The Microbial Degradation for Pectin

Abstract

Pectin considers one of the most plentiful natural components having many applications. It is widely distributed in the middle lamella and cell walls of the terrestrial plant in various concentrations. Pectin is a heteropolysaccharide that involved galacturonic acid and methanol as the main components. Pectin is degraded by the pectinase enzyme, producing several compounds that have industrial applications. This Enzyme is produced by several organisms such as plants, protozoa, nematodes, insects, and microorganisms. However, the microbial source is the most common in commercial production due to its massive applications in various industries. Consequently, this chapter will show the importance of microorganisms to degrade pectin, the different types of microorganisms that can degrade pectin, and their applications.

1. Introduction

Pectin considers one of the most plentiful natural compounds having different applications. It is heteropolysaccharides that compose the main components of the middle lamella and primary cell wall of higher plants and are responsible for the cohesion and the structural integrity of plant tissues [1, 2]. Generally, rhamnogalacturonans and galacturonans are two main chemical components in pectic materials, where C-6 carbon of galactate is oxidized along with arabinogalactans and arabinans. The produced components are colloidal polymeric in nature and structurally heterogeneous, containing a large backbone of anhydrogalacturonic acid units. The carboxyl groups of GalA are partially esterified by methyl residues and totally or moderately neutralized by different ions as potassium, sodium, and ammonium. α-1,4-d-galacturonate units are working as a master and connecting about 2-4% of L-rhamnose units that linked [β- (1 → 2) and β-(1 → 4) to galacturonate units. The side chains include simple carbohydrates (galactan, arabinan, arabinogalactan, fucose, or xylose) but vary in length and composition. They are associated with the central chain through their C1 and C2 atoms [3]. Hence, the main chain of the pectin polymer consists of galacturonic acid (GalA) linked by α-D-1,4- bonds and form three backbone regions [4], Figure 1:

1. SMOOTH REGION OR HOMOGALACTURONAN (HG), represents about 65% of pectin [6] and involves long stretches of (1 → 4)-linked d-galactopyranosyl uronic acid residues [7]. Further modifiable occurs by the process of Methyl esterification at C-6, or acetyl groups at C-2 and C-3 position [8].

2. HAIRY REGION OR RHAMNOGALACTURONAN (RG I) the branched area, represents 20-35% of pectin, involves the side chain of the repeating disaccharide unit [α- 1, 2-rahmnopyranose residues] linked by (1 → 4) disaccharide [9]. Sometimes, lateral chains contain glucuronic acids and fucose found mostly to create the structure more complex.

   • This region is acylated and frequently substituted with arabinans, galactans, and arabinogalactans linked to rhamnose residue [10]. Also, xylose is found for substitutions [11].

   • RGII region, although the name, is not structurally related to RGI. It is a branched pectic domain-containing HG backbone substituted with heteropolymeric side chains involving different sugars [12]. Containing side chain of D-Apiose, 2-Omethyl-D-xylose, and 2-O-methyl-L-Fructose. The galacturonic residues are usually acetylated at the C-2 or C-3 position in rhamnogalacturonan 1 [13].

Figure 1.

(a) pectin Structure (b) Pectin structure in traditional model displaying the homogalacturonan (HG) as the backbone, smooth region, and 60-sugar residue. Feruloyl pectic acids are engaged in organizing cell expansion, resistance to diseases, and lignification initiation. The properties of the pectin the hairy region consisting of Rhamnogalacturonan-I (RG-I) or Rhamnogalacturonan-II (RG-II) units [inspired by Noreen et al., [5].

Most molecules are formed in the series by D-Galacturonic residue. Polymers do not make up a straight string in an aqueous medium, but it is curved and extended with high flexibility. The contrast was found in the configuration of pectin from different sources, and the pectin properties are strongly related to the methylation of galact-uronic acids residues, which is usually 70% [14]. The acidic and neutral pectin has ferulic acid on non-reduced ends of neutralarabinose and or galactose including domains. Pectin carries about one teruloyl residue for every matrix is complicated due to the interaction of the domain structure of pectin between themselves and with other ionized inorganic and organic compounds [15].

The American chemical community, ranked according to the nature of the molecular arrangements of pectic substances into four main groups [16]:

1. PROTOPECTIN is considering the pectin parent were on the restricted hydrolysis yields pectinic acid or pectin [17]. And located in the middle lamella, serving as the glue to hold cells together in the cell walls. Also, it is water-insoluble due to its large molecular weight, formation of ester-bond between carboxylic acid groups (in pectin) and hydroxyl group (in other constituents of the cell wall), and salt bonding between the carboxyl groups (in pectic substances) and basic groups (in proteins).

2. PECTIC ACID or pectate: is a polymer of galacturonan containing a few methoxy groups and is water-soluble.

3. PECTINIC ACID is a polymer of galacturonan containing a significant amount of methoxy groups (up to 75%). Under a suitable condition, can forming gel with sugar and acid.

4. PECTIN (polymethyl galacturonate) is a soluble polymeric material in which almost the carboxyl group of galacturonate units (about 75%) are esterified with a methyl group. Pectin as pectinic acid can be forming a gel with sugar and acid under favorable conditions [18].

2. The depolymerization of the pectin polymer into a simpler form

The pectin depolymerization occurred either physical, chemical, or enzymatic methods, Figure 2.

Figure 2.

The different depolymerization processes of various types of pectin [inspired from Satapathy et al., 2020 [19]].

2.1 Physical process

Includes high-pressure treatment, ultrasonication, radiation, and photolysis. At a pH value lower or higher than 3.5, the acetyl, methoxyl, and neutral sugar groups are eliminated, and the polymer backbone is cleaved.

2.2 Chemical process

Acid or base hydrolysis can catalyze the splitting of chains by the β-elimination reaction. The dissolution takes place at a glycosidic linkage next to an esterified GalA. As a result of the higher methoxylation degree (DM) of pectin becomes extra susceptible to base-catalyzed reactions rather than a low DM pectin. However, by acid hydrolysis (pH < 3.0), pectin hydrolyses with low DM are faster in comparison to pectin with high DM [20].

2.3 Enzymatic process

Wide range of enzymes used for the polymer degradation allowing region-selective depolymerization under mild conditions. So that, using enzymic degradation gain the interest of many researchers.

3. The pectinolytic enzymes

More than a century ago, DeBary at1886 showed pectinase’s importance as a virulence factor in the decomposition of pectin in the plant cell wall [21]. Then, these enzymes have been used for food processing at the domestic level at first, to use in the industrial sector in 1930. Till1960s, pectinases were practically used in fruit juice and wine for clarification [22]. Nowadays, pectinases have received attention worldwide as an eco-friendly biocatalyst that contains a 25% share in the global enzyme market for food and beverages [23, 24]. In 2016, the pectinases market reached 30.0 million $ and was estimated to rise to 35.5 million $ by 2021 [25]. Pectinolytic enzymes or pectinase enzymes are a group of complex enzymes that catalyze the degradation of pectin-containing substances. These enzymes account for 10% of the produced global industrial enzymes [1] and are secreted in plants, insects, Nematoda, protozoa, fungi, yeast, and bacteria [22]. Although plant and microorganisms are the major sources for pectinase enzyme generation, microbial sources (Fungi, yeast, and bacteria) have been selected to be the primary ones due to technical and commercial viability [26].

Pectinolytic enzymes have been classified according to three criteria [27, 28]:

• The used substrate (Pectin, pectic acid, or oligo GalA)

• The cleavage type (trans elimination or hydrolysis)

• The action mode (random cleavage-depolymerizing or endoliquifying enzymes, or end-wise cleavage endo or exo saccharifying enzymes.

For most enzymes, the type of cleavage is random (Endo) or terminal (Exo). The recent classification based on the action mode on pectin, preferred substrates, and products, Table 1, where these enzymes are classified into three types [30]:

E.C. suggested name	Common name	E.C. no.	Mode of action and cleavage	Catalytic reaction
PROTOPECTINASES			Cleavage of non-methylated molecules of galacturonate unit, hydrolyzing the glycosidic bond	Protopectin +H2O → Soluble pectin
ESTERASE ENZYMES
a) Methyl esterase	Pectin esterase	3.1.1.11	Random cleavage of the methyl ester group of galacturonate unit	Pectin +nH2O → Pectate+ Methanol
b) Acetyl esterase	Pectin esterase	3.1.1.11		Pectin +nH2O → Pectate+ n Acetate
DE-POLYMERASE
a) Hydrolases
I) Polygalacturonase (PG)	- Catalyze the hydrolytic cleavage of α-1, 4-glycosidic linkage in pectic acid
- Exo-PG	Pecate polymerase	3.2.1.67	Terminal cleavage from the non-reducing end of the polygalacturonic acid	Pectic acid+H2O → Mono-galacturonates
- Endo-PG	Pecate polymerase	3.2.1.15	Random cleavage of pectic acid	Pectic acid +H2O → Oligo-galacuronates
II) Polymethylgalacturonase (PMG)	- Catalyze the hydrolytic cleavage of α-1,4-glycosidic linkage in pectin
- Exo-PMG	Pectin polymerase		Terminal cleavage from the non- reducing end of pectin	Pectin +H2O → Methyl-mono-galacturonates
- Endo-PMG	Pectin polymerase		Random cleavage	Pectin +H2O → Oligo-methyl-galacturonates
b) Lyases
I) Polygalacturonate Lyase (PGL)	- Catalyze the cleavage of α-1,4-glycosidic linkage in pectic acid by trans-elimination forming unsaturated galacturonates.
- Exo-PGL	Pectate lyase	4.2.2.9	Cleavage of penultimate bonds from non-reducing end	Pectic acid → Unsaturated digalacturonates
- Endo-PGL	Pectate lyase	4.2.2.2	Random cleavage	Pectic acid → Unsaturated oligo-galacturonates
- Oligogalacturonate lyase	Pectate lyase	4.2.2.6	Terminal cleavage	Pectic acid → Unsaturated mono-galacturonate
II) Polymethgalacturonate lyase (PMGL)	Catalyzes cleavage of α-1,4-glycosidic linkage in pectin by trans-elimination forming unsaturated methyl galacturonates at the non-reducing end.
- Exo-PMGL	Pectin lyase		Terminal cleavage	Pectin→ Unsaturated methyl mono-galacturonates
- Endo-PMGL	Pectin lyase	4.2.2.10	Random cleavage	Pectin→ Unsaturated methyl oligo-galacturonates

Table 1.

Classification of pectinases enzymes [inspired from Parissa et al., 2011 [29]].

3.1 Protopectinase

Protopectinase, which catalyzes the solubilization of protopectin in the presence of water to release soluble pectin. The catalyzation process could occur through the reaction at sites having three or more non-methylated molecules of GalA, hydrolyzing the glycosidic bond [31]. According to the catalytic action, these enzymes are classified into two types:

1. Type-A reacts with the smooth region or inner site of insoluble protopectin. It is reported that, in the culture filtrate of many microbes as Trichosporon penicillatum SNO 3 (S-type), Galactomyces reessi (L-type), and Kluyveromyces fragilis IFO 0288 (F-type). F, S, and L types have the same molecular weight of about 30 kDa and are more optimum at pH 5.0. in addition, only F-type is an acidic protein, while others are basic in properties. These enzymes are responsible for a decrease in the viscosity with an enhanced rate of reduction for polygalacturonic acid in the reaction medium. Similarly, Bacillus subtilis IFO 3134 produced R and N-type with 35 and 43 kDa protein, respectively, and highly active at pH 8.0 and temperature 60 °C). These enzymes can help in the reaction of trans-elimination by breaking the glycosidic linkages at the protopectin [32] and categorized according to the pattern of action as endo (random) or exoenzymes (terminal).

2. Type B is responding at the site of polysaccharide chains or outside of the protopectin. It links the chain of polyglacturonic acid and the components of the cell wall [33]. It is isolated from Trametes sunginea (T-Type) and B. subtilis IFO 3134 (Ctype), having different molecular weights 55 and 30 kDa, respectively, and several isoelectric points (T: 8.1 and C: 9.0). The kind of these enzymes is abundantly found in agro-products as orange, lemon, hassaku, carrot, apple, burdock, radish, and sugar beet, acting on protopectin particularly [34].

3.2 Esterase

Esterase, which removes methoxyl and acetyl esters from pectin forming polygalacturonic acid. Where catalyzes the de-esterification of pectins. These enzymes from fungal originate work by arbitrarily eliminating the methyl groups via a multi-chain mechanism, while from plant source work by attacking either the next terminal to a free carboxyl group or the non-reducing-end and progressing linearly through a single-chain mechanism [35]. Esterase enzymes are producing mainly from microorganisms and have a negligible effect on the viscosity of a pectin solution without divalent cation as barium (Ba²⁺), calcium (Ca²⁺), and strontium (Sr²⁺). However, it is reported that the presence of Ca²⁺ ions releases a maximum effect at either small or large preparations [36].

Some of the purified esterase acting against the reducing end. While others are targeting the non-reducing-end of pectin. The molecular weight for these enzymes ranges from 22 to 90 kDa, referring to the difference of the protein confirmations. For the work efficiency of esterase, various ranges of pH (5-11) and temperature (40 – 70°C) should be applied. However, the product optimization of the fungal esterase has a lower pH value versus the bacterial one [37]. Depending on the functional group target, pectinesterase is classified into:

1. Methyl-esterase, which divides the methyl ester group of pectin. Freeing methanol and converting pectin into pectic acid or pectate via a single-chain mechanism. The chain length of pectin polymer is not reduced [38]. Various isoenzymes of pectin methylesterases are isolated and characterized from different sources, considering the functional group’s target [39]. Two types of pectin methylesterase A (PmeA) and B (PmeB), were isolated from Erwinia chrysanthemi [40] and E. chrysanthemi 3937 [41] and were well-studied. The PmeA acting functionally to be extracellular, while the PmeB enzyme acting on the outer membrane.

2. Acetyl-esterase, which catalyzes the hydrolysis of acetyl ester residues of pectin, forms acetate in pectic acid [42].

3.3 Depolymerase

Depolymerase, a range of depolymerizing enzymes degrade the pectic substance through cleaving of α-(1 → 4)-glycosidic bonds in DGalA units either by trans elimination or hydrolysis [43]. Split the -(1,4)-glycosidic bonds in pectins either by hydrolysis (polygalacturonase) or by transelimination (lyases).

1. Hydrolases include polygalacturonase (PG) and polymethylgalacturonase (PMG).

1. Polygalacturonases (PG), that split the glycosidic linkage in the presence of water molecules across the oxygen bridge. Forming a D-galacturonic acid monomer. The structure confirmation of these enzymes loses when it reacts with pectin, which may occur due to the presence of free carboxylic groups in the target molecules. The viscosity of the interaction solution reduces with an increase of reducing end-groups. PG is the most enzyme studied and industrially applied because of its depolymerization specificity via the hydrolysis process [44, 45]. Depending on the pattern of action, PG is categorized into:

• Exo-PG, which targets the terminal groups of the pectic molecule, lowering of chain length gradually.

• Endo-PG, that attacks all chain links arbitrarily, resulting in more incisive and faster consequences.

However, rhamnopolygalacturonase catalyzes cleavage within or at the non-reducing terminals of the rhamnogalacturonan core chains [46, 47]. Various microorganisms can produce PG with several biochemical properties and modes of action. Most PGs stimulate the hydrolysis rate at an optimum temperature ranging from 30 to 50°C with ideal pH that ranges from 3.5 to 5.5. It is reported that almost both exo-PG and endo-PG are synthesized in acidic conditions. While some exo-PG are produced at high basic pH (11.0) by certain bacterial and fungal species as Bacillus sp. KSM-P410, Bacillus licheniformis, and Fusarium oxysporum [48]. Whereas rhamno-PG is more efficient and stable at pH 4.0 and temperature 50°C. The molecular weight average for exo-PG and endo-PG is 38 – 65 kDa, while rhamno-PG is 66 kDa [49].

1. Polymethylgalacturonase (PMG) can catalyze the hydrolytic cleavage of α-1,4-glycosidic linkage in pectin. It is divided according to the action pattern into:

   • Exo-PMG, that targets the terminal groups of the non-reducing end of pectin, releasing methyl mono-galacturonate.

   • Endo-PMG, that attacks all chain links randomly. Resulting in more incisive and faster consequences of oligomethyl-galacturonates.

Lyases (trans eliminases), in which trans-eliminative breakdown for pectinate polymers or pectate through catalyze the Polygalacturonate depolymerization and pectin esterification, by splitting the C-4 of the glycosidic linkage followed by hydrogen removal from the C-5 releasing an unsaturated product with the unsaturated bond between C-4 and C-5. For activation, some cytoplasmic or intracellular lyases, need ions as Ni²⁺, Co²⁺, and Mn²⁺ [50].

According to the acted substrate, lyase is divided into two types [51]:

1. Polygalacturonate lyase (PGL) that requires Ca2+ ions for its activation. Used mainly in baby food products [52]. That classified into:

   • Exo-PGL, target the non-reducing terminal of pectic acid, releasing unsaturated di-galacturonates.

   • Endo-PGL, which works in an unsystematic cleavage fashion on the substrate, producing unsaturated oligogalacturonates.

   • Oligo-D-galactosiduronate lyase, which acts on the terminal position of unsaturated di-galacturonate, released initially by the pectate lyase action, forming mono-galacturonates [53].

2. Polymethylgalacturonate lyase (PMGL), which does not need any metal ions for their activation, although arginine (Arg 236) residues are found at the position of Ca2+ ion as observed in the pectate lyases [54]. Which categorized into:

   • Exo-PMGL, that degrades pectin through stepwise transeliminative cleavage, releasing unsaturated methylmonogalacturonates [55].

   • Endo-PMGL, which acts randomly on the pectin by cleaving α-1,4-glycosidic linkages, producing unsaturated methyloligogalacturonates.

Overall, pectin lyases originated mainly from microorganisms, which lead to change in the biochemical properties according to each microbe. These enzymes working efficiently in the temperature range 40-50°C, and alkaline pH 7.5-10.0. The molecular weight of lyases is ranging from 22-90 kDa, while PMGL reached 89 kDa from Aureobasidium pullulans LV-10 and 90 kDa from Pichia pinus. Whereas the molecular weight for PGL of 55 and 74 kDa was reported in Yersinia enterocolitica, and Bacteroides thetaiotaomicron, respectively. The point of isoelectric for some lyases are ranged from 5.2 to 10.7. While others are still unexplored [56]. Many enzymes act in the adjacent chains of RGI and RGII as exogalactanase, endogalactanase, α- and β-galactosidase, α-L-arabinofuranosidase, exoarabinase, and endoarabinase [57].

4. The microbial producers for pectinases

Pectinolytic enzymes are produced by various plants and microorganisms, while animal cells can’t [58]. Several strains of fungi, yeast, and bacteria are producing different types of pectinolytic enzymes. While the fungal sources provide the largest variety of bulk commercial enzymes and have a broad diversity of applications, well documented by GA [59].

4.1 Fungi

Three fungal classes get the most attention in SSF:

1. Phycomycetes, as genera of Mucor,

2. Ascomycetes, as genera Aspergillus,

3. Basidiomycetes, as genera white-rot fungi [60].

Many fungal strains can produce various types of pectinase enzymes. The produced metabolites by Aspergillus niger are safely used and involved in GRAS (Generally Regarded As Safe), so that it is the most broadly strain used in industries [61]. A. niger can produce various pectinases involving esterase, PGL, and PMGL. Currently, the enzymes of A. niger are used in wine industries and fruit juice [62]. It is reported that almost all the fungi pectinases have acidic nature, so it is applicable to work only in acidic conditions [63]. However, the pectinase production by Aspergillus strains produced higher via SSF than in SmF [64]. Wherever the most commercial pectinases are produced from fungal sources in the industry by Trichoderma and Aspergillus [65].

4.2 Yeast

In 1951, Luh and phaff produced for the first time endoPG from yeast [66]. They identified four species of pectolytic yeasts: Saccharomyces fragilis, Torulopsis kefir, Candida pseudotropicalis (all later renamed as Kluyveromyces marxianus), and Saccharomyces thermantitonum (reclassified as Saccharomyces cerevisiae) [67]. Pectolytic yeasts can be producing PG, lyase, or esterase, depending on the pH, temperature conditions, and substrate availability. For example, Candida, Saccharomyces, and Kluyveromyces can be producing PG (mainly endo-Polygalacturonase), whereas Rhodotorula can be releasing both pectin esterase and PG [68].

S. cerevisiae is considered the most experienced yeast. Initially, it had believed that S. cerevisiae is free of pectolytic enzymes [69]. However, some strains have been shown since then reduction ability for pectin [70]. PG Activity is the main pectolytic activity in S. cerevisiae and was reported in several strains [71]. It is noted that most pectolytic activities have been described in the indigenous yeasts [72]. These yeasts have been mainly discovered during different fermentation [73] or through the clarification and pressing of concentrated juices [74].

4.3 Bacteria

Chawanit Sittidilokratna et al. [75] performed screening of pectinase-producing bacteria and the effectiveness evaluation for bio-pulping. At first, Pectinolytic bacteria were screened from six identified and 118 unknown isolates. Twelve strains gave positive results, including three of Erwinia carotovora subsp. carotovora, two of Erwinia chrysanthemi and seven of Bacillus sp. Crude pectinases were prepared from the selected strains. Then, investigate the activity of three types of pectinase (PG, pectate lyase, and pectin lyase). The results showed the highest PG production from Bacillus sp. strain N10 and E. chrysanthemi strain N05. N10 was isolated from paper mulberry bark, while N05 was isolated from onion [76].Bacillus licheniformis KIBGE-IB21 was isolated from rotten vegetables and produce pectinase at certain conditions [77]. However, the commonly used bacteria for pectinase production are Aeromonas caviae, B. licheniformis, and Lactobacillus [32].

5. Applications of pectinase

Over the years, pectinases have been used in several applications as plant fiber processing, textile, coffee, and tea fermentation, industrial wastewater treatment, oil extraction, wine-making, wood preservation, pectic pre-treatment, degumming and retting of fibers, protoplast formation [78], and fruit industry [79]. With increased knowledge and understanding of the mechanism of pectin-degrading enzymes, pectinases have made their way into other biotechnological processes as purification of plant viruses [80] and paper and pulp making [81]. Today, pectinases represent almost 5% of global enzyme sales with approaches 35 million dollars of the industrial market [82].

The biotechnology application for these enzymes has expanded in recent years. These occurred especially in food and related industries, to increase the product quality, product stabilization, increase the efficiency of extractive processing, improve the flavor and by-product utilization [83], Figure 3.

Figure 3.

Applications of pectinases in the various biotechnological and industrial sectors with the potential purpose [inspired by Garg et al., 2016 [84]].

5.1 Juice processing

Pectinases use in the fruit juice and wine industry since the 1930s [85]. That occurred for reduction maceration and viscosity which contribute to increasing the obtained press juice to the de-peel pulps. Similarly, reducing the viscosity of fruit drinks [86]. Fogarty and Kelly stated that pectinases use in wine clarification [87]. The juice industries produced commercially three different types of juices:

1. Sparking clear juices,

2. cloudy juices, and

3. unicellular product.

So, the objective aim of using pectinase in the three types is different, Table 2.

The juice industries produced	The aim of using pectinase	Examples
Sparking clear juices	To increase the obtained yield during the pressing	Manufacture of apple juice as natural, unclarified, unfiltered, and pulp-containing juice
	To straining of the juices
	To remove the suspended particles
	In apple: mostly used	The production of French cider apples [89]
	To depolymerize the highly esterified pectin [88]
	In cider: enhances the good fermentation to produce high-quality juices and aromatic cider
Cloudy juices	To stabilize the cloud of citrus juices, purees, and nectar through the high levels of PG that have been involved, achieving high carotene and dry matter content of the product	Pectinex Ultra Sp-L is a commercial enzyme used specifically for the preparation of carrot puree [90].
Unicellular product	To transform the organized tissue into suspension of intact cells through maceration process, for nectar and pulpy juices [91]	Such as baby foods, yogurt, and puddings

Table 2.

The objective aim of using pectinase in the industrially produced juice with examples.

5.2 Wine processing

To clarify wine, the enzymes added before fermentation of white wine musts. Musts are made from pressed juice without any skin contact to rapid clarification. Thermovinification technology, during the grape mash heating for few hours releasing large amounts of pectin. So that, adding pectinases is necessary to the heated mash that leading to reduce the juice viscosity. Adding pectolytic enzymes enhances the color, which probably refers to the breakdown of the cell structure, which allows escaping more readily pigments (anthocyanins) [92].

The treated wines enzymatically showed more stability with lowering the filtration time comparing with the untreated wines [93]. The enzymatic treatment raises the levels of alcohol production in fermented grape must, observing an increase in 2-phenyl ethanol and iso-amyl alcohol and a decrease in concentrations of n-propanol [94]. Servili et al. [95] stated that adding pectinases in the wine-making process leads to raising the levels of methanol in wine due to the pectinesterase activity. The methanol concentrations should be regulated due to the toxicity of methanol, therefore in a commercial mixture of pectin esterase should be at lower concentrations [96].

Reddy and Reddy [97] reported that the combined effect of both fermentation by yeast cultures and pectinases treatment on Alcohol production. Consequently, the yield of juice increase when treated with 0.6% of enzyme concentration and conduct the fermentation at Ph 4.5 and 30°C for 12 h.

The most functions of pectinolytic proteins within the wine-making preparation are to bolster the extraction process, maximize juice surrender, encourage filtration and escalating the enhance and color [98]. Enzymatically treated wines appeared more soundness with decreased filtration time in comparison to control wines [99]. Treatment of macerated natural products with pectinolytic chemicals, sometimes recently the expansion of inoculum come about in progressed characteristics of wine [100]. Clarification of must earlier to the onset of alcoholic aging to moves forward the tactile properties of white wine [101]. Bosso [94] detailed the higher levels of liquor generation in matured grape must be pretreated with pectolytic proteins and watched increment in iso-amyl liquor and 2-phenyl ethanol, while a diminish in n-propanol concentrations, Reddy and Reddy [102].

5.3 Textile industry

To treat the natural fibers such as ramie fibers and linen, sometimes pectinases are used to remove the gum before starting the textile making [103]. The old-age practice of retting by textile fibers as hemp, flax, and jute are prepared with the pectinases of certain microorganisms [104]. The traditional methods are using caustic alkaline solution (3-6% aqueous sodium hydroxide) at high temperatures to fulfill the consistent dyeing and finishing. This process can degrade the cotton fiber, needs large amounts of water for rinsing after completing the process, and high energy, releasing a toxic waste product that can damage the environment. For that, use a combination of pectinases and xylanase ensure the low discharge of chemical waste in the environment, producing lower odor, and improving the safety of textile workers with the fabric quality.

To remove the non-cellulosic impurities as pectin, protein, and fats from fiber using pectinase through a novel process called bioscouring. This process is conservation the energy, eco-friendly, and significant results in fiber damage without effects on the cellulose backbone [105].

5.4 Develop seed germination and protoplast formation

Das and Baruah mentioned that Trichoderma reesei isolated from areca nut husk released high polygalacturonase. The areca nut germination was better when treated with cell-free preparation of polygalacturonase enzyme than with distilled water [106].

For protoplast formation of the plant cell, enzymatic and mechanical methods have been used. In the mechanical method, the protoplast formation involved separating plasmolyzed tissue with a sharp-edge knife, releasing the protoplast through de-plasmolysis. The mechanical method is limited with the low yield of the protoplast. Hence, the enzymatic method is mostly used for this purpose. A combination of both pectinases and cellulases has been used for the protoplasts formation from every plant tissue that has not lignin [107]. Pectin-oligosaccharides (POS) have different biological activates involving plant growth promotion and antimicrobial agents. POS is not digestible by humans, where fermented by microbial flora as lactobacillus species and bifidobacteria sp., stimulating their growth. POS is released by chemical and enzymatic degradation of pectic substrates. In enzymatic degradation, pectinase has been applied to break down the α −1, 4 glycosidic linkages of pectin polymer to galacturonic acids and pectin oligosaccharides [108]. Immobilized pectinase has been used for controlled enzymatic reaction with specific characterization and defined range of polymerization degree, avoiding producing large amounts of monosaccharides in batch production [109].

5.5 purification of plant viruses

The highly pure preparation of viruses is necessary for studying their physical, chemical, and biological properties. Various methods of purification can be selected according to the virus type. Pectinase’s enzymes can be used to liberate the virus from the phloem tissues [110].

5.6 Coffee and tea processing

Marcia Soares et al. reported that pectolytic enzymes were used to hasten the removal of the jelly that surrounds the coffee cherry in the processing of green coffee beans, increasing the inferior quality of coffee beans [111]. Using pectinases develops the coffee quality also through converting the mucilage into sugars [112]. Similarly, pectinases treatment enhances the fermentation of tea by breaking down the pectin found in the cell walls of tea leaves, also destroying the foam-forming of the powder tea. It is reported that the used pectinases in tea process from alkaline fungal pectinase type [113].

So, the enzyme enhances the development of color, aroma total soluble solids, dry matter content, and active ingredients as theaflavin, thearubigin, caffeine, and high polymerized substances [114]. Masoud and Jespersen used PG in the fermentation of Coffea arabica produced by Pichia [115]. Adding these strains as started cultures in the coffee fermentation process help in mucilage degradation and the biological control of ochratoxin A which producing by Aspergillus ochraceus during the fermentation [116].

5.7 Oil extraction

Pectinases are applied in disrupting gels to assist the recovery of oils [117]. Vegetable oils of sunflower, olive, palm, coconut, or canola are obtained by extraction with organic solvents as hexane, a potential carcinogen [118]. With pectinase from alkaline type is preferable, lets the extraction of vegetable oils in an aqueous process through cell wall components degradation. Nowadays, the utilize of enzyme preparations involving hemicellulases, cellulases, and pectinase has raised the concentration of phyto-compounds as an antioxidant, polyphenols, essential proteins, lipophilic compounds, and vitamin E content, enhancing its organoleptic quality and storage stability [119, 120]. In 1992, Servili et al. stated that using an endo-PG extract from Cryptococcus albidus improved the olive oil extraction by adding the enzyme during the grinding process of olive [121]. Moreover, citrus oil such as lemon oil can be extracted by pectinases that destroy the emulsifying properties of the pectin of citrus peel [122]. Pharmaceutically, pectinases can improve the oil production from the medicinal plants to use as a treatment for various diseases involving depression, cancer, anxiety, microbial infectious ailments, and wound healing. However, pectinases will be contributing to the cosmetic and perfume industries. Using organic solvent in the extraction process might damage some critical functional groups. So, use pectinases during the extraction process will avoid that by destroying the emulsifying properties of pectin and promote the liquefaction of the cell wall components, releasing a better volume of products [123].

5.8 Assist in cellulose degradation

Pectinase plays a critical role by raising the access of cellulases to their substrates [124]. Spagnulo et al. [125] and Wang and Chang [126] stated that pectinase became the most important enzyme, since hydrolyzing the pectic surface of the lignocellulosic materials. The degradation of cellulose and hemicellulose was favored by the respective enzymes. Thus, pectic enzymes treatment is used for softening the wood-producing commercial softwoods [127].

5.9 Animal feed processing

Using various enzymes in the animal and poultry feed started in the 1980s with adding β-glucanase into barley and then wheat. After that, the xylanase enzyme was tested and achieve the best action in this case. Usually, the preparation of feed enzymes is a cocktail of multi-enzymes containing proteinases, amylases, pectinases, xylanases, and glucanases. Adding enzymes to the animal feed is reduces viscosity, increases nutrient absorption, liberates the blocked nutrients by this fiber, and reduces the feaces amount [128]. Spraying feed with enzymes just before providing the feed, increases the food flexibility management, improving the feed digestibility through various mechanisms as direct hydrolysis, palatability improvements, changes in gut viscosity [129]. Consequently, enzymes supplementation to the animal feed improves significantly the digestive process, weight gain, feed conversion, and digestible energy intake [130].

5.10 Papermaking and pulp industry

With the biotechnology advancement increased dependence of pulp and paper industries. Many enzymes are used almost in papermaking for bio-bleaching and papermaking, such as pectinases, mannanase, and α- galactosidase [131]. Pectinase can degrade the galacturonic acid polymer, lowering the cationic demand of pectin solutions and the filtrate from peroxide bleaching of thermomechanical pulp [132]. Bio-bleaching of eucalyptus kraft pulp obtained by a mixture of alkaline pectinase and xylanases from Streptomyces sp. QG-11–3 [133], Bacillus subtilis, Bacillus pumilus [134], and S. cerevisiae [135]. Bio-bleaching results in less requirement of chemical-bleaching, giving the same pulp brightness, enhance the physical properties of the paper sheet, and reduce the organochlorine compounds in the effluent. Liu et al., lowering the used pectin concentration in bio-bleaching by using cross-linked chitosan beads, leading to a sharp decrease in pectin molecular weight and cationic demand of pectin solution [136].

5.11 Wastepaper recycling

The major problem in wastepaper recycling is deinking process that needs a large amount of environmentally damaging chemicals. Bio-deinking using enzymes is less polluting, gives better quality, and is energy-saving. Pectinases, cellulases, hemicellulases, and ligninolytic enzymes are used for bio-deinking. These enzymes alter bonds near the ink particle, removing the ink from the fiber surface. Then, the resulting ink is removed by washing or floatation [137, 138]. A combination of pectinase and xylanase has been used for bio-deinking of school wastepaper [139]. Hence, bio-deinking lowers the values of biological and chemical oxygen demand in the effluent, reducing the treatment cost for wastewater to be environment friendly [140].

5.12 Wastewater treatment

The wastewater of the vegetable food industries contains pectic substrates. The typical treatment of its wastewater involves multiple steps that are high in cost, have longer times, and pollutes the environment [141]. Thus, using alkaline pectinases to remove the pectic substrate is a good alternative, cost-effective, and eco-friendly method, easing the decomposition by activated sludge treatment [142, 143].

6. Conclusions

The microbial degradation of pectin through various enzymes gains the interest of many researchers. In this chapter, microbial degradation for pectin has created an irreversible renaissance in the current era of innovation and the latest research to find the new utility of microorganisms and their products, a pure fact. This chapter showed the pectin structure generally with its different forms. Then, several methods have been utilized in the pectin depolymerization focusing on the enzymatic process. Followed by, presented the different kinds of the used enzymes that differ according to the pectin forms. After that, the chapter displayed information about the various strains of microbes that can produce pectinolytic enzymes. Finally showed the importance of these enzymes and their industrial applications.