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From in silico to in vitro: modelling and production of trichoderma reesei endoglucanase 1 and its mutant in pichia pastoris

Bayram Akçapınar, Günseli and Gül, Özgür and Sezerman, Uğur (2011) From in silico to in vitro: modelling and production of trichoderma reesei endoglucanase 1 and its mutant in pichia pastoris. (Submitted)

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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.

Item Type:Article
Additional Information:Accepted with minor revisions of few grammer corrections
Uncontrolled Keywords:cellulase; endoglucanase; molecular modelling; site directed mutagenesis; Trichoderma reesei, Pichia pastoris
Subjects:Q Science > Q Science (General)
T Technology > T Technology (General)
ID Code:18197
Deposited By:Uğur Sezerman
Deposited On:29 Dec 2011 20:32
Last Modified:09 Jan 2012 15:24

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