- What is pectin?
- Structure of pectin
- What are Pectinases?
- Microorganisms involved in pectin degradation (pectinolytic microorganisms)
- Enzymes involved in the degradation of pectin
- Factors affecting pectin degradation
- Process (Simple Steps) of pectin degradation
- Mechanisms of microbial degradation of pectin
What is pectin?
- Pectin, a complex heteropolysaccharide, is comprised of linear chains of α-D-galacturonic acid or similar sugar derivatives, commonly found in plant cell walls, serving as a binding material.
- It is often associated with other cell wall polysaccharides like cellulose, hemicelluloses, and lignin.
- The primary cell wall and middle lamella of plant cells contain the highest concentration of pectin, decreasing towards the plasma membrane.
- Pectin plays a crucial role in providing firmness and structure to the cell wall, contributing to intercellular adhesion and mechanical resistance.
- While most natural pectin is water-soluble, some non-soluble or bound forms also exist.
- The solubility of pectin depends on the polymer length and the presence of a methoxy group in its structure.
- Due to its ability to form a thick gel-like structure, pectin finds extensive commercial applications.
- As one of the few biopolymers rich in fermentable dietary fibers, pectin is widely studied.
- Its structural diversity and complexity contribute to multiple applications.
- The term 'pectin' is derived from the Greek word 'pektikos,' meaning curdled or congealed.
Structure of pectin
- Pectins form a group of plant cell wall polysaccharides rich in covalently linked galacturonic acid.
- Approximately 70% of pectin comprises galacturonic acid, with all pectin polysaccharides featuring galacturonic acid linked at the O-1 and O-4 positions.
- Structurally, pectin is categorized into two families: galacturonans and rhamnogalacturonan.
- Galacturonans consist of a backbone composed of α-(1,4)-linked D-galacturonic acid residues, which can be either branched or unbranched.
- In contrast, the backbone of rhamnogalacturonans contains diglycosyl repeating units of α-L-rhamnose-(1,4)-α-D-galacturonic acid.
- Rhamnose residues on the backbone are branched at the O-4 and O-3 positions with polymeric side chains containing arabinose and galactose residues.
- Within pectin, four types of polymeric side chains may exist: arabinans, galactans, type I arabinogalactans, and type II arabinogalactans.
- The chemical structure of pectin is highly intricate, involving as many as 18 different monosaccharides linked by twenty different linkages.
- The overall pectin structure is explained through smooth and hairy regions. Smooth regions comprise linear chains of homo or heteropolymers, while hairy regions contain simple or complex side chains.
- Additionally, various monosaccharides may remain bonded by modified O-ether or O-ester linkages.
1. Galacturonans:
- Homogalacturonans, forming the smooth region, consist of unbranched chains of α-(1,4)-linked G-galacturonic acid residues, potentially methyl or acetyl-esterified. They make up approximately 60% of total pectin across different organisms.
- Heterogalacturonans involve homopolysaccharide chains that are more or less heavily substituted at O-2 and O-3 by monomers or dimers of xylose, resulting in axylogalacturonan. If substituted with complex side chains like rhamnose, they form rhamnogalacturonans.
2. Rhamnogalacturonans:
- Some pectins exist as rhamnogalacturonans, featuring a long chain of alternating L-rhamnose and D-galacturonic acid residues. Rhamnose residues may be replaced by various L-arabinosyl and D-galactosyl-containing side chains. A small number of glucuronic acid and 4-O-methyl glucuronic acid residues may be present.
- Rhamnogalacturonans constitute approximately 20-35% of the total pectin content in nature, with the soybean plant having a particularly high concentration of up to 75%.
What are Pectinases?
- Pectinases constitute a group of enzymes, encompassing at least seven distinct types, that play a crucial role in breaking down pectin derived from diverse sources.
- Due to the varied nature of pectin found in different living organisms, pectinases exhibit considerable diversity.
- The most prevalent and industrially significant pectinases are categorized into different groups based on variations in their substrate, structure, and reaction mechanism.
- Common pectinolytic enzymes include pectinesterases, polygalacturonases, pectin lyases, and pectin depolymerases.
- Pectinases find essential industrial applications in juice extraction, clarification processes, and the maceration of plant tissues.
- In the global carbon cycle, pectinases play a pivotal role as major enzymes involved in natural waste recycling.
- Termed as carbon recycling agents in nature, pectinases break down pectin substances into saturated and unsaturated galacturonans, which can be further catabolized to produce either pyruvate or 3-phosphoglyceraldehyde.
- Beyond these applications, pectinases are utilized in degumming fiber crops, as components in enzyme complexes for animal feed production, in the purification of plant viruses, and for oil extraction.
- Some pectinases serve as virulence factors by aiding in the degradation of pectin within plant cell walls.
- Structurally and mechanistically, pectinases may vary depending on their source; for instance, fungal pectinases differ from bacterial counterparts.
- Bacterial pectinases typically exhibit alkaline characteristics, while fungal pectinases tend to be acidic in nature.
Microorganisms involved in pectin degradation (pectinolytic microorganisms)
Various groups of microorganisms are recognized for producing diverse sets of pectinolytic enzymes, contributing either to nutrient absorption or participating in the pathogenesis of microbial diseases.
1. Pectinolytic Bacteria:
- Bacteria have emerged as significant sources of pectinolytic enzymes, producing distinct sets that contribute to the comprehensive degradation of pectin substrates.
- Common pectinolytic bacteria include Bacillus, Pseudomonas, and Staphylococcus.
- Bacterial pectinolytic activity is primarily observed under aerobic conditions by aerobes, with some activity occurring under anaerobic conditions.
- Certain bacteria, such as Bacillus badius, Bacillus asahin, Bacillus psychrosaccharolyticus, and Pseudomonas aeruginosa, leverage pectinolytic activity in the pathogenesis of various diseases.
- Thermophilic bacteria like Geobacillus sp, Anoxybacillus sp, and Bacteroides, with pectinase activity, play a role in carbon compound recycling in the biosphere.
2. Pectinolytic Fungi:
- Fungi constitute a predominant group of microorganisms engaged in polysaccharide degradation as part of the natural recycling process.
- These fungi inhabit diverse environments and adopt different lifestyles.
- Common fungal groups involved in pectin degradation are Ascomycetes and Deuteromycetes.
- Phanerochaete chrysosporium, a well-studied basidiomycetes (white rot) fungus, is proficient in degrading complex polysaccharides like cellulose, pectin, and chitin.
- Other fungal species contributing to pectin degradation include Magnaporthe oryzae, Giberella zeae, Botrytis fuckeliana, Sclerotinia sclerotiorum, Aspergillus nidulans, Trichoderma virens, Podospora anserine, Rhizopus oryzae, and Aspergillus clavatus.
- The enzymes produced and their mode of action may vary among different fungal species.
Enzymes involved in the degradation of pectin
Pectinases are categorized into different groups based on their source, substrates, and reaction mechanisms:
1. Polygalacturonase:
- Polygalacturonases are enzymes that hydrolyze O-glycosyl bonds within homogalacturonan, breaking down the 1,4-α-D-galactosyluronic linkages between galacturonic residues.
- Most polygalacturonases are endo-enzymes, acting randomly on the linkages to depolymerize or reduce the length of the polymer.
- The primary substrate for endo-polygalacturonase is homogalacturonan, but oligogalacturonides may also serve as substrates depending on their nature.
- Exo-polygalacturonases constitute a class that breaks down polygalacturonates into di- and mono-galacturonates.
- Enzyme activity is measured by quantifying reducing sugars produced through hydrolysis or by the viscous reduction method.
2. Pectinesterase:
- Pectinesterases catalyze the hydrolysis of the methylated carboxylic ester in pectin, yielding pectic acid and methanol.
- Pectin is the primary substrate, although compounds like methyl pectate and methylated oligogalacturonides can also act as substrates.
- (NH4)2SO4, Mg2+, and NaCl enhance or induce pectinesterase activity, while Cu2+ and Hg2+ inhibit it.
- Initially studied in plants, pectinesterases of bacterial and fungal origin have also been identified.
- Most pectinases are specific to esterified pectic substances and may not exhibit activity towards pectates.
3. Pectin Lyases:
- Pectin lyases degrade pectin substances randomly, producing a 4:5 ratio of unsaturated oligomethylgalacturonates.
- These enzymes cleave glycosidic linkages, preferably on polygalacturonic acid through a transelimination reaction.
- Ca2+ ions are essential for pectin lyase activity, making them susceptible to inhibition by chelating agents like EDTA.
- Exo-pectin lyases catalyze substrate cleavage from the non-reducing end of the polymer.
Factors affecting pectin degradation
Pectin degradation, both in natural environments and on artificial growth media, is influenced by various factors, including:
1. Moisture Content:
- Studies on pectin degradation indicate that the rate is significantly accelerated in the presence of free water and complete saturation, while changes in water concentration have minimal effects.
- Excessive water, leading to impaired aeration due to logging, can hinder the rate of pectin degradation.
2. Aeration:
- Pectinolytic microorganisms are predominantly aerobic or facultative aerobic, enhancing the rate of pectin degradation in an oxygen-rich environment.
- Some degradation can occur in low concentrations of CO2, allowing facultative aerobes and anaerobes to remain active.
- A pure oxygen environment (100% O2) may be toxic in certain cases, particularly when readily available energy sources are present.
3. Added Glucose:
- The introduction of glucose into the media or soil can impede the rate of pectin degradation, as microorganisms preferentially utilize the easily metabolizable glucose over pectin as a nutrient source.
- Glucose, being a readily available energy source, causes a delay or reduction in pectin degradation.
- In the absence of glucose or similar sources, pectin degradation is enhanced, as pectin is comparatively less complex than other carbohydrate sources like lignin.
4. Organic Matter:
- Plant fibers rich in pectin support the rate of pectin degradation.
- Organic matter, providing nutrients and minerals for microorganisms, facilitates the formation of biomolecules such as proteins and enzymes.
- An increase in organic matter initially decreases the degradation rate as microorganisms utilize sources like glucose and cellulose. As these sources are depleted, pectin becomes the next nutritional source, leading to an increase in its degradation rate.
Process (Simple Steps) of pectin degradation
The microbial hydrolysis or degradation of pectin in nature involves the following sequential steps:
1. Deesterification:
- The initial enzyme involved in the breakdown of pectin substances is pectin esterase or pectin methyl esterase.
- These enzymes catalyze deesterification by removing the methoxy group from pectin, resulting in the formation of pectic acid and methanol.
- Esterase enzymes, such as pectin acetyl esterases, act before polygalacturonates and pectate lyases, requiring non-esterified substrates for their activity.
- Pectin esterases exhibit a preference for a methyl ester group of galacturonate units next to a non-esterified galacturonate unit. Pectin acetyl esterases specifically hydrolyze the acetyl ester, yielding pectic acid and acetate.
2. Hydrolytic Cleavage:
- The pivotal step in pectin degradation involves the hydrolytic cleavage of the α-1,4-glycosidic linkages present in the backbone of pectin substrates.
- Various enzymes, produced by different microorganisms, target different groups of pectin substrates.
- Polymethylgalacturonases and polygalacturonases act on the α-1,4-glycosidic linkages in highly esterified pectin, resulting in the formation of 6-methyl-D-galacturonate and D-galacturonate, respectively.
- Both of these enzymes can function as endo or exoenzymes, cleaving the pectin backbone either randomly or at the reducing ends.
- Another group of hydrolytic enzymes, pectate lyases, targets the glycosidic linkages of polygalacturonic acid, generating unsaturated products through a transelimination reaction.
Mechanisms of microbial degradation of pectin
The mechanism of microbial pectin degradation varies based on the specific enzymes involved. Here are explanations of the mechanisms of action for enzymes participating in pectin degradation:
1. Mechanism of De-esterification by Pectin Esterases and Pectin Methyl Esterases:
- Pectin esterases operate on pectin substrates through one of three mechanisms:
- Single-Chain Mechanism: Enzyme action occurs on all substrate sites along the polymeric chain.
- Multiple-Chain Mechanism: Involves catalyzing a single reaction that then dissociates the substrate.
- Multiple-Attack Mechanism: Enzyme catalyzes multiple reactions before dissociating from the enzyme-substrate complex.
- Bacterial polyesterases produce products with adjacent galacturonic acid regions through both single-chain and multiple-attack mechanisms.
- Fungal esterases attack randomly through a multiple-chain mechanism.
- In Bacteroides, de-esterification of galacturonan macromolecules follows a multi-attack mechanism, leading to the subsequent decomposition of oligomers and the release of end products.
2. Mechanism of Hydrolytic Cleavage in Polygalacturonases and Pectin Lyases:
- The hydrolytic cleavage of α-1,4-glycosidic bonds in pectin involves positioning the active site amino acids on susceptible glycosidic bonds.
- Active site motifs interact with the substrate on either side of the designated bond through multiple hydrogen bonds, creating strain and distortion on the glycosidic bond.
- Proton transfer between active site amino acids and the glycosidic bond causes cleavage, releasing the first end product and forming a covalent bond between the substrate and the catalytic site nucleophile.
- Another active site residue places a water molecule for a nucleophilic attack on the substrate, resulting in the formation of the second end product and the restoration of the enzyme's active site.
- In Rhizopus oryzae, a set of about 18 polygalacturonases and one β-galactosidase cleave α-1,4-glycosidic linkages through this mechanism using both endo and exoenzymes.
