Regulation and properties of a fungal lipase showing interfacial inactivation by gas bubbles, or droplets of lipid or fatty acid.
Ashbya gossypii can grow on triacyglycerol as carbon source. A degradation rate of 0.05 g x g-1 mycelial dry mass x h-1 was detected for soybean oil. Although this rate was within the sensitivity range of lipase assays no activity was detectable. On the other hand, extracellular lipase activity could be visualized by clearance halos round the growing mycelium when trioleoylglycerol was emulsified as the sole carbon source in agar plates. Variation of the culture conditions revealed that reduced shaking speed and decreased fat content in the medium led to detectable amounts of lipase in the supernatant of flask cultures. A maximal activity of 800 U x l-1 was obtained after 32 h of cultivation in flasks containing 1% yeast extract and incubated at 60 rpm. Because of its pI of 9.0, the enzyme could be purified in a single step by preparative isoelectric focusing. It appeared as a homogeneous protein in analytical isoelectric focusing and SDS/PAGE (M 35,000). The lipase was inactivated within minutes in stirred gas/water, trioleoylglycerol/water or oleic acid/water mixtures. These effects suggested an interface inactivation. This idea was supported by a stability modulation observed with the surfactant Pluronic F-68. Inactivation by oleic acid led to an aggregation of the lipase shown by gel filtration. Growth experiments performed under lipase-stabilizing conditions revealed a negative influence of glucose, glycerol or oleic acid on detectable lipase activity, probably due to a regulation of lipase formation. Inactivation and regulation thus explained the lack of detectable lipase activity in cultures of A. gossypii growing on triacylglycerol.
Stahmann
K P
KP
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Germany. p.stahmann@kfa-juelich.de
Böddecker
T
T
Sahm
H
H
D003470:Culture Media; D004789:Enzyme Activation / Q000187:drug effects; D005227:Fatty Acids / Q000494:pharmacology*; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005740:Gases / Q000494:pharmacology*; D008049:Lipase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008055:Lipids / Q000494:pharmacology*; D008970:Molecular Weight; D004718:Saccharomycetales / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D014280:Triglycerides / Q000494:pharmacology
D003470:Culture Media; D005227:Fatty Acids; D005656:Fungal Proteins; D005740:Gases; D008055:Lipids; D014280:Triglycerides; D008049:Lipase