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Expression of an alkalo-tolerant fungal xylanase enhanced by directed evolution in Pichia pastoris and Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
24146 43501 2009 5 PDF Available
Title
Expression of an alkalo-tolerant fungal xylanase enhanced by directed evolution in Pichia pastoris and Escherichia coli
Abstract

The alkaline stability of the xylanase from Thermomyces lanuginosus was further improved by directed evolution using error-prone PCR mutagenesis. Positive clones were selected by their ability to produce zones of clearing on pH 9 and 12 xylan agar plates. Variant NC38 was able to withstand harsh alkaline conditions retaining 84% activity after exposure at pH 10 for 90 min at 60 °C, while the parent enzyme had 22% activity after 60 min. The alkaline stable variant NC38 was cloned into pBGP1 under the control GAP promoter and pET22b(+) for expression in Pichia pastoris and Escherichia coli BL21, respectively. Best extracellular expression of the recombinant xylanase was observed in P. pastoris (261.7 ± 0.61 U ml−1) whereas intracellular activity was observed in E. coli (47.9 ± 0.28 U ml−1) was low. Total activity obtained in P. pastoris was 545-fold higher than E. coli. The mutated alkaline stable xylanase from P. pastoris was secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.

Keywords
Xylanase; Directed evolution; Yeast expression; Pichia pastoris
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Expression of an alkalo-tolerant fungal xylanase enhanced by directed evolution in Pichia pastoris and Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 141, Issues 1–2, 20 April 2009, Pages 26–30
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