Chitinases: An update

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Review Article Chitinases: An update Rifat Hamid, Minhaj A. Khan, Mahboob Ahmad, Malik Mobeen Ahmad1, Malik Zainul Abdin1, Javed Musarrat2, Saleem Javed

Departments of Biochemistry, 1 Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi, 2Department of Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh, India

ABSTRACT

Address for correspondence: Dr. Saleem Javed, E-mail: saleemjaved70@ yahoo.co.in

Chitin, the second most abundant polysaccharide in nature after cellulose, is found in the exoskeleton of insects, fungi, yeast, and algae, and in the internal structures of other vertebrates. Chitinases are enzymes that degrade chitin. Chitinases contribute to the generation of carbon and nitrogen in the ecosystem. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications, especially the chitinases exploited in agriculture fields to control pathogens. Chitinases have a use in human health care, especially in human diseases like asthma. Chitinases have wide-ranging applications including the preparation of pharmaceutically important chitooligosaccharides and N-acetyl D glucosamine, preparation of single-cell protein, isolation of protoplasts from fungi and yeast, control of pathogenic fungi, treatment of chitinous waste, mosquito control and morphogenesis, etc. In this review, the various types of chitinases and the chitinases found in different organisms such as bacteria, plants, fungi, and mammals are discussed.

Received : 21‑11‑11 Review completed : 16‑03‑12 Accepted : 21‑05‑12

KEY WORDS: Chitinases, chitinolytic enzymes, endochitinase, exochitinases

C

hitin, a linear polymer of β-1, 4-N-acetylglucosamine (GlcNAC), is the second most abundant biopolymer on the planet.[1] Chitin is found in the outer skeleton of insects, fungi, yeasts, algae, crabs, shrimps, and lobsters, and in the internal structures of other invertebrates.[2] The overall weight of shellfish (e.g. crab, krill and shrimp), which is disposed as waste, is approximately 75%, and chitin consists of 20-58% of that dry weight.[3] Amid a broad array of applications, chitin has its use in order to boost up the formation of extracellular chitinase. Chitin and its associated materials have a broad usage in drug delivery, wound healing, dietary fiber, and in waste water treatment.[4] Chitin is a white, hard, inelastic polysaccharide, and is a major contribution to pollution in coastal areas.[5] Chitin has a high percentage of nitrogen (6.89%), which makes it a useful chelating agent.[6] Chitin exists in 2 allomorphic forms i.e. α-chitin and β-chitin. These 2 forms of chitin vary in packing and polarities of adjacent chains in the succeeding sheets.[7,8] Chitin can be degraded by chitinase. The catabolism of chitin takes place in 2 steps, involving the initial cleavage of the chitin polymer by Access this article online Quick Response Code:

Website: www.jpbsonline.org DOI: 10.4103/0975-7406.106559

chitinases into chitin oligosaccharides and further cleavage to N-acetylglucosamine, and monosaccharides by chitobiases.[9] Chitinases (E.C 3.2.2.14) are glycosyl hydrolases with the sizes ranging from 20 kDa to about 90 kDa.[2] They are present in a wide range of organisms such as bacteria, fungi, yeasts, plants, actinomycetes, arthropods, and humans. Chitinases have the ability to degrade chitin directly to low molecular weight chitooligomers, which serve a broad range of industrial, agricultural, and medical functions such as elicitor action and anti-tumor activity.[10] N-acetylglucosamine (GlcNAc) has received special attention for the treatment of osteoarthritis. [11] Chitinases have been receiving an increased attention due to their role in the biocontrol of fungal phytopathogens[12] and harmful insects.[13] Of late, chitinases have also attained a lot of attention as they are thought to play a key role in mosquito control and plant defense systems against chitin-containing pathogens.[13] Chitin and chitinases are used by pathogens (mainly protozoan or metazoan) causing animal and human diseases. Several pathogens contain chitin coats, giving them protection against both external and internal (in a host) environment. Others attack their host using chitinase. In order to establish a successful infection or transmission from one vertebrate to another, they exploit the chitin-containing structures of the host.[14] A number of bacteria have the ability to produce chitinases, including Streptomyces,[15] Alteromonas,[16] Escherchia,[17] Aeromonas.[18] Chitinase-producing bacteria have been isolated from soil, shellfish waste, garden and park waste compost, and hot springs.[10]

How to cite this article: Hamid R, Khan MA, Ahmad M, Ahmad MM, Abdin MZ, Musarrat J, Javed S. Chitinases: An update. J Pharm Bioall Sci 2013;5:21-9.

Journal of Pharmacy and BioAllied Sciences January-March 2013 Vol 5 Issue 1

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Hamid, et al.: Chitinases

Chitinases have been divided into 2 main groups: Endochitinases (E.C 3.2.1.14) and exo-chitinases. The endochitinases randomly split chitin at internal sites, thereby forming the dimer di- cetylchitobiose and soluble low molecular mass multimers of GlcNAc such as chitotriose, and chitotetraose. [19] The exochitinases have been further divided into 2 subcategories: Chitobiosidases (E.C. 3.2.1.29),[20] which are involved in catalyzing the progressive release of di-acetylchitobiose starting at the non-reducing end of the chitin microfibril, and 1-4-β-glucosaminidases (E.C. 3.2.1.30), cleaving the oligomeric products of endochitinases and chitobiosidases, thereby generating monomers of GlcNAc.[19] Based on this nomenclature, the identification of various chitinases in Serratia plymuthica has been carried out for the strains IC1270, IC14, and HRO-C48. It has been reported that, in the strains IC14 and HRO-C48, an endochitinase and a 100 kDa N-acetyl-β-1,4-D-hexosaminidase or chitobiase are produced, while the strain IC1270 produces N-acetyl- β-D- glucosaminidases of 89 and 67 kDa, a 50 kDa chitobiosidases, and an endochitinase with a molecular mass of 59 kDa.[21,22] As far as the amino acid similarity of chitinases from various organisms is considered, 5 classes of chitinases have been proposed, and have been categorized into 2 families, which include families 18 and 19 of glycosyl hydrolases.[23] Family 18 chitinases have a large distribution in organisms, including plants, bacteria, fungi (classes III and V), mammals, and viruses. The sub-classification of chitinases is also based on N-terminal sequence, isoelectric pH, localization of the enzyme, signal peptide, and inducers. The class I chitinases have been found in plants, whereas class II enzymes are contained in plants, fungi, and bacteria. There is no sequence similarity of class III chitinases to enzymes of class I or II. Class I chitinases have similar characteristics to class IV chitinases, including immunological properties, but they are significantly smaller than class I chitinases.[24] Chitinases are a huge and diverse group of enzymes that show differences in their molecular structure, substrate specificity, and catalytic mechanism.[25] It is vital to study the substrate specificity of chitinases as it not only reveals the relationship between the substrate specificity and physiological roles, but also allows one to degrade chitin into novel products having industrial applications.[26] It has been found that, individually, all chitinase classes exhibit different substrate specificities and reaction mechanisms. For example, in the case of tobacco class III chitinases, a considerable level of lysozyme as well as chitinase activity is possessed while class VI chitinases only show chitinase activity.[27] Class I and class II chitinases use an inverting mechanism in order to hydrolyze β-glycosidic linkage while class III chitinases do this through a retaining mechanism. [28,29] It has been established by Sasaki et al. (2006) [30] that class III chitinases do not act against GlcNAc oligomer or polymer, but could be active on an endogenous complex carbohydrate containing a GlcNAc residue, whereas a GlcNAc sequence was the most likely substrate of the class I enzyme. The chitinases of the 2 different families do not share amino acid sequence similarity, and have completely different 3-dimensional (3D) structures and molecular mechanisms. 

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Therefore, they are likely to have evolved from different ancestors. Family 18 consists of a number of conserved repeats of amino acids. It consists of an enzyme core, which has 8 strands of parallel β sheets, forming a barrel laid down α helices, which in turn forms a ring towards the outside.[31] GH family 18 chitinases have the ability to catalyze the transglycosylation reactions. The products formed due to transglycosylation have already been reported for T. harzianum Chit42 and Chit33, as well as for Aspergillus fumigatus ChiB1.[32] A multidomain structure including catalytic domains and both a cysteine-rich chitin-binding domain (different from the catalytic domain) and a serine/threonine-rich glycosylated domain have been found as one of the structural characteristics of chitinases in various animals and microorganisms.[33] The resemblance shown by bacterial and fungal chitinases suggests that the catalytic domains are similar in all of these.[34] A broad study of chitin-binding domains in plant proteins revealed that the 8 cysteins within the chitin-binding domain are greatly conserved. Moreover, plants also possess chitin-binding proteins (CBPs) having a cystein-rich chitin-binding domain, without chitinase activity.[35] As reported by Poole et al., 1993,[36] in bacteria, the chitin-binding domain is different than that found in plants, which contain 8 conserved cystein residues. On the contrary, some amino acids, mainly tryptophan, are amongst the residues that are conserved in their chitin-binding domain of bacteria, and their involvement in the binding of cellulase to cellulose has been revealed. The role of chitinases has also been implicated in the binding of a non-catalytic chitin-binding protein to chitin.[37] It has been identified by Watanabe et al., 1997[38] that there are only 4 amino acids in the catalytic domain that are conserved between bacterial and plant class III chitinases. It is thought that the fungal chitinases attach to their substrate or cell wall with the help of the chitin-binding domain. A 6-cystein conserved region present in the chitin-binding domain of CTS1 and K1Cts1p is probably involved in the protein-protein interaction or tertiary structure through the disulphide bond formation. According to Villagomez et al., 1996, [39] the C-terminal chitin-binding domain in insect chitinases binds to the substrate and it has a characteristic 6-cystein motif similar to nematode chitinases.

Bacterial Chitinases In bacterial chitinases, the chitin-binding domain can either be located in the amino terminal or in the carboxyl terminal domains of the enzyme.[40] Most of the bacterial chitinases, which have been isolated and sequenced so far, are included in family 18 of the glycosyl hydrolases; with the exception of a chitinase (C-1) isolated from S. griseus IIUT 6037 that belongs to the family 19 of the glycosyl hydrolases.[41] Unlike bacterial chitinases, this can hydrolyze only GlcNAc-GlcNAc and GlcNAc-glucosamine linkages; chitinase C-1 of S. griseus HUT 6037 has the capability to hydrolyze glucosamine-GlcNAc and GlcNAc-GlcNAc linkages. Thus, the catalytic site of chitinase C-1 is different from other microbial chitinases. The amino Journal of Pharmacy and BioAllied Sciences January-March 2013 Vol 5 Issue 1

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terminal region of chitinase C-1 share sequences similarity with non-catalytic domains of other bacterial lytic enzymes including chitinases, proteases, and cellulases, and it is postulated that this domain serves for chitin binding.[41] Some other species of bacteria that also produce high levels of chitinolytic enzymes are Serratia.[38,42] Bacterial chitinases having a molecular weight range of 20-60 kDa are smaller than insect chitinases (40-85 kDa) while similar to that of plant chitinases (40-85 kDa).[2] Bacterial chitinases are active over a wide range of pH and temperatures, depending on the source of the bacteria from which they have been isolated. For example, endochitinase from Streptomyces violaceusniger[43] and thermostable chitinase from Streptomyces thermoviolaceus OPC-520[16] have an optimum temperature of, respectively, 28°C and 80°C. Also, the last enzyme has high pH optima in the range of 8.0 to 10.075, while the chitinase isolated from Stenotrophomonas maltophilia C3 has a pH optima in the range of 4.5 to 5.0. Bacterial chitinases also show a broad range of isoelectric points (pI 4.5-8.5).[38] It is considered that in order to supply nitrogen and carbon as a source of nutrients, bacteria mainly produce chitinases.[44] The production of chitinases in bacteria is mainly for the degradation of chitin and its utilization as an energy source. As reported by Chernin, 1997[45] and Downing, 2000,[46] some chitinases of chitinolytic bacteria, such as the chiA gene products from Serratia marcescens and S. plymuthica, are potential agents for the biological control of plant diseases caused by various phytopathogenic fungi. The latter enzymes hydrolyze the chitin present in the fungal cell wall, thereby inhibiting fungal growth. Anti-fungal proteins such as chitinases have a great biotechnological aspect because of their potential use as food and seed preservative agents and for engineering plants for resistance to phytopathogenic fungi.[47] Ordentlich (1988)[48] reported the effectiveness of S. marcescens as a biocontrol agent against Sclerotium rolfsii via its chitinolytic culture filtrate. The vast majority of known bacterial chitinases are grouped into family 18.[49] Since most bacterial chitinases that have been characterized thus far have been classified into group A, it has been speculated that group A chitinase genes are more abundant in nature than enzymes in groups B or C.[50] Bacterial chitinases generally consist of multiple functional domains, such as chitin-binding domain (CBD) and Wibronectin type III-like domain (Fn3 domains), linked to the catalytic domain. The importance of the CBD in the degradation of insoluble chitin has been demonstrated for some bacterial chitinase.[51] In contrast, it has been found that family 19 chitinases are present only in some bacterial strains and plants.[52] The presence of multiple chitinase producing enzymes have been described in various microorganisms such as Aeromonas sp. No. 10S-24,[53] Pseudomonas aeruginosa K-187,[3] Bacillus circulans WL-12.[54] A synergistic action of Chi A, Chi B, and Chi C1 of S. marcescens 2170 has been reported by Suzuki et al. (2002)[42] on chitin degradation. In spite of having comparable catalytic domains, Chi A and Chi B were thought to digest chitin chains in reverse directions, i.e., Chi A from the reducing end and Chi B from the non-reducing end. Journal of Pharmacy and BioAllied Sciences January-March 2013 Vol 5 Issue 1

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Fungal Chitinases Fungal chitinases, like bacterial chitinases, have multiple functions as they play an important role in nutrition, morphogenesis, and fungal development processes. Chitin is a major cell wall component of fungi.[19] Fungal chitinases show a high amino acid homology with class III plant chitinases.[55] Mostly, they belong to the family 18 of the glycosyl hydrolase superfamily.[56] The basic structure of family 18 fungal chitinases consists of 5 domains or regions: (1) catalytic domain, (2) N-terminal signal peptide region, (3) chitin-binding domain, (4) serine/threonine rich-region, and (5) C-terminal extension region. However, serine/threonine rich-region, chitin-binding domain, and C-terminal extension region is absent in most of the fungal chitinases, and these seem to be unnecessary for chitinase activity because naturally-occurring chitinases that lack these regions are still enzymatically active. Fungal chitinases are not as well-classified as the bacterial and plant chitinases, and are identified on the basis of their similarity to family 18 chitinases from bacteria or plants.[57] Therefore, fungal chitinases have been divided into fungal/plant chitinases, which correspond to class III chitinases and show similarity to class V chitinases from plants, fungi, and bacteria.[57] Group C fungal chitinases, which have not yet been characterized, is a novel group of fungal chitinases. It has been predicted that they are as large as 140-170 kDa, and consists of 2 LysM domains and a chitin-binding domain. They have shown resemblance to yeast killer toxins (http://www.fungwall.org/anglet/abstracts. pdf). Chitinases have important physiological and biological roles, which include morphogenetic, autolytic, nutritional, and parasitic roles. For example, disruption of the chitinase gene (CTS1) in the yeast Saccharomyces cerevisiae results in failure of the cells to separate after division and cell clumping, while functional expression of chitosanase and chitinase have been reported to influence morphogenesis in the yeast (Schizosaccharomyces pombe).[58] Lorito et al., 1994, [59] Ulhoa and Peberdy, 1991, [60] have reported the purification and characterization of 3 N-acetylglucosaminidaes (GlcNAcases) from different isolates of Trichoderma and that their molecular masses were similar, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, i.e., SDS-PAGE. Draborg et al., 1995[61] and Peterbauer et al., 1996,[62] reported the cloning of 3 genes of GlcNAcases from Trichoderma sp.: exc1, exc2, and nag1. The genes exc1, exc2 were isolated from T25-1, thereby resulting in the conformation that Trichoderma has 2 different GlcNcases. Lorito et al., 1998,[63] reported the isolation of about 42 kDa endochitinase from Trichoderma. As reported by Yamanaka et al., 1994,[64] Sandor et al., 1998,[65] fungal cell wall chitinases have also been associated with their role in filamentous fungal sporulation since the chitinase inhibitors demethylallosamidin or allosamidin led to the inhibition of fragmentation of hyphae into arthroconidia. Trichoderma spp. have been given the most consideration as biocontrol agents in case of soil borne fungal pathogens amongst various chitinolytic fungi and bacteria.[66-68] The purification and characterisation of chitinases and β-1,3glucanases from Talaromyces flavus and Trichoderma spp. has 23 


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been reported and their function in mycoparasitism of soilborne pathogens i.e. Rhizoctonia solani, S. rolfsii and Fusarium sp. has also been emphasised.[20,69,70] Harman (2000)[71] and Yedidia et al., (2000)[72] reported that the valuable effect of Trichoderma on fungi is because of its direct mycoparasitism and it results in induced resistance and increased development in plants. It has been reported by Yaun and Crawford 1995[73] that the antifungal biocontrol agent, Streptomyces lydicus WYEC108 has the capacity to damage the fungal cell wall hyphae and destroying germinating oospores of Phytium ultimum as well. Earlier studies have shown that chitinase genes (such as ech42, chi33, nag1, chi18-13) from Trichoderma harzianum have a very important role in mycoparasitism.[74,75] It has been reported that disruption of ech42 gene affects mycoparasitism in T. harzianum. [76] The isolation of a novel endochitinase called as CHIT36 from T. harzianum isolate TM was reported by Viterbo et al., 2001.[77] Other important applications of fungal chitinases include the possibility for improving plant resistance with the help of genetic manipulation techniques. The chi42 gene of T. harzianum encodes a powerful endochitinase, which has a much stronger anti-fungal activity against a number of phytopathogenic fungi, and is expressed constitutively in apple, tobacco, and potato. These transgenic plants thereby show a high level of resistance against phytopathogenic fungi.[63] Fungal chitinases are also employed in insect control.

Plant Chitinases Chitinases are constitutively present in plants, stems, seeds, flowers, and tubers. They are developmentally regulated as well as tissue-specific. Taking into account the amino acid sequences, plant chitinases have been categorized into 5 or 6 classes. The key structure of the class I, II, and IV enzymes contains globular domains. While 8 α-helices and 8 β-strands form the class III and V plant chitinases. The former carries out the hydrolysis of the β-1, 4-glycosidic linkage by means of an inverting mechanism, and the latter through a retaining mechanism.[78] Plant chitinases are produced as pathogenesisrelated proteins in plant self defense in response to the attack of phytopathogens, or by contact with elicitors such as chitooligosaccharides or growth regulators such as ethylene. [79] There are some chitinases, which are expressed in response to environmental stresses, (i.e., high salt concentration, cold, and drought). There are also reports of some chitinases, which take part in vital physiological processes of plants, like embryogenesis and ethylene synthesis. [78] Chitinase, which is a polypeptide and a major pathogenesis-related protein, accumulates in the infected plant tissue extracellularly. Garg and Gupta, 2010,[80,81] reported the isolation and purification of chitinase from moth beans against the fungal pathogen Macrophomina Phaseolina strain 2165. The chitinases of plants can be detected during their development in the early stages of growth. The chitinases of plants are generally endochitinases of smaller molecular weight as compared to the chitinases of insects. 

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Insect Chitinases The chitinases found in the insects have been described from Manduca sexta and Bombyx mori. These enzymes have very important roles to play as degradative enzymes during ecdysis where endochitinases randomly break the cuticle to chitooligosaccharides, which are afterwards hydrolyzed by exoenzymes to N-acetyl-glucosamine. The monomer is reused for new cuticle synthesis. Insect chitinases also play defensive roles against their own parasites, and the enzyme production is regulated by hormones during the transformation of the larvae. Allosaminidin is the inhibitor of insect chitinases.[82] Chitinases are also found in crustaceans like shrimps, krills, and prawns.

Mammalian Chitinases Mammalian chitinases belong to the family 18 of glycosyl hydrolases (GH18), which can be divided into chitinase like proteins with no enzymatic activity, and enzymatically active true chitinases.[7] Chitotriosidase was the first mammalian chitinase to be identified.[83] The N-terminal catalytic domain of GH18 family members consists of triose-phosphate isomerase fold, which is characterized by the (β/α)8- barrel structure, and within this barrel, the β4 strand consists of a conserved sequence motif (DXXDXDXE, where D = aspartic acid, E = glutamic acid, and X = any amino acid) forming the active site of the enzyme. Glutamic acid is the key residue donating a proton required for hydrolyzing the β (1-4) glycosidic bond in chitin. [84] In chitinase-like proteins, it is the substitution of this essential glutamic acid to glutamine, leucine, and isoleucine that accounts for the lack of chitinolytic activity. However, as the conserved chitin-binding aromatic residues on the triose- phosphate isomerase barrel remain unaffected, they are still capable of binding to chitin with high affinity.[85]

Methods of Production of Chitinase A number of methods have been used for the production of microbial chitinases, which include “fed-batch fermentation, continuous fermentation, and liquid batch fermentation.” As per the reports of Khan et al., 2010,[86] in the presence of chitin, MgSO4·7H2O and KH2PO4 has a positive effect on chitinase production, whereas yeast extract has a negative effect on chitinase production. It was also reported that there was an enhancement in chitinase secretion with an increase in maltose and chitin concentrations. The components like MgSO4·7H2O, KH2PO4, and yeast extract demonstrated highest chitinase secretion at lower concentration levels. As reported by Bhushan, 1998,[87] and Dahiya, 2005,[88] media constituents for nitrogen and carbon sources and agricultural remains (e.g., wheat bran, rice bran, etc.) influence extracellular chitinase production. He also reported the enhancing effect of glucose on chitinase production when glucose was used in the production medium along with chitin. However, Miyashita et al., 1991, reported that glucose has a repressing effect on the production of chitinase.[89] Bhushan, 1998,[87] reported that chitinase production is also Journal of Pharmacy and BioAllied Sciences January-March 2013 Vol 5 Issue 1


affected by some physical factors such as pH, aeration, and incubation temperature. He also reported that chitinase production was stimulated in Bacillus sp. BG-11 subsequent to addition of amino acids and their analogs, for example tryptophan, tyrosine, glutamine, and arginine in the growth medium at a concentration of (0.1 mM). In order to improve the production of chitinases from different organisms, several methods, such as biphasic cell systems, cell immobilization, solid-state fermentations, etc., have been used.[87,90] There are reports of both natural as well as general enzyme inhibitors, which include oxidizing/reducing agents and organic compounds as well. An antibiotic produced by Streptomyces sp., allosomadin, a competitive inhibitor, has been attained recognition as a specific inhibitor of yeast, insects, fungi, and human serum chitinases. Allosamidin acts as a non-hydrolysable analog of the oxazolinium ion intermediate, thereby exerting its inhibitory effect.[91,92] Psammaplin A has gained recognition in the form of a non-competitive inhibitor of chitinase B from S. marcescens, which belongs to the family 18 chitinase and is a brominated tryrosine-derived compound. As suggested by crystallographic studies, a disordered Psammaplin A molecule binds in vicinity of the active site.[93] Argadin, which was isolated from Clonostachys sp. FO-7314, is another chitinase inhibitor.[94] In order to carry out the cloning and expression of genes from diverse organisms into E. coli, several attempts have been made with S. plymuthica,[95] B. circulans WL-12.[54] ChiA, which is a chitinase gene from family 18, has been cloned from a thermophilic species Rhodothermus marinus and then expressed in E. coli. Hobel et al., 2005,[96] reported that the R. marinus chitinase is the most thermostable chitinase isolated from bacteria. Two chitinase genes of Bacillus, which encode ChiCW and ChiCH, have been cloned into pGEX-6P-1 and later expressed in E. coli in the form of soluble glutathione S-transferase chitinase fusion proteins.[97] Many Streptomyces and non-Streptomyces bacteria are renowned in chitin production and are antagonistic against Sclerotinia minor, the pathogen of the basal drop of lettuce. The two isolates; Streptomyces viridodiasticus and Micromonospora carbonacea have been recognised as high level chitinase producers and therefore, recognisably reduced the growth of S. minor in vitro and under controlled greenhouse conditions resulted in the reduced occurrence of disease.[98]

Uses Chitinases may be used to convert chitin-containing biomass into useful (depolymerized) components. Chitinases can be exploited for their use in control of fungal and insect pathogens of plants.[99,100] Fungal protoplasts have been exploited as a very efficient experimental means to study the synthesis of cell wall, enzyme synthesis and secretion and strain improvement for biotechnological applications.[101] Chitinase activity also acts as an indicator showing the activity of fungi in soil. It has been reported that there is a strong association between chitinase activity and fungal population in the soil. Therefore, it appears that chitinases activity acts as a suitable indicator of Journal of Pharmacy and BioAllied Sciences January-March 2013 Vol 5 Issue 1

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the actively growing fungi in the soil. Miller et al., (1998)[102] by making use of specific methylumbelliferyl substrates reported the correlation of chitinase activity with the content of fungusspecific indicator molecules 18:2ωb phospholipid fatty acid and ergosterol.

Medicinal Functions Chitooligosaccharides have an enormous pharmaceutical potential. They are involved in the signaling for root nodule formation, act as elicitors of plant defense and also have a potential to be used in human medicines (e.g., anti-tumor activity is shown by chitohexaose and chitoheptaose). It was reported by Murao et al., 1999,[103] that chitotriose from colloidal chitin have been prepared using a chitinase from Vibrio alginolyticus. Kobayashi et al., 1997,[104] have reported the use of Bacillus chitinase for the production of chitobiose by combining GlcNAc and a sugar oxazoline derivative. GlcNAc itself is an anti-inflammatory drug, and in the human body, it is synthesized from glucose, then incorporated into glycoproteins and glycosaminoglycans. The GlcNAc administered by oral routes, intravenous (IV), and intramuscular (IM) has been reported to be effective as an anti-inflammatory drug, useful in the treatment of ulcerative colitis and other gastrointestinal inflammation disorders. [105] Horsch et al. (1997)[106] recommended that N-acetylhexosaminidase can be explored for its use as a target for designing antifungals with low molecular weight. According to Laine and Lo, (1996)[107] chitin and chitin binding proteins can be explored for the recognition of fungal infections in humans. Chitinases have a significant function in human health care. An important medical use for chitinases has also been recommended in augmenting the activity of anti-fungal drugs in therapy for fungal diseases.[108] Due to their topical applications, they have a prospective use in anti-fungal creams and lotions. A number of artificial medical articles such as contact lenses, artificial skin, and surgical stitches have been formed from chitin derivatives. These derivatives have an extensive medical use because quite a few of these chitin derivatives are known to be non-toxic, non-allergic, biocompatible, and biodegradable. [109] Chitinases also have some other medical applications as well. For example, first discovery of the involvement of acidic mammalian chitinase (AMCase) in the pathogenesis of asthma was novel and unexpected because of the fact that mammals do not use chitin as an energy source, nor do they produce any chitinous structure.[110] Several lines of evidence have demonstrated the importance of chitinases as an effector of host defense in the mammalian immune system. For example, humans that are deficient in chitotriosidase show an increased rate of microfilarial infection due to suppressed chitinolytic activity, allowing the parasite to thrive within the host. Recombinant human chitotriosidase shows the inhibition of Candida albicans hyphae formation in vitro, thereby, showing anti-fungal activity, and reducing mortality in mouse models of neutropenic candidiasis and aspergillosis. Zhu and co-workers, 1984,[110] reported the first clinically25 


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significant finding related to the role of chitinase in asthma where exaggerated quantities of AMCase were detected in the epithelial cells and macrophages of lung biopsies taken from patients with asthma. Correspondingly, BAL fluid chitinase activity and the AMCase level have also been reported to be induced in the lungs of an ovalbumin-induced mouse asthma model. Moreover, it has been recently found that AMCase, serum, and lung tissue levels of a chitinase-like protein, YKL-40, are increased in patients with asthma. Furthermore, circulating YKL-40 levels correlated positively with thickening of the lung sub-epithelial basement membrane, incidence of use of rescue inhalers, asthma severity, and deterioration in pulmonary function in asthmatic subjects have been studied.[111] Even though mammals cannot carry out the synthesis of chitin, some chitinolytic enzymes or true chitinases (-e.g., acidic mammalian chitinase AMCase and chitotriosidases, or chitinases like proteins (CLPs)) or CBPs (e.g., breast regression protein 39 (BRP-39, chondrocyte protein-39) and Ym-1, Ym-2) have been found in mammals. Mammalian chitinases do not have chitinase activity while AMCase and chitotriosidase have chitinase activity. [112] The enzymatic activity of mammalian chitinases is due to its chitin-binding domain, which consists of 6 cystein residues having chitin binding property.[113] On the contrary, no such typical chitin-binding domain is present in CLPs, but still they show a very high chitin-binding affinity.[114] CHI3L1 does not have chitinase activity because of substitution of an essential glutamic acid residue to leucine,[115] but it has a high affinity for chitooligosaccharides and chitin, which is due to conserved substrate binding cleft.[116] It has also been suggested that YKL-40/BRP-39 has a major role as an active pathogenic mediator in acute colitis during the generation of intestinal bowel disease (IBD). YKL-40 is supposed to play a role in tissue remodeling and inflammation and can bind to chitin, type I collagen, hyaluronan, and heparin even though it lacks chitinolytic activity.[117] Consisting of the body’s first line of defense against external agents, which also includes chitincontaining pathogens, various chitinase family proteins have constitutively shown their expression in macrophages, digestive tract, and in epithelial cells of lungs.[110,117] It has been reported that lungs show an increased expression of AMCases in the development of Th2 inflammation in the human asthmatic airway and in allergic animal models as well.[110] It has also been shown that AMCases have a very important role to play in the IL-13 effector pathway activation and in the pathogenesis of Th2 inflammation. It has been suggested from studies of cancer, arthritis, and liver fibrosis that chitinase 3-like protein 1 (CHI3L1) also has an important role in tissue remodeling and inflammation.[115,118] The chief components of solid waste from shellfish processing are CaCO3, chitin, and protein. Chitinase from S. marcescens was used by their group to hydrolyze the chitinous material and yeast, Pichia kudriavzevii, in order to produce SCP that was acceptable as aquaculture. Hensenula polymorpha, Candida tropicalis, S. cerevisiae, and M. verrucaria have been commonly used for the production of SCP. The chitinase from M. verrucaria and S. cerevisiae have been used to produce SCP from chitinous waste by Wang and Hwang, 2001. [119] 

26

These authors have also reported that M. verrucaria chitinase preparation could be used for chitin hydrolysis, and S. cerevisiae chitinase preparation for SCP. Chitinases find their use in other fields like agriculture and mosquito control. Chitinases can also be exploited as additives in order to supplement to the frequently used insecticides and fungicides so that they can be more potent and at the same time, the concentration of the chemically synthesised active agents in the ingredients can be minimised, which are otherwise harmful to health and environment.[120,121] Chitinases also have applications in the bioconversion of chitin waste to fertiliser.[122] The utilization of microorganisms as biological control agents or their secretions to prevent plant pathogens and insect pests provides us with a striking choice in order to control the plant diseases. Therefore, biological control strategy has become a vital advance in order to make sustainable agriculture possible.[123] The organisms which produce chitinase could also be exploited for their use as biocontrol agents either directly or indirectly by making use of their purified proteins or via gene manipulation.[124]

Future Prospects In the future, there is a possibility of generating chitinases with novel functions. Chitinases can be exploited for their use as food preservatives, thereby increasing the shelf life of the foods. A vast understanding of the biological roles of different chitinases would help us to develop novel therapeutic approaches for several diseases including asthma, and chronic rhinosinusitis. There is a possibility of using chitinases as antitumor drugs since chitohexaose and chitoheptaose has shown an anti-tumor activity. These enzymes can be used for the enhancement of human the immune system. This research can be directed towards the identification of the active sites of chitinases and the novel functions associated with them. We can exploit protein engineering for the production of chitinases with exclusive functions.

Acknowledgement Saleem Javed is thankful to Department of Science and Technology (DST), India for financial support. Rifat Hamid and Mahboob Ahmad are thankful to UGC for fellowship.

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Source of Support: Department of Science and Technology (DST), India, Conflict of Interest: None declared.

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