**2.2** *Candida rugosa*

The synthesis and secretion of lipases in *C. rugosa* have been studied with carbon sources that are known to affect the production of lipase in two opposite ways: glucose (repressor) and oleic acid (inductor; Ferrer et al., 2001). In these studies, lipase production was monitored both by enzyme activity and by immunodetection with specific antibodies. These studies showed that, according to their regulation, lipase-encoding genes might be grouped in two classes, one of which is constitutively expressed and the other is induced by fatty acids. The synthesis of inducible enzymes is inhibited at the level of transcription by the addition of glucose, and, conversely, oleic acid appears to hinder the synthesis of the constitutive lipase (Lotti et al., 1998).

The studies clearly show that different inductors may change the expression profile of individual lipase genes. A differential transcriptional control of *lip* genes had been previously suggested from several studies on the relationship between culture conditions of *C. rugosa* and the lipase/esterase profiles secreted by this organism (Gordillo et al., 1995; Lotti et al., 1998; Linko & Wu, 1996). *Lip* isoenzymes have differences in their catalytic properties (Rua et al., 1993; Diczfalusy et al., 1997; Tang et al., 2000).

Del Río et al. (Del Río et al., 1990) demonstrated the diauxic growth of *C. rugosa* on olive oil. Two stages could be observed in the consumption of the olive oil: the first one was related to the glycerol depletion without lipase production, and the second one was associated with the fatty acids consumption when the enzyme appeared in the medium. According to this observation, the initial presence of a small quantity of lipase would be sufficient to hydrolyze the triacylglycerol to glycerol and fatty acids. Therefore, production of high levels of lipase would be associated with the consumption of fatty acids. Similar results have been obtained by Sokolovska et al. (Sokolovska et al., 1998), who used olive oil and oleic acid for lipase production. It has been observed that the uptake of oleic acid by *C. rugosa* is favored by the presence of extracellular lipases (Montesinos et al., 1996). Based on the observations and hypothesis just described, Serra et al. (Serra et al., 1992) calibrated and validated a model for lipase production on olive oil and free fatty acids in batch fermentation. Sokolovska et al. (Sokolovska et al., 1998) did not observe significant differences in lipase production using these substrates. Montesinos et al. (Montesinos et al., 1996) developed a simple structured mathematical model for lipase production by *C. rugosa* in batch fermentation. Lipase production is induced by extracellular oleic acid present in the medium. The acid is transported into the cell, where it is consumed, transformed, and stored. Lipase is then excreted to the medium, where it is distributed between the available oil-water interface and the aqueous phase. Cell growth is modulated by the intracellular substráte concentration. Model parameters were determined in a calibration step, and then the whole model was experimentally validated with good results. This model was later modified to be applied from batch to fed-batch and continuous lipase production (Montesinos et al., 1997). Finally, it was exploited in simulations and for the design of new operational conditions as discussed next.

Annibale et al. (Annibale et al., 2006) and Brozzoli et al. (Brozzoli et al., 2009) confirmed that lipase production by *Candida* sp. was found to be completely repressed by the presence of simple sugars and induced by using natural oils.

### **2.3** *Pseudomonas* **sp**

462 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

medium. The best results were achieved with soapstock and stearin, reaching values of 62.7 and 37.7 U/gds, respectively, which are higher than the value for olive oil (34.1 U/gds). The use of fatty acids residue inhibited lipase production. This kind of inhibition has already been reported by other authors (Corzo & Revah, 1999; Li et al., 2004). The inhibition effect was not observed for low fatty acid concentrations using palmitic and oleic acid during lipase production by *Candida rugosa* (Dalmau et al., 2000) and *Rhyzopus arrhizus* (Li et al.,

The synthesis and secretion of lipases in *C. rugosa* have been studied with carbon sources that are known to affect the production of lipase in two opposite ways: glucose (repressor) and oleic acid (inductor; Ferrer et al., 2001). In these studies, lipase production was monitored both by enzyme activity and by immunodetection with specific antibodies. These studies showed that, according to their regulation, lipase-encoding genes might be grouped in two classes, one of which is constitutively expressed and the other is induced by fatty acids. The synthesis of inducible enzymes is inhibited at the level of transcription by the addition of glucose, and, conversely, oleic acid appears to hinder the synthesis of the

The studies clearly show that different inductors may change the expression profile of individual lipase genes. A differential transcriptional control of *lip* genes had been previously suggested from several studies on the relationship between culture conditions of *C. rugosa* and the lipase/esterase profiles secreted by this organism (Gordillo et al., 1995; Lotti et al., 1998; Linko & Wu, 1996). *Lip* isoenzymes have differences in their catalytic

Del Río et al. (Del Río et al., 1990) demonstrated the diauxic growth of *C. rugosa* on olive oil. Two stages could be observed in the consumption of the olive oil: the first one was related to the glycerol depletion without lipase production, and the second one was associated with the fatty acids consumption when the enzyme appeared in the medium. According to this observation, the initial presence of a small quantity of lipase would be sufficient to hydrolyze the triacylglycerol to glycerol and fatty acids. Therefore, production of high levels of lipase would be associated with the consumption of fatty acids. Similar results have been obtained by Sokolovska et al. (Sokolovska et al., 1998), who used olive oil and oleic acid for lipase production. It has been observed that the uptake of oleic acid by *C. rugosa* is favored by the presence of extracellular lipases (Montesinos et al., 1996). Based on the observations and hypothesis just described, Serra et al. (Serra et al., 1992) calibrated and validated a model for lipase production on olive oil and free fatty acids in batch fermentation. Sokolovska et al. (Sokolovska et al., 1998) did not observe significant differences in lipase production using these substrates. Montesinos et al. (Montesinos et al., 1996) developed a simple structured mathematical model for lipase production by *C. rugosa* in batch fermentation. Lipase production is induced by extracellular oleic acid present in the medium. The acid is transported into the cell, where it is consumed, transformed, and stored. Lipase is then excreted to the medium, where it is distributed between the available oil-water interface and the aqueous phase. Cell growth is modulated by the intracellular substráte concentration. Model parameters were determined in a calibration step, and then

properties (Rua et al., 1993; Diczfalusy et al., 1997; Tang et al., 2000).

2006), respectively.

**2.2** *Candida rugosa*

constitutive lipase (Lotti et al., 1998).

An extracellular lipase was isolated and purified from the culture broth of *Pseudomonas aeruginosa* SRT 9 (Borkar et al., 2009). Production medium was prepared containing olive oil (1% w/v) as inductor. Marked stability and activity of induced lipase in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. A strain of *Pseudomonas mendocina* producing extracellular lipase was isolated from soil (Dahiya et al., 2010). The bacterium accumulates lipase in culture fluid when grown aerobically at 30 °C for 24 h in a medium composed of olive oil (1%) as substrate. This lipase was capable of hydrolyzing a variety of lipidic substrates and is mainly active towards synthetic triglycerides and fatty acid esters that possess a butyryl group. The medium for lipase production from *Pseudomonas fluorescens* P21 had glucose as carbon source (Cadirci & Yasa, 2010). When glucose was replaced by various lipids, olive oil was the effective lipid for lipase production (3.5 U/l). When glucose in the medium was replaced with olive oil, the lipase yield was increased by 48.9% between 12 and 18 h.
