Improvement of thin-layer chromatography for enzyme assay of geranylgeraniol 18-hydroxylase from<i>Croton stellatopilosus</i>Ohba

Chanama, Manee; Wunnakup, Thaniya; De-Eknamkul, Wanchai; Chanama, Suchart

doi:10.1556/jpc.22.2009.1.9

Cited by 4 publications

(3 citation statements)

References 11 publications

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“…In addition, the activity was determined 1.5-fold higher in 100,000 g microsomal fraction than in the 20,000 g insoluble fraction under the presence of NADPH and aeration (Chanama et al, 2009) …”

Section: Resultsmentioning

confidence: 92%

“…The GGOH 18-hydroxylase was prepared and purified according to the method as previously described (Chanama et al, 2009). Briefly, 30 g of frozen mature leaves were ground rapidly to a powder in liquid nitrogen using a prechilled mortar and pestle.…”

Section: Preparation Of Microsomal Fraction Containing Ggoh 18-hydroxmentioning

confidence: 99%

“…Enzyme activity of GGOH 18-hydroxylase was assayed as previously described (Chanama et al, 2009). The reaction mixture consisted of 83 mM Tricine-NaOH (pH 7.8), 0.8 mM NADPH and 57 µM GGOH (substrate).…”

Section: Ggoh 18-hydroxylase Activity Assaysmentioning

confidence: 99%

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Biochemical and kinetic characterization of geranylgeraniol 18-hydroxylase (CYP97C27) from Croton stellatopilosus Ohba

Chanama¹,

Chanama

2015

Afr. J. Biotechnol.

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Geranylgeraniol 18-hydroxylase (EC 1.14.13.110) that exists solely in Croton stellatopilosus Ohba catalyses the last committed step of plaunotol biosynthetic pathways by conversion of geranylgeraniol (GGOH) to plaunotol. This enzyme and its gene are an attractive target for development of plaunotol production and its detailed biochemical properties need to be understood. Recently, even though the gene (CYP97C27) coding for GGOH 18-hydroxylase has been identified, cloned, and expressed in Escherichia coli system, the enzyme activity has been detected mainly in the insoluble fraction (20,000 g). This means that biochemical and kinetic studies could not be undertaken. However, our previous study indicated that this enzyme activity was easily and specifically detected in the microsomal fraction (100,000 g) of a crude enzyme extract. Therefore, in this report we describe a comprehensive biochemical characterization of GGOH 18-hydroxylase activity in the microsomal fraction from C. stellatopilosus Ohba. The oxygen-dependent enzyme activity of GGOH 18-hydroxylase was inhibited by carbon monoxide and the inhibition was partially reversible upon illumination with white light. Kinetic studies of the GGOH 18-hydroxylase showed high affinity to GGOH and NADPH with apparent K m values of 0.8 and 53 µM, respectively. Furthermore, the enzyme activity was inhibited by P450 inhibitors, including ancymidol, metyrapone, miconazole, potassium cyanide and cytochrome c, with the IC 50 values of 428, 65, 75, 66 and 8 µM, respectively. Based on the biochemical and kinetic characteristics, the GGOH 18-hydroxylase in the microsomal fraction is likely a P450 encoded by CYP97C27 gene as previously described.

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Section: Resultsmentioning

confidence: 92%

Section: Preparation Of Microsomal Fraction Containing Ggoh 18-hydroxmentioning

confidence: 99%

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Biochemical and kinetic characterization of geranylgeraniol 18-hydroxylase (CYP97C27) from Croton stellatopilosus Ohba

Chanama¹,

Chanama

2015

Afr. J. Biotechnol.

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Current Awareness in Phytochemical Analysis

2009

Phytochemical Analysis

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In order to keep subscribers up‐to‐date with the latest developments in their field, John Wiley &Sons are providing a current awareness service in each issue of the journal. The bibliography contains newly published material in the field of phytochemical analysis. Each bibliography is divided into 16 sections: 1 Reviews; 2 General; 3 Nucleic Acids; 4 Amino Acids, Proteins &Enzymes; 5 Carbohydrates; 6 Lipids; 7 Secondary Products ‐ General; Pharmacognosy; 8 Growth Regulators; 9 Industrially‐Important Products ‐ General; Food; Alcoholic beverages; 10 Toxins/Allergens; 11 Pigments; 12 Vitamins; 13 Others. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted.

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geranylgeraniol 18-hydroxylase 1.14.13.110

Schomburg¹,

Schomburg²

2013

Class 1 Oxidoreductases

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Improvement of thin-layer chromatography for enzyme assay of geranylgeraniol 18-hydroxylase fromCroton stellatopilosusOhba

Cited by 4 publications

References 11 publications

Biochemical and kinetic characterization of geranylgeraniol 18-hydroxylase (CYP97C27) from Croton stellatopilosus Ohba

Biochemical and kinetic characterization of geranylgeraniol 18-hydroxylase (CYP97C27) from Croton stellatopilosus Ohba

Current Awareness in Phytochemical Analysis

geranylgeraniol 18-hydroxylase 1.14.13.110

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