**3. Pectolytic enzymes and their classification**

Pectolytic enzymes constitute a wide group of enzyme activities that are responsible for the degradation of the pectic substances summarised in the previous section. During fruit ripening, endogenous pectolytic enzymes act upon insoluble pectic substances and turn them into soluble pectic substances. As a result, the surrounding cell wall loses its grip and firmness, and consequently the plant tissue softens. Analogously, plant pathogens attack their host by secreting many different pectolytic enzymes in addition to cellulases and proteases [23]. This means that pectolytic enzymes exist in different forms depending on the source, the substrates they act on, and the products to be split from the substrate. Generally, pectolytic enzymes are divided into two groups: depolymerizing enzymes and esterases (also named saponifying enzymes) [24]. Depolymerizing enzymes can cleave the α- (1 ! 4) glycosidic bonds in the backbone of the pectin chain, and in this group polygalacturonase (**PG**), pectin lyase (**PNL**), and pectate lyase (**PL**) are included. While pectin methyl esterases (**PE**) (commonly named "pectin esterases") and pectin acetyl esterase (**PAE**) break down ester linkages splitting methoxyl or acetyl groups, liberating the carboxylic groups of pectin polygalacturonic acid residues.

Classification of the plethora of pectolytic activities and their correct naming can be achieved following the consensus recommendations of the International Union of Biochemistry and Molecular Biology [25] and thus, pectolytic enzymes can be grouped as follows:

	- a. *Endo*-polygalacturonase **(***endo***-PG** *EC 3.2.1.15***)** catalyses random hydrolysis of α-(1 ! 4) glycosidic linkages in pectates and other galacturonans. Other names of this enzyme: polygalacturonase, *endo*galacturonase, pectin-depolymerase, pectinase; pectolase, pectinhydrolase, and *endo*-polymethylgalacturonase (*endo*-PMG).
	- b. *Exo-*polygalacturonase **(***exo***-PG** *EC 3.2.1.67***)** catalyses the hydrolysis in a sequential cleavage of the α-(1 ! 4) glycosidic linkage of the

#### **Figure 2.** *Sites of hydrolysis of α-(1* ! *4) glycosidic linkages catalysed by polygalacturonase enzymes.*

non-reducing end of pectates and other galacturonan chains. Other names: galacturan 1,4-α-galacturonidase, poly [(1 ! 4)-α-Dgalacturonide] galacturonohydrolase, poly(galacturonate) hydrolase, *exo-*D-galacturonase, poly (1,4-α-D-galacturonide) galacturonohydrolase, and *exo-*polymethylgalacturonase (*exo*-PMG).

	- a. Pectin lyases **(PNL** *EC 4.2.2.10***)** catalyse the eliminative cleavage of α- (1 ! 4) glycosidic linkages in pectins. Other names: pectin transeliminase, *endo-*pectin lyase, polymethylgalacturonic transeliminase, pectin methyltranseliminase, pectolyase, polymethoxygalacturonide lyase, and polymethylgalacturonate lyases (PMGL). They prefer to act upon highly esterified pectins without the prior action of other enzymes [26] and demethylation of pectins progressively slows their activity. Two activities can be included under this denomination: *endo-*pectin lyase, which comprises most of the studied pectin lyases (*endo-*PNL = *endo-*PMGL) and *exo-*pectin lyase (*exo*-PNL = *exo*-PMGL), which includes scarcely reported enzymes [27].
	- b. Pectate *endo-*lyases (*endo***-PL** *EC 4.2.2.2*) catalyse the cleavage of α -(1 ! 4) glycosidic linkages in pectic acid and pectates. They show specificity for pectates in their anion form over methyl esterified pectins. Other names: *endo-*pectate lyase; polygalacturonic transeliminase; pectic acid transeliminase; polygalacturonate lyase; pectic lyase; α-1,4-D-*endo* polygalacturonic acid lyase, and others. This enzyme type is usually employed for de-gumming natural fibres in the paper and textile industries [28]. It is also used for the preparation of fruit and vegetable maceration products and agriculture wastewater treatment [9].

**Figure 3.** Endo-*pectin lyase catalyzed reaction*

	- a. Pectin esterase **(PE** or PME *EC 3.1.1.11***)** catalyses the hydrolysis of the ester linkage between the methoxyl group and the carboxylic group of galacturonic acid residues in the pectin or pectinic acid backbone, releasing methanol (**Figure 4**). Other names: pectin methylesterase (PME); pectin demethoxylase; pectin methoxylase; pectase and pectinesterase.

The presence of calcium ions maintains fruit firmness by binding to free negatively charged carboxylic acid groups of the pectin molecules that are not methoxylated, forming what is defined as the "egg-box" model with a structure of calcium ion cross-bridges between pectin chains [29]. In this regard, the activity of the pectin esterase can play a role in fruit texture [30].


*Fungal Pectinases in Food Technology DOI: http://dx.doi.org/10.5772/intechopen.100910*

#### **Figure 4.**

*Pectin esterase (EC 3.1.1.11) catalysed hydrolysis of the methoxy group of pectin to yield methanol.*

IV. **Protopectinases.** A mixture of some of the previous enzymes besides other polysaccharidases, such as cellulase or hemicellulase, and protease, can act on the water-insoluble protopectin aggregates, turning them into highly water-soluble pectic substances [9]. This heterogeneous group of enzymes that act on protopectin aggregates is commonly known as protopectinases.

#### **4. Fungal pectolytic enzymes**

Fungi secrete pectolytic enzymes into their growth medium in combination with some other polysaccharide-degrading enzymes, like cellulase, hemicellulase, amylase, and other extracellular secreted enzymes, such as proteases. All these enzymes play an important role in infecting host cells by filamentous fungi. From the biotechnological point of view, extracellular enzymes are easier to obtain than intracellular enzymes, as secreted enzymes are recovered in the culture broth supernatant and simultaneously separated from the remaining cellular biomass of the producer organism. These reasons make filamentous fungi excellent candidates for enzyme production. Nevertheless, in order to use fungal enzymes in the industrial sector, stability and biochemical characteristics of such enzymes produced under various growing conditions must be studied. Purification of an enzyme is needed to estimate its biochemical properties and specificity. On the other hand, the numerous steps that are usually required for the complete purification of an enzyme, consume a long time, largely increase the economic cost and resources. In addition, the purification process could have negative effects on the enzyme activity. Consequently, the balance between technical and economic requirements is mandatory for the industrial production of enzymes.

There are different methods and techniques to separate and isolate pectolytic enzymes from crude extracts. These methods are diverse in their efficiency

and resolution. Precipitation of the enzyme from crude extracts using natural salts (e.g., ammonium sulphate) or organic solvent (e.g., ethanol) followed by column chromatography is a satisfactory procedure to get commercial purified enzymes [10].

One of the early attempts to purify pectolytic enzymes was that of two *exo*-PG isolated from crude extracts of *A*. *niger* after DEAE-cellulose chromatography, using 0.2 M sodium acetate buffer at pH 4.6 as the eluting solvent. The specific activity of both enzymes was increased 209- and 205-fold with 8.6–1% recovery, respectively [31]. PG from *Rhizopus stolonifer* was also separated by ethanol precipitation followed by CM-Sepharose 6B ion-exchange chromatography and the eluate was further purified to reach 10-fold by gel filtration onto Sephadex G-100 [32]. PG and PNL from *Aureobasidium pullulans* LV10 were separated by CM-Sepharose 6B followed by DEAE-cellulose chromatography and gel filtration on Sephadex G-100 [33]. *Endo-*PG from Rohament P, a commercial pectolytic enzyme from *A*. *niger*, was isolated and separated into three isoenzymes by preparative isoelectric focusing onto Bio-gel P-60 [34]. Here it should be noted that multiple purification steps will elevate the enzyme price, thereby, researchers in the field of food processing should pay great attention to make the purification method easy, fast, and inexpensive while increasing as much as possible the enzyme activity.

Immobilised metal ion affinity polysulphone hollow-fibre membranes with a high capacity for protein adsorption were successfully used to separate PNL and PE from a commercial pectolytic preparation [35]. A rapid and simple method to separate pectinases, including PE and PG, from potato enzyme preparations using perfusion chromatography (Poros HS), was introduced by Savary [36]. This method was an economical purification strategy for PE and PG enzymes from crude extract at a commercial scale. Regarding cold-active pectinases isolated from psychrotolerant yeasts, few activities have been characterized that showed pectolytic activity at low temperatures, reaching down to 5°C [37].

The term "pectinase" is widely preferred in the industrial context, and a wide range of pectinase-producing fungi and procedures for recovering and purifying the enzyme can be found in literature and have been successfully applied for industrial purposes [1, 3, 38]. In addition, the production of recombinant pectinases by genetically manipulated fungi has gained the attraction of researchers and biotechnologists [3, 39]. Nevertheless, as mentioned above, the GRAS status awarded by the FDA to the enzyme producer organism is a relevant characteristic when enzyme production is intended for the food industry. **Table 2** shows fungal producers of pectinolytic enzymes that are currently included in the inventory of GRAS notices, being *Aspergillus* the most repeated genus in the GRAS inventory, and *A. niger* the species with the highest number of notices, as mentioned above. With regard to the use of enzymes in foods in the European Union, it is subject to the legislation of its member states, and currently, the European Food Safety Authority (EFSA) is in the process of evaluating the safety of more than 300 food enzymes, whose applications were submitted for approval to be included in a future EU list of authorised food enzymes [40].

### **5. Pectolytic enzymes from fungi and yeast with GRAS status**

**Tables 3** and **4** show polygalacturonases (*EC* 3.2.1.15) and pectin lyases (*EC* 4.2.2.10) from fungal and yeast species that possess the GRAS status and that can be currently found at the NCBI Protein Database. **Table 3** shows the


#### **Table 2.**

*Filamentous fungi and yeast species producers of pectinolytic enzymes that are included in the GRAS inventory of the FDA.*

*exo*-polygalacturonase encoding genes found in fungi: pgxA, pgxB, and pgxC, and those that encode *endo*-polygalacturonases, which are quite numerous: pgaA, pgaB, pgaC, pgaI, and pgaII; whereas only the pgU1 gene was found in GRAS yeast strains. All the polygalacturonases encoded by these genes belong to the glycosyl hydrolases family 28.

The genes that encode pectin lyases found in GRAS fungi are quite numerous as well and pectin lyases from *Aspergillus* strains have been characterised according to their substrate degradation profile [41]. **Table 4** shows the pectin lyase encoding genes that have been sequenced from GRAS fungi: pel1, pel2, pelA, pelB, pelC, pelD, pelE, and pelF. All the encoded proteins belong to the polysaccharide lyase family 6. It is worth mentioning that no pectin lyase has been described in GRAS yeast.

The protein structure of the *endo*-polygalacturonase II of *A. niger* was determined by crystallographic techniques [42] and its sequence is 60% identical to the *endo*-polygalacturonase I. The 1.70 Å resolution crystal structure of *endo*-polygalacturonase I is shown in **Figure 5**. It is folded into a right-handed parallel beta helical structure comprising 10 complete turns. This structure includes a narrow substratebinding cleft, in which the Arg96 residue, previously shown to be critical for the enzyme activity, was shown to interact with the polygalacturonic acid units of the backbone of its substrate [43].

The protein structure of the pectin lyase A and pectin lyase B of *A. niger* were as well resolved in the 90s [44, 45]. *A. niger* pectin lyases shown in **Table 4** share


#### **Table 3.**

*Polygalacturonases (EC 3.2.1.15) included in the NCBI Protein Database that were obtained from fungal and yeast GRAS species.*

### *Fungal Pectinases in Food Technology DOI: http://dx.doi.org/10.5772/intechopen.100910*


#### **Table 4.**

*Pectin lyases (EC 4.4.4.10) included in the NCBI protein database that were obtained from fungal and yeast GRAS species.*

#### **Figure 5.**

*PDB Image of* Endo*-polygalacturonase I from A. niger at 1.7 Å resolution [43]. Available from: https://www. ncbi.nlm.nih.gov/Structure/pdb/1NHC. Code of colours: Beta strands in green, loops in blue, alpha hélix in red.*

46–65% amino acid sequence identity. The 1.70 Å resolution crystal structure of pectin lyase B is shown in **Figure 6** [45] and it shows a parallel beta helical structure, where residues Asp154, Arg176, and Arg236 were expected to play a role in the catalysis [44]. In contrast to the previously shown structure of *endo*-polygalacturonase, the pectin lyase structure shows a number of loops of various sizes and conformations that protrude from the central helix, which bind oligosaccharides and probably confer function to the enzyme [45].
