Research Article 
Isolation of Vacuoles from Fragile Spheroplasts of Candida tropicalis with Lyticase
Arezoo Khorsand and Seyed Vafa Maljaei
Biotechnology, 2008, 7(1), 87-93.
Abstract
In this study, a yeast isolate was recovered from the oral cavity. The yeast cells were converted to spheroplasts using lyticase, instead of zymolyase, which is a cheaper and more available lytic enzyme. Spheroplasts were subjected to metabolic lysis, mechanical disruption and DEAE-dextran-induced-lysis. Vacuoles were then isolated by two methods of sedimentation and flotation. Integrity and purity of isolated vacuoles were checked by phase-contrast microscopy and determination of polyphosphate as a vacuolar marker. Spheroplasts were found more sensitive than have ever reported. So, they were handled very carefully. In contrast to reports on zymolyase, spheroplast yield did not enhance with increasing length of lytic incubation time of yeast cells in lyticase. Among the methods used to lyse the spherplasts, mechanical disruption and treatment with dextran were found more suitable. Despite the reports, dextran seems to affect the released vacuoles of our strain. In addition, both isolation methods yielded isolated vacuoles successfully and a slightly increase in flotation method was probably due to the more spheroplasts lysis when dextran was used or/and less vacuole disruption.
ASCI-ID: 11-360