Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome.
Bottom Line: Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of ∼0.4) were identified, no CE inactive variant was found.Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination.We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found.
Affiliation: CSIC, Institute of Catalysis, 28049 Madrid, Spain. CSIC, Instituto de Agroquímica y Tecnología de Alimentos, 46980 Valencia, Spain.Show MeSH
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Mentions: Above results unambiguously confirmed that mutations at specific hot spots of the polypeptide (i.e. K281, D282, N316 and K317) may play a more effective supportive role in substrate discrimination in the parental 3A6 protein compared with random mutations within the whole protein length, since only one mutant with substrate discrimination ability was identified by epPCR. Figure 2 summarized the effect of single to triple mutations and insert deletion on substrate preference.
Affiliation: CSIC, Institute of Catalysis, 28049 Madrid, Spain. CSIC, Instituto de Agroquímica y Tecnología de Alimentos, 46980 Valencia, Spain.