Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways

Kerstin Petroll, Andrew Care, Martin Waterstraat, Peter Bergquist, Anwar Sunna

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Glucaric acid (GlucA) has been identified as one of the top 10 potential bio-based chemicals for replacement of oil-based chemicals. Several synthetic enzyme pathways have been engineered in bacteria and yeast to produce GlucA from glucose and myo-inositol. However, the yields and titres achieved with these systems remain too low for the requirements of a bio-based GlucA industry. A major limitation for the optimisation of GlucA production via synthetic enzymatic pathways are the laborious analytical procedures required to detect the final product (GlucA) and pathway intermediates. We have developed a novel method for the simple and simultaneous analysis of GlucA and pathway intermediates to address this limitation using mixed mode (MM) HILIC and weak anion exchange chromatography (WAX), referred to as MM HILIC/WAX, coupled with RID. Isocratic mobile phase conditions and the sample solvent were optimised for the separation of GlucA, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), inositol-1-phosphate (I1P), myo-inositol and glucuronic acid (GA). The method showed good repeatability, precision and excellent accuracy with detection and quantitation limits (LOD and LOQ) of 1.5–2 and 577 mM, respectively. The method developed was used for monitoring the enzymatic synthesis of the final step in the GlucA pathway, and showed that GlucA was produced from GA with near 100% conversion and a titre of 9.2 g L−1.
LanguageEnglish
Pages136-145
Number of pages10
JournalAnalytica Chimica Acta
Volume1066
Early online date15 Mar 2019
DOIs
Publication statusE-pub ahead of print - 15 Mar 2019

Fingerprint

Glucaric Acid
Liquid chromatography
Liquid Chromatography
liquid chromatography
acid
Glucuronic Acid
Inositol
glucose
phosphate
analysis
Glucose-6-Phosphate
Chromatography
Yeast
Anions
Limit of Detection
Bacteria
Industry
Oils
Yeasts
chromatography

Cite this

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title = "Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways",
abstract = "Glucaric acid (GlucA) has been identified as one of the top 10 potential bio-based chemicals for replacement of oil-based chemicals. Several synthetic enzyme pathways have been engineered in bacteria and yeast to produce GlucA from glucose and myo-inositol. However, the yields and titres achieved with these systems remain too low for the requirements of a bio-based GlucA industry. A major limitation for the optimisation of GlucA production via synthetic enzymatic pathways are the laborious analytical procedures required to detect the final product (GlucA) and pathway intermediates. We have developed a novel method for the simple and simultaneous analysis of GlucA and pathway intermediates to address this limitation using mixed mode (MM) HILIC and weak anion exchange chromatography (WAX), referred to as MM HILIC/WAX, coupled with RID. Isocratic mobile phase conditions and the sample solvent were optimised for the separation of GlucA, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), inositol-1-phosphate (I1P), myo-inositol and glucuronic acid (GA). The method showed good repeatability, precision and excellent accuracy with detection and quantitation limits (LOD and LOQ) of 1.5–2 and 577 mM, respectively. The method developed was used for monitoring the enzymatic synthesis of the final step in the GlucA pathway, and showed that GlucA was produced from GA with near 100{\%} conversion and a titre of 9.2 g L−1.",
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Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways. / Petroll, Kerstin; Care, Andrew; Waterstraat, Martin; Bergquist, Peter; Sunna, Anwar.

In: Analytica Chimica Acta, Vol. 1066, 20.08.2019, p. 136-145.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways

AU - Petroll,Kerstin

AU - Care,Andrew

AU - Waterstraat,Martin

AU - Bergquist,Peter

AU - Sunna,Anwar

PY - 2019/3/15

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M3 - Article

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SN - 0003-2670

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