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One-Step Purification of Microbially Produced Hydrophobic Terpenes via Process Chromatography.

ABSTRACT: Novel and existing terpenes are already being produced by genetically modified microorganisms, leading to new process challenges for the isolation and purification of these terpenes. Here, eight different chromatographic resins were characterized for the packed bed adsorption of the model terpene ?-caryophyllene, showing their applicability on an Escherichia coli fermentation mixture. The polystyrenic Rensa^® RP (Ø 50 ?m) shows the highest affinity, with a maximum capacity of >100 g L^-1 and the best efficiency, with a height equivalent of a theoretical plate (HETP) of 0.022 cm. With this material, an optimized adsorption-based purification of ?-caryophyllene from a fermentation mixture was developed, with the green solvent ethanol for desorption. A final yield of >80% and a purity of >99% were reached after only one process step with a total productivity of 0.83 g h^-1 L^-1. The product solution was loaded with a volume ratio (feed to column) of >500 and the adapted gradient elution yielded a 40 times higher concentration of ?-caryophyllene. The adsorption-based chromatography represents therefore a serious alternative to the liquid-liquid extraction and achieves desired purities without the utilization of hazardous solvents.

SUBMITTER: Grozdev L

PROVIDER: S-EPMC6681792 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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One-Step Purification of Microbially Produced Hydrophobic Terpenes via Process Chromatography.

Grozdev Ljubomir L Kaiser Johann J Berensmeier Sonja S

Frontiers in bioengineering and biotechnology 20190729

Novel and existing terpenes are already being produced by genetically modified microorganisms, leading to new process challenges for the isolation and purification of these terpenes. Here, eight different chromatographic resins were characterized for the packed bed adsorption of the model terpene β-caryophyllene, showing their applicability on an <i>Escherichia coli</i> fermentation mixture. The polystyrenic Rensa<sup>®</sup> RP (Ø 50 μm) shows the highest affinity, with a maximum capacity of >1 ...[more]

PMID: 31417900

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Project description:BackgroundHydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported.Methods and resultsBacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation.ConclusionsConducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes.

Dataset Information

One-Step Purification of Microbially Produced Hydrophobic Terpenes via Process Chromatography.

Publications

One-Step Purification of Microbially Produced Hydrophobic Terpenes via Process Chromatography.

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