Microbial lipases can be identified and separated on the basis of their physical properties which vary considerably from one enzyme to another, such as thermal stability, temperature and pH optima. The location of the enzyme is also important, for example whether it is intracellular or secreted as an extracellular lipase or whether it cell- associated or cell-bound (Ota et aL, 1968; Gomi et aL, 1984; Jacobsen et aL, 1989 and Janssen et aL, 1994). The properties of the lipases tend to vary with the type of organism. Bacteria usually have pH optima in the alkaline region whilst fungal and yeast lipases tend to exhibit pH optima in the acid region and have a higher thermal stability than many of the bacterial lipases isolated (Suguira et aL, 1974).
Thermostability of lipases is a major concern in industry especially with respect to thermostable lipases produced by psychrotropic bacteria during the storage of heat processed commercially available sterile foods. The most commonly isolated bacteria is
Chapter 6 Microbial lipases
Pseudomonas fluorescens, found in milk and milk derived products, which produces an extracellular, heat stable lipase. The lipase is active at alkaline pH. The lipase has a detrimental effect on the flavour of the products concerned (Adams et a l, 1981 and lizumi et al, 1990). Generally, lipases may be stable up to 65°C although this may not necessarily be the optimum for activity (Sugihara et al, 1991).
As previously mentioned both pH and temperature are crucial for optimum lipase activity. The optimal ranges for each can vary considerably between organisms as well as species (Okumura et a l, 1976; Isobe et al, 1988 and Phillips et a l, 1991). For example, the two lipases produced by Geotrichum candidum have different pH optima dependent on the isoform although the optimum temperature was the same for both (Heidrich et a l, 1991).
The majority of lipases isolated have been extracellular and are excreted through the external membrane of the cell into the culture medium. The culture environment can be optimised for lipase production by variation of the growth conditions which will affect the properties of the enzyme producer. This will also influence the ratio of extracellular to intracellular lipases produced. The actual amounts of the lipase produced is dependent on a variety of external factors such as temperature, pH, nitrogen composition, carbon and lipid sources, concentration of inorganic salts and the availability o f oxygen. Therefore, the composition of the fermentation medium is o f key importance with respect to lipase production (Rivera-Munoz et al, 1991).
It has been demonstrated that lipase production is stimulated by lipids such as butter oil, lard oil, olive oil (Omar et al, 1987; Tan & Gill, 1987 and Suzuki et a l, 1988) as well as certain fatty acids (Iwai et a l, 1973; Suzuki et a l, 1988 and Montesiros et al., 1996). It has also been reported that a minimal level of lipid is required for lipase production which will be of great importance in this study where rape seed oil will be used as the main carbon source (Iwai et a l, 1973). It should be noted that other carbon sources such as glycerol, will support growth and lipase activity although again, this is species dependent (Petrovic et a l, 1990).
Chapter 6 Microbial lipases
The carbon source is also important with respect to not only lipase activity but growth of the organism and secondary metabolite production (chapter 4). Gilbert et al, (1991) showed that the lipase activity of Ps. aeruginosa was weakly induced by carbon and/or energy source limitations but was strongly induced by a wide range of fatty acyl esters including triglycerides and Tweens. However, lipase activity was repressed by long chain fatty acids. Further studies have reported that Tweens and other such anionic surfactants enhance lipase activity in a number of different organisms as well as stimulating the production of other enzymes (Reese et a l, 1969; Nahas, 1988 and Jacobsen, 1989).
The nitrogen source used in the culture medium as well as the presence of amino acids are important in achieving maximum lipase activity. It has been shown that lipase activity increases as the complexity of the nitrogen source used in the medium decreases, and when supplemented with various compounds such as amino acids arginine, lysine, aspartic acid and glutamic acid, lipase activity increases further (Alford
et a l, 1963). Both peptone and ammonium sulphate have been used as nitrogen sources and have been shown to affect lipase activity (Narasaki et a l, 1968; Petrovic et a l, 1990 and Prabhakar & Raju, 1993). The use of either inorganic or organic nitrogen depends on the culture conditions; the source of the nitrogen as well as the organism being grown.
Besides the major medium components there are several co-factors which have been reported to enhance lipase activity and include albumin, lecithin and sodium chloride (Aisaka et al, 1979). It should be noted that the lecithin does not actually increase the net synthesis of the lipase but accelerates the secretion o f the enzyme formed into the culture medium. The addition of some salts such as potassium and sodium have been shown to increase lipase activity although at high concentrations have an inhibitory effect (Petrovic et a l, 1990). A similar effect has been observed with the addition of metal ions such as iron and magnesium.
The final consideration with respect to achieving the maximum lipase production possible, is the manner in which the organism is grown as well as the medium components are important (Rivera-Munoz et a l, 1991). In these studies several filamentous fungi for example. Pénicillium candidum and Pénicillium camembertii were
Chapter 6 Microbial lipases
grown on both solid state and submerged culture. It was found that the lipase activity was higher in submerged culture than in solid media but the actual production of lipase occurred earlier in the solid state system and tended to be more stable. Similar observations were made by Maheura (1984).
One important factor to consider in the production o f lipase by submerged culture is the effect of shear. Lee et al, (1989) investigated the effect of shear inactivation of lipase produced by Candida cylindracae. It was found that the lipase was sensitive to dénaturation by increasing shear-rate although the lipase did not appear sensitive to shear-stress. This indicated that the lipolytic activity was influenced by the length of time the enzyme was exposed to the shear forces.
However, Charm & Wong (1973) who conducted shearing experiments with a variety of other enzymes, such as catalase, reported a loss of enzyme activity as a function of shearing time and shearing rate. These findings are supported by Stahmann et al., (1997) who found that by lowering stirrer speeds and removing baffles during Ashbya gossypii
fermentation, lipase activity increased due to reduced shearing effects. From this and other studies it becomes clear that lipases can be induced but also inhibited by a number of parameters and hence careful manipulation of the environmental conditions is required to achieve optimum lipase activity. This in turn could be related to the growth and morphology of the organism.