Purification and partial characterization of Cu/Zn superoxide dismutase from Kluyveromyces marxianus yeast.

A new thermostable Cu/Zn SOD from a thermotolerant yeast strain Kluyveromyces marxianus NBIMCC 1984 has been purified and characterized. The purification procedure comprises thermal treatment and dialysis, ion-exchange chromatography and chromatofocusing. The methodology is a rapid, efficient and highly specific, generating pure preparation (specific activity 996 U mg of protein(-1)) with a yield of 53%. The purified enzyme is a homodimer with Mw of 34,034 Da and has high N-terminal homology with other yeasts' Cu/Zn SOD enzymes. The protein is characterized with some unique features such as-thermostability (t(1/2) at 70 degrees C=30 min), pH stability in the alkaline range (7.5-8.5) and resistance to inhibitors and variety of chemicals. These characteristics reveal possibilities for wide practical application of K. marxianus Cu/Zn SOD enzyme.

Cu/Zn superoxide dismutase in yeast mitochondria - a general phenomenon.

Fermentative and respiratory yeast strains of genera Saccharomyces, Kluyveromyces, Pichia, Candida and Hansenula have been investigated for mitochondrial localization of Cu/Zn superoxide dismutase (SOD). Pure mitochondrial fractions were obtained and the specific activities of Cu/Zn and Mn SODs were measured in comparison with those in the corresponding cell-free extracts. The Cu/Zn SOD: Mn SOD ratio in mitochondria and crude extracts was calculated and was considered a specific characteristic of all tested strains. Electrophoretical visualization of SOD patterns provided evidence for possible migration of cytosolic Cu/Zn SOD to mitochondria. The characteristic Cu/Zn SOD profile in mitochondria of all tested strains suggested its ubiquity within the fermentative and respiratory yeasts.

Characterization of a non-pigment producing Monascus purpureus mutant strain.

A characterization of a non-pigment producing mutant Monascus purpureus M12 compared with its parental strain Monascus purpureus Went CBS 109.07 has been performed aiming to investigate the relation between pigment biosynthesis and other characteristics of these fungi. A comparison has been made of morphological features, some physiological properties and biochemical activities of both strains. The albino mutant exhibits an anamorph life cycle, high conidia forming capability, slower radial growth rate and temperature sensitivity. The assimilation capacity of both strains for mono-, disaccharides and some alcohols is in the same range (Yx/c 0.2 - 0.35), while the red strain has a higher fermentation capacity. In a selected albino mutant, the growth rate, metabolic activity and capacity for production of typical for Monascus fungi secondary metabolites were reduced considerably. Hydrolytic activity towards natural substrates expressed through glucoamylase and protease was approximately 10 fold lower in the non pigment producing strain (0.05 - 0.08 U/mg protein and 0.01 - 0.07 U/mg protein respectively) compared with the red one. Important qualitative differences between both strains was found in fatty acid composition and in the production of citrinin and monacolin. The mutant strain possessed C17, C20 and C22 fatty acids and did not produce citrinin.

A modified procedure for isolation of yeast mitochondrial DNA.

A modified, rapid and inexpensive method for preparation of mitochondrial DNA (mtDNA), suitable for molecular analysis is proposed. It comprises batch cultivation of Saccharomyces cerevisiae strain NBIMCC 583 on a simple nutrient medium at 28 degrees C; permeabialization of cells from late exponential growth phase with cetyltrimethylamonnium bromide, mechanical disintegration of the cell wall; preparation of a mitochondrial fraction and subsequent isolation and purification of mtDNA. The amount and the purity of the obtained mtDNA have been checked and its application for molecular analysis proven. The main advantages of the proposed procedure for isolation of mtDNA are introduction of simple nutrient medium, replacement of the enzymatic lysis of the cell wall by the cheaper mechanical one, avoidance of ultracentrifugation steps and use of harmful chemical substances.

Superoxide dismutase during glucose repression of Hansenula polymorpha CBS 4732.

Hansenula polymorpha CBS 4732 was studied during cultivation on methanol and different glucose concentrations. Activities of Cu/Zn and Mn superoxide dismutase, catalase and methanol oxidase were investigated. During cultivation on methanol, increased superoxide dismutase and catalase activities and an induced methanol oxidase were achieved. Transfer of a methanol grown culture to medium with a high glucose concentration caused growth inhibition, low consumption of carbon, nitrogen and phosphate substrates, methanol oxidase inactivation as well as decrease of catalase activity (21.8 +/- 0.61 deltaE240 x min(-1) x mg protein(-1)). At the same time, a high value for superoxide dismutase enzyme was found (42.9 +/- 0.98 U x mg protein(-1), 25% of which was represented by Mn superoxide dismutase and 75% - by the Cu/Zn type). During derepression methanol oxidase was negligible (0.005 +/- 0.0001 U x mg protein(-1)), catalase tended to be the same as in the repressed culture, while superoxide dismutase activity increased considerably (63.67 +/- 1.72 U x mg protein(-1), 69% belonging to the Cu/Zn containing enzyme). Apparently, the cycle of growth inhibition and reactivation of Hansenula polymorpha CBS 4732 cells is strongly connected with the activity of the enzyme superoxide dismutase.