Microbial degradation of pectin (Enzymes, Steps, Mechanisms)
Pectin is a type of soluble fiber that is found in the cell walls and intercellular tissues of certain plants, especially fruits and vegetables. Pectin is composed of long chains of sugar molecules, mainly galacturonic acid, that are linked together by glycosidic bonds. Pectin can form a gel-like structure when heated in the presence of water and acid, which makes it a useful thickening agent for jams, jellies, and other food products. Pectin also has various health benefits, such as lowering cholesterol, improving blood sugar control, and providing dietary fiber.
Pectin is derived from the Greek word "pektikos", which means "curdled" or "congealed". Pectin was first isolated and described by the French chemist Henri Braconnot in 1825. Pectin is mainly extracted from citrus fruits and apples, which are rich sources of this fiber. Pectin can also be found in other fruits and vegetables, such as plums, apricots, carrots, beetroots, and tomatoes.
Pectin is a complex and diverse molecule that can vary in its structure, composition, and properties depending on the plant source, the extraction method, and the degree of modification. Pectin can be classified into two main families: homogalacturonans and rhamnogalacturonans. Homogalacturonans are linear chains of galacturonic acid residues that can be methylated or acetylated at different positions. Rhamnogalacturonans are chains of alternating galacturonic acid and rhamnose residues that can have various side chains attached to them, such as arabinose, galactose, xylose, or glucuronic acid.
The structure of pectin determines its gelling ability and its interaction with other molecules. Pectin can form gels by forming hydrogen bonds with water molecules and by forming cross-links with calcium ions or other pectin molecules. The gelling properties of pectin depend on factors such as the degree of methylation, the degree of acetylation, the molecular weight, the pH, the temperature, the presence of sugar, and the presence of other polysaccharides. Pectin can also interact with proteins, lipids, minerals, and phytochemicals in food matrices, affecting their stability, texture, flavor, and nutritional value.
Pectin is not only a food additive but also a dietary component that has various physiological effects in humans. Pectin is a source of soluble fiber that can increase the viscosity and bulk of the intestinal contents, which can modulate digestion and absorption processes. Pectin can also bind to bile acids and cholesterol in the gut, reducing their reabsorption and lowering blood cholesterol levels. Pectin can also slow down the release of glucose into the bloodstream after a meal, improving glycemic control and insulin sensitivity. Furthermore, pectin can act as a prebiotic that stimulates the growth and activity of beneficial bacteria in the colon.
Pectin is a versatile and valuable polysaccharide that has multiple applications in food production and human health. Pectin is widely used as a gelling agent, stabilizer, thickener, emulsifier, texturizer, and water binder in various food products such as jams, jellies, fruit preparations, dairy products, beverages, bakery products, confectionery products, sauces, dressings, meat products, and dietary supplements. Pectin is also used as a drug delivery system for controlled release of active ingredients. Moreover, pectin has potential therapeutic benefits for various chronic diseases such as cardiovascular disease , diabetes , obesity , inflammatory bowel disease , ulcerative colitis , colon cancer , and gastric ulcers .
Pectin is a complex heteropolysaccharide composed of linear chains of α-D-galacturonic acid or other similar sugar derivatives, commonly found in plant cell walls as cementing material. Pectin often remains associated with other cell wall polysaccharides like cellulose, hemicelluloses, and lignin. The highest concentration of pectin is found in the primary cell wall and middle lamella of plant cells with decreasing concentration towards the plasma membrane. Pectin is responsible for providing firmness and structure to the cell wall and also helps in intercellular adhesion and mechanical resistance of the cell. Most of the natural pectin is water-soluble or free; however, some forms of non-soluble or bound pectin can also be found. The degree of solubility of pectin depends on the length of the polymer and the presence of a methoxy group in the structure.
The structural elements of pectin are classified into two families; galacturonans and rhamnogalacturonan. Galactorunans consist of a backbone of α-(1,4)-linked D-galacturonic acid residues. The branched can either be branched or unbranched. The backbone in rhamnogalacturonans, however, contains diglycosyl repeating units of α-L-rhamnose-(1,4)-α-D-galacturonic acid. The rhamnose residues are ramified at the O-4 and O-3 positions with polymeric side chains that include arabinose and galactose residues at other positions. In pectin, four different types of polymeric side chains might exist; arabinans, galactans, type I arabinogalactans, and type II arabinogalactans. The chemical structure of pectin is extremely complicated as it contains as many as 18 different monosaccharides linked together by twenty different linkages.
The overall structure of pectin is explained in terms of smooth and hairy regions. The smooth regions contain linear chains of homo or heteropolymer, whereas the hairy region contains simple or complex side chains. Besides, several other monosaccharides might remain bonded by modified O-ether or O-ester linkages.
The galacturonans found in pectin can either be homogalacturonans or hetero galacturonans.
Homogalacturonans form the smooth region of pectin, that is unbranched chains of α-(1,4)-linked G-galacturonic acid residues that might be methyl or acetyl-esterified. Homogalacturonans accounts for about 60% of all pectin found in different living beings. In the case of heterogalacturonanas, the homopolysaccharide chain is more or less heavily substituted at O-2 and O-3 by monomers or dimers of xylose, resulting in axylogalacturonan.
If the polymer is substituted with complex side chains like rhamnose, it forms rhamnogalacturonans.
Some of the pectins might exist as rhamnogalacturonan consisting of a long chain of alternating L-rhamnose and D-galacturonic acid residues. In some cases, the rhamnose residues might even be replaced by a variety of L-arabinosyl and D-galactosyl-containing side chains. A small number of glucuronic acid and 4-O-methyl glucuronic acid residues might be present. Rhamnogalacturonans account for about 20-35% of the total pectin content in nature, but the amount is as high as 75% in the soybean plant.
Pectinases are a group of enzymes that break down pectin, a complex heteropolysaccharide found in plant cell walls, through hydrolysis, transelimination and deesterification reactions. Pectinases are also known as pectic enzymes, and they include pectinesterases, polygalacturonases, pectin lyases, and pectin depolymerases. Pectinases are useful because pectin is the jelly-like matrix which helps cement plant cells together and in which other cell wall components, such as cellulose fibrils, are embedded. Therefore, pectinases are commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of fruit juice from fruit, including apples and sapota. Pectinases have also been used in wine production since the 1960s.
Pectinases are naturally produced by various plants, fungi, yeasts, insects, bacteria and microbes, but cannot be synthesized by animal or human cells. In plants, pectinases hydrolyze pectin that is found in the cell wall, allowing for new growth and changes to be made. Similarly to their role in plants, pectinases break down pectin during the developmental stage of fungi. Some microorganisms also use pectinases as virulence factors to degrade the plant cell wall and cause diseases.
Pectinases are classified based on how their enzymatic reaction proceeds with various pectic substances (through transelimination or hydrolysis), the preferred substrate (pectin, pectic acid or oligo-n-galacturonate) and if the cleavage that occurs is random or end-wise. Some of the common types of pectinases are:
- Pectinesterases: These enzymes catalyze the hydrolysis of methylated carboxylic ester in pectin to form pectic acid and methanol. They act on highly esterified pectins and prepare them for further degradation by other enzymes.
- Polygalacturonases: These enzymes hydrolyze O-glycosyl bonds in the homogalacturonan to form monomeric units. They act on the 1,4-alpha-D-galactosyluronic linkages between the galacturonic residues. They can be either endo- or exo-enzymes depending on whether they act randomly or from the ends of the polymer chain.
- Pectin lyases: These enzymes degrade pectin substances in a random fashion, yielding unsaturated oligomethylgalacturonates. They cleave glycosidic linkages, preferentially on polygalacturonic acid through transelimination reaction. They require Ca2+ ions for their activity and are inhibited by chelating agents like EDTA.
The role of pectinases in pectin degradation is to depolymerize the complex polysaccharide into smaller units that can be further metabolized by microorganisms or utilized for industrial purposes. Pectin degradation involves three main steps: deesterification, hydrolytic cleavage and transelimination.
- Deesterification: This step is catalyzed by pectinesterases that remove the methyl or acetyl groups from the galacturonic acid residues in pectin, resulting in pectic acid and methanol or acetate. This step increases the solubility and susceptibility of pectin to other enzymes.
- Hydrolytic cleavage: This step is catalyzed by polygalacturonases that break down the alpha-1,4-glycosidic bonds in the homogalacturonan backbone of pectin, resulting in galacturonic acid monomers or oligomers. This step reduces the molecular weight and viscosity of pectin.
- Transelimination: This step is catalyzed by pectin lyases that cleave the glycosidic bonds in polygalacturonic acid through a beta-elimination mechanism, resulting in unsaturated oligogalacturonates with a double bond at C4-C5 position. This step introduces conjugated double bonds into the structure of pectin.
The products of pectin degradation can be further catabolized by microorganisms to produce energy and carbon sources or used for various industrial applications such as food processing, textile degumming, animal feed production, plant virus purification and oil extraction.
Different groups of microorganisms are known to produce multiple sets of pectinolytic enzymes that aid either in the absorption of nutrients or help in the pathogenesis of microbial diseases.
Bacteria have recently become a major source of pectinolytic enzymes where they produce different sets of enzymes that help in the overall degradation of pectin substrates . Some of the common pectinolytic bacteria include organisms like Bacillus, Pseudomonas, and Staphylococcus . Most of the bacterial pectinolytic activity is observed under aerobic conditions by aerobes, whereas some of the activity might be seen under anaerobic conditions. Some bacteria like Bacillus badius, Bacillus asahin, Bacillus psychrosaccharolyticus, and Pseudomonas aeruginosa even utilize the pectinolytic activity in their pathogenesis of different diseases. Common thermophilic bacteria like Geobacillus sp, Anoxybacillus sp, and Bacteroides also exhibit pectinase activity, assisting in the recycling of carbon compounds in the biosphere.
Fungi are the most group of microorganisms involved in the degradation of polysaccharides as a part of the natural recycling process. These fungi might exist in different habitats with different lifestyles. The most common group of fungi involved in pectin degradation are the species belonging to Ascomycetes and Deuteromycetes . Phanerochaete chrsosporium is one of the most studied basidiomycetes (white rot) fungi that degrade most of the complex polysaccharides like cellulose, pectin, and chitin. Other fungal species involved in pectin degradation include Magnaporthe oryzae, Giberella zeae, Botrytis fuckeliana, Sclerotinia sclerotiorum, Aspergillus nidulans, Trichoderma virens, Podospora anserine, Rhizopus oryzae, and Aspergillus clavatus . The type of enzymes produced and their mode of action might differ with the fungal species. Bacterial pectinases tend to be alkaline, whereas fungal pectinases are acidic in nature.
Pectin is a complex polysaccharide that requires a variety of enzymes to be completely degraded into its monomeric units. These enzymes belong to three main families of carbohydrate-active enzymes (CAZy): glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. They differ in their substrate specificity, catalytic mechanism, and action pattern (endo or exo). The following is a brief overview of the main types of pectin-degrading enzymes and their properties.
Pectin esterases (EC 220.127.116.11) are a group of enzymes that catalyze the hydrolysis of methyl or acetyl esters of pectin, forming pectic acid and methanol or acetate. These enzymes are classified as carbohydrate esterases (CE) in the CAZy database and are further divided into subfamilies based on their sequence similarity and substrate preference. For example, CE8 includes pectin methylesterases (PMEs) that act on methyl-esterified pectin, while CE12 includes pectin acetylesterases (PAEs) that act on acetyl-esterified pectin.
Pectin esterases are produced by plants, fungi, bacteria, and some insects. They play important roles in plant development, fruit ripening, plant defense, microbial pathogenesis, and food processing. Pectin esterases can act prior to or in synergy with other pectin-degrading enzymes, such as polygalacturonases and pectate lyases, as they modify the degree of esterification of pectin and affect its solubility and susceptibility to hydrolysis.
Pectin esterases can act by one of three mechanisms: single-chain, multiple-chain, or multiple-attack. In the single-chain mechanism, the enzyme acts on all substrate sites on the polymeric chain without releasing it. In the multiple-chain mechanism, the enzyme catalyzes one reaction and then releases the substrate. In the multiple-attack mechanism, the enzyme catalyzes multiple reactions before releasing the substrate. The mechanism of action determines the pattern of de-esterification and the distribution of free carboxyl groups along the pectin chain.
Polygalacturonases (EC 18.104.22.168) are a group of enzymes that catalyze the hydrolysis of 1,4-alpha-D-galactosidic linkages in homogalacturonan, a major component of pectin. These enzymes are classified as glycoside hydrolases (GH) in the CAZy database and are further divided into subfamilies based on their sequence similarity and catalytic mechanism. For example, GH28 includes endo-polygalacturonases (EPGs) and exo-polygalacturonases (EXPGs) that act by an inverting mechanism, while GH88 includes exo-polygalacturonases (EXPGs) that act by a retaining mechanism.
Polygalacturonases are produced by plants, fungi, bacteria, and some animals. They play important roles in plant cell wall remodeling, fruit softening, plant-microbe interactions, and industrial applications such as juice clarification and textile degumming. Polygalacturonases can act as endo- or exo-enzymes depending on their mode of action on the substrate. Endo-polygalacturonases cleave the glycosidic bonds randomly within the chain, producing oligogalacturonides of varying sizes. Exo-polygalacturonases cleave the glycosidic bonds from the non-reducing or reducing end of the chain, producing monogalacturonates or digalacturonates.
Pectate lyases (EC 22.214.171.124) are a group of enzymes that catalyze the cleavage of 1,4-alpha-D-galactosidic linkages in de-esterified pectin (pectate) by a beta-elimination reaction, producing unsaturated oligogalacturonides with a 4-deoxy-alpha-D-galact-4-enuronosyl group at their non-reducing end. These enzymes are classified as polysaccharide lyases (PL) in the CAZy database and are further divided into subfamilies based on their sequence similarity and substrate specificity. For example, PL1 includes endo-pectate lyases (EPLs) that act on polygalacturonic acid, while PL9 includes endo-pectate lyases (EPLs) that act on rhamnogalacturonan I.
Pectate lyases are produced by fungi, bacteria, and some plants. They play important roles in plant cell wall degradation, microbial pathogenesis, plant defense responses, and industrial applications such as fiber retting and oil extraction. Pectate lyases can act as endo- or exo-enzymes depending on their mode of action on the substrate. Endo-pectate lyases cleave the glycosidic bonds randomly within the chain, producing unsaturated oligogalacturonides of varying sizes. Exo-pectate lyases cleave the glycosidic bonds from the non-reducing end of the chain, producing unsaturated monogalacturonates.
Pectin degradation in nature and on artificial growth media is affected by different factors, some of which are:
- Moisture content: Based on studies done on pectin degradation, it has been observed that the rate of chitin degradation is rapid in the presence of free water and complete saturation. The change in water concentration or moisture content has minimal effect on the rate of pectin degradation. Nevertheless, the rate is impaired if the amount of water increases to the point that causes impairing of aeration due to logging.
- Aeration: Most of the pectinolytic microorganisms are aerobic or facultative aerobic. As a result, the rate of pectin utilization or degradation is enhanced in the increased concentration of O2. Some amount of degradation, however, can be achieved in a low concentration of CO2 as it allows facultative aerobes and anaerobes to remain active. Pure oxygen environment (100% O2) might be toxic in some cases, especially when readily energy sources are available.
- Added glucose: The addition of glucose in the media or soil hampers the rate of pectin degradation as the organisms utilize the readily available source of energy rather than pectin as their source of nutrients. Glucose is a ready energy source which is easy to metabolize. This, in turn, causes a delay or decreased pectin degradation. In the absence of glucose or such similar sources, however, pectin degradation is enhanced as pectin is comparatively less complex when compared to other carbohydrate sources like lignin.
- Organic matter: The presence of plant fibers rich in pectin also supports the rate of pectin degradation. Organic matter rich in nutrients and minerals for the microorganisms helps in the formation of biomolecules like proteins and enzymes. The increase in organic matter increases the substrate concentration. The increased substrate concentration might decrease the degradation rate at first as the organisms utilize sources like glucose and cellulose. As these sources are degraded, pectin becomes the next source of nutrition, which then increases its’ degradation.
- Pectin structure: The structure and composition of pectin also influence its susceptibility to enzymatic degradation. Pectins with different degrees of methylation, acetylation, and branching might require different sets of enzymes for their complete breakdown. Some enzymes might be specific for certain types of linkages or side chains present in pectin. The molecular weight and solubility of pectin also affect its accessibility to enzymes.
The process of pectin degradation by microorganisms involves the sequential action of different types of enzymes that break down the complex structure of pectin into simpler compounds. The process can be summarized as follows:
- De-esterification: The first step is the removal of methyl or acetyl groups from the galacturonic acid residues of pectin by the action of pectin esterases or pectin methyl esterases. These enzymes hydrolyze the ester bonds and release pectic acid and methanol or acetate. This step increases the solubility and susceptibility of pectin to further degradation by other enzymes .
- Hydrolytic cleavage: The second step is the cleavage of the glycosidic bonds between the galacturonic acid residues in the backbone of pectin by the action of polygalacturonases or pectin lyases. These enzymes break down the polymer into smaller oligomers or monomers of galacturonic acid or its derivatives. Polygalacturonases are endo- or exo-hydrolases that use water to cleave the bonds, whereas pectin lyases are endo- or exo-eliminases that use a transelimination mechanism to produce unsaturated products .
- Side chain degradation: The third step is the degradation of the side chains attached to the rhamnose residues in some types of pectin, such as rhamnogalacturonans. These side chains may consist of arabinose, galactose, xylose, or other sugars that are linked by various types of bonds. Different enzymes are involved in this step, such as arabinanases, galactanases, xylogalacturonan hydrolases, arabinogalactan hydrolases, etc. These enzymes hydrolyze or eliminate the bonds between the side chain sugars and release them as monomers or oligomers .
The final products of pectin degradation are mainly galacturonic acid, methanol, acetate, and various sugars that can be further metabolized by microorganisms or absorbed by humans. Some of these products have beneficial effects on human health, such as lowering cholesterol, modulating gut microbiota, enhancing immune response, and preventing constipation . Pectin degradation also has industrial applications, such as improving juice extraction and clarification, enhancing animal feed quality, and producing biofuels .
Microbial degradation of pectin is a complex process that involves different types of enzymes and microorganisms. Pectin is a heterogeneous polysaccharide that consists of various structural elements, such as homogalacturonan, rhamnogalacturonan, and side chains of arabinose and galactose. These elements differ in their degree of methylation, acetylation, and branching, which affect their susceptibility to enzymatic hydrolysis. Therefore, different microorganisms produce different sets of pectinolytic enzymes that target specific regions or bonds in the pectin molecule.
The mechanisms of microbial degradation of pectin can be divided into two main steps: de-esterification and hydrolytic cleavage. De-esterification is the removal of methyl or acetyl groups from the carboxyl groups of galacturonic acid residues in pectin. This step is catalyzed by pectin esterases or pectin methyl esterases, which are produced by various bacteria and fungi. De-esterification increases the solubility and accessibility of pectin to other enzymes, and also affects its gelling properties and interactions with other cell wall components. De-esterification can occur by different mechanisms, such as single-chain, multiple-chain, or multiple-attack mechanisms, depending on the enzyme-substrate complex and the mode of action of the enzyme .
Hydrolytic cleavage is the breaking of glycosidic bonds between galacturonic acid residues in pectin. This step is catalyzed by different types of enzymes, such as polygalacturonases, pectin lyases, and pectate lyases. Polygalacturonases are the most common and widely distributed pectinolytic enzymes, which hydrolyze the α-1,4-glycosidic linkages in homogalacturonan and rhamnogalacturonan. These enzymes can act as endo- or exo-enzymes, depending on whether they cleave the bonds randomly or from the ends of the polymer chain. Polygalacturonases produce oligo- or monogalacturonates as end products . Pectin lyases and pectate lyases are less common but more specific enzymes that cleave the glycosidic bonds in pectin by a transelimination mechanism, resulting in unsaturated products with a double bond between C4 and C5. These enzymes act preferentially on non-methylated or low-methylated regions of pectin .
The mechanism of hydrolytic cleavage involves the positioning of the active site amino acids on the susceptible glycosidic bond, the formation of hydrogen bonds with the substrate residues on either side of the bond, the distortion and proton transfer on the bond, the cleavage of the bond with the release of one product and the formation of a covalent bond between another product and the catalytic site nucleophile, and finally, the nucleophilic attack by a water molecule on the substrate to release the second product and restore the active site .
The microbial degradation of pectin is influenced by various factors, such as moisture content, aeration, added glucose, organic matter, pH, temperature, and enzyme concentration. These factors affect the activity and stability of the enzymes, as well as the availability and solubility of the substrate. The microbial degradation of pectin results in several benefits for both microorganisms and humans. For microorganisms, pectin degradation provides a source of carbon and energy, as well as a means to invade plant tissues or evade plant defenses. For humans, pectin degradation improves the quality and yield of fruit juices and wines, enhances the digestibility and nutritional value of plant foods, modulates the gut microbiota composition and function, regulates the gut inflammatory and immune responses, and influences the bioavailability and efficacy of drugs .
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