Marie Zarevúcka

*Institute of Organic Chemistry and Biochemistry, Czech Republic* 

### **1. Introduction**

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

Zhao, H.; Bie, X;, Lu, F. & Liu, Z.(2005). Lipase Catalyzed Acidolysis of Lard with Capric

Increasing interest in lipases has been observed at the end of the last century, due to their potential application, in (bio)degradation as well as in (bio)synthesis of glycerides. The advantages of the enzymatic hydrolysis over the chemical process consist of less energy requirements and higher quality of the obtained products. Beside this, lipases are also efficient in various reactions such as esterification, transesterification and aminolysis in organic solvents. Examples in the literature are numerous. Lipases are used in different fields such as resolution of racemic mixtures, synthesis of new surfactants and pharmaceuticals, bioconversion of oils and fats and detergency applications.

Lipase activity has been found in different moulds, yeasts and bacteria. Numerous papers have been published on selection of lipase producers and on fermentation process. This kind of information is important in order to identify optimal operation conditions for enzyme production. Previous studies on the physiology of lipase production showed that the mechanisms regulating biosynthesis vary widely in different microorganisms. Obtained results showed that lipase production seems to be constitutive and independent of the addition of lipid substrates to the culture medium. However, their presence can enhance the level of produced lipase activity. On the other hand, it is well known that, in other microorganisms, lipid substrates are necessary for lipase production. These enzymes are generally produced in the presence of a lipid such as oil or triacylglycerols or any other inductor, such as fatty acids. Lipidic carbon sources seem to be essential for obtaining a high lipase yield. The review is focused on the olive oil as lipase inductor.

#### **1.1 Lipases**

Lipases, (triacylglycerol acylhydrolases; EC 3.1.1.3.) are one of the most important classes of hydrolytic enzymes that catalyse both hydrolysis and synthesis of esters. Hydrolysis of a triacylglycerols by lipases can yield di- and monoacylglycerols, glycerol and free fatty acids. Lipases are valuable biocatalysts with diverse applications. Although lipases share only 5% of the industrial enzyme market, they have gained focus as biotechnologically valuable enzymes. They play vital roles in food, detergent and pharmaceutical industries.

Commercial microbial lipases are produced from bacteria, fungi and actinomycetes (Babu & Rao, 2007). Their industrial importance arises from the fact that they act on a variety of substrates promoting a broad range of biocatalytic reactions. Lipases from different sources

although only a small part in proportion to triacylglycerols, includes a very large number of minor compounds, including the phenolics and the sterols. These compounds give olive oil

<sup>+</sup> R3NH2 R1 NHR3

Scheme 1. Processes catalyzed by lipases: (a) enzyme-catalyzed hydrolysis, (b) enzymecatalyzed esterification, (c) enzyme-catalyzed transesterification by acidolysis, (d) enzymecatalyzed transesterification by alcoholysis, (e) enzyme-catalyzed interesterification and (f)

The structure of triacylglycerol molecule is depicted in Fig. 1. The seed triacylglycerols are usually characterized by predominance of C18-unsaturated and polyunsaturated fatty acids, and this distinguishes them from animals fats, which are generally of a more saturated nature. The C18-unsaturated fatty acids (oleic, linoleic, and linolenic) are particularly important and govern, to a large degree, the physical properties of the oil and hence its use

R1 OH

R1 OR2

R1 OR3

O

R3 OR2

O

O

R3 OR2

O

O

+ R2OH

**(a)**

**(b)**

**(c)**

**(d)**

**(e)**

**(f)**

+ H2O

+

+

+ R2OH

R1 OH

R1 OR4

O

O

+ R2OH

O

its unique flavour and contribute greatly to the nutritional benefits.

+ H2O

+ R2OH

+ R3OH

R3 OH

R3 OR4

O

O

+

+

R1 OR2

R1 OH

R1 OR2

R1 OR2

enzyme-catalyzed aminolysis.

and commercial value.

O

R1 OR2

O

O

R1 OR2

O

O

O

show different substrate specificities and they are widely used in industrial applications for biosynthesis (Jaeger & Eggert, 2002).

Most of the lipases, which are used in laboratory investigations and/or in industrial production, are substrate tolerant enzymes, which accept a large variety of natural and synthetic substrates for biotransformation. Microbial lipases are mostly inducible extracellular enzymes, synthesized within the cell and exported to its external surface or environment. Lipases are ubiquitous enzymes which are widely distributed in plants, microbes and higher animals. Microbial sources are superiour to plants and animals for enzyme production and this can be attributed to easy cultivation and genetic manipulation (Hasan et al., 2006). Each microorganism requires a different carbon source to produce lipase at its maximum level.

Microbial lipases are mostly extracellular and their production is greatly influenced by medium composition besides physicochemical factors such as temperature, pH, and dissolved oxygen. The major factor for the expression of lipase activity has always been reported as the carbon source, since lipases are inducible enzymes. These enzymes are generally produced in the presence of a lipid such as oil or triacylglycerol or any other inductor, such as fatty acids, hydrolysable esters, Tweens, bile salts, and glycerol. Lipidic carbon sources seem to be essential for obtaining a high lipase yield. However, nitrogen sources and essential micronutrients should also be carefully considered for growth and production optimization. These nutritional requirements for microbial growth are fulfilled by several alternative media as those based on defined compounds like sugars, oils, and complex components such as peptone, yeast extract, malt extract media, and also agroindustrial residues containing all the components necessary for microorganism development. A mix of these two kinds of media can also be used for the purpose of lipase production. The main studies available in the literature since 2000 covering these subjects are presented below, divided by the kind of microorganisms used (Fernandes et al., 2007; Li et al., 2004; Tan et al., 2003).

### **1.2 Lipase catalytic properties**

Lipase hydrolysis of water-insoluble substrates results from adsorption of the enzyme to the substrate-water interface, which can induce a conformational change in the enzyme structure, causing reaction rates to be influenced by both this adsorptive interaction as well as interaction with substrates. When lipases are active in organic solvents in which substrates are soluble, reactions follow normal enzyme kinetic models (Martinelle & Hult, 1995).

Reactions in which lipases may be involved, both in nature and in laboratory or industrial application, are: (a) enzyme-catalyzed hydrolysis, (b) enzyme-catalyzed esterification, (c) enzyme-catalyzed transesterification by acidolysis, (d) enzyme-catalyzed transesterification by alcoholysis, (e) enzyme-catalyzed interesterification and (f) enzyme-catalyzed aminolysis (Scheme 1).

#### **1.3 Plant oils**

The major components of fats and vegetable oils (98%) are triacylglycerols, which consist of glycerol molecules esterified with three long-chain fatty acids. The remainder of the oil,

show different substrate specificities and they are widely used in industrial applications for

Most of the lipases, which are used in laboratory investigations and/or in industrial production, are substrate tolerant enzymes, which accept a large variety of natural and synthetic substrates for biotransformation. Microbial lipases are mostly inducible extracellular enzymes, synthesized within the cell and exported to its external surface or environment. Lipases are ubiquitous enzymes which are widely distributed in plants, microbes and higher animals. Microbial sources are superiour to plants and animals for enzyme production and this can be attributed to easy cultivation and genetic manipulation (Hasan et al., 2006). Each microorganism requires a different carbon source to produce

Microbial lipases are mostly extracellular and their production is greatly influenced by medium composition besides physicochemical factors such as temperature, pH, and dissolved oxygen. The major factor for the expression of lipase activity has always been reported as the carbon source, since lipases are inducible enzymes. These enzymes are generally produced in the presence of a lipid such as oil or triacylglycerol or any other inductor, such as fatty acids, hydrolysable esters, Tweens, bile salts, and glycerol. Lipidic carbon sources seem to be essential for obtaining a high lipase yield. However, nitrogen sources and essential micronutrients should also be carefully considered for growth and production optimization. These nutritional requirements for microbial growth are fulfilled by several alternative media as those based on defined compounds like sugars, oils, and complex components such as peptone, yeast extract, malt extract media, and also agroindustrial residues containing all the components necessary for microorganism development. A mix of these two kinds of media can also be used for the purpose of lipase production. The main studies available in the literature since 2000 covering these subjects are presented below, divided by the kind of microorganisms used (Fernandes et al., 2007; Li

Lipase hydrolysis of water-insoluble substrates results from adsorption of the enzyme to the substrate-water interface, which can induce a conformational change in the enzyme structure, causing reaction rates to be influenced by both this adsorptive interaction as well as interaction with substrates. When lipases are active in organic solvents in which substrates are soluble,

Reactions in which lipases may be involved, both in nature and in laboratory or industrial application, are: (a) enzyme-catalyzed hydrolysis, (b) enzyme-catalyzed esterification, (c) enzyme-catalyzed transesterification by acidolysis, (d) enzyme-catalyzed transesterification by alcoholysis, (e) enzyme-catalyzed interesterification and (f) enzyme-catalyzed aminolysis

The major components of fats and vegetable oils (98%) are triacylglycerols, which consist of glycerol molecules esterified with three long-chain fatty acids. The remainder of the oil,

reactions follow normal enzyme kinetic models (Martinelle & Hult, 1995).

biosynthesis (Jaeger & Eggert, 2002).

lipase at its maximum level.

et al., 2004; Tan et al., 2003).

(Scheme 1).

**1.3 Plant oils** 

**1.2 Lipase catalytic properties** 

although only a small part in proportion to triacylglycerols, includes a very large number of minor compounds, including the phenolics and the sterols. These compounds give olive oil its unique flavour and contribute greatly to the nutritional benefits.

Scheme 1. Processes catalyzed by lipases: (a) enzyme-catalyzed hydrolysis, (b) enzymecatalyzed esterification, (c) enzyme-catalyzed transesterification by acidolysis, (d) enzymecatalyzed transesterification by alcoholysis, (e) enzyme-catalyzed interesterification and (f) enzyme-catalyzed aminolysis.

The structure of triacylglycerol molecule is depicted in Fig. 1. The seed triacylglycerols are usually characterized by predominance of C18-unsaturated and polyunsaturated fatty acids, and this distinguishes them from animals fats, which are generally of a more saturated nature. The C18-unsaturated fatty acids (oleic, linoleic, and linolenic) are particularly important and govern, to a large degree, the physical properties of the oil and hence its use and commercial value.

*Aspergillus* sp. Cihangir & Sarikaya, 2004; Papanikolaou et

*Bacillus* sp. Sugihara et al., 1991; Eltaweel et al., 2005

*Candida* sp. Annibale et al., 2006; Brozzoli et al., 2009

*Geotrichum candidum* 4013 Stránsky et al., 2007; Brabcová et al., 2010

*Penicillium* sp. Lima et al., 2003; Papanikolaou et al., 2011

*Rhizopus oryzae* Hiol et al., 2000; Salleh et al., 1993; Nunes

*Yarrowia lipolytica* Dominguez et al., 2003; Pignčde et al., 2000;

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

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

mechanisms regulating lipase biosynthesis vary widely in different microorganisms.

et al., 2011

Najjar et al., 2011

al., 2011

**Source of lipase References** 

*Aspergillus niger* Pokorny et al., 1994 *Aspergillus niger MYA 135* Colin et al., 2011

*Bacillus subtilis* NS 8 Olusean et al., 2011 *Burkholderia cepacia* LTEB11 Baron et al., 2011

*Candida cylindracea* (ATCC 14830) Salihu et al., 2011 *Candida rugosa* (DSM 2031) Lakshmi et al., 1999

*Penicillium aurantiogriseum* Lima et al., 2003 *Penicillium cyclopium* Chahinian et al., 2000 *Pseudomonas aeruginosa* KKA-5 Sharon et al., 1998 *Rhizopus arrhizus* Elibol & Oyer, 2000 *Rhizopus delemar* Acikel et al., 2011

Table 1. Sources of lipases induced by olive oil

**2.1** *Aspergillus niger*

*Mycotorula* sp. Peters & Nelson, 1948

*Rhodotorula glutinis* Papaparaskevas et al., 1992 *Serratia rubidaea* Immanuel et al., 2008

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