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CHITIN AND CHITINASE
A General Introduction and Importance in Research
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 contribute to the generation of carbon and
nitrogen in the ecosystem
...
Chitinases have a use in human health care, especially in human diseases like asthma
...

Chitinases
Chitinases are hydrolytic enzymes that break down chitin to its mono- and oligomers
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Chitin
Chitin is a linear stable polymer of 𝛽-1,4-N-acetylglucosamine (GlcNAc), which is 2-acetamido-2deoxy-D-glucose, and is the second highest occurring biopolymer on the planet after cellulose
...
Percentage of chitin found varies with different organisms
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Structures of Chitin
It is a crystalline polysaccharide that exists in nature in three different forms: 𝛼-chitin, 𝛽-chitin, and
γ-chitin
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b)

𝛽-chitin – it is loosely packed as in this chitin, and chains are arranged in parallel fashion
with weaker intermolecular forces which leads to less stable form
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It can be swollen in water as well as dissolved
in formic acid

c) γ-chitin – it is a mixture of both 𝛼-and 𝛽-chitins

Course-Study by Savy P
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It also associates with non-protein part such as calcium carbonate that
forms the structure in shrimp, crab, and lobsters
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Also, chitin
provides protective benefits by forming exoskeleton in arthropods and fungi
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2
...
14) are glycosyl hydrolases, characterized for hydrolyzing 𝛽-1,4 linkage of Nacetyl glucosamine present in chitin chains which vary between the size ranging from 20kDa to
about 90kDa
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These processes involve high cost, low yield, and
corrosion problems due to which cost of its oligomers is much
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CLASSIFICATIONS OF CHITINASES
I
...

a) Endochitinases (EC 3
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1
...


Chitotriose,

ii
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Diacetylchitobiose (Figure1)
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b) Exochitinases have been classified into two categories, namely,
i
...
2
...
29)

which

catalyze

progressive

release

of

diacetylchitobiose from terminal nonreducing end and
ii
...
2
...
30) which cleaves oligomeric products
obtained by endochitinases into monomers of N-acetyl glucosamine (GlcNAc)
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On the basis of aminoacid similarity of chitinases from various organisms, they have been
divided into five classes and have been categorized into families18, 19, and 20 of glycosyl
hydrolases
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It consists of a number of conserved repeats of amino acids and enzyme core,
which has 8 strands of parallel 𝛽sheets, creating a barrel positioned down 𝛼 helices, in turn
forming a ring towards the outside (Figure2)
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c) Family 20 involves N-acetylglucosaminidases from bacteria, certain fungi, and humans
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Minal (MPhil)

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Role of Chitinases in Various Organisms
Mainly, organisms require these chitinolytic enzymes for three different purposes
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Organisms possess a tough layer of chitin and chitinases are expressed during
developmental phases to help in the remodelling of their exoskeleton so as to maintain and
support body size and shape
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Organisms that consume other chitin containing organisms as a source of nutrient express
chitinases to digest the insoluble chitin polymer into absorbable metabolites, which gives
energy
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Organisms that are prone to infection by chitin-coated microorganisms express chitinases
to degrade the protective shield of the infecting pathogens, thereby providing immunity
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Bacteria


Bacteria mainly produce chitinases in order to supply nitrogen and carbon as a source of
nutrients or precursors and parasitism
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Chitinases play an important role in bacterial pathogenesis wherever host
contains chitin
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

All three chitinases belong to family 18 of glycosyl hydrolases with (𝛽/𝛼) 8 TIM-barrel
catalytic domain with approximately six sugar subsites
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CBM modules found in chitinases are distantly related,
and they are characterized by presence of conserved exposed tryptophan residues that interact
with the substrate
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

Marine bacteria such as Vibrio are well studied because they solely live on chitin, which is
highly abundant in marine ecosystems
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The chitin degradation machinery of
Vibrio is proved to be highly efficient
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The interacting sugars undergo conformational changes prior
to hydrolysis by the wild-type enzyme with the help of crystallographic data obtained by four
X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in
the presence and absence of substrates
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Minal (MPhil)

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marine bacterium Vibrio harveyi
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This reflects apparently the need for the bacterium to
take up chitin containing nutrients in aquatic conditions as its sole source of energy
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Fungi


Fungal chitinases play important role in nutrition, morphogenesis, and developmental process
and are known to be produced at various stages during fungus growth
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

The fungal chitinases are divided into three groups, being chitinases A, B, and C on the basis
of sequence and structural similarities
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Type B is
nonprocessive chitinases and they have a CBM or a serine/threonine rich domain on C-terminal
of their catalytic domain
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They have a CBM on the N-terminal of catalytic domain
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LysM containing receptors in plants play an important role in formation of root
nodules in leguminous plants by binding to Nod factor
...




A study on mycoparasitic Trichoderma species showed that these enzymes are involved in
degradation of both self and non-self-cell walls
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

Entomopathogenic fungi can play a major role as a biocontrol agent to control insects and pests
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Nematode parasitic fungi producing chitinase are used as biocontrol agent by
targeting eggs and larva of plant and animal infecting nematodes
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

The chitinases, which are associated with fungal cell wall, have role in filamentous fungal
sporulation
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Trichoderma
have been considered as biocontrol agents in case of soil borne fungal pathogens amongst
various chitinolytic fungi
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have been purified and characterized
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Minal (MPhil)

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C
...
On the basis of sequence similarity insect
chitinases are divided into eight groups, which vary in their molecular weight ranging between
40kDa and 85kDa and their pH optima
...
However, the
last two domains are not necessary for chitinase activity
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

Insect chitinases are involved in degradation of cuticle layer to smaller oligosaccharides by
hydrolyzing and randomly breaking cuticle that is used for synthesis of new cuticle
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

Expression of these enzymes is required to be under stringent control to avoid any premature
exposure, which may lead to growth inhibition
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Chitinases are also reported to be present in venom and salivary gland of some
insects
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D
...
Classes III and V of plant chitinase belong to GH18 family while
the rest of the five classes belong to GH19 family
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Many
plants continuously express chitinases in tuber, stem, and root
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

Chitinase is known to play a major role in defence against chitin containing pathogens like
fungi and insects
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

Chitinases are also known to be expressed during high environmental stress conditions such as
cold and drought
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Some of class III chitinases are reported to play an important role in calcium storage without
having more significant effect on their catalytic activity
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E
...
Chitotriosidase in human macrophages has been
found in higher levels in lysosomal storage diseases as well as in patients with Plasmodium
falciparum malarial infection and chitin binding protein b04 (CBPb04) in bovine serum
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

Human also produces AMCase; a family 18 chitinase is induced during TH2 inflammation
through an interleukin dependent mechanism
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Therefore, mammalian chitinases can be used as a biomarkers for asthma and hence inhibition
of AMCase with chitinase inhibitors such as allosamidin reduces inflammation and can be used
as potential target for asthma therapy
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APPLICATIONS OF CHITINASES

Chitinases have several field applications
...


1
...
Chitooligomers obtained by action of chitinases have a wide range of
biotechnological applications in biochemical, food, and various chemical industries
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Another approach to utilize the chitinous waste effectively is production of single cell protein
(SCP)
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Chitinase producing bacteria and yeast (e
...
, S
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2
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Minal (MPhil)

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for biotechnological applications
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An enzyme of food industry purpose
tannase is produced by Aspergillus niger but tannase binds to its cell wall reducing the yield
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3
...
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
...
So overexpression of a
combination of various chitinases in transgenic plant may aid against fungal pathogens
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Other than being used directly as a biocontrol agent,
chitinase can act as a target for biopesticide as chitin has a major role in insect metamorphosis
as well as in gut of insects
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Chitinases also have applications in the
bioconversion of chitin waste to fertiliser
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Therefore, biological control strategy has
become a vital advance in order to make sustainable agriculture possible
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Bio-Medical Application
a) Chitinases have a significant function in human health care
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According to Laine and Lo, (1996) chitin and chitin binding
proteins can be explored for the recognition of fungal infections in humans
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b) 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
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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 remodelling and inflammation
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c) Chitooligosaccharides also have an enormous pharmaceutical potential to be used in human
medicines because of its following activities: Antitumor activity (shown by chitohexaose
and chitoheptaose), Wound healing property, and Anti-hypertensive activity
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GlcNAc itself is an anti-inflammatory drug, and in
the human body, it is synthesized from glucose, then incorporated into glycoproteins and
glycosaminoglycans
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e) Plasmodium falciparum is also reported to produce chitinase enzyme in the sporogonic
cycle
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Inhibition of chitinase
can stop the sporogonic cycle, so it can be considered a good target
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f) They are involved in the signalling for root nodule formation, act as elicitors of plant
defence
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These derivatives have an extensive
medical use because quite a few of these chitin derivatives are known to be non-toxic, nonallergic, biocompatible, and biodegradable
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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
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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
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j)

Zhu and co-workers, 1984, reported the first clinically 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
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Minal (MPhil)

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reported to be induced in the lungs of an ovalbumin-induced mouse asthma model
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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
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g
...
g
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Mammalian
chitinases do not have chitinase activity while AMCase and chitotriosidase have chitinase
activity
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On the
contrary, no such typical chitin-binding domain is present in CLPs, but still they show a
very high chitin-binding affinity
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l)

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)
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m) Consisting of the body’s first line of defence against external agents, which also includes
chitin containing pathogens, various chitinase family proteins have constitutively shown
their expression in macrophages, digestive tract, and in epithelial cells of lungs
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

Economical and practical utilization of chitinases can be achieved either by high yield or better
catalytic activity
...




Protein engineering of enzyme is providing a way to achieve this goal
...

o

In directed evolution approach, chitinase can be randomly mutated by error prone
PCR and thereby generation of a mutant library
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screened by using fluorescent activated cell sorter and/orin96-wellplates
...

o

In case of site directed mutagenesis, specific amino acid residues residing in the chitin
binding and catalytic sites are mutated
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So, any mutation near glutamate that enhances its
proton donating efficiency can enhance enzymatic activity
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Some residues far from
the active site also play role in binding of chitinase to the enzyme
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Due to this narrow groove, chitinase will
not release the polymeric chain after every cleavage but will continue on cleaving glycosidic
bonds in a processive fashion
...




Many chitinases showed low catalytic activity due to lack of chitin binding domain
...
This could increase the catalytic activity by improving substrate binding of the
engineered chitinase
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Course-Study by Savy P

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