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Increasing the substrate specificity of Bacillus stearothermophillus lactate dehydrogenase by DNA shuffling

Paper ID Volume ID Publish Year Pages File Format Full-Text
4063 206 2009 6 PDF Available
Title
Increasing the substrate specificity of Bacillus stearothermophillus lactate dehydrogenase by DNA shuffling
Abstract

Previously it is suggested that a single mutation (Q102R) in the lactate dehydrogenase (LDH) gene from Bacillus stearothermophillus (bs) could switch the substrate specificity by 3 orders of magnitude from lactate to malate and produce a highly efficient malate dehydrogenase (MDH). In order to examine if random mutation and screening could improve this, a DNA-shuffling method would be used to generate a mutant LDH and recombinant LDH genes which will be later used for transforming Escherichia coli. The recombinant colonies are blotted and screened for their ability to catalyse the oxidation of malate. The most active MDH produced by this method is only slightly more efficient than the rationally designed Q102R variant. In addition to this mutation, the shuffled version incorporates further seven residue changes which are chemically conservative. These experiments demonstrate that the blind shuffling can achieve a huge shift in specificity which was a known, highly effective single-site mutation designed using structural knowledge.

Keywords
Molecular modelling; NAD+-dependent lactate dehydrogenase enzyme; Protein engineering; Recombinant protein; Steady-state kinetic; Substrate specificity
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Increasing the substrate specificity of Bacillus stearothermophillus lactate dehydrogenase by DNA shuffling
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biochemical Engineering Journal - Volume 48, Issue 1, 15 December 2009, Pages 118–123
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
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Full-text PDF Download
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