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A rational design for trypsin-resistant improvement of Armillariella tabescens β-mannanase MAN47 based on molecular structure evaluation

Paper ID Volume ID Publish Year Pages File Format Full-Text
23617 43457 2013 7 PDF Available
Title
A rational design for trypsin-resistant improvement of Armillariella tabescens β-mannanase MAN47 based on molecular structure evaluation
Abstract

Protease resistance of enzymes is required for the feed industry because of the presence of secretary proteases in the digestive tract. In this study, we report a rational method for protease-resistance improvement of enzymes based on molecular structure evaluation. The trypsin-resistance of β-mannanase MAN47 from Armillariella tabescens was investigated. Twelve tryptic sites within it were ordered by their positions in three-dimensional space from external to internal. Except of R144, R192 and R261, which were either conserved or highly related to the catalytic activity, the top external residue K280 and the most internal residue K371 were selected. With conducting computational design via H-bond analysis and molecular dynamics simulations, optimal mutants of K280N and K371Q were predicted. Meanwhile, a triple mutant K280N/K107H/R102N was also predicted. Half-lives of mutants K280N, K280N/K107H/R102N, K371Q and wild-type enzymes which were all pre-treated by trypsin at 40 °C were determined 185 min, 285 min, 102 min and 100 min, respectively. In addition, the temperature–activity and pH–activity profiles revealed that the mutations we designed had no obvious influence on the catalytic activity of the enzyme. Our results proved that trypsin-resistance of an enzyme could be improved by molecular rational evolution of homology modeling and molecular dynamics simulations.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► A rational method for protease-resistance improvement of enzymes was introduced. ► Homology modeling and molecular dynamics simulation were employed in the method. ► Mutating trypsin digestion site located on enzyme surface improved its resistance. ► The mutations we designed had no obvious influence on the catalytic activity.

Keywords
Site-directed mutagenesis; Protease-resistance; Homology modeling; Overlap-extension PCR
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A rational design for trypsin-resistant improvement of Armillariella tabescens β-mannanase MAN47 based on molecular structure evaluation
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 163, Issue 4, 20 February 2013, Pages 401–407
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