Industrial, Agricultural and Pharmaceutical Uses of Chitinases

Introduction:
Chitinases (E.C 3.2.2.14) are glycosyl hydrolase enzymes that break down the linear polymer chitin which is found in fungal cell walls, yeast, algae, shellfish and the exoskeleton of insects; after cellulose chitin would be the most abundant polysaccharide found in nature (Javed et al., 2013). Chitinases are synthesised by bacteria, fungi and digestive glands of animals that have chitin in their diet (Muzzarelli, 2014). Chitin is found in two different forms which differ due to the arrangement of the polymer chains, packing and polarity: α-chitin and β-chitin (Javed et al., 2013). Chitin, shown in figure 1, is an essential structural component in organisms and its presence is associated with extracellular chitinases. The catabolism of chitin occurs in two steps; the first step involves cleavage of chitin into chitin oligosaccharides by chitinase followed by the second step where these oligosaccharides are cleaved to produce N-acetylglucosamine and monosaccharides by chitobiases (Javed et al., 2013).

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Chitinases are found in many different organisms that either need to reshape their own chitin or dissolve and digest the chitin of other organisms. Organisms containing chitinases include plants, yeast, fungi, bacteria and they have even been found in the stomach acid of humans; chitinases in humans were thought to have been involved in digesting chitin at a time when human diets may have been more confined to plant based foods. Before this it was assumed that chitinases were absent in humans and other vertebrates due to the absence of naturally occurring chitin (Ziatabar et al., 2018). Chitinases are important for allowing many plants and animals to perform vital functions such as digestion, growth, chemical attack, immune defence and other metabolic functions. Chitinases are divided into two groups based on function: exochitinases (E.C 3.2.1.30) and endochitinases (E.C 3.2.1.14) and they break down chitin by severing its glycosidic bonds compromising its cell wall and overall structure (Fuglsang et al., 1995). Exochitinases target the β-1,4-glycosidic linkages on the non-reducing end of the chitin chain but endochitinases randomly cleave internal linkages hence their respective names. The two chitinase types are also separated by their mechanism of hydrolysis as well as where the enzyme attacks the chitin chain (Dravid et al., 2015). Chitinases are also classified based on their amino acid similarities and 3D structures from different organisms and have been separated into five classes which in turn are split between families: it was originally thought that these were families 18 and 19 of glycosyl hydrolases (Javed et al., 2013). However, not all chitinases are limited to families 18 and 19 and in fact they are spread between families 18, 19, 23 and 48 of glycosyl hydrolases (Adrangi and Faramarzi, 2013). These five classes are determined based on different characteristics of the different chitinase enzymes such as isoelectric pH, N-terminal sequence, inducers and also the organisms in which each can be found (Javed et al., 2013).
Chitinases are of increasing research interest due to their applications in biotechnology; one example would be their role in agriculture as they can protect plants i.e. crops, by breaking down the chitin containing cell walls in fungal pathogens and the exoskeletons of harmful insects. Upon pathogen attack chitinase production is induced and the enzyme will accumulate in the extracellular space. Mainly endochitinases have antifungal activity but some exochitinases have been shown to have antifungal activity also (Fuglsang et al., 1995). They also have potential roles in human healthcare in the treatment of asthma and even links to allergies (Shuhui, Mok and Wong, 2009). Chitinases also have applications in the food industry and have a role in industrial waste management to break down chitinous waste from marine organisms into more simplified depolymerised products to reduce water pollution (Rathore and Gupta, 2015).
Structures of Chitinases:
Family 18 chitinases are found in viruses, fungi (fungal cells may use chitinase during developmental stages to dissolve their own chitin to reshape the cells), bacteria, animals and some plants. Family 19 chitinases are mostly found in plants with some having been found in bacteria – Streptomyces (Hoell et al., 2006). These two families of chitinases do not share amino acid similarities and have different 3D structures suggesting they have evolved from completely different ancestors (Rathore and Gupta, 2015). Family 18 chitinases consist of numerous amino acid repeats along with the enzyme core which has 8 parallel β-sheets forming a barrel which along with the α-helices forms a ring positioned towards the outside of the 3D complex (Rathore and Gupta, 2015). This structure can be seen in figure 2 below.

Figure 2: Family 18 Bacterial endochitinase interaction with GlcNAc dimer (Rathore and Gupta, 2015).
The first family 19 chitinase to be found in an organism other than plants was chitinase C (ChiC) which was found inStreptomyces griseus. The crystal structure of this chitinase is the only representative of two domain chitinases in this family. The catalytic domain on the C-terminal consists of an alpha-helical fold with a deep groove containing the catalytic site. The N-terminal contains the chitin binding domain. The N-terminal and C-terminal are connected by a linker peptide. The chitin binding domain contains two tryptophan residues on the surface and an all- β protein (Kezuka et al., 2006). The chitin binding domain also contains a cysteine residue which, along with the tryptophan residues is essential for hydrophobic interactions with the substrate (Patel and Goyal, 2017). Figure 3 below shows the ChiC structure.

Figure 3: Overall structure of ChiC, a family 19 chitinase (Kezuka et al., 2006).
 
Industrial uses of Chitinases:
The hydrolysis of chitin by chitinases produces monomers which have vital industrial uses. Chitinases can be a vital tool in waste management as they can break down chitinous waste into smaller, simpler depolymerised molecules that can enter rivers in safer manner therefore solving environmental problem; this strategy of waste management is also cheap and the enzymes are biodegradable (Rathore and Gupta, 2015). Chitinases also can have a role in the food industry, it has been shown that a chitinase from Planococcus rifitoensis can be used as a biological control for a fungus called Botrytis cinerea also known as ‘grey mould’ which causes the decay of grapes and strawberries (Essghaier et al., 2009). The use of chitinases to combat ‘grey mould’ is a massive break through when you consider that over 200 plant species are affected by this and that annual global losses due to grey mould are anywhere between $10 billion and $100 billion. It has been noted that several bacteria have used chitinases as an energy source (van Aalten et al., 2001).
The waste produced by fish and other marine organisms in the sea food industry is high in chitin and the waste itself is a harmful pollutant when entering rivers directly due to a very high biological oxygen demand (BOD) and needs high amounts of demineralization and deproteination requiring a lot of acid. The use of recombinant chitinases will convert the chitinous waste into chito-oligomers which have a wide range of biotechnological uses as well as reducing the pollution entering rivers. Chito-oligomers are non-toxic, water soluble and have vital biological properties such as antibacterial, antitumor effects as well as enhancing animal immunity in diseases (Ibrahim et al., 2016). Chitinase can also be involved in the conversion of chitinous waste into biofertilzers which can then be used in farming to increase crop production. Solid state fermentation is a process where metabolites can be generated by microorganisms which are grown on a purpose based solid growth source and is used mostly in food production. Solid state fermentation can also produce enzymes and has been known to produce chitinase although these reports are scarce (Islam and Datta, 2015).
Chitinase enzymes have proven to be a potential tool in the Biofuel industry. This has extreme relevance in modern society and modern research with a lot of emphasis on renewable energy sources and the aim to move away from depleting natural energy sources. This could provide a more sustainable approach to energy production and would be more economically and environmentally friendly. This approach is centred on an enzymes ability to degrade crystalline structures in polysaccharides such as chitin to release the single chain polymers. A more intense look at accessory proteins involved in this process could really unlock the doors to chitinases producing bioethanol due to the fact that these proteins can significantly increase the efficiency of chitinases (Eijsink et al., 2008). This application does have further to go in its research but it can be seen looking at the chitinase data available that it can stimulate commercial production of lignocellulosic ethanol through the enzymatic conversion of biomass (Eijsink et al., 2008).
Chitinases can also be used to prepare single cell proteins (SCP) which are basically a source of protein coming from single celled organisms such as fungi, yeast and bacteria. They can be used to substitute proteins in human food or animal feeds. This is very useful method of protein generation as microorganisms have a very high replication rate so will generate large amounts of the SCP in little time. With the world’s population increasing more and more it is very possible that agriculture will not be able to meet the food demand, therefore an alternative, reliable method of mass food production like SCP that can operate in harsh conditions, is of vital importance in today’s research. The use of chitinases to generate SCP was first suggested by Revah-Moiseev and Carrod who used chitinase from S. marcescens for the hydrolysis of yeast (Islam and Datta, 2015).
Chitinases have also been used to isolate fungal protoplasts that can be used in the study of their cell wall and how they synthesise and secrete enzymes. This can aid in the research of improving strains for use in biotechnological applications such as medicines (Islam and Datta, 2015).
Figure 4: Industrial applications of chitinase enzymes (Rathore and Gupta, 2015).
Chitinases can be indirectly used to determine the levels of fungal biomass present in soil since more chitinase in the soil would mean a high level of fungal pathogens in the soil which has induced the synthesis of chitinase. This can be useful when studying the populations in a given soil environment although this would be a less popular use of chitinases. Tannase is an enzyme used in food production as it decomposes the tannins in fruit juices such as pomegranate and cranberry. Tannase is known to be produced by the fungus Aspergillus niger but there are issues with its yield because it binds to the cell wall of this fungi. Chitinase can be used to degrade this fungal cell wall thereby releasing the tannase increasing its yield (Barthomeuf, Regerat and Pourrat, 1994).
Agricultural uses of Chitinases:
Chitinases act as biocontrol agents as they are present in plants as part of the plants defence mechanism against fungal pathogens and harmful insects with chitinous exoskeletons. Plants do not have an immune system like humans so heavily rely on mechanisms such as chitinases for protection against pathogens. A lot of research has been done to show that transgenic plants containing overexpressed chitinases will be protected against fungal pathogens because the chitinase can break down the pathogens cell wall as it is made of its substrate chitin. This has major significance in agriculture because crop losses due to pathogens can cost 200-300 billion US dollars annually (Oerke et al., 1994). Most methods to protect crops involve chemicals like pesticides or fungicides which can be harmful to other organisms the environment so chitinase offers a safer, preferable and natural mode of protection. It is also important to note that a huge range of the crops that are affected by fungal pathogens that are heavily relied on by the human population; these crops include tobacco, tomatoes and cabbage.
 An investigation was carried out by Karasuda et al in 2003 to prove whether or not a plant chitinase could be used as a biocontrol agent to replace chemical fungicides effectively and safely. Chitinase E from family 19 of the glycosyl hydrolase enzymes was used in the experiments and was applied via a spray to the surface of a mildew which had been affecting strawberries and their leaves. The results are shown in figure 5 below.
Figure 5: Effect of Chitinase E on Powdery Mildew affecting strawberries. (Karasuda et al., 2003)
Panel 1: Strawberries sprayed with a mixture of Chitinase E and Zymolyase 20T
Panel 2: Strawberries sprayed with a non-enzymatic solution to act as the control
Panel 3: Strawberries sprayed with Chitinase E alone                                                  A = Visual Observations
Panel 4: Strawberries sprayed with Zymolyase 20T alone                                            B = SEM Images
Based on the results in figure 5 it can be visually seen that the chitinase E treatment on the strawberries had a successful impact as a biocontrol agent to replace the chemical fungicide to degrade the mildew and cause no harm to the strawberries. The investigation showed that the chitinase E would be safe and effective as it would be a natural biodegradable method of protection not involving chemicals (Karasuda et al., 2003). The visual observations and Scanning Electron Microscope (SEM) images both show that the chitinase E degraded the powdery mildew but the zymolyase alone did not. The results in panel 2 show that the mixture of chitinase E and zymolyase caused degradation but not to the same extent as chitinase E alone further supporting the theory that the chitinase E alone is breaking down the chitin contained within the hyphae of the mildew on the surfaces of the strawberries (Karasuda et al., 2003).
As well as directly protecting crops by acting as biopesticides, chitinases can also indirectly protect crops by acting as a target for other biopesticides. A pseudo-trisaccharide allosamidin which inhibits chitinase can potentially be used as a biopesticide (Rathore and Gupta, 2015). Chitinase inhibitors are of increasing research interest as they have the potential to act as insecticides, fungicides and even anti-malarials (van Aalten et al., 2001). Not only is chitin the main structural component of fungal cell walls but it also is the main component of insect exoskeletons and also is present in the lining of the gut in insects so chitinases again can degrade the chitin in these insects that are harmful to plants and crops in order to eradicate them. In any insect key structures such as the exoskeleton, peritrophic membrane of the gut and appendages e.g. antennae are made up of mainly chitin and as such these insects contain chitinase enzymes for remodelling these structures in growth and development; therefore any additional chitinases added can cause serious disruption of normal physiological reactions and movements in these organisms such as regeneration of the peritrophic membrane and ecdysis which is also known as moulting and is a key process for many arthropods (Subbanna et al., 2018). Studies have been carried out and have shown that chitinase can induce damage to the peritrophic membrane of the insect gut reducing its ability to be an effective barrier majorly interrupting feeding processes which has a knock on effect to digestion, ability to utilise nutrients and overall growth (Wiwat et al., 2000). This research shows that applying a solution with chitinases alone to essential crop fields can eradicate harmful insect pests saving billions of dollars in crop losses. Numerous studies have been carried out in relation to insect control focussing mainly on caterpillars and sucking pests and although more in depth research is needed, these studies showed a significant decrease in larval development of pests such as Trichoplusia ni and sucking pests like Myzus persicae(Broadway et al., 1998).
Pharmaceutical uses of Chitinases:
Chitinases are thought to have several functions in medicines including chemotherapeutic targets, asthma treatment, allergies and proteins with chitinase activity in humans have been identified as biomarkers in human disease. As mentioned previously, chitinase is an antifungal agent so has uses in treating fungal infections (Oranusi and Trinci, 1985). Chitooligosaccharides also have huge potential in cancer treatments due to their antitumor characteristics. N-acetyl glucosamine also has useful anti-inflammatory properties which could be exploited in medicines also. Chitinases can potentially act as chemotherapeutic targets if we can understand their functional significance and enzyme-substrate reaction mechanisms (Dravid et al., 2015).
It is known that asthma is closely associated with the increased activity of Th2 lymphocytes in an immune response in the airways. In early 2001 a study showed a link between a chitinase-like-protein being upregulated in mouse CD4+ Th2 lymphocytes due to interleukin signalling (Webb, McKenzie and Foster, 2001). This was the first time that chitinases had been linked with inflammation in the airways due to asthma. Chitinases such as TKL-40 and AMCase have plentiful evidence to support their potential for use as biomarkers in Th2 related airway inflammation (Webb, McKenzie and Foster, 2001). Table 1 below shows the potential for such chitinases as biomarkers in asthma treatment due to their increased expression levels during inflammation.
Table 1: Expression levels of chitinases in asthma (Webb, McKenzie and Foster, 2001).

Chitinase
Organism
Expression level

Ym1
Mouse
Increase

Mouse
No change

Ym2
Mouse
Increase

Mouse
Increase

AMCase
Mouse
Increase

Mouse
Increase

Human
Increase

YKL-40
Human
Increase

Chitotriosidase
Mouse
No change

Human
No change

Chitooligosaccharides are potentially very useful cancer treatment methods due to the fact that they have low molecular weight and are water soluble but most importantly have been shown to have antitumor activity. Most chemotherapeutic drugs have nasty side effects so looking for alternatives is a key part of modern cancer research. It has been demonstrated that chitooligosaccharides boost the activity of natural killer cells of lymphocytes in the spleen of mice with cancerous tumours (Kobayashi et al., 1987). The data collected from the experiments ran by Kobayashi et al showed that increasing concentrations of chitooligosaccharides used to treat various human tumour cells showed inhibition of tumour cell growth increasing as concentration of chitooligosaccharides increased. The effect was most noticeable in hepatocellular carcinoma so perhaps treatment of this cancer type would be possible.
As mentioned previously chitins are the substrate for chitinases and will activate the enzyme when their presence is detected. In a study carried out by Hoseini et al, chitin particles were injected into mice and showed that the presence of chitin increased production of TNF-α and IL-10 therefore could be possible that chitin will stimulate the immune system to increase the effectiveness of the immune response (Hoseini et al., 2016). Chitin presence causes the expression of chitinase to be induced which works with the immune system to combat the pathogen. Chitinase activity in humans has a definite defence purpose cleaving pathogens containing chitin as a key structural element in order to eliminate them as a threat.

Figure 6: 3D structure of human chitotriosidase, a chitinase of the GH18 family (Shuhui, Mok and Wong, 2009).
Future Prospective of Chitinases: 300
Looking forward it is clear that chitinases have many vital applications in industry, agriculture and pharmaceuticals but the success of these applications will be heavily influenced by how readily we can produce the different chitinases at a reasonable cost. Their use in the biological control of plant pathogens in agriculture is by far their most effective application currently but further research will bring their full potential out.
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