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Development and validation of an HPLC-based screening method to acquire polyhydroxyalkanoate synthase mutants with altered substrate specificity

Paper ID Volume ID Publish Year Pages File Format Full-Text
21259 43214 2012 7 PDF Available
Title
Development and validation of an HPLC-based screening method to acquire polyhydroxyalkanoate synthase mutants with altered substrate specificity
Abstract

A rapid and convenient method for the compositional analysis of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC) and alkaline sample pretreatment in a 96-well plate format. The reliability of this system was confirmed by the fact that a mutant with a D171G mutation of Aeromonas caviae PHA synthase (PhaCAc), which gained higher reactivity toward 3-hydroxyhexanoate (3HHx), was selected from the D171X mutant library. Together with D171G mutant, several single mutants showing high reactivity toward 3HHx were isolated by the HPLC assay. These new mutants and double mutants combined with an N149S mutation were used to synthesize P(3-hydroxybutyrate-co-3HHx) in Ralstonia eutropha PHB−4 from soybean oil as carbon source, achieving higher levels of 3HHx fraction than the wild-type enzyme. Based on these results, the high-throughput screening system will serve as a powerful tool for exploring new and beneficial mutations responsible for regulating copolymer composition of PHA.

Keywords
Polyhydroxyalkanoates; High-throughput screening; High-performance liquid chromatography (HPLC); Alkaline pretreatment; PHA synthase; Substrate specificity
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Development and validation of an HPLC-based screening method to acquire polyhydroxyalkanoate synthase mutants with altered substrate specificity
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 113, Issue 3, March 2012, Pages 286–292
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