Study of Catalase Enzyme in Methylotrophic Yeasts (original) (raw)

2008, Biotechnology & Biotechnological Equipment

Methylotrophic yeasts possess a respiratory type of metabolism and during growth an accumulation of potentially cytotoxic species (O 2-) and hydrogen peroxide (H 2 O 2) takes place (13, 27). The catalase [E.C.1.11.1.6] and superoxide dismutase [E.C.1.15.1.1] enzymes play a key role in cellular defense against the reactive species (9). The intracellular localization of catalase enzyme has been under debate for many years. It is a topic wildly investigated and the initial localization in peroxisomes has been determined by a series of cytochemical and biochemical studies which also describe its position in other cellular compartments. This approach has been used by number of authors (8, 18, 20, 32). Michailova et al. (20) have isolated a pure heavy mitochondrial fraction from Candida boidinii and have provided evidences for catalase activity. At present it is well documented with Saccharomyces cerevisiae yeast cells that mitochondria possess catalase A enzyme (24). It has been shown that catalase A, although primary considered as a peroxisomal protein, could also be independently target to mitochondria (25). Given this data, it is important to perform broader investigations on catalase enzyme in methylotrophic yeasts. In the present work, we have studied the mitochondrial localization of catalase using three different strains methylotrophic yeasts: Pichia pastoris, Pichia pini and Hansenula polymorpopha and demonstrated that similarly to S. cerevisiae yeast, methylotrophic ones possess constitutive transport of catalase into both organelles. Materials and Methods Microorganisms and growth conditions The yeasts used in this investigation were, as follows: Pichia pastoris X-33 (Invitrogen), Hansenula polymorpha CBS 4732 and Pichia pini NBIMCC 8360. The strains were cultivated in liquid YP medium (1% Yeast Extract, 1% Bacto-Peptone) supplemented either with 2 % glucose (YPD), 1 % methanol (YPM) or 1 % glycerol (YPG) at 30 o C on a reciprocal shaker (204 rpm). Cell-free extract preparation Cells from 6, 12, 20, 30, 48 and 72 h of cultivation were harvested by centrifugation at 800 x g for 10 minutes and washed twice with distilled H 2 O. Cell wall disruption was carried out by spheroplasting according to the procedure of Defontaine et al. (3). The cell debris was removed by centrifugation at 1000 g and 4 • C for 10 min and the cell free extracts were triply frozen and thawed to break open organelles, and centrifuged at 15 000 g and 4 • C for 10 min. The supernatants obtained were used for enzymatic analyses. Subcellular fractionation and Nycodenz gradients For cell fractionation, yeast cells were grown 20 h to reach end of logarithmic phase. Spheroplasts were generated by the procedure of Defontaine et al. (3) and unlysed cells, nuclei and cell debris were removed by centrifugation at 1000 g and 4 • C for 10 min. The supernatant containing the crude yeast cell organelles was again centrifuged at 25 000 g and 4 • C for 20 min, and the crude organelle fraction was resuspended in a total