Industrial-scale production and rapid purification of an archaeal beta-glycosidase expressed in Saccharomyces cerevisiae.
The application of enzymes isolated from extreme thermophiles in biotechnological processes is hampered by their unconventional fermentation conditions. The expression, in mesophilic hosts, of genes encoding for thermophilic proteins enables these difficulties to be overcome and permits the production of enzymes in high yield by using conventional fermentation plants and an efficient enzyme purification utilizing heat precipitation of host proteins. The beta-glycosidase gene from Sulfolobus solfataricus, a thermoacidophilic archaeon growing at 87 degrees C and pH 3.5, has been cloned and expressed in Saccharomyces cerevisiae (baker's yeast). The fermentation of a S. cerevisiae strain on a 100-litre scale and the two-step purification of the expressed beta-glycosidase by cell autolysis and extracts thermal precipitation is described. This procedure, after 72 h of autolysis, gave a yield 56-fold higher with respect to that obtained with the beta-glycosidase from S. solfataricus.
Morana
A
A
Institute of Biochemistry of Macromolecules, II University of Naples, Italy.
Moracci
M
M
Ottombrino
A
A
Ciaramella
M
M
Rossi
M
M
De Rosa
M
M
D001709:Biotechnology; D003001:Cloning, Molecular; D005285:Fermentation; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D001617:beta-Glucosidase