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ISSN
1672-9145
Acta Biochim Biophys Sin
2004, 36(9): 603–608
CN 31-1940/Q
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 XU1,3, Lan-Ying LIU2, and Fu-Kun ZHAO1,3*
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
—————–
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]