title: Rationally selected single-site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates.
abstract: Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via site-directed mutagenesis. Using a rational mutagenesis approach based on structural bioinformatics and evolutionary analysis, two positions (F16S and Y95F) were identified as priority sites for mutagenesis. The mutant and parent enzymes were expressed and secreted from Pichia pastoris and the single site mutants F16S and Y95F showed 1.7- and 4.0-fold increases in k(cat) and 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, respectively, while maintaining thermostability. Similar to the parent enzyme, the two variants were active between pH 4.0 and 8.0 and showed optimal activity at temperature 70°C at pH 5.0. The purified enzymes were active at 50°C for over 12 h and retained at least 80% of initial activity for 2 h at 70°C. In contrast to the improved hydrolysis seen with the single mutation enzymes, no improvement was observed with a third variant carrying a combination of both mutations, which instead showed a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.
authors.affiliation: Department of Chemical Engineering and Materials Science, The Henry Samueli School of Engineering, University of California, Irvine, California, USA.
authors.forename: Sneha
authors.initials: S
authors.lastname: Srikrishnan
authors.forename: Arlo
authors.initials: A
authors.lastname: Randall
authors.forename: Pierre
authors.initials: P
authors.lastname: Baldi
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Da Silva
mesh_terms: D019943:Amino Acid Substitution; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020125:Mutation, Missense; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D013696:Temperature; D055327:Thermoascus / Q000201:enzymology* / Q000235:genetics
chemical_list: D050505:Mutant Proteins; D002482:Cellulose; D002480:Cellulase