Purification and characterization of thermostable chitinases from bacillus lichemiformis MB-2

PURIFICATION AND CHARACTERIZATION
OF THEMOSTABLE CHITINASES FROM
BaciUus licheni/ornris MB-2

ARlS TOHARISMAN

SCHOOL OF GRADUATE PROGRAM
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2004

ABSTRACT
ARIS TOHARISMAN. Purification and Characterization of Themstable
C h i t k s h m Bacillus Eicheniformis MB-2. Under the supervision of MAGGY
T. SUHARTONO, ANTONIUS SUWANTO, and TRESNAWATI
PURWADARSA

Chitinases are of great biotechnological interest and have received remarkable
attention. A themophilic bacterium, Bacillus lichenifomis MB-2,from Tompaso
hot spring, North Sulawesi Indonesia, secreted thermostable chitinases into
culture media. Preliminary study showed that the enzymes occur in multiple

forms and were stable at a high temperature and a broad range of pH.
The
objectives of this research were to purify and characterize thermostable chitinases
from B. lichenrformis MB-2.
The extracellular chitinases were isolated
by successive column
chromatographies on Phenyl Sepharose CL4B, DEAE Sephacel, and Superdex
G-75. The purified enzymes had molecular weight of 67 (Chi-67) and 102 kDa
(Chi-102), estimated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis). Chi-67 and Chi-102 had an endo- and exo- chitinolytic
activities, respectively. The optimal temperature of Chi-67 was 70°C, whereas
Chi-102 was 60 "C. The optimal pH for both enzymes was 6.0. Chi-67 wets
stable below 60°C and over a broad pH range of 4-1 1 for 1 h. In contrast, Chi102 was not stable at temperature above 50 "C and only stable at its optimum pH
for 1 h.
Chi-47 was resistant to denaturation by urea, Tween-20 and Triton-X, but
unstable toward organic solvents such as propanol, ethanol, DMSO (dimethyl
sdfoxide), and PEG (polyethylene glycol), indicating that hydrophobic interaction
of proteins plays an important role in maintaining enzyme activity. Ionic
interactions are also important for Chi-67 fold as the activity was reduced by
guanidine hydrochloride and NaCl (1 M). Chi- 102 was relatively more stable

toward various organic solvents than Chi-67.
Both enzymes hydrolyzed colloidal chitin, glycol chitin, or glycol chitosan,
but were less active to regenerated chitin, fine powder of chitin, or methyl
cellulose. The Michaelis constants &) for colloidal chitin, glycol chitin, 4methylumbelliferyl
N',
W-diacetylchitobioside
[MUF(GICNAC)~], 4methyiumbelliferyl N', N', M-triacetylchitotrioside [MCTF(G~CNAC)~]
for Chi-67
were
3.08 mg ml-', 0.315 mg ml-', 0.02 pmol d-'and 0.02 pmol dl,
respectively. Meanwhile, the K, for colloidal chitin, glycol chitin and
MUF(GlcNAc) for Chi-102 were 2.00 mg ml-', 1.32 rng ml-', and 0.03 m M ml-',
respectively. The first 13 N-tertninal amino acids of Chi-67 were determined to
be SGKNYKIIGYYPS, which is identical to chitinase from B. licImenIfomris PR1 (accession no. AA022144) and B. circuImrs No. 4.1 (accession no. A M 2 3 368).

DECLARATION

I hereby declare that this thesis is my own work and has not been accepted for the
award of any other degree or diplorna in any university or other institute of higher

leadng.

To the best of my knowledge and belief, this thesis contains no material
previously published by any other person except where due acknowledgment has
been made.

Bogor, May 20U4

ARlS TOHARISMAN

PURIFICATION AND CHARACTERIZATION
OF THERMOSTABLE CHITINASES FROM
BaciNus lichenifomis MB-2

BY
ARIS TOHARISMAN

A DISSERTATION

Submitted to Bogor Agricultural University

in partial fulfillment of the requirements for
the Doctorate Degree in Food Sciences

GRADUATE PROGRAM
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2004

Title

: Purification and Characterization of

Thermostable
Chitinases
lichen(ftorrnis MB-2
Name

from

BaciNus

: Aris Toharisman

Student Number : PO9600004

Study Program

:

Food Sciences

Approved by:

.

1 Advisor Board

Prof. Dr. Ir. Antonius Suwanto
Member

2. Head of Food Sciences Study Program

Examination date: May 11,2004

Dr. Tresnawati Purwadaria
MemtKr

of Graduate Program

BIOGRAPHY
Aris Toharisman was born on January 19, 1966 in Kuningan, West Java, the
fourth of five children of M. Suwarman and Hj. C, Suwarman. The author

finished his study at Senior High School (SMAN I Surnedang) in 1984 and
started his undergraduate work at Bogor Agricultural University (IPB). The

author received a Bachelor of Science degree in Agriculture from IPB

and a

Master of Applied Science degree in Biotechnology from the University of New

South Wales Australia in 1989 and 1997, respectively. Since 1989, the author
has been working at the Indonesian Sugar Research Institute, involved in sugarindustry waste treatment and its co-product utilization research. September 2000,
the author started his PhD program at Food Sciences Department of PB,with
the scholarship from Indonesian Department of Agriculture. The author had also

got a scholarship for 5 months, March-July 2003, fromDeutscher Akademischer
Austausch Dienst (DAAD) to do Sandwich in Bioscience Programme at
Department of

Germany.

General Zoology and Endocrinology, the University of Ulm,

The author had done the project entitled Purification and

Chmterization of Chitimes.

Foremost, I would like to express my sincere gratitude to my major supervisor:

Prof. Dr. Maggy T. Suhartono, for her thoughtful and invaluable guidance during
my study.

It was a great pleasure to me to conduct research under her

supervision. I am deeply indebted to my other supervisors, Prof. Dr. Antonius
Suwmto and Dr. Tresnawati Purwadaria,
stimulaiing suggestions.

Special

for their encouragement

thank is due

to

and

Prof. Margarethe Spindler-

Barth for having accepted me to do research at Department of General Zoology

and Endocrinology, UniversiGt Ulm, Germany. She gave me great help and
constant support during my work in her laboratow along the way.

I am grateful to

all the staff and students of the Microbiology and

Biochemistry Laboratory, Research Center for Biotechnology,

Bogor

Agricultural University: especially to ibu Ika Malikha, ibu Eni Sutnartini, ibu
Endang Yuli, ibu Rosita Lintang, pak Sumardi, ibu Yuyun, ibu Tati, mbak Nur

Azizah, mbak Emma, Yanti Lim, Winda, Ace, Dim, teh Ida, Pudin, and Yanti
for their assistance and supports.

I thank ibu Ekowati Chasanah for the

cooperative spirit and fruitful discussion during my research.

I am thankful to Prof. Dr. Margarethe Spindler-Bartb p u p especially to
Michael Wagemann, Natalja Mobius and Karin Dengler for dl their assistance on
laboratory works. Special thank goes to Dr. Yaya Rukayadi for sending me

valuable journals and other information and Dra Tami Indiyanti, MSc (Indonesian

Research Institute, PUSPIPTEK Serpong) for allowing me to try Acta Purifier.

I am grateful to both the Agricultural Department for the scholarship given to
me and to the Director of the Indonesian Sugar Research Institute for giving me

the chance to study at the IPB. Also, I greatly appreciate hancial support fiom

Bioproducts Research Center, Yonsei University (Korea) and the scholarship
fiom Deutscher Akadernischer Austausch Dienst (DAAD).

Many thanks to my familes for their emotional and spiritual supports.

I

must thank my wife. She has put a lot of enthusiasm and energy into eliminating
many short comings in various ways.

Many more persons participated in various ways to ensure my research

succeeded and I am thankful to them all.
F W , I want to express my gratitude to Allah SWT whose blessings made
this effort fruitful.

Bogor, May 2004

LIST OF TABLES
Variation in chitin contents at d e r e n t organisms ..................
Worldwide market for chitin derived products in year 2002 ........
Applications of chitin and its derivatives .............................

Nomenclature of chitholytic and cellulolytic enzymes ............

................
Properties of Some Bacterial C h i t h s ..............................
The pH stability of some chitinases ..................................
Activators and inhibitors of some kcterial chitinases .............
Kinetic parameters of some chitinases ...............................
Classification of extremophiles and example of ....................

Purification steps of some thermostable chithases

applications of some their enzyme
Proposed structural thennostabiiizingmechanisms of protein .....

Chitinolytic index of the isolates fiom Tompaso grown at 5 S°C ..
Ammonium sulhte precipitation of chitinases produced .........
by Bacillus licheniformis MB-2
Relative activity of crude-extract chitinases towards ...............
Various chitin substrates

Chitinase precipitation using 80% ammonium sulfate at
various pH

...........

Yield and purity of chitimes treated with .........................
several methods
Effect of salts removal of precipitated chitinases ..................
on enzyme yield and purity

Yield and purity ocf chitinases after binding .........................
with 3 different substrates

Effect of heat treatment on pudlcation results of c h i t k s ....
Protein recovery from HIC columns eluted with ...................
various conditions

Purification steps of endochitinase produced by .....................
B. lichenifomis MB-2
Purification steps of exochitinase produced by .......................
B. lichenifomis MB-2
Effect of denaturants, organic solvents, inhiiitors and
others compounds on enzyme activity

...........

6-2

Relative activity of Chi-67 and Chi-102 towards ...........
various chitin substrates

1 18

6-3

Kinetic parameters of Chi-67 for natural and artificial chitins .....

123

6-4

Kinetic parameters of Chi- 102 for natural and arti6cimtl chitins ...

123

7- 1

Chracteristics of crude-extract, Chi-67 and Chi- 102 chitinases ...

129

7-2

Characteristics of MB-2 pure chitinases and two other chitinases.

130

7-3

Comparison of N-terminal amino acid residues of chitinase ......
from 3. Iicheniformis MB-2 to other microorganisms

136

Time course of chitinases production by B, licheniformis MB-2
in medium containing 0.5%colloidal chitin (pH 7.0) at 5 5 O C

......

Chitinase yield of pellet and supematant precipitated ................
at different ammonium sulfate concentration
Activities of endochitinase and exochitinase after ......................
exchange buffer using ultrafiltration at 1 kDa
(1, fdtrate; 2, effluent) and 10 kDa (3, filtrate; 4, effluent)

Percentage of chitbases bound by various chitin substrates
Elution profile of chitinase &om Phenyl Sepharose CL4B ...........
c o b chromatography with gradients of ammonium sulfate
at pH 7.0

Elution profle of chitinase from Phenyl Sepharose C L 4 B .........
column chromatography with gradients of ammonium sulfate
at pH 8.0
Elution profile of chitinase h m PhenyI Sepharose CL-4B ............
column chromatography with gradients of ammonium sulhte
at pH 8.0 and decreasing pH gradient

Elution profile of chit& from Phenyl Sepharose CL-4B ..........
column chromatography with gradients of ammonium sulfate
at pH 8.0 and increasing pH gradient
Elution profile of chitinas.from Phenyl Sepharose CL-4B ..........
column chromatography with gradients of ammonium sulfate
at pH 8.0 and increasing urea gradient
Elution profle of chitinase fiom Phenyl Sepharose CL-4B ..........
column chromatography with gradients of ammonium sulfate
at pH 8.0 and increasing propanol gradient
Elution profile of chit& from Phenyl Sepharose CL-4B ..........
column chromatography with gradients of ammonium sulfate
at pH 8.0 and increasing Tween-20 gradient

Elution proiile of chitinase &om DEAE Sephacef column ...........
chromatography with gradients NaCl at pH 8.0 (-) and increasing
Tween-20 gradient
Elution profle of chitinase fiom Sephadex G-75 column ..............
chromatography

The scheme of chitinase pu&cation steps ..............................
SDS-PAGE of peak fractions fiom different purification steps ......

SDS-PAGE and native PAGE of exochitinase fractions .............
fiom different p d c a t i o n steps

Effwt of temperature on Chi-67 and Chi- 1 02 activities ...............
Effsct of pH on Chi-67 and Chi-102 activities .........................

Heat stability of Chi-67 ...................................................

Heat stability of Chi- 102 .................................................

..............
Effect of metal ions on Chi-67 and Chi-102 activities ...............
pH stability ofchi-67 and Chi-102 aRer 1 h and 4 h
Synergism effect of Chi-67 and Chi- 102 for various
chitin substrates

..................

Chitinax activities on flourogenic substrates as a function of
incubation time
Chitinase activities on flourogenic substrates as a function
of enzyme-

........

...........

Lineweaver-Burk plots of Chi-67 using MUF(G~CNAC)~...........
and MUF(GICNAC)~
as substrates

LIST OF APPENDICES

Sequences of 16s rRNA of MB-2 (1478 bases)

.........................

Phylogenetic Tree of Bacteria-Producing Chitinases

....................

Sequence Producing Alignments of Chi-67 N-terminal

...............

Conserved Domain Search of Chi-67 ....................................
Amino Acid Composition of Chi-67 .....................................

Predicted of Chi-67 Catalytic Domain Structure h m .................
GenBank Database
Predicted of Chi-67 Substrate Binding Domain Structure .............
fiom GenBank Database
Predicted of Chi-67 Fn 111-likeDomain Structure from
GenBank Database

................

1. INTRODUCTION
Chitin, an insoluble polysaccharide consisting of P-(14) linked N-ace@-Dglucosamine (GicNAc) units, is the second most abundant polysacc&de
nature, after cellulose.

in

It is widely distributed as structural component of

crustaceans, insects, and other arthropods, as we1 as component of the cell walls
of most fungi and some algae. About 10" tons of chitin is produced annually in

the aquatic biosphere alone, however, only 0.1% of this material is cmently being
converted to valuable products.

Chitin and its derivatives exhibit interesting properties and constitute a
valuable materials for biomedical, cosmetic, food and agricultural applications.
Currently they are used as immunoadjuvants, drug delivery systems, dietary

fibers, agrochemicals, and flocculants of wastewater sludge. In the f a d areas
chitin and its derivatives are used for clarification of beverages, food wraps,
recovery of solid materials from f a d processing wastes and water, and

irnmobiiization of enzymes. Chitin oligomers may promote the growth of Bifidus
bacteria and suppress spoilage organisms,

reduce toxins and detrimental

enzymes, prevent diarrhea and constipation, lower serum cholesterol, protect liver
function, and protect against cancer (Shahidi el al, 1999).

Enzymatic degradation of chitin is performed by chitinases and appears to
occur in two steps. An endochitinase (EC 3.2.1.14) reduces the polymer to
oligomers, which are subsequently degraded to monomers by exwhitinax-

chitobiase (P-N-acetylhexosaminidase, EC 3.2.1.52). Chitinases are found in a

wide variety of organisms such as bacteria, fungi, insects, plants and animals.

The enzymes are also known to be synthesized by some of the
coelenterates, nematodes, mollusks and +pods

protozoans,

(Muzzarelli, 2002).

Chitinases are of great biotechnological interest and have received remarkable
attention. First, these enzymes may be used to convert chitin-containing biomass
into biologically reusable forms such as oligosaccharides, chitobiox and

N-

acetylglucosamine. Secondly, chitinases may be exploited for the control of
fungal and insect pathogens of plants. Thirdly, chitinase inhibitors potentially
inhibit growth of chitin-containing pathogens

and plague insects that need

chitinases for normal development. Chitinase inhibitors have generated a lot of
interest given their potential as insecticides, fungicides, and antimalarials. Efforts

are going on throughout the world to enhance the production and purity of
chitinases. Some characteristics of chitinases have been reported but only a few
thermostable chitinolytic enzymes are known.
Exploration of extremozymes with their unusual stability towards heat, organic

solvents, extreme pHs, detergents, and resistance to common protein denaturing
agents has attracted quite a number of researchers as such features confer a

number of advantages with r e s p t to industrial applications.

Usage of

themostable enzymes in biotechnological processes will allow faster reaction
times and reduction of contaminations, reduce energy costs in large scale
fermentations, less viscosity and kter solubility of chemicals intended to be

~ r d u at
d high temperatures,and reduce pathogen contamination and efficient
product recovery p m s s e s such as distillation. Investigation of thermostable
enzymes is fundamental for the elucidation of the structural basis for their

stabilization, specificity, and catalytic properties. Through such an understanding,

it may be possible to engineer proteins designed for the conditions required in
various industrial processes. Additionally, such enzymes are also suited for
studying structure-function relationships and mechanisms for promoting and
maintaining tertiary structures at extreme conditions.

Thermophilic microorganisms that grow optimally at temperature of a b u t 50
to over 90 "C are potential sources of thermostable chitinases.

The natural

environment for thermophilic microorganisms are multifaceted throughout
Indonesia, including crater, terrestrial hot spring, and deep-sea thermal vents.

Many microorganisms producing various thermostable enzymes such as
protease (Mubarik er al, 2000; Wahyuntari et al, 2000), keratinase (Lintang et al,

2002), chitin deacetylase (Toharisman et al, 2001, 2002a), chitinase (Natsir et al,

2002; Suhartono et al, 20031, and chitosanase (Azizah et al, 2002; Haliza et al,
2002) have been isolated in our laboratory. Furthermore, we screened more than
50 therrnophiles from Tompaso hot spring (North Sulawesi), able to hydrolyse

chitin. Among them, isolate MB-2 identified as Bacillus licheniformis MB-2,
secreted a variety of thermostable enzymes including chitinases into culture
media. Preliminary study with zymograrn analysis showed that partial purified

chitinases occur in multiple forms and were stable at a high temperature and a
broad range of pH.

However, these chitinases have not yet been purified and

fkrther characterized.

The objectives of the research are to purify and characterize thermostable
chitinases from B. licheniformis MB-2. Pure enzymes are required for research
and analysis in biochemical and clinical chemistry, whereas information of
enzyme characteristics are needed for industriallcommercid applications as well

as understanding of structural-function of protein (enzyme). Thus, there is a
rapidly increasing demand for enzymes of high purity with comprehensive

characteristics. Characterization of the purified enzymes includes analysis of
substrates specificity, optimum pH and temperature, heat stability, the effect of

cations, stability of enzymes
substances,

towards

specific inhibitors and

denaturant

analysis of molecular weight, enzyme kinetics, and N-terminal

amino acid sequencing.

This research should greatly contribute for developing marine industry in
Indonesia, as the country presently has 170 shrimp processing units with capacity

of 500 000 tons per year. It is predicted that Indonesia produces chitin of about
100 000 tons annually. Enzymatic conversion of chitinous materials into valuable
and marketable products may drive the development of marine industries in

Indonesia,

2. LITERATURE REVIEW
2.1. Chitin
2.1.1. Structure and Distribution

Chitin is a polymer formed primarily of repeating units of P-1-4-linked 2
wtamido-2deoxy-b-D-glucopyranoseresidues (GlcNAL). Its structure is similar
to

the structure of cellulose, except that acety1arnino groups have replaced the

hydroxyl groups in position C-2 (Fig 2-1).

Chitin polymer tends to form

microfibrils of about 3 nrn in diameter that are stabilized by hydrogen b n d s
formed between the amine and carbonyl groups (Gooday, 1994).

Chitin
I

Figure 2-1. Structure of chitin, chitosan and cellulose
(Skjak-Braek and Sanford, 1989)

X-ray diffraction analysis suggested that micrwrystalline structure chitin

occurs in a-,p- and y- arrangements. in the a form, all chains exhibit an antiparallel orientation; in the

0 form, the chains are arranged in a parallel manner;

and in the y form sets of two parallel strands dtemate with single anti-parallel
strands.

The anti-parallel arrangement allows tight packaging into chitin

microfibrib, consisting of 20 single chitin chains that are stabilized by a high
number of hydrogen bonds formed within and between the molecules. In contrast,
in the p- and y-chains, packing tightness and numbers of inter-chain hydrogen
bonds are reduced, resulting in an increased number of hydrogen bonds with
water. The high degree of hydration and reduced packaging tightness result in

more flexible and soft chitinous structures (Goosen, 1997).

The most abundant form of chitin is a-chitin. It is found in the hydrozoa,
nematodes, rotifers, mollusks and arthropods. 0-chitin, a less stable and more

degradable form of chitin, is occurred in mollusks, squid pen, diatoms, cuttlefish
bone, insect exoskeletons and cocoons, and is major component of cell walls. The
y form is found in stomach lining of squid (Gooday, 1990).

Chitin has a very wide distribution among organisms (Table 2-1) and slightly

different structure and associated proteins and minerals.

Variations in the

amounts of chitin may depend on physiological parameters in natural
environments.

Chitin is always found cross linked to other structural

components such as protein and glucan. In insects and other invertebrates, chitin
is always aswiated with specific proteins, whereas in fungal walls it is found to

covalently bonded to glucans, either directly or via peptide bridges (Gooday,
1994).

Table 2.1. Variation in chitin contents at different organisms
(Skjak-Braek and Sandford, 1 989)
Sources

Pacentage of chitin

Fungi

5-30

Worm

20-38

Scorpion

30

Spider

38

Cockroach

35

Silkworm

44

Crab

25-50

Squid

3-20

Shrimp

45-70

Chitin varies in crystallinity,degree of covalent bonding to other components
such as glucms, and degree of deaeetylation. Degree of chitin acetylation is
between 0 to 1OOO/o, but the natural chitin is approximately 16% deacetylated.

Deacetylation of above 80% chitin units

is generally termed as chitosan

(Goosen, 1997).
Chitosan is composed primarily of GlcNAc and GlcN (2-amino-2-deoxy-P-~glucopyranose) residues, Unlike most polysaccharides, chitosan has three types

of reactiond functional groups, an amino group as well as both primary and
secondary hydroxyl groups at the C-2, C-3, and C-6 positions, respectively.
Chemical modifications of these groups provide numerous useful materials in

different field of applications. The molecular weight of chitosan is about 0.1-0.5 x
10' Dalton, whereas native chitin is about 1 - 2 x1o6 Dalton (Goosen, 1997).

In 2002 about

id

tons of chitin were recovered industrially h m marine

industry, approximately 65% of which was converted into glucosamine, 25% into

chitosan and 10% into chitooligosacchrtrides. The world market of chitin and its
derivatives was estimated to reach $137 million (Table 2-2).

Table 2-2. Worldwide market for chitin derived products in year 2002
(Sandford, 2003)
I

Quantity
(Tom)

Price
($/ton)

Value
($ million)

Chitom

6 700

10 000

67

Glucosamine

4 000

10 000

40

500

60 000

30

Derivative

Oligosaccharides

J

2.1.2. Application

During the past 20 years, a substantial amount of work has been published on
chitin and its derivates and their potential use in various applications such as in

food, agriculture, medicine, biotechnology, pharmacology, and waste treatment

(Table 2-3).

In food processing, chitin and its derivates may be used for clarification of
wine, beer and juices, deacidification of fruit juices, formation of biodegradable
films, production of flavor compounds, preservation of foods from microbial

deterioration, recovery of waste material from focd processing discards, and

reducing warmed over flavor in precooked meats (Shahidi et al, 1999). They are
used as food supplement and may reduce plasma cholesterol and triglycerides due
to their ability to bind dietary lipids (Koide, 1998).

Chitin has been reported to regulate some interactions between plant

pathogenic fungi and their plant host. The fungi-resistant properties of chitin
derivates have resulted in their application as fertilizer, soil stabilizer, and seed
protector (Kohle et al, 1984; Kauss et al, 1989; Shirnosaka et el, 1993 and 1995;

Leger et a/, 1996).

h the biomedical area, chitin and its derivates have been observed to accelerate
wound healing properties and the attainment of an attractive skin surface. It has
been suggested that the mechanism by chitin and its derivates act at wound site

involves activation of neutrophiles and macrophages. These compounds stimulate
the migration of plymorphonuclear and mononuclear cells and accelerate the

reformation of connective tissue and angiogenesis (Bianco et al, 2000). Because
of its high oxygen permeability, the chitin derivate was used as a material for
contact and intramular lenses. It has also been found to expedite blocd clotting

and can form complexes with other natural polymers, such as collagen and

keratin,to form materials with unique biomedical properties (Sandford, 1989).
The fact that chitin and its derivates are biodegradable and biocompatible

makes them particularly appropriate for use in drug delivery systems (Thacharodi

and Rao, 1995; Genta et al, 1997; Zecchi et al, 1997). This property is extremely
valuable in cancer chemotherapy since the agents are often highly toxic and
require long periods of time for administration (Dodane and Vilivalam, 1998).

Because of their binding and ionic properties, chitin derivates can be used as a
flocculating agent to remove heavy metals and other contaminants h m
wastewater. Current applications in this area include treatment of sewage
effluents, paper mill wastes, metal-finishing residues, and radioactive wastes
(Guibal er al, 1997).

Table 2-3. Applications of chitin and its derivatives

1

Areas

1

Applications

Nutritional uses

Dietary supplement
Fiber source

Food

Neutriceutical
Flavor preservative
Flavor enhancer
Texture-enhancing agent
Emulsifying
Beverage clarifier

Biomedicine

Wound healing
Burn healing
Contact lenses
B l d dialysis membranes
Antitumor

Skin and hair care

Moisturizing creams and lotions
Hair care products

Environment and agriculture

Water treatment
Seed treatment
Fertilizer and fungicide

Others

Paper finishing
Sorption of dyes
Solid state batteries
Feed additive
Contact lens
Chromatographic supports

1

1

Chitin

oligomers

or

chitin

oligosaccharides

[chitooligosaccharides),

customarily used for saccharides having the degree of polymerization of 2-10,
are biologically active compounds. The products have activities as elicitors,

antibacterial agents, immuno-enhancers and 1ysozyme inducers (Aiba, 1994;
Shahidi et a],1999; Wang el al, 2002a md 2002b). Aiba (1994) reported that
(GlcNAc), (n=2-5) are useful in agriculture and medicine, meanwhile (GlcNAc),
activate macrophages and the immune system. Another chitooligosaccharide,
(GICNAC)~,
is claimed to be a potent anti-metastatic agent against mouse bearing

Lewis lung carcinoma (Roseman et at, 1999). Monomer of chitin, GlcNAc, is a
valuable pharmacological agent in the treatment of a wide variety of ailments
including gastritis, fwd allergies, inflammatory bowel disease (IBD) and

diverticulitis. It does not have m y established negative side effects (Haynes et
al, 1999).

2.13. Biodegradation of Chitin
Chitin degradation towards chitosoligosaccharides, GlcNAc, GlcN and other
derivatives can be obtained by various treatments. The most used method is

chemical treatment using strong acid at high temperatures for extended of time.
Unfortunately, it is not easily controlled and environmentally unsafe. The product

has a broad range of molecular weight and a heterogeneous extent of

deacetylation, so it is not suitable in food, cosmetics and pharmaceuticals
industries, which need high purity grade and uniform prducts. In addition, the
method i s disadvantageous due to the occurrence of side reactions, energy-

intensive need and the generation of acid waste (Synowiecki and Al-Khateeb,

1997; Yoon et al, 1998; Kolodziejska et al, 2000). Enzymatically process, on the

other hand, could be employed under mild conditions, would not yield side
product, and results in specific products with good quality.

There are two possible pathways of chitin biodegradation (Fig 2-2, Gooday,
1990). First, it involves the initial hydrolysis of 1,4 P-glycosidic bond of chitin,
Second, it is the deacetylation of chitin to chitosan. The fonner is accomplished

by chitholytic enzymes and the latter by chitin deacetylase and chitosanase.

Chitin d e q l a w (EC 3.5.1.41)

Cbitinase [eC 3.2.1.141

CHlTOSAN

~ O ~ C # ) M W

Cbitolana~(EC 3.2.1 -132)

GIN&-MC @C 3.2.1.30)

f

7

N - A m mWAMmE

I

cmTOSAN 0LICK)MERS

I

Figure 2-2. Enzymatic pathways of chitin degradation (Gooday, 1990)

Chitinolytic enzymes catalyze the hydrolysis of chitin by cleaving the bond
between the C1 and C4 of two consecutive N-acetylglucosamines, chitin
deacetylase (EC 3 -5.1.41 ) modify chitin into chitosan molecule through

deacetylation mechanism, while chitosanase (EC 3 -2.1.13 2 or 3 -2.1-99) hydrolyze
chitosan to chitosan oligomers (Somashekar and Joseph, 19%; Cohen-Kupiec and

Chet, 1998).

23. Chitinase
2.2.1. Biologica1 Role

Different organisms p d u c e a wide variety of chitinases that exhibit different
substrate specificities and other properties useful for various functions. In
bacteria, chitinases play roles in nutrition (Bati ZU et al, 2000; Tsujibo et al, 2000;

Folders et al, 2001) and parasitism (Grenier et al, 1993; Wenuganen, 1996;
Khaeruni, 1998; Kobayashi et al, 2002; Lutz et al, 2003; Malik et al, 2003a and
2003b), whereas in fungi, protozoa and invertebrates, they role in rnorphogenesis
(Bakkers ef al, 1997). Plant chitinases function in a self-defense mechanism
against pathogenic fungi (Hou et al, 1998; Minic et 01, 1998; Vander er 01, 1998;

Velazhahan et a!, 2000). Baculovirus, which are used for biological control of

insect pests, also produce chitinase for pathogenesis (You et al, 2003). Chitinase
activity in human serum has recently been described. The possible role suggested
is a defense against fungal pathogens (Escott and Adams, 1995; Langer el 01,
2002).

Chitinase from marine organisms play a crucial role in the recycling of

chitinous materials for maintenance of the ecosystem in the marine environment.
Many bacteria and fungi containing chitinolytic enzymes convert chitin into
carbon and nitrogen that can serve as energy source (Folders et a/,2001).

Chitinase plays an important role in insect growth and development. It
involves in molting and cuticle turnover by hydrolyzing the structural polymer
chitin, a principal component of the insect exoskeleton and gut lining. Chitinase
activity has been identified in the molting fluids and midguts of several insects

including Bornbyx mori, Manducu sexta, Ae&s aegypti, and the wasp Chelonus
(Royer et al, 2002). Chitinase is also important in the life cycle of arthropods
(Spindler et al, 1997).
Plants do not have an immune system and instead use various defense
mechanisms to protect their vegetative and reproductive organs against pathogen
infection. One response to pathogenic attack involves expression of pathogenesis-

related proteins such as chitinases (Andersen et al, 1997). The enzymes are
capable of releasing chitin oligomers that elicit a series of defense reaction from
the fungus invading the plant (Nishizawa et a/, 1999; Staehelin et a), 2000).

Purified barley and bean chitinases inhibited the growth of fungal hyphae (Leah at
al, 1991). It has also been demonstrated that transgenic plants that over express

chitinases have increased resistance to fungal attack (Wiendi, 2002). In root
nodules, chitinases may protect the symbiotically infected zone from external

pathogens (Minic et al, 1998).

2.2.2. Application

Chitinases have many industrial and agricultural applications such as
preparation of chitooligosacc~des, biocontrol of plant pathogenic fungi,

production of single-cell protein, fungal protoplast technology and chitochemical
studies (Shaikh and Deshpande 1993; Patil et al, 2000; Gimerez-Pecci et al,

2002). Chitinases can be exploited to produce chitmligosaccharides from chitin.
A chitinase from Vibrio alginolyticus was used to prepare chitopentaose and

chitotriose fiom colloidal chitin ( M m el al, 1992). Strepdomyces chitinase was
utilized for the enzymatic hydrolysis of colloidal chitin. The chitobiose produced
was subjected to chemical modifications to produce novel disaccharides
derivatives of 2-acetamido-2-deoxy-~al1opposemoieties that are potential

intermediates for the synthesis of an enzyme inhibitor (Ohtakara et al, 1W).
Fungal formulations containing chitinases are perceived as safe alternatives to

chemical pesticides for insect treatment. Entomophatogenic fungi e.g. Beauveria
bassima, Metharizizrm anisoplue and Verticillium Iecanii (Leger ef a/, 1996;

Freimoser et al, 2003) are parasites of various pests including potato beetle,
sugarcane frog hopper and aphids. A numlxr of soil bacteria produce chitinolytic
enzymes that can also be used to inhibit fungal infection (Lodto et a/, 1996).

The enzymatic conversion of waste chitin to yeast single-cell protein (SCP) has

been investigated. Revah-Moiseev and Carroad (1981) used the S. marcescens
chitinase system to hydrolase the chitin and Pichia kuclriavezevii to yield the SCP.

Vyas and Deshpande (1 99 1) showed that the M. verrucaria chitinase complex and
S. cerevisiae can also be used for SCP production h m chitinous waste. The total
protein and nucleic acid contents of produced SCP were 61% and 3.1%,
respectively.
Since chitin is the major structural component in the cell walls of most fungi,

chitinolflc enzymes play a significant role in protoplast isolation. H d y n et a1
(198 1 ) evaluated various commercial mycolytic preparations for protoplast

isolation and found that high chitinase levels permit effective mycelia

degradation.

The lectins, due

to

their specific monosaccharide-binding properties, can be

used to locate sugar residues in thin section of plant and fungi. Similarly,

hydrolytic enzymes like chitinase can be also employed to Iucate fungal pathogens

that possessd chitinous cell wall. The chitinase-gold complex can be used for
this purpose (Peters and Latka, 1997).

2.23. Classification

Based on amino acid sequences homology of glycosyl hydrolases, chitinases
were grouped into two evolutionarily unrelated groups, designed as families 18

and 19 (Henrissat and Davies, 1997). Family 18 chitinases include most of the

chitinases from bacteria, fmg, insects, plants (class I11 and V chitinases), and

animals. Family 19 chitinases include classes I, ll and IV of the plant chitinases
(Meins et ai, 1992; Gijzen et al, 2001) and a chitinase from Streptomyces griseus

(Ohno et a/, 1996). Class I and IV of plant chitimes consist of the sequence with
a highly conserved main structure and an N-terminal Cys-rich domain. Class IV

chitinases are smaller than class I chitinases due to deletions in both the cysteinerich domain and the catalytic domain.

Class I1 chitinases are structurally

homologous to class I and JV but lack the Cys-rich domain. Class III chitinases
show little sequence similarity to the enzymes in classes I, II, or lV and are more
prevalent in fungi and bacteria than in higher plants. They also seem to possess
reduced antifungal activity compared to the class II enzymes, presumably because

they possess a different substrate specificity (Roberts and Selitrenikoff, 1988;

Collinge et al, 1993).

Family 18 chitinase is further classified into 3 groups: A, B, and C, based on

the amino acid sequence of individual catalytic domains (Fig 2-3). Chitinases in
group A have an insertion domain between the seventh and eight P-strands of the

(P/(~)~-barrel
basic structure, whereas chitinases in group B and C do not have
such an insertion domain (Uchiyama et a],2001 ;Suzuki et al, 2002).

~trea#rrrwe
dimmiridis
~
: ex*

Chi 0 1

SrrcIUmn w s dintru :chiunasc 63

Aemmmm sp. IOS-2.4 : chili-

f1

Fig 2-3. Classification of family 18 chitinase based on the amino acid sequence of
individual catalytic domains, Shadow boxes indicate the homologous
regions of individual chitinases to the d y t i c domain of B. circulans
chitinase A1 (Group A), 3. circulans chitinase D (Group B), or
Strepiomyces erythraeus chitinase (Group C).
Arrow (+) indicates
fibmnectin type III-like domain (Watanabe et al, 1992)

Based on the mode of action, chitinases were classified into endochitinases
and exochitinases.

Endochitham (EC 3.2.1.14) cleave chitin randomly at

internal site generating soluble low molecular mass muftimers of GlcNAc such as
chitotetraose, chitotriose, and the dirner of di-acetylchitobiose. Exochitinase can

be divided into two subcategories: chitobiosidases (EC 3.2.1.29) which catalyze
the progressive release of di-acetylchitobiose starting at the non-reducing end of

the chitin microfibril; and 1-4-~~N-acetylglucosamidase (EC 3.2.1 -30) which
cleave the oligomeric product of endochitinase and chitobiosidases generating
monomers of GlcNAc (Cohen-Kupiec and Chet, 1998). This class5cation is
almost parallel to the cellulolytic complex (Table 2-4).

Table 2-4. Nomenclature of chitinokytic and celluloZytic enzymes
(Patil et al, 2000)

Mode of action

Cellulolytic enzymes

Chitinolytic enzymes

Random hydrolysis of the Endochitinase (1,4, P-ply-N- Endo- 1,CP-glucanase
chain
(EC 3.2.1.4)
a&y1gluco saminidase,
EC 3.2.1.14)
Hydrolysis of terminal
non reducing sugar

Cellobiase,
Earlier classification as
chitobiase
(EC 3.2.1-29); -gluwsidase
P-D-Acetylgluco~dase (EC 3.2.1.21)
(EC 3.2.1.30)

Successive removal of

Present classikation as

sugar unit from the non

p-N-acetylhexoddase
(EC 3.2.1.52)

reducing end

Chitobiohydrolase (?)
Successive removal of
dimer sugar fiom the non
reducing end

Exoglucanase,
exo- l,4-b-glucosidase
(EC 3.2.1.74)

,

Exo-cellobiohydrolase;
cellulose 1,4-Pcellobiosidase (EC
3.2.1.91)

The classification of endo and exochitinase depends mainly on the substrate.
For example, the Streptomyces chitinase complex degrades pure crystdine

P-

chitin of diatom spines only from the non-reducing ends to yield
diacetylchitobiose, whereas colloidal chitin is degraded to a mixture of oligomers
and diacetylchitobiose. Some 1-4-P-N-acetylgluwsamidases
can also act weakly
as exochitinases cleaving monomer units f k m the non-reducing ends of chitin
chain (Gooday, 1994).

23.4. Structure and Catalytic Mechanism

Crystal structure of chitinases has been successively reported. The crystal

structure of 26 kDa chitinase from barley seeds (Hordem vulgare L.), which is
classifid into family 19, was first solved. After that, the structures of a 60 kDa
c h i w e A from S. murcescens and a 29 kDa chitinase from rubber tree (Hevea
brasiliensis) were reported. Both are family 18 enzymes.
Comparison between the structures of family 18 and 19 chitinases revealed a

clear different in their structures. The family 18 chitinase has a typical (dB)s
barrel structure composed of eight a-helices and an eight stranded &sheet (Fig 24).

In addition to the main barrel domain, it has an N-termid p-strand-rich

domain and a small (a+P) domain. The crystal structures of other family 18
chitinases exhibit similar barrel structure. Apparently, in family 18 chitinases, the
sequence homology results in the similarity in three-dimensional structure
(Fukamizo, 2000; van Aalten et al, 2001; Watanabe el al, 2003). On the other

hand, barley chitiaase (family 19) is composed of two loks, each of which is rich
in a-helical structure (Fig 2-5). From a docking calculation of the chitinase and

(GICNAC)~,
the substrate binding cleft is estimated to lie between the two lobes.

The hypothetical binding cleft is composed of two a-helices and three-stranded p-

sheet (Fukamizo, 2000).

Fig 2-4. A ribbon drawing of a representative family 18 chitinase fiom S.
marcescens, an a l p barrel structure with eight parallel strands of sheet
and eight return helices. Glu 315 is the catalytic acid, The N-terminal
147 residues form a chitin-anchoring domain; this appears at the lower
left of the chit& barrel domain (Robertus and Monzingo, 1999)

Fig 2-5. A ribbon drawing of barley chitinase (family 19 chitinase) revealing a
mixture of secondary structure, including 10 a-helical segments and one
three-stranded P sheet. The structure shows a globular protein with high
a-helical content and an elongated cleft nmning the length of the protein,
presumably for substrate binding and catalysis (Robertus and Monzingo,
1 999)

Family 18 bacterial chitinases contain the consensus sequences SXGG and
DXXDXDXE. The sequences SXGG and DXXDXDXE are substrate-binding
and active sites, respectively. Several runs of conserved amino acids for family

18

h m Coccidioides immitis (Yang et al, 1997), Trichderma harzianurn

(Garcia et al, 1994), Aphanocladium album (Blaiseau and Lafay, 1992), and
Serratia murcescens (Brurberg et al, 1994) are shown in Fig 2-6. A section of

signature sequence for the family 19 is shown in Fig 2-7, This represents the
chitinase from barley, Hordeurn vulgare (Leah et al, 19911, potato, Solmum

tuberosum (Gaynor, 1988), Arabidopsis thulium (Sarnac ef

02,

1 9901, and pea,

Pisum sativum (Chang et al, 1995).

In addition to the difference in 3D structure, chitinases of the two families
show the difference in catalytic mechanism (Fig 2-8 and Fig 2-91, Family 118
chitinase hydrolyze the glycosidic bond with

retention

of the anorneric

configuration (Sasaki et al, 2002), whereas family 19 chitinases with inversion
(Ohno et al, 1996). Substrate assisted catalysis is the most widely accepted model

for the catalytic mechanism of family 18 (Brameld et al, 1998), whereas a general

acid and base mechanism has been suggested to be the catalytic mechanism of
family 19 (Ohno er al, 1996). Family 1 8 c h i t h s hydrolyse GlcNAc-GlcNAc

and GlcNAc-GlcN, meanwhile family 19 chitinases hydrolyse GlcNAc-GlcNAc

and GlcN-GlcNAc linkage (Ohno et al, 1996; Mitsutorni eb ai, 1997). Family 18
chitinases are sensitive to allosarnidin, but a family 19 chitinase from higher
plants has been shown to k insensitive (Koga et aI, 1999).

Chi-ci
Chi-th
Chi-aa
Chi-sm

130
LSIGGWTYSPNF
LSIGGWTWSTNF
LSlGGWTWSTNF
PSIGGWTLSDPF

170

FDGIDIDWEYPED
FDGIDlDWEYPAD
FDGDIDWEYPAD
FDGVDIDWEFPGG

Fig 2-6. Conserved amino acid on the active site of family 18 chitinases. The
sequence numbers correspond to Coccidioides immitis (Chi-ci); Chith, Trichoderma harzinnum; Chi-aa, Aphanocladium album; Chi-sm,
S. marcescem

60

70

80

100

90

Chi-hv
Chi-st

KREVAAFLAQTSHE'ZTGGWATAPDGAFAWGYCFKQERGASSDYCTPSAQWPCAPGK
KREIAAFLA QTSHEmCCWASAPDGPYAWGYCFLRERGNPGDYCPPSQQWPCAPGK

Chi-at
Chi-ps

KReVMFGQTSHETLY;GWATAPDCPYSWGYCFKQEQNPD YCEPSATWPCASGE
KREIAAFLG QTSHEITGGWPTAPDGPYAWGYCFLR EQNP SWCQASSEFPCASGK

-

Fig 2-7. Conserved amino acid on the active site of family 19 chitinases. The
sequence numbers correspand to barley chitinase, Hordeum vulgare
(Chi-hv); Chi-st, Solanurn iuberosum; Chi-at, Arabidopsis thuliana;
Chi-ps, Pasum safivum

The catalytic mechanism of W y - 18 chitinase was first reported by Watanabe
et al (1990) for chitinase A1 h m B. circulam.

The Glu204 residue was

considered to be a proton donor in its catalysis since site-directed mutagenesis of
the residue completely e h t e d its activity. The glutamic residue was also
found to & conserved in family 18. In S, marcescem chitinase A and B, the

catalytic carboxylate corresponding to Glu204 of B. circulans chitinase AT is
Glu3 15 and Gh 144, respectively (Vaaje-Kolstad et al, 2004).

In the consensus region of the catalytic domain of family-18 chitinases, there
are several consewed carboxylic amino acid residues. For example, Asp200 and

Asp202 in chitinase A1 from 3. circulam,Asp3 1 1 and Asp313 in S. marcescens

(Watanabe et al, 1992 and 1993). The location of these residues does not
correspond to that of the second carboxyhte in lysozyrne (Asp52) or in family 19

barley chitinase (Glu89). Thus, the second carbxylate cannot be identikd in any
h d y 18 chitiwise. Therefore, the family 18 c h i t b s should have a different

mechanism of catalysis. Recent studies on the family 18 chitimes indicate that

the catalytic reaction of the enzymes takes place through a substrate-assisted
mechanism (van M e n el al, 2000).

Fig 2-8. Glycosyl hydrolases catalyzed by family 18 chitinase following
substrate-assisted catalysis. The oxocarbonium ion intermediate is
stabilized by an anchimeric assistance of the sugar N-acetyl group
after donating a proton from the catalytic carboxylate (Fukamizo,
2000)

Fig 2-9. Glycosyl hydrolases catalyzed by barley chitinase (family 19)
following single displacement mechanism. Glu 67 as the general
acid acts as the proton donor, and Glu 89 as the general base
activates the water molecule which then attacks the C 1 atoms of the
intermediate sugar residue at site (-1) (Fukamizo, 2000)

As shown in Fig 2-8, a putative oxocarboniurn ion intermediate is stabilized by
an anchimeric assistance of the sugar N-acetyl group after donation of a proton

from the catalytic carboxylate to the leaving group. Such a stabilization might
occur either through a charge interaction between the C 1 carbon and the carbonyl
oxygen.

The mechanism does not require the second carboxylate and can

rationalize the anomer retaining reaction of the enzyme without the second
carboxylate (Fukarnizo, 2000).
The catalytic mechanism of family 19 chitinase is shown in Fig 2-9. At first,

the general acid, Glu67, protonates the
oxacarbonium ion intermediate.

P- 1,4-glycosidic oxygen atom forming an

The water molecule, activated by the general

base (Glu89), attacks the C1 atom of the intermediate state for the a-side to

complete the reaction. The separated location of the two catalytic residues might
be permit the water molecule to be located in between the anomeric C1 atom and
the carboxyl oxygen of the general base (Glu89). This location of the water
molecule would result in the anomeric inversion of the reaction products. From

the molecular dynamic simulations, however, Glu89 was found not only to
activate the nucleophilicity of the water molecule but also to act as a stabilizer of
the carbonium ion intermediate (Brameld and Goddard, 1 998). In addition, the

simulation study indicated that the (G~CNAC)~
substrate binds to barley chitinase
with all sugar residues in a chair conformation, no sugar residue distortion was
found in family 19 chitinase complexd with the substrate (Fukarnizo, 2000).

2.2.5. Chitin Binding Domain

Chitinases generally consist of multiple functional domains, such as C-terminal

domains which binds especially to insoluble chitin (chitin binding domains,
ChBD), fibronectin type III-like donins (FnIIIDs), and a N-terminal catalytic
domain. The fimtion of each dolllain except for the ChBD has not been yet

elucidated (Morimoto

at

al, 1997; Gao et al, 2003).

The ChDB has been

demonstrated to be important in the degradation of insoluble chitin (Wu et al,

2001 ; Jee el d,2002; Royer et al, 2002; Orikoshi er al, 2003).

B, circulans WL-1 2 produces ChiAl , Chic 1 and ChiD1 as the initial products
of the three chitinase genes (Alam et a!, 1996). ChiAl has the highest hydrolyzmg
activity against insoluble chitin. This chitinase comprises the catalytic domain

(CatD), two FnlIIDs, and the C-terminal ChBD (Watanabe et 01, 1 992; 2003), The
deletion of FnIIIDs did not affect chitin binding but decreased hydrolysis of
insoluble substrate like colloidal chitin. However, hydrolysis of soluble substrate

either carboxymethy1 or chitin-oligomer was not significantly affected.

In

addition to its involvement in binding to insoluble substrate, the type I11 dornain
could possibly IE involved in the exo-hydrolytic mechanism (Watanabe et al,
1992; Tsujib at al, 1998b).

Suzuki et a1 (1999) identified the chitinase C1 and C2 fiom S. marcescens
2170. Chitinase C 1 lacks a signal sequence and consists of a catalytic domain

belonging to glycoside hydrolase family 18, FnlIID and a C-terminal. Chitinase

C2 corresponds to the catalytic domain of C1 and is probably generated by
proteolytic removal of the FnIIID and ChBD. The loss of the C-terminal portion
reduced the hydrolytic activity towards powdered chitin and regenerated chitin,

but not towards colloidal chitin and glycol chitin, illustrating the importance of

the ChBD for the efficient hydrolysis of crystalline chitin.
Unlike cellulose binding domain (CBD), the tertiary structure of ChsD

remains unclear. Chitin differs chemically from cellulose only in that each C2

hydroxyl (-OH) group in cellulose is replaced by an acetamide (-NHCOCH3)
group in chitin, hence the mechanism by which ChBD binds to chitin was

expected to be similar to the mechanism by which CBD bind to ceILulose
(Ikegami el al, 2000).

The most accepted model for the binding of CBD to cellulose is that aromatic
rings arranged in the flat face of a CBD are stacked on every other pyranose ring
of plysaccharides through hydrophobic interactions. The involvement of

arormdic residues in the interactions has been observed using NMR, site-directed

mutagenesis, and chemical modscation. In CBD, the stWWst motif is widely
c o r n e d