Enzymatic Determination of Methanol with Alcohol Oxidase, Peroxidase, and the Chromogen 2,2‘-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and Its Application to the Determination of the Methyl Ester Content of Pectins

Mangos, Thomas J.; Haas, Michael J.

doi:10.1021/jf960274z

Cited by 28 publications

(27 citation statements)

References 13 publications

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“…Methanol input rate for methanol-limited chemostat cultures is 41.0 nmol/(ml ⅐ min). The methanol concentration in the effluent was below the detection limit (ϳ50 M) of our assay (23), indicating essentially complete consumption of the methanol. For a steady-state OD 600 of 0.99 (the average of nine cultures), the consumption rate therefore is 41.5 nmol/ (ml ⅐ OD 600 ⅐ min).…”

Section: H-labeling Experiments Incorporation Of Deuterium (mentioning

confidence: 80%

“…M. extorquens AM1 was routinely grown at 28°C in the minimal medium described previously (11), either in batch cultures, with 120 mM methanol or 15 mM succinate, or in chemostat cultures in a benchtop fermentor essentially as described previously (9,37), with a dilution rate of 0.100 h Ϫ1 and a substrate concentration in the feed of either 25 mM methanol or 3.7 mM succinate plus 12.5 mM methanol. Methanol was measured in the effluent as described previously (23). Wild-type chemostat cultures maintained a steady-state optical density at 600 nm (OD 600 ) of approximately 1.0.…”

Section: Methodsmentioning

confidence: 99%

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Formate as the Main Branch Point for Methylotrophic Metabolism in Methylobacterium extorquens AM1

Crowther¹,

Kosály²,

2008

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In serine cycle methylotrophs, methylene tetrahydrofolate (H 4 F) is the entry point of reduced one-carbon compounds into the serine cycle for carbon assimilation during methylotrophic metabolism. In these bacteria, two routes are possible for generating methylene H 4 F from formaldehyde during methylotrophic growth: one involving the reaction of formaldehyde with H 4 F to generate methylene H 4 F and the other involving conversion of formaldehyde to formate via methylene tetrahydromethanopterin-dependent enzymes and conversion of formate to methylene H 4 F via H 4 F-dependent enzymes. Evidence has suggested that the direct condensation reaction is the main source of methylene H 4 F during methylotrophic metabolism. However, mutants lacking enzymes that interconvert methylene H 4 F and formate are unable to grow on methanol, suggesting that this route for methylene H 4 F synthesis should have a significant role in biomass production during methylotrophic metabolism. This problem was investigated in Methylobacterium extorquens AM1. Evidence was obtained suggesting that the existing deuterium assay might overestimate the flux through the direct condensation reaction. To test this possibility, it was shown that only minor assimilation into biomass occurred in mutants lacking the methylene H 4 F synthesis pathway through formate. These results suggested that the methylene H 4 F synthesis pathway through formate dominates assimilatory flux. A revised kinetic model was used to validate this possibility, showing that physiologically plausible parameters in this model can account for the metabolic fluxes observed in vivo. These results all support the suggestion that formate, not formaldehyde, is the main branch point for methylotrophic metabolism in M. extorquens AM1.Methylobacterium extorquens AM1 is a facultative methylotroph that has served as a model system for understanding methylotrophic metabolism for many decades (1,3,33). One outstanding question in methylotrophic metabolism involves the route by which C 1 compounds are incorporated into assimilatory metabolism. In this bacterium, methanol or methylamine is oxidized via formaldehyde and formate to CO 2 for energy metabolism and carbon is assimilated via the serine cycle ( Fig.

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Section: H-labeling Experiments Incorporation Of Deuterium (mentioning

confidence: 80%

Section: Methodsmentioning

confidence: 99%

Formate as the Main Branch Point for Methylotrophic Metabolism in Methylobacterium extorquens AM1

Crowther¹,

Kosály²,

2008

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“…It has been reported that exposure of an AOD preparation from Pichia pastoris to air produced low levels of H 2 O 2 (apparent autoxidation), even in the absence of added substrate. 9 The autoxidation is possibly caused by a unique property that the protein has 65 free-SH groups per molecule. 10 Therefore, the intercept might correspond to the amount of H 2 O 2 formed in the autoxidation.…”

Section: Determination Of Ethanolmentioning

confidence: 99%

Immobilized Enzyme-Based Microtiter Plate Assay for Ethanol in Alcoholic Beverages

1999

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A method for the determination of ethanol using alcohol oxidase (AOD), peroxidase (POD), and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was developed in which those enzymes were separately immobilized on the inside surface of each well of a 96-well microtiter plate. Prior to immobilization, the surface was activated with glutaraldehyde. The activation conditions remarkably affected the activity of the immobilized AOD. A sample solution containing ethanol was first added into the well with immobilized AOD (AOD-well). After 30 min, an aliquot (20 µl) was transferred into the well with immobilized POD containing 180 µl of 2 mM ABTS and the absorbance change at 415 nm for 10 min was measured with a microplate reader. The calibration curve was linear over the range 0.10 -1.0 mM, and the relative standard deviation (n=5) was 0.8% for 1 mM ethanol. The ethanol concentration in alcoholic beverages, obtained by the present method, agreed well with that by the F-kit method. The AOD-well was reusable for 20 successive determinations.

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“…Opening and closing of the specwell as the enzymatic determination of methanol with trophotometer lid and pipetting and multiple flushing AOD, POD, and ABTS, exhibit optimum activities at of the pipet tip to disperse the pectinesterase was done pH 7.5 (21). Therefore, this pH was used for the assay in 10 s. A rapid, reversible change in absorbance accomdescribed here.…”

Section: Addition Of Pectinesterase To Determine Demethoxylaexaminatimentioning

confidence: 99%

“…To gain such information, a sensitive and sim-of the AOD procedure (21). As with the previous published method (19) it uses alcohol oxidase from the ple assay for pectinesterase is desired.…”

mentioning

confidence: 99%

A Spectrophotometric Assay for the Enzymatic Demethoxylation of Pectins and the Determination of Pectinesterase Activity

Mangos¹,

Haas²

1997

Analytical Biochemistry

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Enzymatic Determination of Methanol with Alcohol Oxidase, Peroxidase, and the Chromogen 2,2‘-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and Its Application to the Determination of the Methyl Ester Content of Pectins

Cited by 28 publications

References 13 publications

Formate as the Main Branch Point for Methylotrophic Metabolism in Methylobacterium extorquens AM1

Formate as the Main Branch Point for Methylotrophic Metabolism in Methylobacterium extorquens AM1

Immobilized Enzyme-Based Microtiter Plate Assay for Ethanol in Alcoholic Beverages

A Spectrophotometric Assay for the Enzymatic Demethoxylation of Pectins and the Determination of Pectinesterase Activity

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