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From in silico to in vitro: Modelling and production of Trichoderma reesei endoglucanase 1 and its mutant in Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
23693 43463 2012 8 PDF Available
Title
From in silico to in vitro: Modelling and production of Trichoderma reesei endoglucanase 1 and its mutant in Pichia pastoris
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.

► We inserted ten aminoacid long loops into endoglucanase I of T. reesei. ► We analyzed the impact of the loops on the enzyme stability via MD and MM. ► Best loop mutation predicted in silico was introduced to EGI via SDM. ► EGI and its mutant were successfully expressed in P. pastoris for the first time. ► We created a hotspot via loop insertion for directed crosslinking of EGI.

Keywords
Cellulase; Endoglucanase; Molecular modelling; Site directed mutagenesis; Trichoderma reesei; Pichia pastoris
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From in silico to in vitro: Modelling and production of Trichoderma reesei endoglucanase 1 and its mutant in Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 159, Issues 1–2, 31 May 2012, Pages 61–68
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us