Chitin is one of the most abundant renewable biopolymer on earth that can be obtained as a cheap renewable biopolymer from marine sources [1]. It is biocompatible, biodegradable and bio-absorbable, with antibacterial and wound-healing abilities and low immunogenicity; therefore there have been many reports on its biomedical applications [2]. Accordingly, a very broad range of applications in different fields such as food technology, material science, microbiology, agriculture, wastewater treatment, drug delivery systems, tissue engineering, bionanotechnology have been reported.

Henri Braconnot, a French professor of natural history, discovered chitin in 1811 after the discovery of a “material particularly resistant to usual chemicals” by A. Hachett, an English scientist in 1799, and in 1843 Lassaigne demonstrated the presence of nitrogen in chitin [3]. Henri Braconnot’s name for chitin was fungine. In 1823, Odier found the same material in insects and plants and named it chitine [4]. Since all chitin-based materials are derivatives of chitin, in this paper the word chitin is used generally to describe both chitin and its derivatives unless otherwise mentioned in the text.

Chitin, a poly-beta-1,4-N-acetylglucosamine (GlcNAc), is the main component of arthropod exoskeletons, tendons, and the linings of their respiratory, excretory, and digestive systems [5,6]. It is also found in the reflective material (iridophores) both in the epidermis and the eyes of arthropods and cephalopods (phylum: Mollusca) [6]. Chitin is also an important component of the cell wall of fungi [7]. Moreover, there is one report - based on lectin binding, endo-chitinase binding and enzymatic degradation studies - that the epidermal cuticle of a vertebrate named Paralipophrys trigloides (fish) is chitinous [8]. Therefore, chitin is not only an essential component of invertebrates but may also be present in vertebrates. Unlike cellulose, chitin can be a source of nitrogen as well as carbon (C:N = 8:1) [9].

Chitin contains 6–7% nitrogen and in its deacetylated form, chitosan contains 7–9.5% nitrogen. In chitosan, between 60 to 80% of the acetyl groups available in chitin are removed [10]. There are three forms of chitin: α, β, and γ chitin. The α-form, which is mainly obtained from crab and shrimp shells, is widely distributed. Both α and β chitin/chitosan are commercially available. The α-chitin chains are aligned in anti-parallel fashion. The anti-parallel arrangement in α-chitin gives rise to strong hydrogen bonding and consequently makes it more stable [11]. The β-form mainly obtained from mollusks such as squid, is arranged in parallel, whereas the γ-form contains two parallel and one anti-parallel strands of chitin [12]. Conversion from the β-form to the α-form is possible, but not the reverse [13–15]. γ chitin can be converted to α chitin by treatment with lithium thiocyanate [16].

Chitin is insoluble in water due to its intermolecular hydrogen bonds [17]. But water-soluble chitin-based derivatives such as chitosan or carboxymethyl chitin can be obtained. One of their most important features is the ability (flexibility) to be shaped into different forms such as fibers, hydrogels, beads, sponges, and membranes [18]. The origin of chitin affects its crystallinity, purity, polymer chain arrangement, and dictates its properties [19].

Chitin biosynthesis has been studied in a large variety of organisms. The enzyme for this synthesis is called chitin synthase (CS). Three CSs have been found in Saccharomyces cerevisiae CS I, CS II, and CS III. They are different from each other in terms of function and catalytic activity. Most of chitin is synthesized by CS III [20]. Fungal CS are grouped into two families and five classes [21]. There are two CS genes in insects [22,23]. Nematodes possess two chitin synthase genes, which are differentially expressed [24]. Therefore, there seems to be specialization among chitin synthases in different organisms where different enzymes carry out different functions. CSs use UDP-N-acetylglucosamine as substrate to produce chitin fibrils [25]. This was proven in an experiment done on Mucor rouxii [26]. The genomes of some chloroviruses contain a chitin synthase gene (cs). By introducing the gene of chlorovirus CVK2 into Chlorella cells, an algae that does not possess a chitin synthase gene, this microorganism could be engineered to produce chitin [27].

Several enzymes, so called DG, have been isolated from the Xenopus laevis embryos that are differentially expressed during the gastrulation stage. One of these enzymes is DG42. It is expressed from midblastula to the end of the neuralation stages. DG42 synthesizes a Nod-like chitin oligosaccharide. Nod is the enzyme responsible for producing chito-oligosaccharide in Rhizobium [28]. It seems that while this enzyme is expressed during early embryogenesis the amount of hyaluronic acid (HA) increases. It has been suggested that chitin oligosaccharides are present at the reducing ends of HA chains. Thus, it has been proposed that chitin oligosaccharide produced by this enzyme acts as a primer for synthesizing HA. Homologs of DG42 have been found in zebrafish and mice [29,30]. They also synthesize Nod-like chitin oligosaccharide during early embryogenesis. However, it remains controversial whether DGs are chitin synthase or hyaluronan synthase. Since DG42 has been isolated from frogs, zebrafish and most importantly mice, further study is needed to investigate whether or not this enzyme is widespread throughout mammals including humans, and if DGs is shown to synthesize chitin in animal, the role of chito-oligosaccharide in embryonic development should be further investigated.

Chitinases, which hydrolyze chitin, are present in a wide range of organisms including viruses [31], bacteria [32], fungi [33], insects [34], higher plants [35,36], and mammals [37]. Most organisms (bacteria, plants and insects) have large families of chitinases with distinct functions, including digestion, cuticle turnover, and cell differentiation. Based on amino acid sequence similarity they can be grouped into glycosyl hydrolase families (GH) 18 and 19, which are structurally unrelated. The catalytic mechanism of chitinases family 18 involves substrate-assisted catalysis, which retains the anomeric configuration of the product [38]. They are ubiquitous with an (alpha/beta) 8-barrel fold structure in the catalytic domain [39]. Family 19, glycosyl hydrolases, share a homologous catalytic domain. They consist mainly of alpha helices. Their catalytic mechanism is a general acid-base mechanism that inverts the anomeric configuration of the hydrolyzed GlcNAc residue [38]. Family 19 chitinases have mostly been identified in plants [40]. Both GH 18 and 19 chitinases have signal peptides, indicating that the enzymes are secreted and function outside the cells.

Family 18 of glycosyl hydrolases include active chitinases and inactive chitinase-like proteins or chito-lectins, which lack endogenous chitin and have been found widely in mammals [41]. Recent comparative genomic analysis to address the evolutionary history of the GH18 multiprotein family from early eukaryotes to mammals, revealed that the GH18 chitinase involved in an emerging interface of innate and adaptive immunity during early vertebrate history [41]. Chito-lectins are inactive due to lack of some critical residues in their catalytic sites. Several of these chitinase-like proteins have been identified such as oviductin or oviduct-specific glycoprotein [42], YKL-39 [43], HC gp39 and YKL-40 [44–46] in human, YM1 [47], YM2 [48], ECFL [49], breast regression protein 39 (BRP39) [50] in mice, and gp38K [51] in pigs. Their functions are unknown, however, it is speculated that they may be involved in fertilization [52], chemotaxis [49], and tissue remodeling [53]. Besides these inactive chitinases, two active chitinases have been found in humans, these are chitotriosidase [54] and acidic mammalian chitinase (AMCase) [55]. Chitotriosidase can cut both chitin oligomer and colloidal chitin; its optimum pH is broad, and it is mainly secreted from macrophages [56]. AMCase is active in acidic pH, it is mostly expressed in stomach and lung, its gene has 11 exons and is located on chromosome 1 [57]. Interestingly, Glyco_18-containing proteins are established biomarkers for human diseases, and both human chitinases and chitinase-like proteins have been used as indicators of inflammation and cancer [58].

Bacterial chitinases belong to both family GH18 and 19. Most of their structure-function studies came from the studies of the enzymes from Serratia marcescens [59–61], B. circulans [62–67], and Vibrio spp. [68–70].

Recently, chitinases have gained interest in different biotechnological applications due to their ability to degrade chitin in the fungal cell wall and insect exoskeleton, leading to their use as antimicrobial or insecticidal agents. [71–73]. Another interesting application of chitinase is for bioconversion of chitin, a cheap biomaterial, into pharmacological active products, namely N-acetylglucosamine and chito-oligosaccharides [74–76]. Production of chitin derivatives with suitable enzymes is more appropriate for sustaining the environment than using chemical reactions [77]. Other interesting applications include the preparation of protoplasts from filamentous fungi [75], bio-control of insects and mosquitoes as well as the production of single cell protein [75,76]. Thus, there have been many reports on cloning, expression and characterization of chitinases from various organisms, including bacteria, fungi, plant and animals [75,78–80].

Kawabata et al. identified a molecule, named tachycitin, from hemocytes of a horseshoe crab with chitin binding activity. This is a small molecule that is 73 residues long and possesses five disulfide bonds [81]. A major protein in the chitin-walled cyst of protozoan Entamoeba histolytica also contains chitin binding domains. This protein is a lectin named EiJacob1 [82]. In addition, a chitin binding lectin has been isolated from Stinging Nettle Rhizomes [83]. Several genes encoding proteins with chitin binding domains from arthropods have been identified by whole genome sequence analysis [84]. Two proteins with chitin-binding domains have been identified from silkworm, Bombyx mori [85]. In addition, it has been shown that Tribolium castaneum, red flour beetle, contains genes encoding proteins peritrophin A-type chitin-binding domains [86].

Chitin binding proteins have also been isolated from plants. They belong to the lectin superfamily and are secreted from the plants as a part of their defense system [87]. Most of plant chitin binding proteins have a conserved motif, called havein domain or chitin binding domain [88].

A chitin binding protein with antifungal activity has been isolated and characterized from Streptomyces tendae Tü901. This protein binds to the surface of germinated conidia and tips of growing hyphae, changing the growth polarity of the fungi [89]. Other organisms in which chitin binding proteins have been identified include sweet potato hornworm, Agrius convolvuli [90], Amaranthus caudatus [91], Ginkgo biloba [92], and Streptomyces reticuli [93]. There is a conserved motif, known as RR consensus motif in the arthropods cuticular proteins, which along with its extended forms, are considered as chitin binding motifs [84,94,95].

Various chitinolytic microorganisms have been shown to produce non-hydrolytic accessory proteins that increase enzyme efficiency [96], such as the Gram-negative soil bacterium Serratia marcescens, which uses three different family 18 chitinases to degrade chitin. It has been reported that a small non-catalytic protein, CBP21, which binds to the insoluble crystalline substrate, leading to structural changes in the substrate and increased substrate accessibility, is required for efficient chitin degradation [97].

It is difficult to obtain pure carbohydrates, especially chitin, through conventional techniques. Bacterial cells have been engineered in an effort to overcome this problem [98]. E. coli has been engineered to produce chitobiose. This method took advantage of NodC, which is a chito-oligosaccharide synthase, and genetically engineered chitinase to make a cell factory with the ability to produce chito-oligosaccharides [99]. Recombinant chito-oligosaccharides have also been obtained using E. coli cells which expressed nodC or nodBC genes [100]. By expressing different combinations of nod genes in E. coli, O-acetylated and sulfated chito-oligosaccharide have been produced [101].

There are several reports indicating that chitin plays a role not only in those organisms in which its role is expected, such as in fungi and crustaceans, but also in other organisms. It has been shown that Rhizobia-mediating nitrogen-fixing nodules possess a signal molecule necessary for the root nodulation process termed lipo-chitin oligosaccharide, which comprises a β-1,4-linked N-acetylglucosamine (GlcNAc) tetra- or penta-saccharide [28,102]. N-acetylglucosamine has been reported to play a role during development of moulds as well [103].

The presence of intracellular and extracellular GlcNAc-containing compounds, in the forms of N-glycans and intracellular glycans, indicates the role of chitin and its derivatives from an evolutionary point of view [104]. Finding a role for chitin derivatives in animals was an important discovery. Semino et al. (1997) proved the importance of chitin oligosaccharides in the embryogenesis of zebrafish and carp [105]. GlcNAc containing muscle cell surface molecules have been shown to possess the ability to guide neurites through extracellular matrix [106]. It has been reported that the addition of O-linked GlcNAc (O-GlcNAc) to the proteins in nucleus and cytoplasm is different from classical O-glycosylation of the secretory pathways [107]. This reversible post-translational modification [108] modifies serine and threonine residues [109] and seems to play an important role in apoptosis [110], death signaling of mouse embryonic neuroepithelial cell [111], neurodegenerative disorders [112], signal transduction [113,114], synaptic plasticity [115], transcription and translation [116,117], stability of target proteins [118], proteasomal function [119], cancer [120], diabetes [121], subcellular localization [122], and nuclear transport [123]. O-GlcNAc is also important in the cardiovascular [124], and immune systems [125]. Considering the fact mentioned in part 4 about the role of chitin synthase and chitin-oligomer in mice, which is close to human species, and that humans have chitinase genes, it would not be surprising if future studies found one or more roles for chitinous materials in humans at some point in human evolution and/or embryonic development.

Recent trends in the field of chitin research have focused on oligosaccharides, which are more soluble and have several attractive biological effects. N-acetylchitooligosaccharide and chitooligosaccharide (COS) are originated from chitin and chitosan, respectively. Oligomers of chitin and chtitosan can be obtained both chemically and enzymatically [126]. Their degree of polymerization (DP) is usually < 20.

It has been shown that chito-oligosaccharide accelerates the wound healing effects of Poly vinyl alcohol (PVA) if it is used in the early stages of the healing process [204]. It could inhibit the growth of Actinobacillus actinomycetemcomitans, indicating that it has antimicrobial activity [205]. N-acetylchitooligosaccharide causes an increase in biophotons emission from suspension-cultured rice cells. Biophotons are very weak light emitted from biological processes/systems. All organisms, including plants, constantly produce biophotons as part of their vital activities. Photon emissions are elevated by environmental stresses and disease responses induced by pathogen attack. It has been shown that photon emissions from rice cells elicited by N-acetylchitohexaose are closely associated with the ROS-generating system, and are regulated by Ca2+ signaling and protein phosphorylation via phosphatidic acid (PA), an intermediate of phospholipid signaling [206]. Chito-oligosaccharide has shown inhibitory effects on tumor growth and metastasis of lung cancer in mice [207]. N-acetylchitohexaose (chitin derivertive) and chitohexaose (chitosan derivertive) can also induce production of interleukins 1 and 2 and, consequently, help to improve the function of macrophages, natural killers, cytotoxic T cells, and polymorphonuclear leukocytes, in defense mechanisms. The anti-metastatic activity of acetylchitohexaose against Lewis lung carcinoma in mice [208] has been demonstrated. There are many reports indicating antitumor activity of chito-oligosaccharides [209]. It has been shown that oligosaccharides that contain N-acetylglucosamine play an important role in the interaction between HIV and T-helper during pathogenesis of this virus [210]. N-acetylchitooligosaccharide was used as analogue to study lysozyme [211]. Thus, these materials can be used to study protein-carbohydrate interaction and associated enzymatic activities.

After two centuries of research on chitin, this biopolymer now has applications in numerous fields, as described in many review articles [140,212–216]. However, there is still room for further chitin research. Chitin is a potential energy source as well as gene and drug delivery carrier, and in the emerging field of nanobiotechnology. The evolutionary effects of chitin is emerging, but is not yet fully discovered. To take advantage of the mechanical characteristics of chitin, it should be used directly and without chemical modification. To achieve this goal, more research, especially on fundamental aspects, needs to be done.

Much attention has being paid to chitinase research [75], but its substrate seems to have been neglected or, at least, underestimated. If we are going to use the huge amount of chitin produced annually in nature, chitin research needs an increase in funding to develop equipment to harvest chitin from its diverse sources, improve the purity of obtained chitin, and produce novel materials with new applications from this environmentally friendly biopolymer.

Despite the multiple potential applications of chitin, we believe that the most promising in the future are (i) applications in nanobiotechnology, which involves drug and gene delivery and scaffold for tissue engineering, and (ii) applications of chito-oligosaccharides in medicine and agriculture.