In this study the effectiveness of linear dsDNA plasmids isolation from D. hansenii yeast cells after application of enzymatic protoplastization methods, mechanical disintegration techniques and chemical cell lysis was evaluated. Lytikase from Arthrobacter luteus and a mixture of lysing enzymes from Trichoderma harzianum used in the protoplastization process enabled the efficient isolation of the linear plasmids. Lytikase appeared to be the fastest cell wall digestive enzyme, while the advantage of the T. harzianum enzymes was the purity of the resulting DNA preparation. The use of β-glucuronidase did not cause efficient lysis of the cell wall, thus it has been considered as unsuitable for this process. All mechanical methods (grinding with glass beads or micropestles, freezing in liquid nitrogen and disintegration with Micro-Dismembrator) proved to be fully effective for plasmids isolation in contrast to chemical techniques. The alkaline lysis with SDS, conventionally used for the isolation of circular plasmids caused the loss of plasmids in preparation, while the CelLytic Y Cell Lysis Reagent led to obtain only strongly contaminated samples.

The object of this study was to evaluate the ability of 113 yeast strains isolated from Polish blue-veined cheese for the production of extracellular and intracellular proteolytic and lipolytic enzymes. Examined yeast strains belonged to 7 species: Candida famata, C. sphaerica, C. intermedia, C. kefyr, Geotrichum penicillatum, Yarrowia lipolytica and Saccharomyces kluyveri. The cultivation was performed in the shake-flasks experiment in medium YCG containing yeast extract – glucose – casein. Extracellular proteolytic activity was determined at pH 3.0 and pH 7.5 towards hemoglobin and casein, respectively. The activities of carboxypeptidase, diand tripeptidase and aminopeptidase were tested as well. In addition, extracellular lipolytic activity was investigated by well-diffusion test. It was demonstrated that differences in the extracellular and intracellular activity of lipolytic and proteolytic enzymes depended not only on the species, but also on yeast strain within a species. All the yeast species under evaluation produced extracellular lipases and their activity ranged from 7 U·mg^-1 (for C. famata) to 90 U·mg^-1 (for Y. lipolytica). Intracellular lipolytic activity was observed for all yeast strains (with the exception of two belonging to C. intermedia and C. famata species) however, the maximum level of 78 U·mg^-1 was obtained for Y. lipolytica and C. sphaerica strains. Y. lipolytica strains showed comparatively high activities of extraand intracellular proteases active towards casein (47 and 93 U·mg^-1, respectively) and the highest activities of extracellular proteases hydrolyzing hemoglobin (417–457 U·mg^-1). Moreover, Y. lipolytica yeast strains had a high carboxy-, di and tripeptidase activities.

Presentation reviewed occurrence of allergenic sensitizing prolifins and storage proteins in fruit and vegetables. Cross reactivity between allergens and homologus proteins in polens of commonly occurring plants was noted. Finally, direction of research leading to identification of hypoallergenic cultivars of fruit and vegetables was discussed.