PERMEABILIZATION OF THE METHYLOTROPHIC YEAST PICHIA-PINUS FOR INTRACELLULAR ENZYME ANALYSIS - A QUANTITATIVE STUDY

A quantitative in situ assay of methanol oxidizing enzymes in permeabilized suspensions of the methylotrophic yeast Pichia pinus is described. Activities of the intracellular enzymes alcohol oxidase, formaldehyde dehydrogenase and formate dehydrogenase were readily measured in cell suspensions treated with 0.1% surfactant, digitonin or cetyltrimethylammoniumbromide (CTAB) for 15 min. Recovery of enzyme activities from the permeabilized cell suspension was higher than from disrupted cells. The permeabilizing ability of surfactant was dependent on its concentration, being highest at 0.1%. Cell permeabilization caused a marked decrease in dry weight and protein content of cells whereas no significant immidiate leakage of the intracellular enzymes alcohol oxidase, formaldehyde dehydrogenase and formate dehydrogenase was observed. However, 20-h storage of permeabilized suspension at 8 degrees C resulted in marked leakage of dehydrogenases but not of alcohol oxidase from the cells. Addition of CTAB at 0.01% and digitonin at 0.01% and 0.1% to the cell-free extract for 1 h did not inhibit formaldehyde dehydrogenase and alcohol oxidase, whereas incubation with CTAB at 0.1% for the same time reduced activity of these enzymes to 62% and 37% from the initial, respectively. Properties of alcohol oxidase (K-m towards methanol and substrate specificity) in permeabilized cell suspensions were similar to these measured in cell-free extract. Treatment of cell suspension with surfactants reduced the viability of cells dependent on the type of surfactant and its concentration.