study on characterization of chitinase from streptomyces

i


Nguyen Thanh Huong






STUDY ON CHARACTERIZATION OF CHITINASE
FROM STREPTOMYCES




MASTER THESIS MAJOR BIOTECHNOLOGY

HANOI – 2011


UNIVERSITY OF LIEGE
***
VIETNAM NATIONAL UNIVERSITY, HANOI
INSTITUTE OF MICROBIOLOGY
AND BIOTECHNOLOGY
***

ii

Nguyen Thanh Huong

STUDY ON CHARACTERIZATION OF CHITINASE
FROM STREPTOMYCES



Speciality: Biotechnology
Code: 60 42 80


MASTER THESIS MAJOR BIOTECHNOLOGY


SUPERVISOR: Dr. DUONG VAN HOP



HANOI – 2011


LIEGE UNIVERSITY
***
VIETNAM NATIONAL UNIVERSITY, HANOI
INSTITUTE OF MICROBIOLOGY
AND BIOTECHNOLOGY
***

iii

ABBREVIATIONS
DNA - Deoxyribonucleic acid
DNS - Dinitrosalicylic acid
E.coli - Escherichia coli
GlcNAc - N-acetyl-D-glucosamine
M1 - Medium 1
Mr - Molecular mass
PCR - Polymerase chain reaction
PR - protein - pathogenesis-related proteins
rDNA - Ribosomal DNA
SEM - Scanning electron microscope
SDS - PAGE - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
TLC - Thin layer chromatography
VTCC - Vietnam Type Culture Collection
YS - Yeast extract - starch
iv

LIST OF FIGURES
Figure
Title
Page
Figure 1.
Natural sources of chitin
4
Figure 2.
Chemical structures of cellulose and chitin

6
Figure 3.
Structure of chitin and chitosan
13
Figure 4.
Phylogenetic relationships of family 19 chitinases
17
Figure 5.
Amino acid sequence of a chitinase from Streptomyces
erythraeus
18
Figure 6.
Purification of the pea antifungal hydrolases
21
Figure 7.
Production of recombinant chitinase from Trichoderma
virens UKM-1 in E.coli
23
Figure 8.
Calibration curve of N - acetyl – Glucosamine
33
Figure 9.
Clear zones’ diameters illustrated Chitinase activity of
Streptomyces strains
41
Figure 10.
Morphology of strain VN08-A0438 : conoly (A) and spores
(B)
45
Figure 11.

Extraction of the total DNA (A) and amplification of 16S
rDNA (B) from strain VN08A-438
47
Figure 12.
Phylogenetic tree contruction for VN08-A0438 strain
48
Figure 13.
Effect of some parameters on chitinase activity of
Streptomyces VN08-A0438
50
Figure 14.
Chromatographygram of chitinase enzyme on Sephadex
G100 (A) and bioassay of the active fraction (B)
52
Figure 15.
Zymogram (A) and SDS-PAGE (B) of chitinase
53
Figure 16.
Effect of temperature and pH on chitinase activity
54
Figure 17.
TLC analyzing of final chitinase reaction products
55
v

LIST OF TABLES
Table
Title
Page
Chapter 1

Table 1.1.
Chitin content of some organisms
5
Table 1.2.
Comparison of the characteristics of purified chitinase from
others reported Enterobacter sp
22
Chapter 2


Table 2.1.
List of instruments
29
Chapter 3


Table 3.1.
Primarily screening chitinase activities of 500 Streptomyces
strains
62
Table 3.2.
Summary chitinase activities of 500 Streptomyces strains
42
Table 3.3.
Chitinase avtivity of 60 selected Streptomyces strains
43
Table 3.4.
Effect of sugar on the growth of strain VN08-A0438

46
Table 3.5.
Summary of partly purification
51





v

TABLE OF CONTENTS

ABSTRACT 7
CHAPTER 1. INTRODUCTION 10
1.1. Chitin and application of chitin and chitinoligosaccharides 10
1.1.1. Application of chitin in Agriculture and Environment 12
1.1.2. Application of chitin in Medicine 14
1.1.3. Application of chitin in cosmetic and industry 15
1.2. Compositions and methods for producing chitin 17
1.3. Chitinase 20
1.3.1. Main chitinase sources 20
1.3.2. Chitinase from Streptomyces and other sources 22
1.3.3. Purification of chitinase 26
1.3.4. Recombinant chitinase 28
1.3.5. Diversity of chitinase 29
1.4. Potential of chitin product application in Vietnam 31
1.5. All domestic related studies 32
CHAPTER 2. MATERIALS AND METHODS 35
2.1. Analytical instruments 35

2.2. Microbes 35
2.3. Media 35
2.4. Methodology 36
2.4.1. Screening of chitinase-producing Streptomyces and culture conditions 36
2.4.2. Selecting good chitinese producers by chitinase activity assay 36
2.4.3. Identification of Streptomyces strain 39

vi

2.4.4. Effect of culture conditions (temperature, pH, aeration, carbon, nitro sources) for chitinase
fermentation from Streptomyces 42
2.4.5. Purification of chitinase 43
2.4.6. SDS-PAGE and activity gel (zymogram) 44
2.4.7. Characterization of the partly purified chitinase 45
CHAPTER 3. RESULTS AND DISCUSSION 47
3.1. Screening of chitinase-producing Streptomyces 47
3.1.1. Primary screening good Streptomyces strains for chitinase production 47
3.1.2. Chitinase activities of 60 Streptomyces strains in liquid medium 48
3.2. Identification of Streptomyces strain VN08-A0438 50
3.2.1. Morphology of strain VN08-A0438 50
3.2.2. Studying carbon sources assimilation of the culture 51
3.2.3. Some physiological criteria of the culture 53
3.2.4. 16S rDNA sequencing of Streptomyces VN08-A0438 53
3.3. Selecting medium and conditions for chitinase production 55
3.4. Purification of chitinase 57
3.5. Characterization of the partly purified chitinase 59
CONCLUSION 62
FURTHER STUDIES 62
BIBLIOGRAPHY 63

7

ABSTRACT
In this study, a total of 500 Streptomyces strains isolated from soil in Hoang
Lien Son national park (Sa Pa, Vietnam) were subjected to a screening for their
chitinase activities. Through two screening steps, Streptomyces strain VN08-
A0438 had the highest chitinase activity so it was selected for next studies.
Taxonomical studies based on the morphology, physiological criteria and 16S
rDNA gene sequencing indicated that strain VN08-A0438 was belonging genus
Streptomyces and was proposed as Streptomyces chromofuscus.
Besides that, selecting conditions for chitinase production from strain VN08-
A0438 were studied, focused on some key factors on chitinase production:
optimum temperature, pH, aeration, fermentation time, carbon and nitrogen
sources. The culture grew well on medium with carbon source as glucose - 5 g,
colloidol chitin 5 g and nitrogen source as (NH
4
)
2
SO
4
- 2 g, at 35
o
C, pH 6.5 with
shacking rate 200 rpm for 5 days.
Chitinase from Streptomyces sp. VN08-A0438 was purified by ammonium
sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and
Sephadex G-100 gel filtration. Treatment of chitinase (80% ammonium sulfate
saturation) gave highest specific activity (40U/mg protein). The high chitinase
activity was found in fractions from 45 to 70. The sample was concentrated by
evaporation at room temperature to 10 folds, and loaded on SDS-PAGE and

activity gel. Characterization of the partly purified chitinase was also checked,
including effect of pH, temperature, and Thin layer chromatography (TLC) for
detecting the enzymatic product. Enzyme was stable at pH 5-5.5 and 55
o
C. The
TLC chromatogram showed that there were a number of three enzymes involved:
endochitinase with chitobias and chitinooligosacharide as the main products,
exochitinase with N-acetyl glucosamine and chitinooligosaccharide as the main
products, chitobiase with N-acetyl glucozamin as the final products.

8

TÓM TẮT
Tên luận văn: Nghiên cứu đặc tính chitinase từ xạ khuẩn.
Người hướng dẫn: TS. Dương Văn Hợp
Viện Vi sinh vật và Công nghệ Sing học,
Đại học Quốc gia Hà Nội.
Ngành: Công nghệ sinh học Chuyên ngành: Công nghệ sinh học
Mã số: 60 42 80
Trong đề tài này, năm trăm chủng xạ khuẩn Streptomyces được phân lập từ
Vườn Quốc gia Hoàng Liên Sơn được tiến hành tuyển chọn và xác định hoạt tính
chitinase. Thông qua 2 quá trình sàng lọc cơ bản, chủng xạ khuẩn mang kí hiệu
VN08-A0438 là chủng có hoạt tính sinh chitinase cao nhất, vì vậy chủng này được
lựa chọn để phục vụ cho các mục đích tiếp theo của nghiên cứu này.
Cũng trong nghiên cứu này, chủng VN08-A0438 được tiến hành định loại
dựa trên đặc điểm hình thái, hóa sinh và giải trình tự 16S rDNA, kết quả cho thấy
xạ khuẩn phân lập được là chủng Streptomyces chromofuscus.
Bên cạnh đó, chúng tôi cũng tiến hành các thí nghiệm xác định điều kiện tối
ưu cho sự phát triển và sinh chitinase của chủng Streptomyces chromofuscus
VN08-A0438 bao gồm các thiết lập về nhiệt độ, pH, chế độ thoáng khí, thời gian

lên men, thử nghiệm các nguồn cácbon và nitơ khác nhau. Kết quả phân tích cho
thấy chủng này phát triển tốt ở môi trường có nguồn cacbon là glucoza (5g/l),
colloidol chitin (5g/l) và (NH4)
2
SO
4
(2 g/l) được sử dụng là nguồn cung cấp nitơ,
điều kiện nhiệt độ 35
0
C, pH = 6.5 và lắc 200 vòng/phút trong 5 ngày.
Chitinase của chủng Streptomyces VN08-A0438 sau đó được tinh sạch sơ bộ
bằng kết tủa amôn sunphat. Chitinase cũng được nghiên cứu đặc tính về độ bền
nhiệt, pH và sắc ký bản mỏng (TLC). Kết quả phân tích cho thấy chitinase bền ở
pH 5.5 và nhiệt độ 55
0
C. Kết quả phân tích TLC cho thấy sản phẩm tinh sạch có
chứa toàn bộ mono-; di- và oligomers.


9

FOREWORD
Chitin is insoluble polysaccharide composed of linear chains of β-1,4- N-
acetylglucosamine (GlcNAc) residue that are highly cross-linked by hydrogen
bonds. It is found in the outer skeleton of insects, fungi, yeasts, algae, crabs,
shrimps, lobsters and in the internal structures of other invertebrates. As for many
other enzymatic substrate, chitin is being used as a strong promoter to boost up
extracellular chitinases formation. Chitinases are capable of degrading chitin
directly to low molecular weight of chitooligomers, which have broad range of a
agricultural, industrial and medical functions such as anti-tumor activity and

elicitor action.
Chitinases (EC 3.2.1.14) are glycosyl hydrolases group of enzymes that vary
widely in size (20 kDa to about 90 kDa). Bacterial chitinases have a molecular
weight range of ~20-60 kDa, which is similar to that of plant chitinases (~25-40
kDa) and are smaller than insect chitinases (~40-85 kDa). Chitinases can be
produced by many bacteria, including Aeromonas, Alteromonas, Bacillus, Serratia,
Streptomyces, Enterobacter, Vibrio and Escherichia. Chitinase-producing bacteria
were isolated from different environments including soil, garden and park waste
compost and shellfish.
During the last decade, chitinases have received increased attention because
of their wide range of applications. The enzyme could either be used directly in the
biological control on microorganisms.
To contribute to purify chitinase from Streptomyces strains and detect their
characterization, we have implemented topic: “Study on characterization of
chitinase from Streptomyces”.


10

CHAPTER 1. INTRODUCTION
1.1. Chitin and application of chitin and chitinoligosaccharides
Historically, chitin was first discovered in the sediment of a fungus extracted
by Braconnot in 1821. The substance is named "Fungine" to remember its origin.
In 1823, Odier isolated a substance from the beetle, which he called chitin or
"chiton" (Greek meaning is armor). However, he did not detect the presence of
nitrogen in this substance. Both Odier and Braconnot eventually concluded that
chitin has the same formula as cellulose [41].
Chitin (C
8
H

13
O
5
N)
n
is one of the natural polysaccharides including a
copolymer of N-acetyl-D-glucosamine and D-glucosamine residues. These two
components are linked together by β-1,4 glycosidic bonds. Chitin is popular and
can be found in a variety of species such as in shells of crustaceans, in cuticles of
insects or in the cell wall of fungi and some algae [15]. Being an amorphous solid,
chitin has typical properties of these groups like largely insoluble in water, dilute
acids and alkali as well. Although chitin is well known in numerous commercial
uses, greater commercial benefits can be found by changing it into a deacetylated
product named chitosan [7].

Figure 1. Natural sources of chitin [33],[36],[37],[40].
Chitin has a fibrous shape and it is an extremely insoluble material. With the
exception of cellulose, chitin is the most abundant biopolymers globally with an

11

estimated yearly production account for 1010 to 1011 tons. Structural similarities
between chitin and cellulose are illustrated in Fig. 1. Chitin occupies a large
percentage in the structural component of most fungi and algae cell walls, insect
exoskeletons, the shells of crustaceans, and the microfilarial sheath of nematodes.
Nevertheless, chitin is soluble in most of the organic solvents [41]. In addition,
chitins in animal tissues are frequently calcified, such as in the case of shellfish
[16]. In a detailed instance, the proportion of chitin from shrimp and crab are
usually 0.06 and 0.17 g/ml respectively. It means that chitin in shrimp is more
porous than in crab. Chitin in mollusks is 2.6 times as porous as in crab. A thermal

conductivity showed that the proportions of chitin and chitosan from crustaceans
are very high (0.39g/cm
3
). In crustaceans, the proportion of commercial chitin and
chitosan show some differences. This may be due to the difference in crustacean
species or processing methods. In addition, the deacetyl level also increases their
proportion [41]. The content of crude chitin varies between species, as illustrated
in table 1 [16].
Table 1.1. Chitin content of some organisms.

The amount of acetylation of the D-glucosamine (GlcN) residues in chitin
made it notable. Polymer consisting of 70% or higher acetylating are considered

12

chitin whistle those with less than 30% are called chitosan. In fact, the vast
majority of chitin produced annual in biosphere are degraded by chitinase [27].

Figure 2. Chemical structures of cellulose and chitin.
1.1.1. Application of chitin in Agriculture and Environment
According to scientists, chitin is a useful substance that helps plants develop.
It has been known to take part in a popular phenomenon named defense
mechanisms in plants as an extreme good inducer. Plants productivity and life
expectancy also witnessed a remarkable increase by using Chitin as a specific
fertilizer. Chitin is also regulated in agriculture use within the USA by US
Environmental Protection Agency. Besides, in agriculture and horticulture,
chitosan-a substance derived from chitin, can be used as a bio-control elictor [30].
Chitin oligosaccharides are also well-known by their abilities in “fast turning
on” plant’s defense mechanisms against some invasion by fungi, therefore,
enhance the plant disease resistance. Similarly, in some symbioses such as beans

and clover plants, symbiotic bacteria which usually live around plant’s root, can
release chitin oligosaccharides in order to give a sign of root nodules formation,
sites for nitrogen-fixation [31].
Chitosan is a form of de-acetylated chitin and have a better potential in
biodegradation than chitin [38]. It is known to offer a natural alternative to the use
of medical products. Hence, environment and human can be protected better.
Chitosan can trigger plant defensive mechanisms as a vaccine in human, stimulate
plant growth and induce unexpected effects of certain enzymes (synthesis of
phytoalexins, chitinases, pectinases, glucanases, and lignin). The approach using

13

chitosan - an organic compound opens a real promising of bio-control tool-
chitosan.
In addition to the growth-stimulation properties and fungi, chitosans are used for:
Seed-coating
Frost protection
Bloom and fruit-setting stimulation
Timed release of product into the soil (fertilizers, organic control agents,
nutrients)
Protective coating for fruits and vegetables [39].
* The role of chitin in environment
Scientists believe that chitin is used for environmental treatment because of
its features: natural origin and being biodegrable. Most of physicochemical-type
treatments result in environmental problems such as vulnerable and pollution,
therefore, different approaches using “go green methods” are necessary. Hence,
“chitosan method” can be the suitable choice for several points of view being
indicated below.
By integrating a natural polymer made of crustaceans into an existing
system, two purposes would be achieved: (i) increasing the effectiveness of water

treatment and (ii) reducing or eliminating harmful synthetic chemical compounds
such as aluminum sulphate and synthetic polymers. Some chitosan’s characteristics
that can be enumerated for ecological solution are:
Natural and biodegradable
A powerful competitor for synthetic chemical products
Potentially reduces the use of alum by up to 60% and eliminates 100% of the
polymers from the treated water
Improves system performance (suspended solids and chemical oxygen
demand)
Significantly reduces odor [39].

14

1.1.2. Application of chitin in Medicine
Scientists estimated the extremely high cost of producing pure
oligosaccharides in laboratories. With the cost of $5 to $15 per milligram, a normal
experiment taking place in a laboratory can cost many thousands of dollars.
Although chitin oligosaccharides may have potential use in human medicine, this
costly experiment is considered as the main barrier to popularize knowledge about
chitin oligosaccharides in medicine. Fortunately, it is known that numerous
bacteria species can easily transfer substances into others and chitin
oligosaccharide is not an exception. Thanks to natural enzymes, chitin
oligosaccharides can be produced quickly and environmentally while, over a
period of 30 years, in laboratories, this process requires intensive use of acid and
bases [31].
Occupations associated with high environmental chitin levels, such as
shellfish processors, are prone to high incidences of asthma. Recent studies have
suggested that chitin may play a role in a possible pathway in human allergic
disease. Specifically, mice treated with chitin develop an allergic response,
characterized by a build-up of expressing innate immune cells. In these treated

mice, additional treatment with a chitinase enzyme abolishes the response [30].

* For biopharmaceutical uses
It is estimated that the number of chitosan applications in health fields is
plentiful. Such properties (bacteriostatic, immunologic, antitumoral, cicatrizant,
hemostatic and anticoagulant) are of great value. Take a human disease for
example, because of its biocompatibility with human tissue, chitosan’s cicatrizant
properties have illustrated its role as a component, notably in all types of dressings
(artificial skin, corneal dressings, etc.), surgical sutures, dental implants, and in
rebuilding bones and gums. A specific technique that is developing nowadays is
using chitosan instead of human or animal’s skin (artificial skin) and producing
surgical sutures that can be absorbed after operations and corneal contact lenses.
Finally, chitosan can be used to delivers and time-releases drugs used to treat

15

animals and humans. It is said that chitosan application in medicine can more and
more developed unless having the human regulation in pharmaceutical-grade
requirements. Possible applications include:
Ointments for wounds
Surgical sutures
Ophthalmology
Orthopedics
Pharmaceutical products (delivery agent)
Contact lenses [39].
1.1.3. Application of chitin in cosmetic and industry
* In industry
It is said that the role of chitin in industry is of great value. Chitin
participates in plenty of important industrial processes. Chitin has been previously
used as an additive to thicken and stabilized foods. Besides, chitin acts as a binder

in dyes, fabrics and adhesives. Industrial separation membranes and ion-exchange
resins can be made from chitin. In paper production, chitin is known to be a
substance improving paper’s size and strength.
In surgical thread, chitin with its strong and flexible properties is very
favorable. Its biodegradibility means it wears away with time as the wound heals.
Its unusual properties that accelerate healing of wounds in humans can make it
easy to produce artificial thread [30].

* In food
In Europe, United States and Japan, chitosan has widely used in food
production, preservation and in diet diagrams. Because of its “lipid trap”

16

properties, chitin acts as an important dietetic breakthrough. When chitosan goes
into human digestive system, that human body cannot digest this substance makes
it acts as a fiber, a crucial diet component. Research has found that 20 to 30% of
cholesterol can link with chitosan, hence, reduce the amount of cholesterol in
human blood. In fish sauce preparation, chitosan is used due to its thickening and
stabilizing properties. It is also known to be used in other dishes that hold their
consistency well. Finally, due to the flocculating property, chitosan acts as a
flocculating agent that can be used to clarify beverages. Moreover, chitosan is also
phytosanitary and based on this property, human can use chitosan in food
protection. Chitosan can be changed into liquid forms and then sprayed in dilute
form on foods such as fruits and vegetables, creating a protective, antibacterial,
fungi static film. This action is so popular that Japanese use it as an effective
method of fruit protective measure. There are many other applications in the areas
of nutraceutical and nutritional supplements, particularly for the broad range of
chitosans that have been chemically or enzymatically modified.
Principal commercial applications include:

Preservatives
Food stabilizers
Animal feed additives
Anti-cholesterol additives (fat traps) [39].
* In cosmetics
Chitosan is applied popularly in cosmetics. Its abilities in skin treatment
have been recorded. Chitosan forms a protective, moisturizing and elastic film on
the surface of the human skin, binds numerous failures, spots or ingredients on the
skin. Therefore, chitosan is applied in cosmetics in the name of formulating
moisturizing agents such as sunscreens and organic acids protector… With these
characteristics, chitosan can enhance skin bioactivity and effectiveness. Besides,
due to its antibacterial properties, chitosan is widely used in the composition of

17

skin-care creams, shampoos and hair spray. It has been certified that there have
been numerous patents registered and new applications are just beginning to appear
including the most highly prized moisturizing and chitosan’s antibacterial
properties. Applications include:
Maintain skin moisture
Treat acne
Tone skin
Protect the epidermis
Reduce static electricity in hair
Fight dandruff
Improve suppleness of hair
Make hair softer [39].
1.2. Compositions and methods for producing chitin
Seafood has recently shown its potential value in contributing to
biochemical sources. With the percentage of 30%, edible proportions of seafood

play an important part in human diet and become a delicacy praised by many
gastronomists. However, the remainder that counts for 70% (mostly include shells)
is an extremely waste. As stated in recent researches, thanks to scientists’ efforts,
numerous biochemical substances such as chitin - a natural biopolymer with
unique properties, pigments, seafood peptones, etc., can be produced from seafood.
Nevertheless, despite that chitin is a highly applicable polymer, it is still a
relatively “new” polymer in research and food processing applications [16].
Chitin is common in most of flora and fauna species. The traditional sources
of chitin can be found in shellfish waste from shrimp, Antarctic Krill, crab and
lobster processing [5]. It is also said that the amount of chitin in these species
widely varies from trace quantities up to 40% of the body weight of the organism.
Among that, the crustacean waste is the most important chitin source for
commercial use due to its high chitin content and ready availability [35].

18

Worldwide, ca. 75,000 tons of dried shrimp shells are produced annually, and these
could easily yield 3,000 tons of chitin [1].
* Chemical methods
It is almost easy and quick to produce chitin from shrimp waste by chemical
methods. A 4% NaOH solution which is used for deproteination and 4% HCl for
demineralization can be used to isolate chitin in this treatment. However, people
believed that this process may not be considered as a good recovery option because
of expensive cost and non - environmental friendly. This leads to other approaches
in producing chitin and biological method that uses the more environmental
friendly technologies such as partial fermentation using lactic acid bacteria for the
production of chitin [14].
* Physical methods
It can be stated that physical methods are of great value in producing chitin
from seafood. Researches demonstrated that shrimp waste contains about 23%

chitin and annually, 80,000 tons of this waste was released in India. Recently,
scientists has tested and indicated that irradiation of shrimp shells with gamma
irradiation dose of 25 kGy reduces the time of reaction of deproteinization from 8
hr to 1 hr, resulting in tremendous amount of energy and cost-saving in the process
[27]. Radiation method is illustrated in scheme 2.
Scheme 1: Conventional method
Shrimp shells → Demineralization with 2 N HCl for 48 hr →
Deproteinization with 1 N NaOH at 1000
o
C for 8 hours → Chitin.
Scheme 2: Radiation method
Shrimp shells → Irradiated to 25 kGy with gamma radiation
→Demineralization with 2 N HCl for 48 hr → Deproteinization with 1 N NaOH at
1000
o
C for 1 hour → Chitin.

19

Studies reported that in radiation process, chitin structure is changed and
undergoes chain scission in a manner similar to cellulose as the backbones of these
two substances are very similar.
The only concern of chitin resulted from its insolubility property restricts its
applications. However, chitin can be easily converted into chitosan by using 50%
w/w NaOH whereby acetyl group of chitin is converted into a free amino group.
Chitosan is readily soluble in acidic media. Chemical structures of both chitin and
chitosan are shown in Fig. 3.

Figure 3. Structure of chitin and chitosan [13].
Physical method in producing chitosan from chitin is more effective in

comparison with chemical method because of its simple and fast features in
degradation. It is clear that radiation process can be operated in ambient
temperature in either dry or solution form without any additive. In physical
method, not only contaminations of additive and thermally decomposed materials
cannot be found, but also the molecular weight distribution is narrower than that of
the conventional method [13].
* Biological methods (Enzymatic methods)
Proteases can be used for the deproteinization of crustacean shells for the
production of chitin or chitosan. Enzymes as tuna protease were used at pH 8.6 and
37.5
o
C, papain at pH 5.5-6.0 at 37
o
C, or bacterial protease at pH 7.0 and 60
o
C for

20

over 60 hr. After treatment with enzyme, the amount of protein still associate with
chitin was about 5%. In 1988, Shimahara and Takiguchi used bacterial protease
from Pseudomonas maltophilia in a culture medium with crustacean shell, and
observed that after 24h, the protein content remaining in the shells was only about
1%.
Chitin deacetylase, the enzyme responsible for the deacetylation of chitin to
chitosan, shows a pH optimum at 5.5 and is markedly inhibited by acetate. It
showed no effect against chitin in its isolated form, but was active against a soluble
glycol chitin substrate.
According to Kauss and Bauch (1988), chitin deacetylase extracted from
Colletotrichum lindemuthianum exhibited similar properties but had a pH optimum

at 8.5 and was not inhibited by sodium acetate. The enzyme is found in the cell
extracts and also secreted by this plant pathogen, so it is more easily isolated from
the culture filtrate.
One important fact about chitin deacetylase is that it is ineffective against
preformed chitin but it readily attacks nascent chitin chains. In other words,
chitosan is made by deacetylation of chitin provided that the deacetylation process
occurs in tandem with chitin synthesis, those requirements being met when both
chitin synthetase and chitin deacetylase are present simultaneousfy. Seemingly, the
two enzymes operate in tandem, one polyrnerizing GlcNAc unit from UDP-
GlcNAc, the other removing acetate moieties from the nascent chains (Bartnicki-
Garcia, 1989) [16].
1.3. Chitinase
1.3.1. Main chitinase sources
In biotechnology, chitooligomers that are well-known for their
biotechnological value were generated in a degradation process catalyzed by
chitinase. Chitinase (EC 3.2.1.14) is a member of the glycoside hydrolyse family,
and are characterized by their ability to catalyze the hydrolytic cleavage of chitin
[27]. Chitinase cleaves a bond between the C1 of N-acetylmuramic acid and C4

21

of N-acetylglucosamine in the bacterial peptidoglycan (by displaying a more or
less pronounced lysozyme activity EC 3.2.1.17). A chitinase was first described
in 1911 by Bernard, and after that, in 1929 Karrer and Hofmann found chitinase
in snail [4]. Chitinase has been detected in a wide variety of organisms including
organisms

that do not contain chitin, such as bacteria, fungi, viruses, plants and
insects [27] and play important physiological and ecological

roles [34] such as
energy extraction from the environment, modification of the chitin components in
fungi, anthropods and the stress response systems in plants [33].
The roles of chitinases in different organisms are diverse. Bacteria produce
chitinases to digest chitin for use as a carbon and energy sources, while fungi
produce this enzyme to modify the important cell wall component chitin and
invertebrates require chitinases for the partial degradation of old exoskeletons. In
plants, however, chitinases are part of the plants defense mechanisms against
fungal pathogens [26].
The two types of chitinase families differ not only in 3D structure but also in
their biochemical properties. For instance, family 18 chitinases hydrolyse

the
glycosidic bond with retention of the anomeric configuration while family

19
chitinases hydrolyse with inversion. Family 18 chitinases are sensitive

to
allosamidin, but a family 19 chitinases from higher plants

have been shown to be
insensitive. Family

18 chitinases hydrolyse GlcNAc- GlcNAc and GlcNAc-GlcN
linkages,

whereas family 19 chitinases hydrolyse GlcNAc-GlcNAc and GlcN-
GlcNAc. Because of their catalytic mechanisms, these

differences are probably
common among all members of the

two families. Substrate - assisted catalysis is
the most widely

accepted model for the catalytic mechanism of family 18
chitinases, whereas a general

acid-and-base mechanism has been suggested to be
the catalytic

mechanism for family 19 chitinases [34].
Recently, chitinase can be produced from marine bacteria and fungi on a
laboratory bioreactor and pilot plant scales based on three major modes of
operation: batch, fed-batch and continous. Batch growth refers to culturing in a
vessel with an initial charge of medium that is not altered by further nutrient

22

addition or removal. This form of cultivation is simple and widely used both in the
laboratory and industrially. In fed-batch culture, nutrients are continuously or
semi-continuously fed, while effluent is removed discontinuously. Fed-batch
operation permits the substrate concentration to be maintained at some pre-
determined level. Continuous culture provides constant reactor conditions for
growth and product formation and supplies uniform-quality products [20].
Naturally, chitinases are produced by microorganisms and its production is
influenced by environment conditions such as temperature, nutrients resources and
soil pH [8].
1.3.2. Chitinase from Streptomyces and other sources

1.3.2.1. Chitinase from Streptomyces
Streptomyces species are important soil microorganisms. Chitinase C from
Streptomyces griseus HUT6037, described in

1997, is the first family 19 chitinase
found in an organism

other than higher plants [34].
Family 19 chitinase was almost found in plant (plant class I, II, IV chitinase)
with among the aception, the Streptomyces griseus Chitinase C. S. griseus
chitinase C exhibited strong antifungal activity, whereas the other bacterial
chitinases which belong to family 19 did not. This notes that antifungal activity
may be a general property of family 19 chitinases. Two relationships between
Streptomyces family 19 chitinase and plant family 19 chitinase were constructed. A
common ancestral chitinase was already present prior to divergence of plant and
bacteria, and family 19 chitinase then evolved independently in plants and bacteria.
It means that family 19 chitinase in Streptomyces and plant develop from an origin
(figure 4). Some strains of Streptomyces are plant pathogens, and family 19
chitinase from Streptomyces can be acquired from plants by horizontal gene
transfer [34].

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Figure 4. Phylogenetic relationships of family 19 chitinases.
Exo-and endoactivities were described in Streptomyces plicatus;
Streptomyces erythraeus chitinase had a Mr of 30,000, a pI of 3.7 and showed
optimal activity at pH 5.0 in the presence of a ≤ 0.2 M buffer. Using
chitooligosaccharides and their derivatives, the binding mode of the Streptomyces
erythraeus chitinase to the substrate seems similar to that of hen egg white or

Streptomyces erythraeus lysozymes. The Streptomyces erythraeus chitinase
consisted of 290 amino acid residues (Mr = 30,000) and has two disulfide bridges
at Cys 45-Cys 49 and Cys 265-Cys 272. Chitinase from Streptomyces plicatus was
cloned (figure 3). The partial DNA sequence showed that the protein possesses a
signal sequence of 30 amino acids. This chitinase didn’t exhibit sequence
homology with the Streptomyces erythraeus chitinase [4].

24

Cellulose, chitin, xylan, for example, are the most abundant carbon polymers
on Earth and are major constitients of the soil. The Streptomycetes depend on these
subtrates for growth, and glucose kinase and not cAMP seems to be involved in
catabolite control in Streptomyces [3].

Figure 5. Amino acid sequence of a chitinase from Streptomyces erythraeus.

1.3.2.2. Chitinase from other sources
* Plant chitinase
All plant chitinases are small proteins with Mr from 25.000 to 40.000 units.
Plant chitinases are classified in three classes: class I, II and III chitinase. All three
classes can be present in the same plant. Many plant chitinase are PR-proteins.
They are induced in the presence of pathogens, or pathogen extracts, and also after
a stress. Plant chitinases are potent inhibitors of fungal growth, but other enzymes
are induced simultaneously [4].
* Fungal chitinase