Microbial degradation of chitin (Enzymes, Steps, Mechanisms)

Chitin is a natural polysaccharide that is widely distributed in nature, found in the cell walls of fungi, the exoskeletons of arthropods, and certain structures of other invertebrates . It is also synthesized by some fish and amphibians. Chitin is the second most abundant biopolymer in the world, after cellulose , and it has various industrial and medical applications as a flocculating agent, wound-healing agent, thickener, stabilizer, ion-exchange resin, membrane, binder, and sizing and strengthening agent .

Chitin is a polymer of N-acetylglucosamine units linked together by β- (1→4)-glycosidic bonds . It has a similar structure to cellulose, except that one hydroxyl group on each monomer is replaced by an acetyl amine group . This modification allows for increased hydrogen bonding between adjacent polymers, giving the chitin-polymer matrix increased strength. Chitin can exist in different forms or conformations depending on the orientation of the polymer chains and the degree of acetylation . The most common forms are α-chitin, β-chitin, and γ-chitin.

Chitin is a versatile biomaterial that can be modified chemically or biologically to produce derivatives with different properties and functions . One of the most important derivatives is chitosan, which is obtained by deacetylation of chitin . Chitosan is more soluble than chitin and has antimicrobial and hydrating properties that make it useful for biomedical applications .

Chitin is a biodegradable and biocompatible polymer that does not accumulate in the biosphere as it is degraded by various microorganisms that produce chitinolytic enzymes . These enzymes break down the glycosidic bonds in chitin and release smaller oligomers or monomers that can be utilized by the microorganisms or other organisms as a source of carbon and nitrogen . Chitin degradation plays an important role in the carbon and nitrogen cycles in nature and also affects the immune responses of plants and animals.

In summary, chitin is a remarkable biopolymer that has many biological and ecological roles as well as potential applications in various fields. In this article, we will discuss the structure of chitin and its different forms, the enzymes involved in chitin degradation, the microorganisms that degrade chitin, the factors affecting chitin degradation, the process of chitin degradation, and the mechanisms of microbial degradation of chitin.

Chitin is a polysaccharide that consists of repeating units of N-acetylglucosamine, an amino sugar derived from glucose. These units are linked by β-1,4-glycosidic bonds, similar to those in cellulose. Chitin is a structural component of the cell walls of fungi, the exoskeletons of arthropods, and some other invertebrate structures.

Chitin has a linear structure that forms a three-dimensional α-helix configuration. The stability of this structure is due to the hydrogen bonding between the N-acetyl groups of adjacent chains. However, chitin can also exist in different forms or allomorphs, depending on the source and the orientation of the microfibrils. These allomorphs are α-chitin, β-chitin, and γ-chitin.

• α-chitin is the most common and abundant form of chitin, found in crustaceans, insects, and most fungi. In this form, the chitin chains are antiparallel to each other, forming an orthorhombic lattice. This arrangement results in strong hydrogen bonds in both the a and b directions, while the c direction has weak forces. α-chitin has two types of hydrogen bonds: intrasheet bonds between the carbonyl group of amide I and amide II, and intersheet bonds between the CH2OH group and the carbonyl group. α-chitin has the highest decomposition temperature (330°C) among the chitin allomorphs, due to its high degree of crystallinity and hydrogen bonding.
• β-chitin is found in squid pens, tubeworms, and some algae. In this form, the chitin chains are parallel to each other, forming a monoclinic lattice. This arrangement results in only intrasheet hydrogen bonds, which are weaker than those in α-chitin. β-chitin has the lowest decomposition temperature (230°C) among the chitin allomorphs, due to its low degree of crystallinity and hydrogen bonding.
• γ-chitin is a less common form of chitin, found in some fungi and bacteria. In this form, the chitin chains alternate between parallel and antiparallel orientations, forming a hexagonal lattice. This arrangement results in both intrasheet and intersheet hydrogen bonds, similar to those in α-chitin. γ-chitin has an intermediate decomposition temperature (310°C) between α-chitin and β-chitin, due to its intermediate degree of crystallinity and hydrogen bonding.

The different forms of chitin have different physical and chemical properties, such as solubility, crystallinity, mechanical strength, and biodegradability. These properties affect their applications in various fields, such as medicine, biotechnology, agriculture, and industry.

Chitinases are a group of enzymes that catalyze the hydrolysis of chitin, a structural polysaccharide of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues. Chitinases are widely distributed in nature, found in bacteria, fungi, plants, animals, and even viruses. Chitinases play an important role in chitin biodegradation, which affects the global carbon and nitrogen cycles. Chitinases also have various biotechnological applications, such as biocontrol agents, biofertilizers, biomedical materials, and food processing.

Chitinases can be classified into different families based on their amino acid sequence similarities, catalytic mechanisms, and substrate specificities. The most studied families are family 18 and family 19 chitinases, which use a retaining and an inverting mechanism of hydrolysis, respectively. Family 18 chitinases are found in archaea, bacteria, eukaryotes, and viruses, while family 19 chitinases are mainly found in plants and some bacteria. Family 20 chitinases are another group of enzymes that hydrolyze GlcNAc-containing substrates, such as peptidoglycan and chitooligosaccharides.

Chitinases can also be divided into two types based on their mode of action: endo-chitinases and exo-chitinases. Endo-chitinases cleave the glycosidic bonds randomly along the chitin chain, producing soluble oligosaccharides of various lengths. Exo-chitinases act on the non-reducing ends of the chitin chain, releasing diacetylchitobiose or GlcNAc monomers. Some chitinases can also act on chitosan, a deacetylated derivative of chitin that has biomedical applications. These enzymes are called chitosanases and belong to different families than chitinases.

The role of chitinases in chitin degradation is to break down the insoluble and recalcitrant polymer into smaller and more soluble units that can be further utilized by the microorganisms or other organisms. Chitin degradation involves two main steps: depolymerization and deacetylation. Depolymerization is the process of reducing the polymer length by the action of chitinases, resulting in the formation of GlcNAc units or oligomers. Deacetylation is the process of removing the acetyl groups from GlcNAc units by the action of chitin deacetylases, resulting in the formation of glucosamine units and acetic acid. The end products of chitin degradation can serve as nutrient sources or signaling molecules for the chitinolytic organisms or other organisms.

Chitin degradation is a complex and dynamic process that involves multiple enzymes and factors. The expression and activity of chitinases are regulated by various environmental conditions, such as temperature, pH, oxygen availability, substrate concentration, and presence of other carbon sources. The interaction and synergy between different chitinases and other enzymes are also important for efficient chitin degradation. Moreover, the structure and properties of chitin can affect its susceptibility to enzymatic hydrolysis. For example, different forms of chitin (α, β, γ) have different degrees of crystallinity and hydrogen bonding that influence their accessibility to chitinases.

In summary, chitinases are key enzymes in the degradation of chitin, a major biopolymer in nature. Chitinases have diverse structures, mechanisms, and functions that enable them to hydrolyze chitin into smaller units. Chitin degradation is a multifactorial process that depends on various enzymatic and environmental factors. Chitin degradation has significant ecological and biotechnological implications for carbon and nitrogen cycling, biocontrol, biomedicine, and food processing.

Chitin is a widely distributed polysaccharide in nature, found in the cell walls of fungi, the exoskeletons of arthropods, and some structures of other invertebrates. Chitin degradation is an important process that recycles carbon and nitrogen from this abundant biopolymer and also provides a source of nutrients for many microorganisms. Chitin degradation is carried out by a diverse group of microorganisms that produce different enzymes and mechanisms to break down the β-1,4-linked N-acetylglucosamine units of chitin. Some of the major groups of microorganisms involved in chitin degradation are:

• Chitinolytic bacteria: Chitinolytic bacteria are capable of producing chitinases, enzymes that break down chitin into smaller, more easily metabolized molecules. Chitinolytic bacteria are widely distributed in different habitats, such as soil, freshwater, marine, and animal guts. Chitinolytic bacteria belong to many genera of Gram-negative and Gram-positive bacteria, but not to Archaebacteria. Some examples of chitinolytic bacteria are Vibrio, Photobacterium, Aeromonas, Cytophaga, Streptomyces, Bacillus, Clostridium, and Chromobacterium . Chitinolytic bacteria can degrade chitin by either a chitinoclastic mechanism (solely by hydrolysis of glycosidic bonds) or a deacetylation mechanism (by conversion of chitin to chitosan and then hydrolysis) .
• Chitinolytic fungi: Chitinolytic fungi are also important players in chitin degradation. Chitinolytic fungi are mainly found in soil, where they compete with bacteria for chitin substrates. Chitinolytic fungi belong to different groups, such as Mucorales, Deuteromycetes, Ascomycetes, and Basidiomycetes. Some examples of chitinolytic fungi are Mortierella, Aspergillus, Verticillium, Thielavia, Trichoderma, Penicillium, and Humicola . Chitinolytic fungi produce a complex system of extracellular chitinases that can degrade chitin by a chitinoclastic mechanism. Some fungi can also produce chitin deacetylases that convert chitin to chitosan and then degrade it by a deacetylation mechanism .
• Slime molds: Slime molds are another group of organisms that can break down chitin. Slime molds are eukaryotic protists that can exist as single cells or as multicellular aggregates. Slime molds are found in soil and decaying organic matter. Slime molds produce a complex of extracellular chitinases that can degrade chitin by a chitinoclastic mechanism . Some examples of slime molds that can degrade chitin are Physarum polycephalum and Plasmodium.
• Protozoa and algae: Protozoa and algae are also involved in chitin degradation, although to a lesser extent than bacteria and fungi. Protozoa and algae are unicellular eukaryotes that can be found in various aquatic and terrestrial habitats. Protozoa and algae can degrade chitin by engulfing chitinous food particles and digesting them intracellularly with the help of their own or symbiotic chitinases . Some examples of protozoa and algae that can degrade chitin are Hartmanella, Schizopyrenus, and Nitzschia alba.

There are different classes and families of chitinases that act on different stages of chitin degradation and might even utilize different mechanisms of degradation. The important families of chitinases include family 18, 19, and 20 chitinases. These chitinases might differ in their source and their structural components.

Family 18 chitinases

Family 18 chitinases are retaining enzymes that include both chitinases and chitosanases. These are found in many organisms, including archaea, bacteria, eukaryotes, and viruses. This family of enzymes is widely studied.

These chitinases are further classified into subclass A, B, and C on the basis of their amino acid sequence similarities.

In some of the chitinases of the family 18, only a catalytic domain is found, whereas others might have one or more carbohydrate-binding modules.

The mechanism of catalysis in family 18 chitinases has some modifications compared to the typical double retaining mechanism. Instead of using a carboxylate side chain of the enzyme as the catalytic nucleophile, family 18 chitinases use the acetamido group of the C-1 sugar.

Family 19 chitinases

Family 19 chitinases are different from family 18 chitinases as they use an inverting mechanism leading to α-anomeric hydrolysis rather than the retaining mechanism.

Traditionally, family 19 chitinases were known to exist only in plants, but some bacterial chitinases are also added to the family over the years.

The family 19 chitinases consist of catalytic domains that have a lysozyme-like fold with shallow substrate-binding grooves that are not rich in aromatic residues.

Due to the lack of information about the structure of these enzymes, information on their interaction with the substrate is also limited.

Chitin deacetylases

Chitin deacetylases include enzymes like peptidoglycan N-acetyl glucosamine deacetylase and peptidoglycan N-acetylmuramic acid deacetylase that remove the acetyl groups in the substrates.

These enzymes are essential as they reduce the branching in the structure, which reduces the steric hindrance for other exo and endoenzymes.

Chitin deacetylases are mostly found in bacteria and fungi, where they play a role in modifying cell wall components or producing bioactive compounds.

Chitin deacetylases belong to polysaccharide deacetylase (PD) superfamily that share a common catalytic mechanism involving a zinc ion and two conserved histidine residues.

Chitin degradation is a complex process that involves various enzymes, microorganisms, and environmental conditions. Several factors can influence the rate and extent of chitin degradation, such as:

• Enzymatic activity: Chitinases, enzymes that break down chitin, can be produced by various microorganisms, fungi, and animals. The type, number, and specificity of chitinases can affect the efficiency and mode of chitin degradation. For example, endochitinases cleave chitin at random sites, while exochitinases release diacetylchitobiose from the ends of the polymer. Chitin deacetylases remove the acetyl groups from chitin, converting it to chitosan, which can be further degraded by chitosanases .

• pH: Chitin degradation can be affected by the pH of the environment. Different chitinases have different optimal pH ranges for their activity. For instance, some bacterial chitinases work best at acidic pH, while some fungal chitinases prefer alkaline pH. The pH can also affect the solubility and stability of chitin and its derivatives.

• Temperature: Like pH, temperature can also affect chitinase activity and chitin solubility. Higher temperatures can increase the rate of enzymatic reactions, but also cause thermal denaturation of enzymes and substrates. Temperature can also influence the microbial community composition and diversity, as different microorganisms have different temperature preferences and tolerances .

• Moisture content: Chitin is insoluble in water, so moisture content can play a role in chitin degradation. Higher moisture content can facilitate the diffusion of enzymes and substrates, as well as the growth and activity of microorganisms. However, excess moisture can also impair aeration and oxygen availability, which can affect aerobic chitin degraders .

• Organic matter: The presence of organic matter rich in chitin or other carbon sources can also influence chitin degradation. Organic matter can provide substrates and nutrients for microorganisms, as well as complexing agents that can enhance chitin solubility. However, organic matter can also compete with chitin for enzyme binding sites or microbial utilization. Moreover, organic matter can affect other factors such as pH, temperature, and moisture content .
  
  Process of chitin degradation

Chitin degradation is the process of breaking down chitin, a complex polysaccharide composed of N-acetylglucosamine units, into simpler and smaller molecules that can be utilized by various organisms. Chitin degradation is essential for the recycling of carbon and nitrogen in the marine environment, where chitin is abundant in the exoskeletons of crustaceans, insects, and other invertebrates.

Chitin degradation involves two main steps: depolymerization and deacetylation. Depolymerization is the hydrolysis of the β-1,4-glycosidic bonds that link the N-acetylglucosamine units together, resulting in the formation of oligomers and monomers. Deacetylation is the removal of the acetyl groups from the N-acetylglucosamine units, producing glucosamine and acetic acid.

Chitin degradation is mediated by a variety of enzymes, collectively known as chitinases, that are produced by different microorganisms, such as bacteria, fungi, algae, and protozoa. Chitinases are classified into different families and classes based on their amino acid sequence similarity, substrate specificity, and catalytic mechanism. The most important families of chitinases are family 18, 19, and 20.

Family 18 chitinases are retaining enzymes that use a double displacement mechanism to cleave the glycosidic bonds without changing the anomeric configuration of the sugar units. They include both endo- and exo-chitinases that act on different positions of the chitin chain. Endo-chitinases randomly cleave the internal bonds of the chitin chain, producing chitooligomers of various lengths. Exo-chitinases sequentially cleave the terminal bonds of the chitin chain from either the reducing or non-reducing end, producing mainly diacetylchitobiose or N-acetylglucosamine.

Family 19 chitinases are inverting enzymes that use a single displacement mechanism to cleave the glycosidic bonds with inversion of the anomeric configuration of the sugar units. They are mostly found in plants and some bacteria and act as endo-chitinases.

Family 20 chitinases are also retaining enzymes that use a double displacement mechanism to cleave the glycosidic bonds without changing the anomeric configuration of the sugar units. They include N-acetylglucosaminidases that hydrolyze the oligomers and monomers produced by other chitinases into N-acetylglucosamine.

Chitin deacetylases are another group of enzymes that catalyze the removal of acetyl groups from N-acetylglucosamine units, converting chitin into chitosan. Chitosan is a partially deacetylated derivative of chitin that has different physical and chemical properties and biotechnological applications. Chitosan can be further degraded by chitosanases that hydrolyze the β-1,4-glycosidic bonds between glucosamine units.

The process of chitin degradation can be influenced by several factors, such as moisture content, added glucose, aeration, organic matter, pH, temperature, and microbial community composition. These factors can affect the availability and accessibility of chitin as a substrate, the expression and activity of chitinolytic enzymes, and the interactions and cross-feeding among different microorganisms involved in chitin degradation.

Chitin degradation is an important ecological process that contributes to nutrient cycling, carbon sequestration, organic matter remineralization, microbial diversity, and biogeochemical transformations in marine ecosystems.

Chitin is a biopolymer that is widely distributed in nature, especially in the cell walls of fungi and the exoskeletons of arthropods. Chitin is also a renewable and biodegradable resource that has many potential applications in various fields. However, chitin is resistant to degradation by most vertebrates due to the lack of chitinolytic enzymes. Therefore, chitin degradation mainly depends on the activity of microorganisms that produce various chitinases and chitosanases.

Microorganisms that degrade chitin can be found in different habitats, such as soil, water, and animal guts. These microorganisms can utilize chitin as a source of carbon, nitrogen, and energy, and also as a signal for colonization and pathogenesis. Microbial degradation of chitin can occur by one of the two mechanisms: chitinoclastic or deacetylation.

Chitinoclastic mechanism

The chitinoclastic mechanism of chitin degradation involves the hydrolysis of the β-1,4-glycosidic bonds between the N-acetylglucosamine units of chitin by chitinases. Chitinases are a group of glycosyl hydrolases that can be classified into two types: endo-chitinases and exo-chitinases.

Endo-chitinases cleave the chitin chains at random internal sites, producing soluble oligomers of various sizes, such as diacetylchitobiose, triacetylchitotriose, and tetraacetylchitotetraose. Exo-chitinases act on the non-reducing ends of the chitin chains, releasing diacetylchitobiose units. These units can be further hydrolyzed by β-N-acetylglucosaminidases to produce N-acetylglucosamine monomers.

Chitinases are widely distributed among bacteria, fungi, plants, animals, and viruses. They belong to different families based on their amino acid sequence similarity and catalytic mechanism. For example, family 18 and 19 chitinases are retaining enzymes that use a double-displacement mechanism to preserve the anomeric configuration of the substrate. Family 18 chitinases are found in archaea, bacteria, fungi, eukaryotes, and viruses, while family 19 chitinases are mostly found in plants and some bacteria. Family 20 chitinases are inverting enzymes that use a single-displacement mechanism to invert the anomeric configuration of the substrate. Family 20 chitinases include N-acetylglucosaminidases from bacteria, fungi, and humans.

The structure and function of chitinases are influenced by various factors, such as substrate specificity, pH, temperature, metal ions, inhibitors, and modulators. Chitinases can also interact with other proteins or domains to form complex systems that enhance their activity or specificity. For example, some bacterial chitinases have carbohydrate-binding modules (CBMs) that bind to insoluble chitin and facilitate its hydrolysis.

Deacetylation mechanism

The deacetylation mechanism of chitin degradation involves the removal of acetyl groups from the N-acetylglucosamine units of chitin by chitin deacetylases. Chitin deacetylases are a group of enzymes that catalyze the conversion of chitin to chitosan, which is a partially or fully deacetylated derivative of chitin.

Chitosan has different physicochemical properties than chitin, such as higher solubility in acidic solutions and higher reactivity with other molecules. Chitosan also has various biological activities, such as antimicrobial, antifungal, immunostimulatory, and wound-healing properties. Therefore, chitosan has many potential applications in medicine, agriculture, biotechnology, and environmental engineering.

Chitosan can be further degraded by chitosanases, which are enzymes that hydrolyze the β-1,4-glycosidic bonds between the glucosamine units of chitosan. Chitosanases are also glycosyl hydrolases that belong to different families based on their sequence similarity and catalytic mechanism. For example, family 46 and 75 chitosanases are retaining enzymes that use a double-displacement mechanism to preserve the anomeric configuration of the substrate. Family 8 and 80 chitosanases are inverting enzymes that use a single-displacement mechanism to invert the anomeric configuration of the substrate.

Chitosan degradation produces glucosamine oligomers or monomers that can be further metabolized by microorganisms for various purposes. For example, some bacteria can use glucosamine as a carbon and nitrogen source, while some fungi can use glucosamine as a signal for morphogenesis and pathogenicity.

Conclusion

Microbial degradation of chitin is a complex and diverse process that involves various enzymes, mechanisms, and factors. Microorganisms that degrade chitin can benefit from the utilization of chitin as a nutrient source, a signal molecule, or a precursor for other compounds. Microbial degradation of chitin also plays an important role in the global carbon and nitrogen cycles, as well as in the bioremediation of chitin-containing wastes. Understanding the molecular and ecological aspects of microbial chitin degradation can help to explore the potential applications of chitin and its derivatives in various fields.

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