z-logo
Premium
Rationally selected single‐site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates
Author(s) -
Srikrishnan Sneha,
Randall Arlo,
Baldi Pierre,
Da Silva Nancy A.
Publication year - 2012
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.24414
Subject(s) - thermostability , mutagenesis , mutant , enzyme , site directed mutagenesis , biochemistry , enzyme kinetics , hydrolysis , pichia pastoris , cellulase , chemistry , directed evolution , active site , saturated mutagenesis , recombinant dna , gene
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. Biotechnol. Bioeng. 2012; 109:1595–1599. © 2011 Wiley Periodicals, Inc.

This content is not available in your region!

Continue researching here.

Having issues? You can contact us here