Cold adaptation of a mesophilic cellulase, EG III from Trichoderma reesei, by directed evolution
Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase III (EG III) from Trichoderma reesei, and obtained a cold adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG III with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes Fmore thermolabile than its parent. In addition, activation energies of w-3 and wild type EG III calculated from Arrhenius equation are 13.3 kJ · molt-1 and 26.2 kJ · molt-1, respectively. Therefore, the increased specific activity of w-3 at lower temperatures could result from increased Kcat value and decreased activation energy.
Keywordscold adaptation directed evolution endoglucanase III Trichoderma reesei
Unable to display preview. Download preview PDF.
- 7.Johnston, J. R., Molecular Genetics of Yeast, Oxford: Oxford University Press, 1994.Google Scholar
- 8.Somogyi, M., Notes on sugar determination, J. Biol. Chem., 1952, 195: 19–23.Google Scholar
- 9.Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989.Google Scholar