Metabolically engineered methylotrophic yeast cells and enzymes as sensor biorecognition elements

Abstract

An extended definition of the term metabolic engineering is given and its successful use in the construction of biorecognition elements of sensors is demonstrated. It is shown that genetic and chemical modifications of methylotrophic yeast cells provide directed changes in their physiological responses towards methanol, ethanol and formaldehyde resulting in enhanced selectivity and shorter time response of the corresponding potentiometric and amperometric biosensors.

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

“…As shown by us previously (Gonchar et al, 2002), chemical modification of the cells resulted in increased permeability of the cellular barrier to low-molecular weight compounds. As in the case of the immobilized AOX at the outlet of the columns containing both types of cells, the FA concentration in the gas phase was 0.02 ppm.…”

“…(1) CSA-33 (gcr1) ("constitutive synthesis of alcohol oxidase") with impaired glucose catabolite repression of AOX synthesis and (2) C-105 (gcr1 catX) with additional mutation inactivating catalase (Gonchar et al, 2001(Gonchar et al, , 2002.…”

“…FdDH is widely used for bioanalytical purposes (Ali et al, 2006;Gonchar et al, 2002;Hämmerle et al, 2010;Herschkovitz et al, 2000;Kawamura et al, 2005;Korpan et al, 1993Korpan et al, , 2000Vastarella and Nicastri, 2005;Winter and Cammann, 1989).…”

A reagentless bienzyme amperometric biosensor based on alcohol oxidase/peroxidase and an Os-complex modified electrodeposition paint

“…Highly purified alcohol oxidase (AOX) was isolated from a cell-free extract of the genetically constructed mutant strain Hansenula polymorpha C-105 (gcr1 catX) [17,18] by means of a two-step ammonium sulphate fractionation (at 40 and 60% of saturation) [19], followed by dialysis and ion exchange chromatography on DEAE-Toyopearl 650 M. The first stages of the purification process were carried out in the presence of protease inhibitors (1 mM EDTA and 0.4 mM phenylmethylsulfonyl fluoride, PMSF). The final preparation of the enzyme had a specific activity 30 U mg −1 protein and was homogeneous during electrophoresis in a polyacrylamide gel in the presence of SDS.…”

“…In this communication, the development of a novel alcohol bi-enzyme biosensor is reported which is based on AOX isolated from a genetically constructed mutant strain Hansenula polymorpha C-105 [17,18] in combination with a non-manual electrochemically induced immobilization procedure using a new type of Os-complex modified electrodeposition paints (EDP) for the immobilization of horseradish peroxidase in a first layer and a cathodic EDP for the immobilization and stabilization of AOX in a second layer. The used redox EDP assures fast electron transfer between the integrated peroxidase and the electrode surface at a low working potential.…”