Secretion of Extracellular Lipases by Cundidu ZipoZyticu

Lipolytic activity was associated with Candida lipolytica in several early studies (Long, 1936; Long & Hammer, 1938, 1939), and the influence of culture conditions on lipase production has been described by Peters & Nelson (1948). Studies on the intracellular lipases (Lloyd et al., 1971) indicated that C. lipoiytica grown in liquid media with glucose as a carbon source contained five distinct lipases that appeared at different stages of the growth cycle. The extracellular lipases have not been examined in detail. The present communication reports on the secretion of an inducible lipase when the organism is grown on short-chain triacylglycerols. The organism was normally grown in continuous culture, by using the M-9 medium described by Roberts et al. (1957) with either a triacylglycerol (0.1 %), glucose (1 %), or glucose (1 %) with corn-steep liquor (2%) as carbon source. Lipases were assayed by the fluorimetric method of Kramer & Guilbault (1963) and were detected in gels by the method of Nachlas & Seligman (1949). In batch culture, with triacetylglycerol as a carbon source, extracellular lipase production reached a maximum rate in the early exponential phase and declined to about 10% of this value by the mid-exponential phase. In continuous culture the concentration of extracellular lipase fell with increasing dilution rates, but the intracellular lipases were not affected. This is in agreement with previous observations that lipase production is associated with slow growth and the mycelial, Gram-negative form of the organism rather than the unicellular Gram-positive form. Polyacrylamide-gel disc electrophoresis, on a 7.5 % gels, revealed a complex pattern of extracellular and intracellular lipases, with some variability between samples. Only with triacetylglycerol as carbon source was a simple pattern observed with a single extracellular lipase and three or four intracellular lipases, of which the most active had the same mobility as the extracellular enzyme. With glucose as carbon source no lipases could be detected in the cells or the medium. When corn-steep liquor was added, three lipases were detected in the medium, the major enzyme having the same mobility as the enzyme for triacetylglycerol, whereas five bands with lipase activity were usually found in electrophoresis of the cellular proteins. With trioleoylglycerol up to four faint bands of lipase activity were found by electrophoresis of the medium, but four very active enzymes, with different mobilities to the extracellular lipase, were noted among the cellular proteins. It seems likely that lipases are induced by appropriate triacylglycerols, those with short-chain fatty acids inducing extracellular lipases, those with long-chain fatty acids inducing intracellular lipases. The variability found in gel electrophoresis may be due to the glycoprotein nature of the exoenzymes (Eylar, 1965). Electrophoresis of the medium from cells grown on triacetylglycerol gave gels H ith only one protein band when stained with Coomassie Blue, which corresponded to the region of lipase activity. The specificities of the lipases were determined by using a series of fluorescein esters, from the diacetate to the dilaurate. The extracellular enzyme from cells grown on triacetylglycerol was only active against short-chain esters up to tributyrylglycerol, and should perhaps be called an esterase. Other lipases tested showed more activity against longer-chain esters. The transport of the triacetylglycerol extracellular enzyme (esterase) from the cell was followed by labelling the enzyme with radioactive glycine. Cells were grown on the triacetylglycerol medium containing 200Ci of [2-3H]glycine (0.75pmol) in lOml of medium. They were collected by centrifugation and resuspended in 2ml of either * Present address: Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow G61 IBD, U.K.