**4. Pectic enzymes in nature: Microbial pectinases**

Pectic enzymes are widely distributed in nature and are produced by bacteria, yeast, fungi (Fig. 2A) and plants. (Lang & Dornenburg, 2000; Whitaker, 1990). In plants, pectic enzymes are very important since they play a role in elongation and cellular growth as well as in fruit ripening (Sakai, 1992; Ward & Moo-Young, 1989; Whitaker, 1990). Pectolytic activity of microorganisms plays a significant role, firstly, in the pathogenesis of plants since these enzymes are the first to attack the tissue (Collmer & Keen, 1986; Whitaker, 1990). In addition, they are also involved in the process of symbiosis and the decay of vegetable residues (Hoondal et al., 2002; Lang & Dornenburg, 2000).

increase in their viscosity which hinders its clarification, filtration and concentration (Alkorta et al., 1998). The degradation of pectic substances in mashed fruit purees is achieved through the addition of pectolytic enzymes resulting in an increase in juice yield and its clarification as well as a decrease in viscosity. Treatment with pectinases also provides filtering of the product (de Gregorio et al., 2002; Fernandez-Gonzalez et al., 2004;

**pH** 

*Bacillus* sp NT-33 Polygalacturonase 10.5 75 Cao et al., 1992 *Bacillus* sp DT7 Pectin lyase 8 60 Kashyap et al., 2000

*Aspergillus ficuum* Pectin lyase 5 50 Yadav et al., 2008

*Sclerotium rolfsii* Endopolygalacturonase 3.5 55 Chane & Shewal,

*Penicillium paxilli* Pectin lyase 5 35 Szajer & Szajer,

The fruit and vegetable juice industry uses mainly acidic pectinases of fungal origin, principally from *Aspergillus* spp*.* Commercial preparations are mixtures of polygalacturonases, pectate lyases and pectin esterases. Pectate lyases can act on the esterified pectin while the polygalacturonases act on the desesterified pectin thus it might require previous action of the pectin esterases. Pectic enzymes treatments vary depending

In this type of product, pectolytic enzymes are added to increase the yield in juice during the pressing and for removing matter in suspension. In the case of apple juice, the most commonly used enzymes are those that can depolymerize the highly esterified apple pectin. Apple juice can be obtained through a two-step process consisting of a first treatment of the crushed apple mush with pectinases to obtain the premium juice followed by pomace liquefaction treatment made with a mixture of different pectinases and cellulases for the

After washing and crushing, the apples are pressed to obtain the juice. Pectic enzymes are used to facilitate the pressing and juice extraction and to assist in the separation of a flocculant precipitate by means of sedimentation, filtration or centrifugation. If a cloudy product is required, the juice is pasteurised after pressing to inactivate residual enzymes. Centrifugation removes the large-size remains leaving small particles in suspension.

Endopolygalacturonase 3.5-5 50 Boirin et al., 1996

Endopolygalacturonase 5.5 45 Blanco et al., 1994

Endopolygalacturonase 4.5 55 Serrat et al., 2002

**Optimal temperature (°C)** 

**Reference** 

199

1995

1982

Ribeiro et al., 2010; Sarioglu et al., 2001; Souza et al., 2003).

**Microorganism Enzyme Optimal** 

Table 2. Biochemical properties of some pectinases

**5.1.1 Clear juices (i.e., apple, pear, grape)**

complete extraction of the juice (Will et al., 2000).

**Bacteria** 

**Fungi** 

**Yeasts**  *Saccharomyces cerevisiae* 

*Kluyveromyces marxianus* 

on the type of juice (Fig. 2B):

*Penicillium frequentans* 

Fig. 2. A: Pectic enzymes produced by different microorganisms growing on minimal medium supplemented with polygalacturonic acid and detected by a clear halo around the colonies. 1, *Xanthomonas campestris* CECT 97; 2, *Kluyveromyces marxianus* CECT 1043; 3, *Aspergillus niger* CECT 2088; 4, *Botryotinia fuckeliana* CECT 20518. B: Cloudy (peach) and clear (apple) juices. CECT: Spanish Type Culture Collection

The microbial world has shown to be very heterogeneous in its ability to synthesize different types of pectolytic enzymes with different mechanisms of action and biochemical properties (Favela-Torres et al., 2005; Gummadi & Panda, 2003). Pectic enzymes are produced by both prokaryotic microorganisms, which primarily synthesize alkaline pectinases, and by eukaryotic microorganisms, mostly fungi that synthesize acid pectinases (Hoondal et al., 2002; Jayani et al., 2005; Kashyap et al., 2001). Furthermore, the production of these enzymes has also been described in yeast (Alimardani-Theuil et al., 2011; Blanco et al., 1999).

There are many studies that have been conducted related to the characterization of different microbial pectic enzymes concerning their mechanisms of action and biochemical properties. The optimal pHs that these enzymes may act range between 3.5-11, while the optimal temperatures vary between 40-75 °C (Gummadi & Panda, 2003; Kashyap et al., 2001). Table 2 shows an example of the diversity of pectic enzymes produced by microorganisms. Given the features of the substrate on which they act and the effect that is required of them, acidic and depolymerizing pectinases are of great interest for the food industry although some applications such as the extraction of oils requires the alkaline ones (Hoondal et al., 2002).
