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.

5-16