role in the xylose metabolic pathway (Scheme 1) (Ingram et al., 1998; Jeffries and Jin, 2004). Raising the ther- mostability of XDH has a significant impact on fermentation efficiency in the biomass to bio-ethanol conversion system. However, the present protein engineering approaches mentioned above have difficulties when using PsXDH. That is, the random mutagenesis method demands an appropriate selection system (Giver et al., 1998), lack of three-dimensional (3D) structure of PsXDH precludes rational design (Mooers et al., 2003) and with- out any reports on XDHs from thermophilic microorganisms, we cannot employ the consensus method. In a recent report, we constructed PsXDH with structural zinc (C4 mutant) by site-directed substitution of Ser96, Ser99, and Tyr102 with cysteine based on the fact that some members of the polyol dehydrogenase (PDH) family contain structural zinc, which is bound tetrahedrally to four sulfur atoms of 0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.02.006