Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha

Abstract

Alcohol oxidase (AOX) has been purified 8-fold from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha C-105 (gcr1 catX) with impaired glucose-induced catabolite repression and completely devoid of catalase. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and HPLC. Some physicochemical and biochemical properties of AOX were studied in detail: molecular weight (approximately 620 kD), isoelectric point (pI 6.1), and UV-VIS, circular dichroism (CD), and fluorescence spectra. The content of different secondary structure motifs of the enzyme has been calculated from the CD spectra using a computer program. It was found that the native protein contains about 50% alpha-helix, 25% beta-sheet, and about 20% random structures. The kinetic parameters for different substrates, such as methanol, ethanol, and formaldehyde, were measured using a Clark oxygen electrode. The rate of enzymatic oxidation of formaldehyde by alcohol oxidase from H. polymorpha is only twice lower compared to the best substrate of the enzyme, methanol.

Modern methods of wine quality analysis

“…В Інституті біології клітини (ІБК) НАНУ було cконструйовано унікальний надпродуцент алкоголь-оксидази (AO) -штам термотолерантних метило-трофних дріжджів Hansenula polymorpha C-105 (gcr1 catX) та розроблено ефективну технологію одержан-ня високоочищеного препарату АО [33].…”

“…1) для кількісного визначення етанолу, гліце-ролу, L-лактату, L-аргініну. Основним біорозпізнаю-чими елементами біосенсорів на етанол, лактат і ар-гінін були відповідні високооочищені ферменти, ви-ділені із клітин дріжджів H. рolymorpha за власними технологіями [33,43,[50][51][52][53][54] або сами клітини [53]. Для покращення фізико-хімічних характеристик біо-сенсорів (стабільності, чутливості) ферменти іммобілізували на наночастинках (НЧ) благородних металів.…”

Immobilized formaldehyde-metabolizing enzymes from Hansenula polymorpha for removal and control of airborne formaldehyde

“…Isolation and purification of the enzymes 2.4.1. AOX from mutant overproducing H. polymorpha yeast cells AOX was isolated from cell-free extracts of the yeast H. polymorpha C-105 (gcr1 catX) strain cultivated in glucose medium (Gonchar et al, 2001;Shleev et al, 2006). Purification of the enzyme was carried out using a two-step precipitation with ammonium sulfate (at 40 and 60% saturation) in the presence of 1 mM EDTA and 0.4 mM PMSF to inhibit proteases.…”

“…AOX activity was measured as described previously (Shleev et al, 2006). FdDH activity was determined by the rate of NADH formation monitored spectrophotometrically at 340 nm (Schutte et al, 1976) under the following conditions: 25 • C, 1 mM FA, 1 mM NAD + , and 2 mM GSH in PB (50 mM phosphate buffer, pH 8.0).…”

Formaldehyde Oxidizing Enzymes and Genetically Modified Yeast Hansenula polymorpha Cells in Monitoring and Removal of Formaldehyde

“…This unexpected effect is the first reported example of specific "chemical enhancement" of the pH-SFET biosensor response. A highly stable and sensitive amperometric bi-enzyme biosensor (Smutok et al, 2006) was developed for assay of ethanol, as well as of FA, using the highly-purified AOX preparation (Shleev et al, 2006), isolated from the yeast cells of H. polymorpha C-105. The sensor's layer was created with a non-manual electrochemically-induced immobilization procedure using a new type of Os-complex modified electrodeposition paints (EDP) for horseradish peroxidase placing in a first layer and a cathodic EDP for AOX immobilization and stabilization in a second layer.…”