Microbial degradation of Pectin – Enzymes, Steps, Mechanisms

Pectin is a carbohydrate that occurs naturally in plant cell walls. Long chains of galacturonic acid molecules are joined together to form this complex polysaccharide. Apples, citrus fruits, and berries are good sources of pectin since it is a natural gelling factor in food.

Pectin’s gel-forming properties make it ideal for use in the preparation of jams, jellies, and other fruit preserves when combined with sugar and acid. Certain dairy products, drinks, and food sources of dietary fiber all use pectin in their manufacture.

Pectin comes in a variety of forms, each having its own methylation profile (a chemical modification). Low-methoxyl pectin takes less sugar and acid to make a gel than high-methoxyl pectin and is typically used in sugar-reduced or no-sugar-added jams and jellies.

What is Pectin?

• Pectin is a heteropolysaccharide, a structural acid found in the primary lamella, the middle lamella, and the cell walls of terrestrial plants. Henri Braconnot isolated and described galacturonic acid (a sugar acid derived from galactose) as the primary chemical component of pectin in 1825.
• Pectin is a white-to-light-brown powder manufactured from citrus fruits for use as an edible gelling agent, particularly in jams and jellies, dessert fillings, medicines, and sweets; as a food stabilizer in fruit juices and milk drinks; and as a source of nutritional fiber.
• Pectin is formed of complex polysaccharides that are present in the primary cell walls of a plant, and are plentiful in the green sections of terrestrial plants.
• Pectin is the major component of the middle lamella, where it binds cells. Golgi apparatus-produced vesicles deposit pectin into the cell wall through exocytosis. Pectin varies in quantity, structure, and chemical composition between plants, within a plant through time, and between plant components.
• Pectin is an essential cell wall polymer that enables the elongation of the primary cell wall and plant development. Pectin is degraded during fruit ripening by the enzymes pectinase and pectinesterase, which causes the fruit to soften as the central lamellae break down and cells become separated.
• A similar process of cell separation caused by the breakdown of pectin occurs in the abscission zone of the petioles of deciduous plants at leaf fall.
• Pectin is a natural component of the human diet, but its contribution to nutrition is negligible. If approximately 500 g of fruits and vegetables are consumed daily, the estimated daily pectin intake from fruits and vegetables is approximately 5 g.
• In human digestion, pectin binds to cholesterol in the gastrointestinal stream and traps carbs to limit glucose absorption. Pectin is thus a soluble dietary fiber. Pectin was found to increase the incidence of diabetes in non-obese diabetic (NOD) mice.
• Due to the degradation of natural pectin (which is esterified with methanol) in the colon, the content of methanol in the human body increased by as much as an order of magnitude after eating fruit, according to a study.
• Pectin has been discovered to play a role in mending the DNA of certain types of plant seeds, particularly those of desert plants.
• Pectinaceous surface pellicles, which are rich in pectin, produce a mucilage layer that helps the cell repair its DNA by retaining water.
• Intake of pectin has been demonstrated to marginally (3–7%) reduce blood LDL cholesterol levels. Pectins derived from apples and citrus fruits were more effective than those derived from orange pulp fibre.
• The mechanism appears to be an increase in intestinal viscosity, which leads to a reduction in cholesterol absorption from bile or diet. In the large intestine and colon, bacteria breakdown pectin and produce health-promoting short-chain fatty acids (prebiotic effect).

Properties of pectin

• Pectin is a heteropolysaccharide present in the middle lamella and cell wall of all higher plants. Pectin consists of two primary components; its primary structure is homogalacturonan (HG).
• HG is a linear polymer of D-galacturonic acid with α-(1–4) linkages. It is generated by α 1-4-linked galacturonic acid (GalA).
• The carboxyl group of galactoronic acid is the most vital characteristic of HG. The HG backbone can be disrupted by neutral sugar side chains to form the rhamnogalacturon I (RGI) and rhamnogalacturon II (RGII) regions.
• RGI is composed of 1, 4-linked D GalA and 1, 2-linked α L rhamnose, along with neutral sugars. The RGI region is thought to be the attachment point for neutral sugars (galactose, arabinose, glucose, and other sugars).
• Moreover, the other chain side contains arabinogalactan (I and II) and xylogalacturons, which are predominantly attached to protein. The resulting connections can be acid and alkaline stable.
• RGI region consists of repeated disaccharides of α-D-galacturonic acid and (1,2)-α—L-rhamnose as backbone combined with neutral sugars primarily galactose, D-xylose, and L-arabinose.

Tyeps of pectin

There are several types of pectin, which differ in their chemical composition and gelling properties. Here are some of the main types:

1. High-methoxyl pectin (HM pectin): This type of pectin has a high degree of esterification, meaning that many of the carboxyl groups on the galacturonic acid units are methylated. HM pectin requires the presence of sugar and acid to form a gel, and the amount of sugar required depends on the degree of esterification. HM pectin is commonly used in high-sugar jams and jellies.
2. Low-methoxyl pectin (LM pectin): This type of pectin has a lower degree of esterification than HM pectin, which means that it requires less sugar and acid to form a gel. LM pectin is often used in low-sugar or no-sugar-added jams and jellies. LM pectin may also be used in dairy products and other applications where a firm gel is desired.
3. Amide pectin: This type of pectin has amide groups instead of ester groups, which gives it different gelling properties than traditional pectin. Amide pectin forms a gel at a higher pH and with less sugar than HM or LM pectin, which makes it useful for applications such as fruit fillings and toppings.
4. Amidated low-methoxyl pectin (ALM pectin): This type of pectin is a combination of amide and low-methoxyl pectin. It can form a gel at a lower pH than amide pectin and requires less sugar than LM pectin. ALM pectin is often used in dairy products and low-pH applications such as fruit-based beverages.
5. Modified pectin: Pectin can be modified through chemical or enzymatic processes to alter its properties. Modified pectin may have improved gelling properties, stability, or solubility, depending on the modification. Modified pectin may be used in a variety of applications, including dairy products, beverages, and baked goods.

Importance of pectin substances

Pectin is an important substance with various applications in the food industry and beyond. Here are some of its key roles and benefits:

1. Gelling agent: Pectin is commonly used as a gelling agent in food, particularly in the production of jams, jellies, and fruit preserves. When heated with sugar and acid, pectin can form a gel that gives these products their characteristic texture.
2. Stabilizer: Pectin can help stabilize food products, particularly acidic ones such as yogurt, by preventing separation or curdling.
3. Dietary fiber: Pectin is a type of dietary fiber that can have beneficial effects on digestion and gut health. It can help regulate bowel movements and lower cholesterol levels.
4. Health benefits: Some studies suggest that pectin may have other health benefits, such as reducing inflammation, improving blood sugar control, and boosting immunity.
5. Industrial applications: Pectin has applications beyond the food industry, such as in the production of pharmaceuticals, cosmetics, and textiles.

Overall, pectin is a versatile substance with a wide range of applications and benefits.

Structure of pectin

• Galacturonic acid is abundant in pectins, commonly known as pectic polysaccharides. Within the pectic category, numerous different polysaccharides have been found and characterized. Homogalacturonans are α-(1–4)-linked D-galacturonic acid chains.
• Substituted galacturonans are distinguished by the presence of saccharide appendant residues (such as D-xylose or D-apiose in the cases of xylogalacturonan and apiogalacturonan, respectively) branching from a backbone of D-galacturonic acid residues.
• Rhamnogalacturonan I (RG-I) pectins comprise a backbone of the repeating disaccharide: galactose. 4)-α-D-galacturonic acid-(1,2) (1,2) -α-L-rhamnose-(1. Several rhamnose residues branch out into various neutral sugar sidechains. The neutral sugars consist primarily of D-galactose, L-arabinose, and D-xylose, with the types and amounts variable depending on the pectin source.
• Rhamnogalacturonan II (RG-II), a rare, complex, highly branched polysaccharide, is an additional structural type of pectin.
• Some publications classify Rhamnogalacturonan II as a member of the category of substituted galacturonans since its backbone consists solely of D-galacturonic acid units.
• Depending on its origin and extraction methods, the molecular weight of isolated pectin ranges from 60,000 to 130,000 g/mol.
• In nature, approximately 80% of galacturonic acid’s carboxyl groups are esterified with methanol. Its fraction is lowered to a varied degree after pectin extraction. Pectins are categorized as high- versus low-methoxy pectins (short HM-pectins against LM-pectins), depending on whether or not more than fifty percent of the galacturonic acid is esterified.
• The ratio of esterified to non-esterified galacturonic acid determines the behaviour of pectin in food applications – HM-pectins can form a gel under acidic conditions in the presence of high sugar concentrations, while LM-pectins form gels by interaction with divalent cations, particularly Ca2+, according to the idealized ‘egg box’ model, in which ionic bridges are formed between calcium ions and the ionised carboxyl groups of the galacturonic acid.
• Individual pectin chains are held together by hydrogen bonds and hydrophobic interactions in high-methoxy pectins with a soluble solids content greater than 60% and a pH between 2.8 and 3.6. These linkages are formed when sugar binds water, causing pectin filaments to adhere.
• These molecules construct the macromolecular gel by forming a three-dimensional molecular net. Low-water-activity gel or sugar-acid-pectin gel describes the gelling mechanism.
• Low-methoxy pectins require calcium to form a gel, but they do so at lower soluble solids and a higher pH than high-methoxy pectins. Typically, low-methoxy pectins create gels with a pH range between 2.6 and 7.0 and a solubility range between 10 and 70%.
• The non-esterified galacturonic acid units may be free acids (carboxyl groups) or sodium, potassium, or calcium salts. The salts of partially esterified pectins are referred to as pectinates; if the degree of esterification is below 5%, the salts are referred to as pectates; the insoluble acid form is pectic acid.
• Several plants, including sugar beet, potatoes, and pears, also contain pectins with acetylated galacturonic acid in addition to methyl esters. Pectin’s stabilizing and emulsifying properties are enhanced by acetylation, but gel formation is inhibited.
• Pectin has been altered to become amidated pectin. Some galacturonic acid is transformed with ammonia to carboxylic acid amide in this step. These pectins are more tolerant of the various calcium amounts that may be encountered during use.
• Thiolated pectin has significantly enhanced gelling characteristics due to the ability of this thiomer to generate disulfide bonds. This high gelling capabilities are beneficial for a variety of pharmaceutical and food industry applications.
• For the preparation of a pectin-gel, the materials must be heated to dissolve the pectin. The formation of a gel commences upon cooling below the gelling temperature. Syneresis or a granular texture occur from excessive gel formation, whereas overly soft gels are produced by insufficient gel production.
• Amidated pectins have the same properties as low-ester pectins, but require less calcium and are more tolerant of excess calcium. In addition, amidated pectin gels are thermoreversible; they can be heated and then resolidify upon cooling, whereas traditional pectin gels stay liquid after chilling.
• Low-ester pectins gel at a lower temperature than high-ester pectins. Nonetheless, when the degree of esterification decreases, gelling interactions with calcium rise. Similarly, lower pH levels or higher soluble solids (mostly sugars) enhance gelling rates. Hence, suitable pectins can be used for jams, jellies, and confectionery jellies with a higher sugar content.

• Pectin is a complex heterogeneous structural polysaccharide that constitutes a significant portion of the primary cell walls of all terrestrial plants. Pectin is a galacturonic acid-rich glycan matrix that attaches and cross-links the load-bearing cellulosolic and hemicellulosolic fibers of the cell wall.
• There are three types of pectic species that differ in the abundances and chemistry of their respective monosaccharide subunits: rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan.
• Homogalacturonan is the principal target of the enzymes considered in this study; hence, it will be referred to as “pectin” below.
• At its most fundamental level, pectin consists of α-1,4-linked galacturonic acid subunits. Due to its distinctive axial C-4 structure, this connection generates an accordion-like shape between adjacent residues.
• Depending on the degree of hydration and the presence of cations, the total fiber can adopt either a 21 or 31 helix structure. Variability in the possible three-dimensional structure of pectic fibers was supported by the galacturonate pentasaccharide-Pel cocrystal structure, which revealed that the substrate consisted of a mixture of 21 and 32 helical conformations.
• Heterogeneity in the chemical composition of pectin can be caused by xylose decorations at C-3, methoxyl and acetyl group esterifications at C-6 and C-2/C-3, respectively, and the presence of divalent cations like calcium.
• Different structural and functional features of the polysaccharide are related with these chemical changes. Specifically, methylesterification of the C-6 uronate group enhances “gellation” by neutralizing the negative charges of monosaccharide subunits and permitting a more compact packing of pectic chains.
• During tissue remodeling and pectin degradation, the enzymatic removal of methoxyl groups restores the intrinsic charges on the carboxylates, resulting in the conversion of pectin to polygalacturonate. Numerous outstanding reviews provide additional information on the chemistry of pectin structure.

Pectin Substrates

Pectic compounds are complex glycosidic macromolecules with a large molecular mass that are present in higher plants. They are found in the primary cell wall and make up the majority of the middle lamellae, a thin extracellular adhesive layer that forms between the walls of adjacent young cells. In short, they contribute significantly to the structural integrity and cohesion of plant tissues. There are three primary categories of pectic polysaccharides that contain D-galacturonic acid to varying degrees.

1. Homogalacturonan (HG)

• D-galacturonic acid can be converted into a linear polymer called HG, which can then be acetylated and/or methyl esterified. One name for these is “pectin smooth regions.”
• Pectin contains at least 75% of its carboxyl groups methylated, while pectinic acid has less than 75%, and pectic acid or polygalacturonic acid has no methyl esterified carboxyl groups, leading to three distinct classes of the molecule. Pectin is often used interchangeably with other pectic substances.

2. Rhamnogalacturonan I (RGI)

• RG I is composed of the disaccharide rhamnose-galacturonic acid, which repeats. Both galacturonic residues are able to transport side chains of neutral sugars, including galactose, arabinose, and xylose .

3. Rhamnogalacturonan II (RGII)

• Despite its name, RGII is a homogalacturonan chain with galacturonic residues connected to complicated side chains. Vincken and colleagues have presented a structural model for the pectin molecule in which HG and RGII are long side chains of the RGI backbone.
• Each RG chain is also known as a hairy region of the pectin molecule. In unripe fruit, pectin exists as a water-insoluble pectic molecule called protopectin, which is attached to cellulose microfibrils and gives cell walls their stiffness.
• During ripening, fruit enzymes break the pectin backbone and side chains, resulting in a more soluble molecule. When parts of HG are cross-linked to form a three-dimensional crystalline network that traps water and solutes, pectic subsances tend to form a gel structure.
• Temperature, pectin type, esterification degree, acetylation degree, pH, sugar and other solutes, and primarily the interaction between calcium ions and pectin unesterified carboxyl groups determine gelling qualities.
• In high-ester pectins, the junction zones are generated by the cross-linking of HG via hydrogen bridges and hydrophobic interactions between methoxyl groups, both of which are facilitated by a high sugar content and a low pH.
• Pectic polysaccharides have been utilized as bioactive food additives and as detoxification agents. It is a suitable dietary supplement for infants.

What are Pectinases?

Pectinases are a group of enzymes that break down pectin, a complex carbohydrate found in the cell walls of plants. Pectinases are produced by various microorganisms, including fungi and bacteria, and are also found in some plants.

There are several types of pectinases, each with a different function:

1. Pectinesterases: These enzymes cleave the ester bonds in pectin, releasing methanol and creating free carboxyl groups. This can affect the gelling properties of pectin.
2. Polygalacturonases: These enzymes break down the glycosidic bonds in the linear chains of pectin, causing it to break down into smaller fragments.
3. Pectin lyases: These enzymes cleave the backbone of pectin by breaking the α(1-4) linkages between galacturonic acid units. This can also cause pectin to break down into smaller fragments.
4. Rhamnogalacturonases: These enzymes break down rhamnogalacturonan, a type of pectin that contains additional sugars such as rhamnose and galactose.

Pectinases have various industrial applications, particularly in the food industry. They are used to extract juice from fruits, to clarify fruit juices and wines, and to break down pectin in the production of jams, jellies, and other fruit products. Pectinases are also used in the production of textiles, paper, and other industrial products.

Microorganisms involved in pectin degradation – pectinolytic microorganisms

Pectinolytic microorganisms are microorganisms that are capable of breaking down pectin, the complex carbohydrate found in the cell walls of plants. These microorganisms play an important role in the decomposition of plant material in nature, as well as in various industrial processes.

Here are some of the main types of pectinolytic microorganisms:

1. Fungi: Many fungi are capable of producing pectinases, including species of Aspergillus, Penicillium, and Trichoderma. These fungi are often used in the production of pectinases for industrial applications.
2. Bacteria: Several types of bacteria are known to produce pectinases, including species of Bacillus, Clostridium, and Pseudomonas. Some of these bacteria are commonly found in soil and water, while others are used in industrial processes such as the production of fruit juices and wines.
3. Yeasts: Some yeasts are capable of producing pectinases, including species of Candida and Saccharomyces. These yeasts may be used in the production of fermented foods and beverages.
4. Algae: Some species of algae are capable of producing pectinases, including species of Chlorella and Chlamydomonas. These algae may be involved in the decomposition of plant material in aquatic environments.

Pectinolytic microorganisms are important for the degradation of plant material in nature, as they help to break down the tough cell walls of plants and release nutrients back into the environment. They are also important for various industrial processes, particularly in the food and beverage industry, where they are used to break down pectin in the production of fruit products such as juices, jams, and jellies.

Enzymes involved in the degradation of pectin – Pectinolytic enzymes

Pectinases are an enzyme group that catalyzes pectic material breakdown through depolymerization (hydrolases and lyases) and deesterification (esterases) processes. The well-known pectinolytic enzymes are homogalacturonan degrading enzymes.

1. Protopectinases

• Protopectinases solubilize protopectin generating highly polymerized soluble pectin. They are categorized into two types: one reacts with the polygalacturonic acid area of protopectin, A type; the other with the polysaccharide chains that may connect the polygalacturonic acid chain and cell wall elements, B type.

2. Pectin Methyl Esterases (PME)

• Pectin methyl esterase or pectinesterase (EC 3.1.1.11) catalyzes deesterification of the methoxyl group of pectin generating pectic acid and methanol.
• The enzyme operates preferentially on a methyl ester group of galacturonate unit close to a non-esterified galacturonate unit.
• It operates before polygalacturonases and pectate lyases which need non-esterified substrates. It is classified into carbohydrate esterase family 8.

3. Pectin Acetyl Esterases (PAE)

• Pectin acetyl esterase (EC 3.1.1.-) converts pectin’s acetyl ester into pectic acid and acetate. It belongs to families 12 and 13 of carbohydrate esterases.

4. Polymethylgalacturonases (PMG)

• Polymethylgalacturonase catalyzes the hydrolytic breakage of a-1,4-glycosidic linkages in the pectin backbone, preferably highly esterified pectin, resulting in the formation of 6-methyl-D-galacturonate.

5. Polygalacturonases (PG)

• Polygalacturonase catalyzes the hydrolysis of a-1,4-glycosidic bonds in polygalacturonic acid to generate D-galacturonate. It belongs to family 28 of glycosyl-hydrolases.
• Both groups of hydrolase enzymes (PMG and PG) are endo- and exo-active. Endo-PG (EC 3.2.1.15) and endo-PMG catalyze random substrate cleavage, whereas exo-PG (EC 3.2.1.67) and exo-PMG catalyze hydrolytic substrate cleavage at the nonreducing substrate end, yielding monogalacturonate or digalacturonate in some situations.
• Fungi are the primary producers of hydrolases, which are more active in acidic or neutral environments at temperatures between 40 and 60 degrees Celsius.

6. Pectate Lyases (PGL)

• Pectate lyase cleaves glycosidic bonds on polygalacturonic acid preferentially, producing an unsaturated product (Delta-4,5-D-galacturonate) via a transelimination process. Ca2+ ions are an absolute need for PGL.
• Chelating substances such as EDTA substantially reduce the enzyme’s activity. Pectate lyases are categorized as endo-PGL (EC 4.2.2.2), which acts randomly towards substrate, and exo-PGL (EC 4.2.2.9), which catalyzes substrate cleavage from the nonreducing end.

7. Pectin Lyases (PL)

• Pectin lyase catalyzes the random cleavage of pectin, preferentially highly esterified pectin, resulting in the transelimination of glycosidic bonds and the formation of unsaturated methyloligogalacturonates.
• Ca2+ is not a necessity for PLs, but they are activated by it and other cations. Up to now, all described pectin lyases are endo-PLs (EC 4.2.2.10).
• Van Alebeek and colleagues undertook a comprehensive analysis of the mechanism of action of pectin lyase A from Aspergillus niger, which generates mono-, di-, tri-, and tetragalacturonates, in addition to unsaturated di-, tri-, and tetragalacturonates, from methyloligogalacturonates.
• In no experiment were unsaturated monogalacturonates detected in the reaction products. Both lyase groups belong to the polysaccharideslyase family 1 classification. Fungal lyases are most active in acidic and neutral environments, whereas bacterial lyases are more active in alkaline environments. Enzymes that cleave the rhamnogalacturonan chain are necessary for the complete breakdown of pectin substrate.

8. Rhamnogalacturonan Rhamnohydrolases

• RG rhamnohydrolase, rhamnogalacturonan a-Lrhamnopyranohydrolase or a-L-rhamnosidase (EC 3.2.1.40) catalyzes hydrolytic cleavage of the rhamnogalacturonan chain at nonreducing end yielding rhamnose. The glycosyl-hydrolase families 28, 78, and 106 classify these enzymes.

9. Rhamnogalcturonan Galacturonohydrolases

• RG galacturonohydrolase (EC 3.2.1.-) catalyzes hydrolytic cleavage of the rhamnogalacturonan chain at nonreducing end producing monogalacturonate. It is classified into glycosyl-hydrolase family 28.

10. Rhamnogalacturonan Hydrolases

• RG hydrolase randomly hydrolyses the rhamnogalacturonan chain producing oligogalacturonates.

11. Rhamnogalacturonan Lyases

• RG lyase (EC 4.2.2.-) catalyzes the random transelimination of the rhamnose-galcturonate linkage from the rhamnoga lacturonan chain, yielding an unsaturated galacturonate at the nonreducing end of one oligomer and a second oligomer with a rhamnose as the reducing end residue. These enzymes belong to families 4 and 11 of polysaccharides-lyases.

12. Rhamnogalacturonan Acetylesterases

• RG acetylesterase (EC 3.1.1.-) catalyzes hydrolytic cleavage of acetyl groups from rhamnogalacturonan chain. It is classified into carbohydrate esterase family 12.

13. Xylogalacturonan Hydrolase

• Xylogalacturonase (EC 3.2.1.-) catalyzes hydrolytic cleavage of glycosidic linkages between two galacturonate residues in xylose-substituted rhamnogalacturonan chain, producing xylose-galacturonate dimers. These enzymes are classified into glycosyl-hydrolase family 28.

Factors affecting pectin degradation

The degradation of pectin, the complex carbohydrate found in the cell walls of plants, is influenced by several factors. Here are some of the main factors that can affect pectin degradation:

1. pH: The pH of the environment can have a significant impact on pectin degradation. Most pectinases work best in slightly acidic conditions, with an optimal pH range of around 4.5 to 6.5.
2. Temperature: The temperature of the environment can also affect pectin degradation. Pectinases typically work best at moderate temperatures, with an optimal range of around 40-60°C.
3. Substrate concentration: The concentration of pectin in the environment can affect the rate of pectin degradation. In general, higher concentrations of pectin will lead to faster degradation rates, up to a certain point where the concentration becomes inhibitory.
4. Enzyme concentration: The concentration of pectinases in the environment can also affect the rate of pectin degradation. Higher concentrations of enzymes will generally lead to faster degradation rates, up to a certain point where the enzymes become saturated.
5. Co-factors: Some pectinases require co-factors such as calcium or magnesium ions to function properly. The presence or absence of these co-factors can affect the activity of the enzymes.
6. Inhibitors: Some compounds, such as heavy metals or certain chemicals, can inhibit the activity of pectinases and slow down or stop pectin degradation.
7. Microbial community: The specific microorganisms present in the environment can also affect pectin degradation. Different microorganisms produce different types and amounts of pectinases, which can affect the overall rate and extent of pectin degradation.

Understanding these factors is important for optimizing pectin degradation in various applications, such as the production of fruit products or the decomposition of plant material in nature.

Process of pectin degradation

The complex process of pectin degradation in nature is a perplexing series of steps that involve microbial activity. The intricacy of this process is characterized by a bursty sequence of enzymatic reactions that occur in a specific order.

1. Step 1 – Deesterification: The first step is deesterification, which is catalyzed by pectin esterases or pectin methyl esterases. These enzymes initiate the breakdown of pectin substances by catalyzing the deesterification of the methoxy group of pectin, resulting in the formation of pectic acid and methanol. The esterase enzymes need non-esterified substrates and prefer a methyl ester group of galacturonate units next to a non-esterified galacturonate unit. Esterases like pectin acetyl esterases also play a crucial role in this step by hydrolyzing the acetyl ester of pectin, leading to the formation of pectic acid and acetate.
2. Step 2 – Hydrolytic cleavage: The second step is the hydrolytic cleavage of the α-1,4-glycosidic linkages that exist in the backbone of the pectin substrates. This step is the most important part of pectin degradation, and it involves the action of different enzymes produced by various microorganisms. These enzymes act on a different group of pectin substrates. Polymethylgalacturonases and polygalacturonases act on the α-1,4-glycosidic linkages on highly esterified pectin, leading to the formation of 6-methyl-D-galacturonate and D-galacturonate, respectively. Both of these enzymes can act as either endo or exoenzymes, cleaving the pectin backbone either randomly or through the reducing ends. Another group of hydrolytic enzymes is pectate lyases that act on the glycosidic linkages of polygalacturonic acid, forming unsaturated products through transelimination reaction.

In conclusion, the process of pectin degradation is a highly complex series of steps that involve a bursty sequence of enzymatic reactions. Understanding the intricacies of this process is essential in various fields such as food science, microbiology, and biotechnology.

Mechanisms of microbial degradation of pectin

The perplexing mechanisms of microbial degradation of pectin are highly dependent on the type of enzyme involved in the process. Multiple mechanisms of action have been identified for enzymes responsible for pectin degradation. One such mechanism is the de-esterification mechanism carried out by pectin esterases and pectin methyl esterases.

Pectin esterases act on pectin substrates using one of three mechanisms. These include the single-chain mechanism, the multiple-chain mechanism, and the multiple-attack mechanism. Bacterial polyesterases utilize both the single-chain and multiple-attack mechanisms to produce products with adjacent regions of galacturonic acids. Fungal esterases, on the other hand, attack randomly using a multiple-chain mechanism. During this random attack, de-esterification causes the release of protons, which further promote the action of endopolygalacturonases.

In Bacteroides, for example, the mechanism of de-esterification of galacturonan macromolecules occurs through a multi-attack mechanism. This is then followed by the decomposition of the oligomers to release the end products.

Another mechanism involved in the degradation of pectin is the hydrolytic cleavage mechanism carried out by polygalacturonases and pectin lyases. This process begins with the positioning of the active site amino acids on the susceptible glycosidic bonds. The motifs on the active sites interact with the substrate on either side of the designated bond through multiple hydrogen bonds. These hydrogen bonds create sufficient strain and distortion on the susceptible glycosidic bond.

The distortion is followed by proton transfer between the amino acids of the active site and the glycosidic bond. This causes the cleavage of glycosidic bonds with the release of the first end product, followed by the subsequent formation of a covalent bond between the substrate and the catalytic site nucleophile. Another active site residue on the enzyme then places a water molecule for a nucleophilic attack on the substrate. The nucleophilic attack results in the formation of the second end product, along with the restoration of the active site of the enzyme.

For example, in Rhizopus oryzae, hydrolytic cleavage involves about 18 polygalacturonases and one β-galactosidase. These enzymes cleave the α-1,4-glycosidic linkages by the above-mentioned mechanism by endo and exoenzymes.

Microbial producers for pectinases

Pectinases are enzymes that break down pectin, a complex polysaccharide found in plant cell walls. There are several microbial producers of pectinases, including bacteria, fungi, and yeast.

• Bacteria: Bacterial producers of pectinases include Bacillus, Pseudomonas, and Erwinia. These bacteria are commonly found in soil and can be isolated from plant material. Bacillus species are particularly useful for producing pectinases as they can grow rapidly and produce large amounts of enzyme.
• Fungi: Fungal producers of pectinases include Aspergillus, Penicillium, and Trichoderma. These fungi are common in soil and can also be isolated from plant material. Aspergillus species are widely used for producing pectinases due to their ability to produce high levels of enzyme and tolerate a wide range of pH and temperature conditions.
• Yeast: Yeast producers of pectinases include Candida and Saccharomyces. These yeasts can also be isolated from soil and plant material. Candida species are commonly used for producing pectinases as they can produce high levels of enzyme and are able to grow in a variety of nutrient conditions.

Overall, microbial producers of pectinases are widely available and can be easily cultured in the laboratory. These enzymes have a wide range of applications, including in the food and beverage industry for fruit juice clarification and in the textile industry for enzymatic scouring of cotton fibers.

Applications of pectinase

1. Food Industry: Pectinase is commonly used in the food industry to break down pectin in fruits and vegetables. This process helps to increase the yield of juice, enhance the flavor and aroma, and improve the texture of the final product. Pectinase is also used in the production of baked goods, wine, beer, and dairy products.
   • Juice processing: Since the 1930s, pectinases have been utilised in the fruit juice and wine industries. This resulted in a decrease in maceration and viscosity, which contributed to a higher ratio of obtained press juice to de-peeled pulps. Similarly, decreasing fruit drink viscosity.
   • Wine processing: Enzymes are introduced to white wine musts before fermentation in order to clarify wine. Musts are produced from pressed juice with no touch with the skin for quick clarity.
2. Textile Industry: Pectinase is used in the textile industry to remove pectinaceous substances from natural fibers such as cotton, wool, and silk. The enzyme helps to increase the absorbency and dyeability of the fibers, resulting in a higher quality fabric. Before beginning textile production, pectinases are occasionally used to remove gum from natural fibres such as ramie fibres and linen.
3. Paper Industry: Pectinase is used in the paper industry to improve the quality and strength of paper. The enzyme is added to the pulp during the manufacturing process to break down pectin and other impurities, resulting in a smoother and more uniform product.
4. Biofuel Production: Pectinase is used in the production of biofuels such as ethanol and butanol. The enzyme helps to break down pectin in cellulosic biomass, making it easier to extract the sugars needed for fermentation.
5. Pharmaceutical Industry: Pectinase is used in the pharmaceutical industry to extract plant-based drugs and enzymes. The enzyme helps to break down the plant cell walls and release the desired compounds, which can then be purified and used in various medical applications.
6. Develop seed germination and protoplast formation: Enzymatic and mechanical approaches have been utilised for protoplast production in plant cells. In the mechanical approach, protoplast creation consisted of detaching plasmolyzed tissue with a sharp-edged knife and de-plasmolyzing the tissue to release the protoplast. The mechanical approach is hampered by the low protoplast yield. Hence, the enzymatic approach is primarily utilised for this function. For the generation of protoplasts from nonlignin-containing plant tissues, a combination of pectinases and cellulases has been employed.
7. Purification of plant viruses: For researching the physical, chemical, and biological aspects of viruses, a very pure preparation is necessary. Depending on the type of virus, various purifying procedures might be chosen. The enzymes of pectinase can be employed to release the virus from phloem tissues.
8. Coffee and tea processing: In the preparation of green coffee beans, pectolytic enzymes were utilised to expedite the removal of the jelly that surrounds the coffee cherry, hence boosting the substandard quality of the coffee beans. Using pectinases improves coffee quality by turning mucilage into sugars. In a similar manner, pectinase treatment improves the fermentation of tea by degrading the pectin contained in the cell walls of tea leaves, while preventing the formation of froth in powdered tea. According to reports, the pectinases utilised in tea processing are of the alkaline fungal pectinase type.
9. Oil extraction: Pectinases are used to disrupt gels in order to facilitate the recovery of oils. Sunflower, olive, palm, coconut, and canola oils are extracted with organic solvents such as hexane, a possible carcinogen. Using alkaline-type pectinase, vegetable oils can be extracted in an aqueous process by the destruction of cell wall components.
10. Assist in cellulose degradation: Pectinase serves a key role by enhancing the access of cellulases to their substrates.
11. Animal feed processing: Employing various enzymes in the animal and poultry feed started in the 1980s with putting β-glucanase into barley and eventually wheat. After that, the xylanase enzyme was tested and achieve the best action in this circumstance. Generally, the formulation of feed enzymes is a cocktail of multi-enzymes containing proteinases, amylases, pectinases, xylanases, and glucanases. Adding enzymes to the animal feed is decreases viscosity, promotes nutrient absorption, liberates the blocked nutrients by this fibre, and reduces the feaces amount.
12. Wastepaper recycling: The biggest challenge in wastepaper recycling is deinking procedure that involves a considerable volume of environmentally hazardous chemicals. Bio-deinking utilising enzymes is less polluting, delivers greater quality, and is energy-saving. Pectinases, cellulases, hemicellulases, and ligninolytic enzymes are employed for bio-deinking. These enzymes change bonds near the ink particle, eliminating the ink from the fibre surface. Afterwards, the resultant ink is removed by washing or floating.
13. Wastewater treatment: The wastewater of the vegetable food industries contains pectic substrates. The conventional treatment of its wastewater includes numerous procedures that are costly in cost, have longer timeframes, and pollutes the environment. Hence, utilising alkaline pectinases to remove the pectic substrate is an excellent alternative, cost-effective, and eco-friendly way, facilitating the decomposition by activated sludge treatment.

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References

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• Mosaad Khattab, A. (2022). The Microbial Degradation for Pectin. Pectins – The New-Old Polysaccharides. doi: 10.5772/intechopen.100247
• Haile, S., Masi, C. & Tafesse, M. Isolation and characterization of pectinase-producing bacteria (Serratia marcescens) from avocado peel waste for juice clarification. BMC Microbiol 22, 145 (2022). https://doi.org/10.1186/s12866-022-02536-8
• Abbott DW, Boraston AB. Structural biology of pectin degradation by Enterobacteriaceae. Microbiol Mol Biol Rev. 2008 Jun;72(2):301-16, table of contents. doi: 10.1128/MMBR.00038-07. PMID: 18535148; PMCID: PMC2415742.
• Manucharova, Natalia. (2009). The microbial destruction of chitin, pectin, and cellulose in soils. Eurasian Soil Sci. 42. 1526-1532. 10.1134/S1064229309130146.
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