Cytochemical study of catalase activity in Candida boidinii during incubation on methanol

Catalase activity of the methanol-assimilating yeast Candida boidinii M-363 was determined cytochemically and biochemically. Electron microscopic investigations on ultrathin sections were made on cells from 16, 24, and 48h batch cultures in nutrient medium with methanol (or glucose as a control) as the sole source of carbon and energy. The electron-dense oxidation product of 3,3′-diaminobenzidine was found predominantly in the mitochondrial cristae and membranes. The mitochondria were increased in number, enlarged, sometimes aggregated, with variable form and size and they characteristically developed when the strain was grown on methanol. The significant development of these organelles and their intensive DAB staining correlated with the considerable increase in catalase activity. Biochemically, catalase in the cell-free extract was determined to be maximal along the exponential growth phase of the strain during its incubation on methanol. Enzyme analysis of the heavy mitochondrial fraction showed that it possessed catalase activity but not peroxidase activity. The results showed that not only peroxisomes but also mitochondria may be structurally and functionally responsible for the high catalase activity of some methanol-assimilating yeasts. What is more, the contribution of the mitochondria to the utilization of methanol may be significant.     

Mutant Hansenula polymorpha strain with constitutive alcohol oxidase and peroxisome biosynthesis

A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains--DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol x min(-1) x mg(-1) protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.     

Investigations of mannan and glucan in the cell wall ofCandida boidinii cultivated on methanol

The polysaccharide components (mannan and glucan) in the cell wall ofCandida boidinii M 363 grown on methanol and glucose as control were investigated using electron microscopy, cytochemical and biochemical methods. An ultrastructural rearrangement of the polymers in the cell wall of yeasts cultivated on methanol in comparison to those cultivated on glucose was established. The morphological changes correlate to the quantitative changes in the polysaccharide constituents of the cell wall. The forming and the role of thiosemihydrocarbazide (TSHC) — negative zones in theCandida boidinii cell wall cultivated on methanol media are discussed.     

Cytochemical study of catalase activity in Candida boidinii during incubation on methanol

Catalase activity of the methanol-assimilating yeast Candida boidinii M-363 was determined cytochemically and biochemically. Electron microscopic investigations on ultrathin sections were made on cells from 16, 24, and 48h batch cultures in nutrient medium with methanol (or glucose as a control) as the sole source of carbon and energy. The electron-dense oxidation product of 3,3-diaminobenzidine was found predominantly in the mitochondrial cristae and membranes. The mitochondria were increased in number, enlarged, sometimes aggregated, with variable form and size and they characteristically developed when the strain was grown on methanol. The significant development of these organelles and their intensive DAB staining correlated with the considerable increase in catalase activity. Biochemically, catalase in the cell-free extract was determined to be maximal along the exponential growth phase of the strain during its incubation on methanol. Enzyme analysis of the heavy mitochondrial fraction showed that it possessed catalase activity but not peroxidase activity. The results showed that not only peroxisomes but also mitochondria may be structurally and functionally responsible for the high catalase activity of some methanol-assimilating yeasts. What is more, the contribution of the mitochondria to the utilization of methanol may be significant.     

Screening of fungi for phytase production

Over 200 fungal strains were screened for phytase production using a two-step procedure. The best strain, an Aspergillus sp., produced phytase almost equally active at both pH 5.0 and 2.5. Synthesis of the enzyme was limited by content of inorganic phosphorus above 20mg/dm.