Regulation and Properties of A Fungal Lipase Showing Interfacial Inactivation by Gas Bubbles, or Droplets of Lipid or Fatty Acid

Abstract

Ashbya gossypii can grow on triacylglycerol as carbon source. A degradation rate of 0.05 gXg-' mycelial dry massxh-' 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 UX1-I 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 (Mr 35 000). The lipase was inactivated within minutes in stirred gadwater, trioleoylglycerol/water or oleic acidwater mixtures. These effects suggested an interface inactivation. This idea was supported by a stability modulation observed with the surfactant PluronicR 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.Keywords: lipase; enzyme stability ; interface ; fungi ; Ashbya gossypii.Lipases are widely distributed among animals, plants and microorganisms [ l , 21. All the enzymes studied so far have in common that they are water-soluble but only maximally active when adsorbed to the interface between water and their waterinsoluble substrate. This property, also relevant for other interfacial enzymes [3], is known as interfacial activation [4]. It can be interpreted in two ways: firstly as an activation of the substrate and secondly as a change in the enzyme's conformation [5]. The major aspect of this paper is a strong effect of the interface on a lipase resulting in complete loss of its enzymatic activity. The enzyme in question is an extracellular lipase formed by the filamentous fungus Ashbya gossypii.The hemiascomycete A. gossypii (Ashby & Nowell) Guilliermond (1 928) is an important overproducer of vitamin B,. Since 1968 it has been used in technical processes on an industrial scale [6]. As a carbon source, carbohydrates are suitable but plant oils are even better 171. An unsolved problem in the metabolism of the fungus was its first step, namely the degradation of the triacylglycerol. Up to now, it has not been possible to detect an extracellular lipase in cultures degrading fat. Its existence could only be concluded from growth experiments with pure trioleoylglycerol and the subseq...

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