pectins and alcohol-oxidase

The methylotrophic yeast Pichia methanolica was able to grow on pectic compounds, pectin and polygalacturonate, as sole carbon sources. Under the growth conditions used, P. methanolica exhibited increased levels of pectin methylesterase, and pectin-depolymerizing and methanol-metabolizing enzyme activities. On the other hand, P. methanolica has two alcohol oxidase (AOD) genes, MOD1 and MOD2. On growth on pectin, the P. methanolica mod1Delta and mod1Deltamod2Delta strains showed a severe defect in the growth yield, although the mod2Delta strain could grow on polygalacturonate to the same extent as the wild-type strain. The expression of MOD1 was detected in pectin-grown cells, but the MOD2-gene expression detected by pectin was much lower than that of MOD1. Moreover, pectin could induce peroxisome proliferation in P. methanolica, like methanol and oleic acid. These findings showed that P. methanolica was able to utilize the methylester moiety of pectin by means of methanol-metabolic enzymes in peroxisomes, and that the functional AOD subunit for pectin utilization was Mod1p in P. methanolica.

Topics: Alcohol Oxidoreductases; Carboxylic Ester Hydrolases; Electrophoresis, Polyacrylamide Gel; Gene Deletion; Gene Expression; Methanol; Pectins; Peroxisomes; Pichia; RNA, Fungal; RNA, Messenger; Transcription, Genetic

Thermoascus aurantiacus showed the best growth on medium containing pectin as a carbon source. The enzyme involved in the production of catalase in the fungus was alcohol oxidase. Formaldehyde dehydrogenase and formate dehydrogenase, in addition to alcohol oxidase and catalase, were detected in the cells grown on pectin. Alcohol oxidase was alkali resistant (pH 7 to 11), and was comparatively heat stable (55 degrees C).

Topics: Alcohol Oxidoreductases; Aldehyde Oxidoreductases; Bioreactors; Catalase; Cell Culture Techniques; Culture Media; Eurotiales; Formate Dehydrogenases; Pectins

Pectin methylesterase (PME; EC 3.1.1.11) activities are widespread in bacteria, fungi, and plants. PME-mediated changes in cell wall pectin structure play important roles in plant development. Genome sequencing projects have revealed the existence of large PME multigene families in higher plants. Additional complexity for PME regulation arises from the presence of specific PME inhibitor proteins (PMEI) in plant cells. Several assay procedures for the determination of PME activity have been reported. However, previous protocols suffered from various limitations. Here we report a protocol for a coupled enzyme assay based on methanol oxidation via alcohol oxidase (AO; EC 1.1.3.13) and subsequent oxidation of formaldehyde by formaldehyde dehydrogenase (FDH; EC 1.2.1.3). This simple and robust assay allows the continuous monitoring of PME activity in the neutral pH range. Furthermore, as plant PMEIs do not interfer with AO and FDH activities, this assay is suitable for the characterization of the inhibition kinetics of PMEI.

Topics: Alcohol Dehydrogenase; Alcohol Oxidoreductases; Aldehyde Oxidoreductases; Bacteria; Carboxylic Ester Hydrolases; Cell Wall; Citrus sinensis; Enzyme Inhibitors; Formaldehyde; Fruit; Fungi; Hydrogen-Ion Concentration; Methanol; Pectins; Spectrophotometry

The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

Topics: Alcohol Oxidoreductases; Aldehyde-Ketone Transferases; Candida; Carboxylic Ester Hydrolases; Genes, Fungal; Kinetics; Methanol; Mutagenesis; Pectins; Peroxisomes; Polygalacturonase; Polysaccharide-Lyases; Substrate Specificity

A rapid spectrophotometric method for the determination of pectinesterase activity is presented. In this assay, methanol released from pectin by pectinesterase is oxidized with alcohol oxidase to form hydrogen peroxide and formaldehyde. Hydrogen peroxide is then quantitated with peroxidase and the chromogen 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Since both reactions exhibit the same pH optimum it was possible to couple the methanol assay directly to the action of pectinesterase for the real-time determination of this enzyme. The assay is reliable and sensitive, being capable of quantitating a minimum pectinesterase activity of 0.0625 unit (1 unit = 1 microM methanol released per minute). It is also capable of detecting the enzymatic demethoxylation of galactopyranosyl uronic acid methyl esters of pectin down to a minimum concentration of 1.56 nM of methanol per milliliter using a pectin substrate with a methoxy content of 10% (w/w) at a concentration of 0.5 microgram/ml.

Topics: Alcohol Oxidoreductases; Benzothiazoles; Carboxylic Ester Hydrolases; Chromatography, Ion Exchange; Formaldehyde; Hydrogen Peroxide; Indicators and Reagents; Pectins; Peroxidase; Spectrophotometry; Sulfonic Acids