INTRODUCTION:

Actinomycetes are one of the main and very widely distributed population groups of soil. Geographical location including organic matter content, soil pH,soil temperature, moisture content, aeration, soil type and cultivation etc. determines the number and forms of actinomycetes present in a specific soil¹. Many of the Actinomycetes are free-living, which are distributed extensively in both terrestrial and aquatic environments². The title Actinomycetes is originated from the Greek words “aktis” or “aktin” for “ray” and “mukes” for “fungi” since the hyphae grow by the mixture of tip extension and branching. Usually, they were deemed as an intermediate type among fungi and bacteria. Actinobacteria produce mycelium and reproduce by sporulation since they apparently resemble filamentous fungi. Though, like bacteria, cells are thin with peptidoglycan cell walls and prokaryotic nucleoids³. The adequate exclusive features delineate the Actinobacteria into ‘Kingdom bacteria’. They are spore-producing aerobic, gram-positive bacteria with a high GC rich genome and characterized with a non-septate distinctive substrate and aerial mycelium.

Actinomycetes were the most widely used group of secondary metabolites produced in medical and agricultural use by microorganisms and commercial-importance enzymes. Actinomycetes are a good source for new antibiotics, enzyme inhibitors, enzymes, vitamins, and immune-modifiers especially Streptomyces⁴.

The unique biodiversity of Western Ghats is conserved and protected by wildlife sanctuaries, national parks. Western Ghats biosphere reserves situated in states, hill ranges run through, like Karnataka, Gujarat, Tamil Nadu, Maharashtra, and Kerala. It is one of the global biodiversity hotspots covering an area of 180,000 km2 and harbours with numerous species of plants, animals, and microbes. Western Ghats forest regions are largely underexplored, though in recent times few studies were carried out for bio prospection. The Chitinase producing Streptomyces spp. are less characterized from the Western Ghats.

Streptomyces are prominent microorganisms of the saprophytic soil, popular antibiotics, and extracellular producer enzymes. Soil and marine actinomycetes have important hunger survival abilities. During a late growth cycle, antibiotics and protein inhibitors evolve when familiar regulation processes, including transcriptional control, are ineffective⁵. There are several reports of isolation of chitinases from actinomycetes for example biocontrol agent antifungal S. lydicus WYEC108 has been able to not only destroy Pythium ultimum germinating oospores but also to damage the fungal hyphae’s cell walls⁶. WYEC108 also developed high chitinase levels in a growing media when induced by Chitinase fungal cell walls. Therefore, negligible enzyme levels have been observed if S. lydicus WYEC108 has been produced without chitin. Production of chitinase from S. lydicus WYEC108 has been caused by chitin of colloidal, chit oligosaccharides, and N-acetylglucosamine. Therefore, high (not low) glucose and CMC (carboxy methyl cellulose) levels have been repressed in the synthesis⁷. Chitinase isolated from the soil bacterial strain S. griseus MG3 having high activity over a broad range of pH, such as alkaline and acidic pH was demonstrated⁸. This property together with the ability of the crude enzyme inhibitors, MG3 is highly useful for implementation as a biocontrol agent in the increasing of all fungi pathogens tested.

Chitin degrading enzymes other than chitinases like β–N– acetyl hexosaminidase (hexosaminidase) chitin deacetylase and chitosanases have acquired a lot of importance because of their biotechnological applications. Organisms that manufacture chitinase can be utilized directly or indirectly as biocontrol agents using their purified proteins⁹. It was estimated that the loss of crops due to disease-causing pathogens is 25 percent of the total yield in Western countries and in most of the developing countries, it is around 50%¹⁰. Fungal infections alone result in a third of these diseases. As we know that the nonspecific toxicity and adverse effects of commercial fungicides on the environment have made us think about other lines of pest control strategies like the use of chitinases/chitinolytic enzymes as antifungal agents to control pathogens.

The chitinase [EC.3.2.1.14] group is the N-acetyl D-glucosamine Residue group of enzymes responsible for Chitin hydrolysis, a linear polymer of β(1,4) nitrogen. Chitinases are widespread in nature and play a key role in chitin degradation. They play a pivotal role in performing many biological functions and widely distributed in plants and microbes including bacteria, fungi and actinomycetes in plants, chitinases are involved in defense against pathogens.

Actinobacteria:

Actinomycetes are largely found in the superficial layer of soils with alkaline and rich organic matter and gradually decreases when increasing the depth. They are ubiquitous microbial groups broadly distributed in nature such as soils, fresh and saltwater, and also in the air. The majority are inhabitants of the soil (Kuster, 1968), although they are broadly dispersed in diverse extreme habitats including deep-sea vent sediments¹¹. Western Ghats are known to be an active hot spot region with huge biodiversity wealth. Huge source of potent microorganisms that could be potentially exploited for various industrial and medicinal applications in the microbial variety of Western Ghats¹²

The phylum Actinobacteria is included in the fourth and fifth volumes of Bergey’s Manual of Determinative Bacteriology, which represent the largest taxonomic units including 5 subclasses, 6orders and 14 suborders¹³. All Actinomycetes are encompassed by the Actinomycetales order and divided into four families Actinomycetaceae, Actinoplanaceae, Mycobacteriaceae and Streptomycetaceae. The two generally defined Actinobacterial genera are Streptomyces and Micromonospora. The genus Streptomyces is extensively known as the largest natural bioactive product reservoirs such as antibiotics, enzymes, immunosuppressive agents, antitumor agents, etc. Studies on actinomycetes have enhanced significantly in the last decade and research has taken the phase in many parts of India¹⁴.

Actinomycetes represent an important part of soil micro-flora and are widely distributed in different types of soil¹⁵. They usually develop as hyphae such as fungi liable for the characteristic “earthy” smell of the newly transformed healthy soil. Actinomycetes are develop in the form of mycelia, their natural existence mostly restricted in the soils¹⁶. The actinomycetes are of particular biotechnological interest because they are known to generate chemical diverse compounds with a broad range of biological activities. Among the filamentous Actinomycetes, approximately 75% of metabolites are obtained from species of the genus Streptomyces. They constitute an important microbial population factor in the majority of soils and can generate extracellular enzymes that can decompose different materials. In 1943, Waksman and Henrici introduced the genus Streptomyces¹⁷. Concerning the number and species diversity, Streptomyces signifies the largest taxonomic unit in Actinomycetes. Currently, more than 500 species of the Streptomyces genus have been defined and also unveils extensive phylogenetic spread. They are non-acid- Fast, gram-positive, aerobic bacteria with around 70% GC content in the genome¹⁸. Due to the development of spectacular diversity of secondary metabolites, Streptomyces is considered as the chief competent chemist in nature with boundless interest in industry and medicine.

Streptomyces - A Source of Industrial Enzymes:

The Actinobacterial members especially Streptomyces are the dominant, auspicious producers of innumerable antibiotics, secondary metabolites, and industrial enzymes¹⁹. They have the ability to bio transform and bio convert certain organic compounds, urban and agricultural wastes into economic valued products. Streptomyces, a successful reservoir of a diverse collection of industrially important enzymes actively participated in the putrefaction of various organic compounds and the produced enzymes are commonly exploited in leather, textile, food, detergent, pharmaceutical, and medical sectors^16,17. To encounter the increasing demand for industrial enzymes, there is a need for endless research and innovations to find novel highly efficient enzymes with cost-effective and environmentally friendly production. Further, the large accessibility of genomic and proteomic data leads to the high throughput production of high-value enzymes from Streptomyces strains.

Chitin Formation:

In nature chitin is one of the most essential biopolymers. It is a strongly stored enzyme present in all chitin synthesizing organisms. The enzyme makes use of UDP GlcNAc (UDP‐N‐acetyl glucosamine) as an activated sugar donor contributing to the formation of the polymer of chitin^18,19. The first step in chitin formation is trehalose hydrolysis which is accompanied by glucose phosphorylation. Transmutation results in the formation of phosphorylated fructose. Amination and acetylation follow and then the final conversion of the product results in the formation of nucleotide phosphate- acetylated amino sugar²⁰.

Chitin- Enzymatic Degradation:

The recalcitrant nature of chitin forces enzymes to develop which could efficiently degrade these carbohydrate species to generate chitin enzymes like chitinases for various reasons. For instance, in chitinase species (e.g., crustaceans) chitinases are required, due to the growth requires chitin degrading enzymes to reshape them. In addition, higher plants develop chitinases that use the enzymes in their defence themselves toward pathogenic actions by deteriorating chitin in bacteria as well as fungi cell walls.

Chitin enzymatic degradation may take 2 distinct pathways, a chitinolytic pathways or through chitosan. The chitin degradation strategy provides direct hydrolysis of the glycosidic bonds within GlcNAc units by chitinases²¹. The recent discovery of lytic polysaccharide monooxygenases, in synergy with Chitinases, also performs chitinolytic activity. Additionally, chitin could be decomposed by first being solubilised by deacetylation. This procedure is performed by chitin deacetylation and chitosanases are hydrolysed with the derived substrate (chitosan).

Chitinase Classes and structure:

In terms of considering the amino acid similarity between chitinases from different species, 5 families of chitinases have been suggested and classified as two families, namely family 18 and family 19. Family 18 chitinases including plants, fungi, bacteria are generally distributed in species (classes III and V), viruses, and mammals. The chitinases are also sub-categorized as per the N-terminal sequence, localization of the enzyme, isoelectric pH, inducers, and signal peptides.. Chitinases of Class I have similar features to class IV Chitinases, such as immunological properties, but are substantially less than chitinases of Class I²².

The structure of a number of GH enzymes is modularly possessing a catalytic and one or more non‐catalytic domains that can or cannot play a part in the substrate binding (fig 1). The sequenced bacterial chitinases till now do not show any connection concerning the percentage of comparative work performed by comparison between Catalytic and Citin-binding fields. It seems that the binding domain of chitin among bacterial chitinases is conserved evolutionarily²³.

A wide analysis of chitin-binding domains in plant proteins has shown that eight cysteines are greatly preserved in the Chitin binding domain. In addition, plants also have CBPs (chitin-binding proteins) with a high cysteine chitin-binding domain, without the activity of chitinase. In bacteria, the binding domain is dissimilar from the one obtained from plants that contain eight preserved cysteine residues²⁴. Instead, some amino acids, most of them tryptophane, are among those that remain in the Chitin-binding region of the bacteria and have been shown to include cellulase-cellulose binding. The function of chitinases was also involved in the binding of the Chitin binding non-catalytic protein to chitin.

Figure 1: 3D Structure of a Chitinase (Source: Fusetti et al., 2002).

(Grey ribbon ‐‐the backbone, Blue –domain, Purple sticks ‐‐Solvent‐expose aromatic side chains lining the active site cleft, Orange –NAG)

Sources of Chitinases:

Glycosyl hydrolases of 20 kDa to approximately 90 kDa are chitinase-size (E.C 3.2.2.14)²⁵. They are observed in a number of species including actinomycetes, arthropods, bacteria, yeasts, fungi, humans, and plants. Chitinases can directly degrade chitin to low molecular chitooligomers with a wide variety of commercial, agricultural, and medical functions including elicitor activity and anti-tumor activities. Bacterial chitinases are harvested in order to satisfy nutritional requirements, so chitin could be useful as a source of nitrogen and carbon²⁵. Many bacteria produce as well as secrete chitinolytic enzymes following induction with chitin in the media in which they grow. The Plant chitinolytic enzymes were classified into 5 or 6 groups depending on their amino acid sequence. The chitinases from the class I, II, IV possess a key structural unit having two rich globular domains. The class III and V chitinases encompass a major unit possessing eight α and β strands and a TIM barrel. Dependent upon the amino acid sequence of their GH 18 modules, fungal chitinases have been classified into three separate subgroups, that is, A, B and C. The chitinases were used to manage insects and pests. The insect chitinases have been reported to be cleaned from the molting fluid and integument of the tobacco hornworm, Manduca sexta, venom gland of endoparasite wasp, silkworm and Chelonus i.e. Bombyx mori as well. The insect chitinases as well as many others have been characterized by their chemical, physical and kinetic characteristics²⁷. It was only recently that chitinases were identified in various human tissues and their function was related to the prevention of parasite infections along with some allergic conditions²⁸.

Mechanism of Hydrolysis of Chitinases:

The hydrolysis of the glycosidic bond which can either result in the maintenance or reversed the anomeric configuration is a nucleophilic replacement for the anomeric carbon. Since acid catalysis is the basis of both the hydrolysis reactions, the active site should have a couple of carboxylic acids. The carboxylic acid which receives a proton acts as a proton acceptor (an inverting mechanism). The proton donor is located in both systems within a hydrogen bonding gap with glycosidic oxygen.

Family-18 chitinases can act in a holding process when the water entry leads to hydrolysis findings and formation of oxazoline ring on the substratum (between N-acetyl group carbonyl oxygen and C-1 of GlcNAc). With the creation of the ring of oxazoline and the lack of steric obstacles, water is expected to join the β-anomer from the other side so as to keep the C-1 configuration²⁹.

Family 19 chitinases (referred as “class I, class II, class III, class IV, class V, class VI”) are primarily plant-producing enzymes that are used to protect plants against fungal pathogens and insect pathogens by defeating the cell wall containing their chitin. Family nineteen Chitinases operate by inverting process. In this class the steric impediment inhibits the creation of the structure of the oxazoline ring and in this instance, the arrangement is reversed to the α-anomer and therefore accommodating the oxazoline ring is created.³⁰

Microbial Production Methods of Chitinase:

Microbial production of chitinase was reported to produce in both solid-state and submerged fermentation. The suitability of the fermentation methods may vary from organism to organism which is involved in the fermentation.

There are few reports on the production of chitinase under SSF employing various microbes and different agro-based by-products.The production of chitinase under SSF using sugarcane bagasse by Serratia marcescens and dos³¹. Screened for extracellular chitinase development in solid-state fermentation, fourteen Penicillium strains on wheat bran as crude chitin blend medium.For submerged fermentation, various microbes were used to produce chitinase³². Bacterial isolates such as Bacillus thuringiensis, Vibrio alginolyticus, Bacillus pabuli, Bacillus lichiniformis , Nocardia orientails and Serratia marcescens were reported to produce considerable titre of chitinase under submerged fermentation.

It is useful to analyse and comparison the activity of chitinase on an independent system basis with a responsive, quick, and exact basis to analyse the diversity of species and recommend enzymes that require chitinase degradation. Chitinase upon catalysing the hydrolysis of chitin releases N-acetyl-D-glucosamine as the end product. The activity of Chitinase was evaluated using different approaches. Colorimetric, radiochemical, and viscosimetric approaches are the most widely used. In the viscosimetric approach, the evolution of the activity of chitinase could be performed by tracking the viscosity shift in derivatives of solubilized chitin, including carboxymethyl chitin, 6-O-hydroxypropyl-chitin, and ethylene glycol chitin. The viscosimetric approach is more resistant to end chitinases than other approaches.^33,34 Recombinant techniques can be used to increase the production of chitinases for industrial purposes^35,36

CONCLUSIONS AND FUTURE PROSPECTS:

Chitinase, an important commercial enzyme produced by viruses, fungi, and insects, has a wide range of applications, including waste management, single-cell protein production, protoplast generation, biocontrol, etc. Its use as an agricultural biological control agent has far-reaching effects. As an alternative non-toxic chemical and pesticide³⁷.On an industrial scale, underwater fermentation is an optional form of chitinase production, but a larger product can be obtained using solid state fermentation that requires further research. Because of the numerous applications of chitinase, their demand is expected to rise in the near future.

CONFLICT OF INTEREST:

The author have no conflicts of interest regarding this investigation.

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Received on 25.06.2022 Modified on 15.11.2022

Research J. Pharm. and Tech 2023; 16(11):5021-5026.

DOI: 10.52711/0974-360X.2023.00813