title: Neurospora crassa tox-1 Gene Encodes a pH- and Temperature-Tolerant Mini-Cellulase.
abstract: 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.
authors.forename: Yue
authors.initials: Y
authors.lastname: Xiao
authors.forename: Qiongsi
authors.initials: Q
authors.lastname: Zhang
authors.forename: Yiquan
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xi
authors.initials: X
authors.lastname: Luo
authors.forename: Yuchuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Genetics and Biochemistry, Clemson University , Clemson, South Carolina 29634, United States.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Cao
authors.affiliation: 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.
authors.forename: Vito De
authors.initials: VD
authors.lastname: Pinto
authors.forename: Qiuyun
authors.initials: Q
authors.lastname: Liu
authors.forename: Gang
authors.initials: G
authors.lastname: Li
mesh_terms: D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D012997:Solvents; D013501:Surface-Active Agents; D002480:Cellulase
keywords: conservative amino acid replacements; enzyme assays; mini-cellulase; pH and thermal tolerance