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J Agric Food Chem. 2016 Jun 15;64(23):4751-7. doi: 10.1021/acs.jafc.6b00043. Epub 2016 Jun 6.

Neurospora crassa tox-1 Gene Encodes a pH- and Temperature-Tolerant Mini-Cellulase.

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Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China.
Department of Genetics and Biochemistry, Clemson University , Clemson, South Carolina 29634, United States.
Department of Biomedicine and Biotechnology BIOMETEC, Section of Biology and Genetics, University of Catania , 95125 Catania, Italy.
National Institute for Biomembranes and Biosystems, Section of Catania, 95125 Catania, Italy.


Cellulases that endure extreme conditions are essential in various industrial sectors. This study reports a mini-cellulase gene tox-1 from Neurospora crassa. The gene tox-1 was cloned in Escherichia coli after chimerization with the YebF gene and substitutions of certain isoleucine and valine with leucine residues. The yeast transformants could grow on rice straw-agar medium. The 44-amino acid peptide and its two mutant variants displayed potent cellulase activities in Congo Red assay and enzymatic assays. Conservative replacements with leucine have substantially increased the stabilities and half-lives of the peptides at alkaline pH and low and high temperatures and also the tolerance to organic solvents and surfactants, on the basis of activities toward cellose. The small size of the mini-cellulase would allow for commercially viable automatic chemical peptide synthesis. This work suggests that conservative leucine replacements may serve as a general strategy in the engineering of more robust enzymes with special features with little loss of activities.


conservative amino acid replacements; enzyme assays; mini-cellulase; pH and thermal tolerance

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