**6.1 Extraction, clarification and filtration**

The addition of pectinase to the must reduces its viscosity and causes the grouping of suspended particles in larger aggregates that can be removed by sedimentation. If the enzymes are added to the pulp before pressing, must yield increases, facilitating the pressing and enhancing the colour. Macerating enzyme preparations for this purpose contain pectinases as well as cellulases (Ribereau-Gayon et al., 2006). A high level of polygalacturonase is very effective for clarification but may require the prior action of pectin lyase activity. For this reason the enzyme preparations with a high content of pectin lyase are desirable when very fast racking is required to prevent any problems related to must oxidation, development of endogenous microbiota and nutrient loss. In addition, wines made with pectic-enzyme-treated grape must significantly reduces filtration times (Blanco et al., 1997; Blunt, 2000).

#### **6.2 Intensification and stabilization of colour**

In the case of musts obtained from red grapes, the degradation of cell walls in the skin of the grapes through pectolytic enzyme treatment results in an increased release of phenolic

Internationale de la Vigne et du Vin) regulates the origin of these enzymes for the wine industry. Commercial products contain mainly a mix of polygalacturonase, pectin lyase and pectin methyl esterase. The use of different strains of *Aspergillus* and modification of substrates and culture conditions can lead to mixtures enriched in one type of enzyme. Companies, including AEB, Gist-Brocades, Novo Nordisk and Lallemand, offer different products with pectic enzymes in different proportions to suit the needs of each process. However, these commercial pectic preparations are not always optimal for each process, and its use is not without side effects and controversy. Thus, while pectin methyl esterase activity present in the samples may be necessary for the action of polygalacturonase in the case of pectin with a high degree of esterification, its action may lead to an undesirable increase of methanol in the products (Vilanova et al., 2000). Moreover, although the pectic enzymes are the most abundant in commercial mixtures, they can also contain other undesirable activities, such as in the case of making wine, polyphenoloxidases or cinnamyl

203

At present especially in recent years, the accumulated knowledge of new microbial pectolytic enzymes as well as methodological and technological advances can address the production and use of these enzymes with a different approach. The diversity of applications and conditions in which these enzymes must work also demand a large number of different enzymes capable of acting in such conditions. Even more interesting would be having more robust, broad-spectrum enzymes which allow for a more versatile

Considering the variety of enzymes, traditionally the majority of studies refer to the pectinases of *Erwinia* and *Bacillus* within the bacteria and various fungi especially *Aspergillus* (de Vries and Visser, 2001; de Vries et al., 2002; Gummadi & Panda, 2003; Hoondal et al., 2002; Jayani et al., 2005; Yadav et al., 2009) although there has been a major advance in the description and characterization of pectic enzymes produced by yeast in the last 15 years (Alimardani-Theuil et al., 2011; Blanco et al., 1999; Rodríguez-Gámez & Serrat, 2008). In recent years there has been also a growing interest in studying pectic enzymes with very interesting properties from the point of view of their application. These include thermostable pectinases (Kar & Ray, 2011; Swain & Ray, 2010) or pectinases with optimal activity at low temperatures (Cabeza et al., 2011; Merin et al., 2011; Nakawagua et al., 2004;

The possibility of producing different types of pectolytic enzymes separately and for later preparation of their mixtures in the proper proportions would allow to provide more suitable commercial preparations for each application and lacking undesirable activities. In this sense, microorganisms such as some strains of yeast *Saccharomyces cerevisiae* and *Kluyveromyces marxianus* which produce only one type of enzyme (Blanco et al., 1999; Serrat et al., 2002) or that constitutively synthesize it (Serrat et al., 2002) are of great interest. Similarly, obtaining constitutive mutants from producing strains allow the optimization of production and contribute to making cost-effective production processes (Favela-Torres et al., 2006; Trigui-Lahiani et al., 2008). Furthermore, these strains can be used to produce pectinases that accumulate together with other metabolites in the culture broth, which contributes favourable to the overall economy of the process (Serrat et al., 2011). Heterologous expression of enzymes in prokaryotic or eukaryotic systems is a technique of great interest for the production of a single type of enzyme. Table 3 shows some of the many pectic enzymes, both from bacteria, yeast and fungi, which are rightly expressed in

esterases (Mantovani et al., 2005; van Rensburg & Pretorius, 2000).

use in different applications.

Padma et al., 2011).

compounds responsible for colour (Busse-Valverde et al., 2011; Pinelo et al., 2006). Early work related to the use of pectinases to enhance the colour of the wines were a bit confusing because Ough et al. (1975) were able to intensify the colour of wine by the enzyme treatment, but Wightman et al. (1997) found that some enzyme preparations containing pectic enzymes reduced red wine colour. Subsequent studies performed with two different enzyme preparations (Watson et al., 1999) confirmed that both allow to produce wines with higher concentrations of anthocyanins and total phenols and have a higher colour intensity. Similarly, in 1994 the Australian Wine Research Institute conducted a study with different commercial preparations of pectic enzymes and in all cases concluded that its use leads to faster and better colour extraction during maceration, pressing and fermentation as well as improve the clarification of the product (van Rensburg & Pretorius, 2000).

#### **6.3 Boosting aroma and flavour**

The aromatic profile of wines consists of two components: the varietal aromas characteristic of the variety of grape used and the aromas originated by the yeast during fermentation (Piñeiro et al., 2006; Vilanova & Sieiro, 2006). In many cases the grape variety used completely determines the aroma of the wine, especially in young wines. The volatile compounds of grapes include monoterpenes, C13 nor-isoprenoides, benzene derivatives and aliphatic alcohols. The aromatic components of the grape may appear as free forms, which contribute directly to its scent or bound forms, of greater concentration, to sugars and nonvolatile. Nonodorous glycoside flavour precursors accumulate in the grape especially in the skin during the ripening process (Bayonove, 1993; Williams et al., 1989; Winterhalter & Skouroumounis, 1997). The hydrolysis of these precursors via beta-glucosidases releases the olfactory active aglycones (Günata et al., 1988; Williams et al., 1989). Pectic enzymes help break down the cell walls of the grapes and thus to extract the aromatic precursors. The addition of pectic enzymes during the extraction or fermentation of the must results in an increase in aromatic precursors susceptible to being attacked by beta-glucosidases from the must, those produced by yeasts and bacteria during fermentation or those which are included in commercial enzyme preparations, thereby enhancing the aroma of wines (Comitini et al., 2011; du Toit et al., 2011; Gómez-Plaza et al., 2000; Pinelo et al., 2006).

#### **7. Biotechnology production of microbial pectic enzymes**

As discussed until now, there are numerous applications of microbial pectic enzymes in food and wine-making industry. Not surprisingly, the sales volume of these enzymes represents 25% of the enzymes that are commercialized in the food and alcoholic beverages industry. These many applications require one or more types of pectinases that must act in very different condition according to the process in which they are involved. For example, while in the extraction and clarification of fruit juice enzymes can be used at temperatures between 45-95 ºC (Kashyap et al., 2001), the wine industry employs temperatures below 15- 10 °C (Gómez-Plaza et al., 2000). Although most food applications pectinases have to act in a medium acid, in others, such as oil extraction, they should perform in a medium alkaline (Hoondal et al., 2002).

So far, commercial pectic enzymes are prepared only from cultures of filamentous fungi, mainly of the genus *Aspergillus*. In fact, in the European Union, the OIV (Organisation

compounds responsible for colour (Busse-Valverde et al., 2011; Pinelo et al., 2006). Early work related to the use of pectinases to enhance the colour of the wines were a bit confusing because Ough et al. (1975) were able to intensify the colour of wine by the enzyme treatment, but Wightman et al. (1997) found that some enzyme preparations containing pectic enzymes reduced red wine colour. Subsequent studies performed with two different enzyme preparations (Watson et al., 1999) confirmed that both allow to produce wines with higher concentrations of anthocyanins and total phenols and have a higher colour intensity. Similarly, in 1994 the Australian Wine Research Institute conducted a study with different commercial preparations of pectic enzymes and in all cases concluded that its use leads to faster and better colour extraction during maceration, pressing and fermentation as well as

The aromatic profile of wines consists of two components: the varietal aromas characteristic of the variety of grape used and the aromas originated by the yeast during fermentation (Piñeiro et al., 2006; Vilanova & Sieiro, 2006). In many cases the grape variety used completely determines the aroma of the wine, especially in young wines. The volatile compounds of grapes include monoterpenes, C13 nor-isoprenoides, benzene derivatives and aliphatic alcohols. The aromatic components of the grape may appear as free forms, which contribute directly to its scent or bound forms, of greater concentration, to sugars and nonvolatile. Nonodorous glycoside flavour precursors accumulate in the grape especially in the skin during the ripening process (Bayonove, 1993; Williams et al., 1989; Winterhalter & Skouroumounis, 1997). The hydrolysis of these precursors via beta-glucosidases releases the olfactory active aglycones (Günata et al., 1988; Williams et al., 1989). Pectic enzymes help break down the cell walls of the grapes and thus to extract the aromatic precursors. The addition of pectic enzymes during the extraction or fermentation of the must results in an increase in aromatic precursors susceptible to being attacked by beta-glucosidases from the must, those produced by yeasts and bacteria during fermentation or those which are included in commercial enzyme preparations, thereby enhancing the aroma of wines

(Comitini et al., 2011; du Toit et al., 2011; Gómez-Plaza et al., 2000; Pinelo et al., 2006).

As discussed until now, there are numerous applications of microbial pectic enzymes in food and wine-making industry. Not surprisingly, the sales volume of these enzymes represents 25% of the enzymes that are commercialized in the food and alcoholic beverages industry. These many applications require one or more types of pectinases that must act in very different condition according to the process in which they are involved. For example, while in the extraction and clarification of fruit juice enzymes can be used at temperatures between 45-95 ºC (Kashyap et al., 2001), the wine industry employs temperatures below 15- 10 °C (Gómez-Plaza et al., 2000). Although most food applications pectinases have to act in a medium acid, in others, such as oil extraction, they should perform in a medium alkaline

So far, commercial pectic enzymes are prepared only from cultures of filamentous fungi, mainly of the genus *Aspergillus*. In fact, in the European Union, the OIV (Organisation

**7. Biotechnology production of microbial pectic enzymes** 

improve the clarification of the product (van Rensburg & Pretorius, 2000).

**6.3 Boosting aroma and flavour** 

(Hoondal et al., 2002).

Internationale de la Vigne et du Vin) regulates the origin of these enzymes for the wine industry. Commercial products contain mainly a mix of polygalacturonase, pectin lyase and pectin methyl esterase. The use of different strains of *Aspergillus* and modification of substrates and culture conditions can lead to mixtures enriched in one type of enzyme. Companies, including AEB, Gist-Brocades, Novo Nordisk and Lallemand, offer different products with pectic enzymes in different proportions to suit the needs of each process.

However, these commercial pectic preparations are not always optimal for each process, and its use is not without side effects and controversy. Thus, while pectin methyl esterase activity present in the samples may be necessary for the action of polygalacturonase in the case of pectin with a high degree of esterification, its action may lead to an undesirable increase of methanol in the products (Vilanova et al., 2000). Moreover, although the pectic enzymes are the most abundant in commercial mixtures, they can also contain other undesirable activities, such as in the case of making wine, polyphenoloxidases or cinnamyl esterases (Mantovani et al., 2005; van Rensburg & Pretorius, 2000).

At present especially in recent years, the accumulated knowledge of new microbial pectolytic enzymes as well as methodological and technological advances can address the production and use of these enzymes with a different approach. The diversity of applications and conditions in which these enzymes must work also demand a large number of different enzymes capable of acting in such conditions. Even more interesting would be having more robust, broad-spectrum enzymes which allow for a more versatile use in different applications.

Considering the variety of enzymes, traditionally the majority of studies refer to the pectinases of *Erwinia* and *Bacillus* within the bacteria and various fungi especially *Aspergillus* (de Vries and Visser, 2001; de Vries et al., 2002; Gummadi & Panda, 2003; Hoondal et al., 2002; Jayani et al., 2005; Yadav et al., 2009) although there has been a major advance in the description and characterization of pectic enzymes produced by yeast in the last 15 years (Alimardani-Theuil et al., 2011; Blanco et al., 1999; Rodríguez-Gámez & Serrat, 2008). In recent years there has been also a growing interest in studying pectic enzymes with very interesting properties from the point of view of their application. These include thermostable pectinases (Kar & Ray, 2011; Swain & Ray, 2010) or pectinases with optimal activity at low temperatures (Cabeza et al., 2011; Merin et al., 2011; Nakawagua et al., 2004; Padma et al., 2011).

The possibility of producing different types of pectolytic enzymes separately and for later preparation of their mixtures in the proper proportions would allow to provide more suitable commercial preparations for each application and lacking undesirable activities.

In this sense, microorganisms such as some strains of yeast *Saccharomyces cerevisiae* and *Kluyveromyces marxianus* which produce only one type of enzyme (Blanco et al., 1999; Serrat et al., 2002) or that constitutively synthesize it (Serrat et al., 2002) are of great interest. Similarly, obtaining constitutive mutants from producing strains allow the optimization of production and contribute to making cost-effective production processes (Favela-Torres et al., 2006; Trigui-Lahiani et al., 2008). Furthermore, these strains can be used to produce pectinases that accumulate together with other metabolites in the culture broth, which contributes favourable to the overall economy of the process (Serrat et al., 2011). Heterologous expression of enzymes in prokaryotic or eukaryotic systems is a technique of great interest for the production of a single type of enzyme. Table 3 shows some of the many pectic enzymes, both from bacteria, yeast and fungi, which are rightly expressed in

extraction and clarification of fruit juices and oils as well as being important in the fermentation of coffee, cocoa and tea. Also, in the wine industry they play an important role by contributing to the release of the molecules responsible for aroma and colour, two of the

205

Traditionally, this industry uses different mixtures of pectolytic enzymes derived from fungi cultures, mainly of the genus *Aspergillus*, not always completely adequate for the processes they must carry out because of the type and concentration of different enzyme activities that they contain, not without undesirable effects due to other non-pectic enzymes that may be

The exploration of microbial biodiversity has allowed, especially in recent years, to identify and characterize new pectic-enzyme-producing microorganisms with different biochemical characteristics, some potentially very interesting from the point of view of their application. Also, it has been technically possible, on the one hand, to select wild strains and constitutive mutants that produce a single enzyme, and, on the other hand, the heterologous expression in bacteria and yeast of numerous genes which encode pectic enzymes, obtaining producing

All this opens the possibility of producing different pectic enzymes individually and preparing commercial mixtures of these, adapted to each process. Research focused on protein engineering in order to obtain pectic enzymes more robust and versatile as well as the optimization of production processes with new strains are necessary for the successful

Alimardani-Theuil, P., Gainvors-Claise, A. & Duchiron, F. (2011). Yeasts: An attractive

Alkorta, I., Garbisu, C., Llama, M.J. & Serra, J.L. (1998). Industrial applications of pectic

Amorim, H.V. & Amorim, V.L. (1977). Coffee enzyme and coffee quality. In: *Enzymes in* 

Bayanove, C. (1993). Les composés terpeniques. In : *Les acquisitions récentes en* 

Blanco, P., Sieiro, C. & Villa, T.G. (1999). Production of pectic enzymes in yeasts. *FEMS* 

Blanco, P., Sieiro, C., Díaz, A. & Villa, T.G. (1994). Production and partial characterization

Blanco, P., Sieiro, C., Díaz, A., Reboredo, N.M. & Villa, T.G. (1997). Grape juice

Blanco, P., Sieiro, C., Reboredo, N.M. & Villa, T.G. (1998). Cloning, molecular

source of pectinases-From gene expression to potential applications: A review.

*food and beverage processing*, R.L. Ori & A.J. st Angelo, (Eds.), pp. 27-56, American

*chromatographie du vin. Applications á l´ analyse sensorielle des vins*, B. Doneche,

of an endopolygalacturonase from *Saccharomyces cerevisiae*. *Canadian Journal of* 

biodegradation by polygalacturonases from *Saccharomyces cerevisiae. International* 

characterization, and expression of an endo-polygalacturonase-encoding gene

completion of this new approach for the production and use of microbial pectinases.

enzymes: a review. *Process Biochemistry*, Vol.33, pp. 21-28

Chemical Society, ISBN: 084120375X, Indiana, USA

(Ed.), pp. 99-119, ISBN: 2877773647, Paris, France

*Biodeterioration and Biodegradation,* Vol.40, pp. 115-118

*Microbiology Letters*, Vol.175, pp. 1-9

*Microbiology*, Vol.40, pp. 974-977

*Process Biochemistry,* Vol.46, pp. 1525-1537

major components that characterize a wine.

present in the mixtures.

strains of interest.

**9. References** 

*Escherichia coli* and different species of yeast. Although *E. coli* is unable to carry out posttranslational modifications of proteins, fungal pectolytic enzymes expressed in these bacteria are active (Wang et al., 2011). However, one of the most interesting strategies seems to be the expression of pectinase genes in yeast, particularly in *Pichia pastoris*, in which very high levels of constitutive expression has been achieved (Sieiro et al., 2009). In some cases changes in glycosylation patterns conducted by yeast did not affect the activity and characteristics of recombinant pectinases (Sieiro et al., 2009), while other changes do occur with respect to the characteristics of the native protein, which even lead to enzymes with interesting properties for certain applications (Lang & Looman, 1995; Sieiro et al., 2003).


Table 3. Microbial pectic enzymes expressed in different host strains
