scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the extent of epoxidation and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at the Leu377 site of SaGGR. We show this TLC-based screen can distinguish between fourfold differences in enzyme activity for select mutants and validated those results by GC–MS. Conclusions With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR).Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS.Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An Automated Workflow to Screen Alkene Reductases Using High-throughput Thin Layer Chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS. Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS. Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

O-Methylierung von Adrenalin, 3.4-Dihydroxybenzoesäure und 6.7-Dihydroxycumarin in Sproßpilzen /O-Methylation of Epinephrine, 3,4-Dihydroxybenzoic Acid and 6,7-Dihydroxycoumarin in Yeasts

1976 ◽  
Vol 31 (9-10) ◽  
pp. 509-513 ◽  
Author(s):  
D Müller-Enoch ◽  
H Thomas ◽  
W Streng ◽  
W Ildfeuer ◽  
O Haferkamp
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Isotope Dilution ◽  
Dihydroxybenzoic Acid ◽  
Dilution Technique ◽  
Isotope Dilution Technique ◽  
Methyl Derivatives ◽  
Layer Chromatography ◽  
Methoxybenzoic Acid

Abstract In the yeasts C. albicans, C. tropicalis, and C. stellatoidea but not in C. krusei, R. rubra, and S. cerevisiae enzyme activity was found by which - as by the catechol-O-methyltransferase (EC 2.1.1.6) found in the liver - the O-methylation of epinephrine to metanephrine and paranephrine, of 3,4-dihydroxybenzoic acid to 4-hydroxy-3-methoxybenzoic acid and 3-hydroxy-4-methoxybenzoic acid, and of 6,7-dihydroxycoumarin to 7-hydroxy-6-methoxycoumarin and 6-hydroxy-7-methoxy-coumarin is catalysed. When the substrates 3,4-dihydroxybenzoic acid, or 6,7-dihydroxycoumarin or epinephrine were incubated in the presence of S-adenosyl-ʟ-[methyl-14C] methionine and S-adenosylmethionine hydrogensulfate with a 100 000 X g supernatant of C. albicans, C. tropicalis or C. stellatoidea the cor­responding O-methylethers were detected in the extracts of the incubation medium by thin-layer chromatography. Final identification of the isomeric radioactive O-methylethers obtained from 3,4-dihydroxy­ benzoic acid and 6,7-dihydroxycoumarin was performed after thin-layer chromatographic separation by the reversed isotope dilution technique. The radioactive m-and p-O-methyl derivatives from epinephrine were separated by thin-layer chromatography and then cleaved with periodate to the corresponding aldehydes which were also identified mainly by the reversed isotope dilution technique.

scholarly journals Visualization of Aspalathin in Rooibos (Aspalathus linearis) Plant and Herbal Tea Extracts Using Thin-Layer Chromatography

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 938 ◽  
Author(s):  
Emily Amor Stander ◽  
Wesley Williams ◽  
Fanie Rautenbach ◽  
Marilize Le Roes-Hill ◽  
Yamkela Mgwatyu ◽  
...  
Keyword(s):  
Quality Control ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput Screening ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Herbal Tea ◽  
Limit Of Quantification ◽  
Aspalathus Linearis ◽  
Layer Chromatography

Aspalathin, the main polyphenol of rooibos (Aspalathus linearis), is associated with diverse health promoting properties of the tea. During fermentation, aspalathin is oxidized and concentrations are significantly reduced. Standardized methods for quality control of rooibos products do not investigate aspalathin, since current techniques of aspalathin detection require expensive equipment and expertise. Here, we describe a simple and fast thin-layer chromatography (TLC) method that can reproducibly visualize aspalathin in rooibos herbal tea and plant extracts at a limit of detection (LOD) equal to 178.7 ng and a limit of quantification (LOQ) equal to 541.6 ng. Aspalathin is a rare compound, so far only found in A. linearis and its (rare) sister species A. pendula. Therefore, aspalathin could serve as a marker compound for authentication and quality control of rooibos products, and the described TLC method represents a cost-effective approach for high-throughput screening of plant and herbal tea extracts.

PROGESTERONE, 20α-DIHYDROPROGRESTERONE AND 20β-DIHYDROPROGESTERONE LEVELS IN DIFFERENT COMPARTMENTS FROM THE HUMAN FOETO-PLACENTAL UNIT

1975 ◽  
Vol 80 (3) ◽  
pp. 558-568 ◽  
Author(s):  
B. Runnebaum ◽  
H. Runnebaum ◽  
I. Stöber ◽  
J. Zander
Keyword(s):  
Liquid Chromatography ◽  
Mass Spectrum ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Reductase Activity ◽  
Placental Tissue ◽  
Gas Liquid Chromatography ◽  
Wet Weight ◽  
Layer Chromatography ◽  
Foetal Blood

ABSTRACT The endogenous levels of progesterone, 20α-dihydroprogesterone and 20β-dihydroprogesterone in different foetal tissues, in placental tissue and in foetal blood during pregnancy (week 9–25) were determined by gas-liquid chromatography or by radioimmunoassay. The identification of these steroids in foetal tissues was based on the behaviour in paper and thin-layer chromatography, the formation of different derivatives, and the retention times in gas-liquid chromatography. For additional identification of progesterone and 20α-dihydroprogesterone a mass spectrum was obtained. The average concentrations of progesterone (P), 20α-dihydroprogesterone (20α-DHP) and 20β-dihydroprogesterone (20β-DHP) in foetal tissues were as follows (ng/g wet weight tissue): brain, P 119.0 ± 45.5, 20α-DHP 86.9 ± 44.7 and 20β-DHP 9.7; lungs, P 257.7 ± 195.7, 20α-DHP 60.3 ± 26.4 and 20β-DHP 7.8; liver, P 103.7 ± 84.6, 20α-DHP 95.2 ± 89.8 and 20β-DHP 17.8; adrenals, P 295.7 ± 90.0 and 20α-DHP 254.5 ± 194.6; kidneys, P 214.2 ± 155.3 and 20α-DHP 76.6 ± 43.0; intestine, P 273.2 ± 166.7, 20α-DHP 77.9 ± 51.2 and 20β-DHP 8.7; residual tissues, P 246.3 ± 178.3 and 20α-DHP 54.8 ± 44.5 The average concentrations in placental tissue and in foetal plasma (week 12–18) were as follows (ng/g wet weight tissue or ng/ml plasma): placenta, P 2619. ± 2153.6 and 20α-DHP 461.0 ± 155.9; foetal plasma, P 378.7 ± 231.8 and 20α-DHP 283.0 ± 108.9 These results indicate that some foetal organs contain a high 20α-reductase activity at least during the first half of pregnancy.

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