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Molecular Methods for the Diagnosis of Fungal Infections
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Adam M. Bressler, Christine J. Morrison
Enzymatic disruption generally uses zymolyase11,14,15 or lyticase12,16–19 to degrade chitin and glucan in fungal cell walls. The spheroplasts produced can then be osmotically, chemically, or mechanically disrupted. Because the cell walls of filamentous fungi are more resistant to enzymatic breakage compared to their yeast counterparts, most researchers use detergents or an alkalinization step to increase the DNA yield.15,19 Various combinations of detergents, proteinase K, RNase (for DNA targets), DNase (for RNA targets), and boiling steps usually follow enzymatic digestion of the cell walls with zymolyase or lyticase.11,14,15,17
Molecular Diagnostics: Present and Future
Published in Johan A. Maertens, Kieren A. Marr, Diagnosis of Fungal Infections, 2007
Holger Hebart, Juergen Loeffler, Hermann Einsele
The release of fungal DNA can be achieved by enzymatic or mechanical approaches. Many investigators have used zymolyase and lyticase, ß-l,3-glucanases to generate fungal spheroplasts (19). However, an efficient release of DNA from many molds, such as Aspergillus niger, Aspergillus terreus, Mucorales, and Fusarium spp. requires additional preparation steps such as boiling of the samples with NaOH (20), high-speed cell disruption, grinding with mortar and pestle, or repeated freeze-thawing using liquid nitrogen (21). Zymolyase, an enzyme extracted from brewery sewage, may contain traces of yeast DNA. Therefore, recombinant enzymes, such as recombinant lyticase should be preferably used for diagnostic assays.
Yeast-inspired drug delivery: biotechnology meets bioengineering and synthetic biology
Published in Expert Opinion on Drug Delivery, 2019
Chinnu Sabu, Panakkal Mufeedha, Kannissery Pramod
The nonmechanical chemical method includes the use of detergents and solvents. The detergent causes direct damage to the cell wall and further releases the intracellular components. Sometimes, the method leads to denaturation of the protein. Their usage in large scale is limited due to additional purification steps. Solvents can cause cell disruption by the extraction of lipid contents. By appropriate choice of solvent, the suitable product can be obtained. The enzymatic method causes decomposition of the cell wall with the aid of digestive enzymes. Zymolase is one of the commonly used for disruption of yeast cell wall. Other enzymes used are cellulases, pectinases, xylanases, and chitinases. Moreover, the complication of the process and the high cost of enzyme limits the use [41].
The force-from-lipid principle and its origin, a ‘what is true for E. coli is true for the elephant’ refrain
Published in Journal of Neurogenetics, 2022
Zymolyase strips the cell wall and converts yeast cells into spheroplasts, exposing their plasma membrane for patch-clamp examination. Yoshiro and Boris started generating yeast spheroplasts in 1985 and were soon joined by Mike Gustin to first describe a 20-pS K+ conductance, showing that even a walled microbe has channel activities similar to those found in nerves (Gustin, Martinac, Saimi, Culbertson, & Kung, 1986). Soon, joined by Xin-Liang Zhou, they also discovered a 36-pS cation-nonspecific mechanosensitive (MS) channel activity in yeast spheroplast membrane and suggested its possible role in osmoregulation (Gustin, Zhou, Martinac, & Kung, 1988).
Ethanol production from cassava starch by protoplast fusants of Wickerhamomyces anomalus and Galactomyces candidum
Published in Egyptian Journal of Basic and Applied Sciences, 2020
Tolulope Modupe Adeleye, Sharafadeen Olateju Kareem, Mobolaji O. Bankole, Olusegun Atanda, Abideen I. Adeogun
The two parent yeast isolates were grown aerobically in a 250 ml flask containing 50 ml of YPD medium for 30 h. Cells were harvested by centrifuging 5 ml cultures at 500 rpm for 5 min. The cells were subsequently washed three times with sterile distilled water and were suspended in zymolase protoplasting solution (2-mercapto-ethanol), 50 mM phosphate buffer (pH 7.5) and 0.5 mg zymolase 60,000 per ml (Sigma Co. UK). The suspension was incubated at 30ºC for 1 h with occasional shaking. Protoplasts were harvested by centrifugation at 1000 rpm for 10 min. They were subsequently washed thrice with the protoplast buffer containing 0.08 M sorbitol (KPS).