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Directed evolution of thermotolerant malic enzyme for improved malate production

Paper ID Volume ID Publish Year Pages File Format Full-Text
20804 43192 2014 6 PDF Available
Title
Directed evolution of thermotolerant malic enzyme for improved malate production
Abstract

The directed evolution of the thermotolerant NADP(H)-dependent malic enzyme from Thermococcus kodakarensis was conducted to alter the cofactor preference of the enzyme from NADP(H) to NAD(H). The construction and screening of two generations of mutant libraries led to the isolation of a triple mutant that exhibited 6-fold higher kcat/Km with NAD+ than the wild type. We serendipitously found that, in addition to the change in the cofactor preference, the reaction specificity of the mutant enzyme was altered. The reductive carboxylation of pyruvate to malate catalyzed by the wild type enzyme is accompanied by HCO3−HCO3−-independent reduction of pyruvate and gives lactate as a byproduct. The reaction specificity of the triple mutant was significantly shifted to malate production and the mutant gave a less amount of the byproduct than the wild type. When the triple mutant enzyme was used as a catalyst for pyruvate carboxylation with NADH, the enzyme gave 1.2 times higher concentration of malate than the wild type with NADPH. Single-point mutation analysis revealed that the substitution of Arg221 with Gly is responsible for the shift in reaction specificity. This finding may shed light on the catalytic mechanisms of malic enzymes and other related CO2- and/or HCO3−HCO3−-fixing enzymes.

Keywords
Malic enzyme; Directed evolution; Cofactor preference; Reaction specificity; HCO3−HCO3− fixation
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Directed evolution of thermotolerant malic enzyme for improved malate production
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 117, Issue 2, February 2014, Pages 147–152
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
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Any Questions? feel free to contact us