**2. Lipase biosynthesis**

Lipase production requires carbon and nitrogen sources as required by any fermentation process. Most of the lipases production studies do not use simple sugars as carbon sources. They rather use lipid substrates as sole carbon sources (Zhang et al., 2009a; Zhang et al., 2009b; Hun et al., 2003). Lipase production is rarely constitutive and the quantity of the extracellular lipase produced is meagre (Lee et al., 2001). Hence inductors like vegetables oils (Kumar et al., 2005), Tween 20, Tween 80 (Li et al., 2004), hexadecane (Boekema et al., 2007), and synthetic like tributyrin and tripalmitin, are used. Generally, production of lipases increases when the relative percentage of C18:n fatty acid esters in the respective vegetable oil is increased; this indicates the importance of such substances in the synthesis and secretion of the enzyme (Lakshmi et al., 1999). Among vegetable oils, olive oil has also been referred as one of the best inductors of lipase production (Table 1; Sokolovska et al., 1998). To elucidate some aspects of the induction effect of lipid related substrates on lipase production, mixed carbon sources consisting of a soluble compound selected for its growthpromoting capacity and a fatty acid selected as inductor of the enzyme production were used (Dalmau et al., 2000). This strategy allows for the microorganism to use both substrates in a sequential or simultaneous way, depending on its metabolism. Biomass production can be higher in most cases, no increase in lipolytic activity can be observed. This suggested a possible competing effect of some soluble carbon sources or a close relation between extracellular lipase activity production and consumption of fatty acids.

*sn-***1**

C

<sup>O</sup> OCR <sup>H</sup>

O

Ilyas-Qureshi, 1965; Gunstone et al., 1965)

**2. Lipase biosynthesis** 

<sup>2</sup> CH OCR

*sn-***3**

Fig. 1. The particular fatty acids in the plant triacylglycerols are not distributed randomly between the different *sn*-carbon atoms. It is a general rule that saturated species of fatty acids are confined to the positions *sn*-1 and *sn*-3 with some enrichment in the first position, and that the polyunsaturated C18 fatty acids are located mainly at position *sn*-2 (Gunstone &

Lipase production requires carbon and nitrogen sources as required by any fermentation process. Most of the lipases production studies do not use simple sugars as carbon sources. They rather use lipid substrates as sole carbon sources (Zhang et al., 2009a; Zhang et al., 2009b; Hun et al., 2003). Lipase production is rarely constitutive and the quantity of the extracellular lipase produced is meagre (Lee et al., 2001). Hence inductors like vegetables oils (Kumar et al., 2005), Tween 20, Tween 80 (Li et al., 2004), hexadecane (Boekema et al., 2007), and synthetic like tributyrin and tripalmitin, are used. Generally, production of lipases increases when the relative percentage of C18:n fatty acid esters in the respective vegetable oil is increased; this indicates the importance of such substances in the synthesis and secretion of the enzyme (Lakshmi et al., 1999). Among vegetable oils, olive oil has also been referred as one of the best inductors of lipase production (Table 1; Sokolovska et al., 1998). To elucidate some aspects of the induction effect of lipid related substrates on lipase production, mixed carbon sources consisting of a soluble compound selected for its growthpromoting capacity and a fatty acid selected as inductor of the enzyme production were used (Dalmau et al., 2000). This strategy allows for the microorganism to use both substrates in a sequential or simultaneous way, depending on its metabolism. Biomass production can be higher in most cases, no increase in lipolytic activity can be observed. This suggested a possible competing effect of some soluble carbon sources or a close relation between

extracellular lipase activity production and consumption of fatty acids.

H2 COCR

O

*sn-***2**


Table 1. Sources of lipases induced by olive oil

The induction process can be accomplished by adding edible oils such as butter fat, olive, canola and fish oils to the fermentation medium. It is well known that certain lipids in the culture medium can influence the production and activity of lipases from microorganisms. Generally, the activity of intra and extracellular lipases increases with increasing lipid concentrations, although excessive levels in the growth medium may be cytotoxic. The mechanisms regulating lipase biosynthesis vary widely in different microorganisms.

#### **2.1** *Aspergillus niger*

Lipases from *Aspergillus niger* were induced by solid-state fermentation using, as substrate, agroindustrial residue supplemented with by-products from corn oil refining process or olive oil. Based on the values of lipase activity obtained after 48 hour fermentation byproducts from corn oil refining were tested as inductors in the preparation of fermentation

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

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

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.

The influence on lipase induction in *Mucor hiemalis* of different types of triacylglycerols containing mainly oleic acid (olive oil), erucic acid (mustard oil), or saturated fatty acids of 8 to 16 carbons (coconut oil) was studied (Akhtar et al., 1980). The fungus produced a significant amount of lipase in the presence of glucose, but the lipase activity increased markedly when olive oil was added to the medium at the beginning of the fermentation. Among the various sources of triacylglycerols used as the carbon source, olive oil was found to be most effective in inducing the lipase. The lipase of *M. hiemalis* can be considered to be

While supplementation with olive oil gave the best lipase results, the highest values of glucoamylase and protease activities (de Azeredo et al., 1999) were achieved with starch enrichment. This indicates that the type of carbon source used as supplementation plays a determinant role in the kind of major enzymes that will be produced by *P*. *restrictum*. Enriching the babassu cake with different carbon sources favours the synthesis of different enzymes: olive oil supplementation results in high lipase activities, while starch supplementation results in high glucoamylase activities. Therefore, according to the application desired, the basal medium may be differentially enriched to give high yields of

operational conditions as discussed next.

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

**2.4** *Mucor hiemalis*

both constitutive and inducible.

**2.5** *Penicillium restrictum* 

the desired enzyme.

simple sugars and induced by using natural oils.

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., 2006), respectively.
