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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(9): 603–608 CN 31-1940/Q

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pH-Dependent Stability of EGX, A Multi-functional Cellulase from Mollusca, Ampullaria crossean

Wen-Ying LI², Ji WANG¹, Yan-Hong LI¹, Ming DING¹, Gen-Jun XU^1,3, Lan-Ying LIU², and Fu-Kun ZHAO^1,3*

¹Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China;
²College of Life Science, Jilin University, Changchun 130023, China;
³College of Life Science, Zhejiang University of Science and Technology, Hangzhou 310018, China

Abstract The cellulase activity and stability of EGX, a multi-functional cellulase previously purified from the mollusca Ampullaria crossean, was systematically studied under different pH. The pH induced con-formation and stability change of EGX have been investigated by using the intrinsic fluorescence, ANS fluorescence and CD spectrum. It has been found that the conformation and activity of this cellulase were strongly dependent on the pH. EGX was stable for both the enzyme activity and the conformation from pH 5.6 to pH 7.4. As shown by intrinsic and ANS fluorescence, no red shift of emission maximum occurred and a negligible intensity change was observed at pH 5.6-7.4. The activity of EGX remained about 80% in pH 5.6-7.4 and obviously decreased out of side the pH range. Urea-induced changes in EGX at pH 5.4 and pH 8.0 were measured by intrinsic fluorescence and CD spectrum. At pH 5.4, a significantly red shift of emission maximum occurred when the concentration of urea was 5 M compared to the concentration was 3 M at pH 8.0. The a-helix at pH 5.4 was 40.51% in the absence of urea and 31.04% in the presence of 4 M urea. At pH 8.0 the a-helix was 7.23% in the presence of 4 M urea. The data indicated that EGX was much susceptible to urea-induced unfolding at pH 8.0 and much stable at pH 5.4. The greater pH dependent stability of EGX may allow the enzyme to adequately catalyze the hydrolysis of cellulosic materials under natural or industrial extreme conditions.

Key words multi-functional cellulase; stability; Ampullaria crossean; fluorescence spectroscopy; pH-dependent structural change; CD spectrum

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Received: June 14, 2004 Accepted: July 26, 2004

This work was supported by the grants from the National Natural Science Foundation of China (No. 30370336), the Major State Basic Research Development Program of China (No. 2003CB716006), and the Creation Foundation from Shanghai Institutes for Biological Sciences

Abbreviations: bis-ANS, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid; pNPC, p-nitrophenyl b-D-cellobioside; pNP, p-nitrophenol; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis

*Corresponding author: Tel, 86-21-54921155; Fax, 86-21-54921011; E-mail, [email protected]