1. From in silico to in vitro: modelling and production of Trichoderma reesei endoglucanase 1 and its mutant in Pichia pastoris.
- Author
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Bayram Akcapinar G, Gul O, and Sezerman UO
- Subjects
- Bioreactors, Carboxymethylcellulose Sodium, Cellulase chemistry, Cellulase genetics, Computer Simulation, Enzyme Stability, Fungal Proteins chemistry, Fungal Proteins genetics, Hydrogen-Ion Concentration, Hydrolysis, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Mutation, Pichia metabolism, Protein Engineering, Recombinant Proteins chemistry, Recombinant Proteins genetics, Temperature, Trichoderma genetics, Trichoderma metabolism, Cellulase metabolism, Fungal Proteins metabolism, Pichia genetics, Recombinant Proteins metabolism, Trichoderma enzymology
- Abstract
In this study, a major cellulase, namely endoglucanase 1 (EGI) from Trichoderma reesei was mutated by the introduction of four different lysine and glycine rich loops to create a hotspot for directed crosslinking of EGI away from the active site. The impact of the inserted loops on the stability of the enzyme was analyzed using molecular dynamics (MD) and the effect on the active site was studied using molecular mechanics (MM) simulations. The best loop mutation predicted in silico (EGI_L5) was introduced to EGI via site directed mutagenesis. The loop mutant EGI_L5 and EGI were both expressed in Pichia pastoris. Enzymes were characterized and their activities against soluble substrates such as CMC and 4-MUC were determined. Both enzymes exhibited similar pH and temperature activity and thermal stability profiles. Moreover, specific activity of EGI_L5 against 4-MUC was found to be the same as the native enzyme., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
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