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Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060

Paper ID Volume ID Publish Year Pages File Format Full-Text
20108 43158 2016 6 PDF Available
Title
Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060
Abstract

Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C (t1/2). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations.

Keywords
Glycerol kinase; Thermostabilization; Cellulomonas; Ancestral mutation; Epistasis effect; Diagnostic enzyme
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Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 121, Issue 5, May 2016, Pages 497–502
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