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
*** Nguyen Thanh Huong


ABSTRACT Error! Bookmark not defined.
CHAPTER 1. INTRODUCTION Error! Bookmark not defined.
1.1. Chitin and application of chitin and chitinoligosaccharides Error! Bookmark not defined.
1.1.1. Application of chitin in Agriculture and Environment Error! Bookmark not defined.
1.1.2. Application of chitin in Medicine Error! Bookmark not defined.
1.1.3. Application of chitin in cosmetic and industry Error! Bookmark not defined.
1.2. Compositions and methods for producing chitin Error! Bookmark not defined.
1.3. Chitinase Error! Bookmark not defined.
1.3.1. Main chitinase sources Error! Bookmark not defined.
1.3.2. Chitinase from Streptomyces and other sources Error! Bookmark not defined.
1.3.3. Purification of chitinase Error! Bookmark not defined.
1.3.4. Recombinant chitinase Error! Bookmark not defined.
1.3.5. Diversity of chitinase Error! Bookmark not defined.
1.4. Potential of chitin product application in Vietnam Error! Bookmark not defined.
1.5. All domestic related studies Error! Bookmark not defined.
CHAPTER 2. MATERIALS AND METHODS Error! Bookmark not defined.
2.1. Analytical instruments Error! Bookmark not defined.
2.2. Microbes Error! Bookmark not defined.
2.3. Media Error! Bookmark not defined.
2.4. Methodology Error! Bookmark not defined.
2.4.1. Screening of chitinase-producing Streptomyces and culture conditions Error! Bookmark not
defined.
2.4.2. Selecting good chitinese producers by chitinase activity assayError! Bookmark not defined.
2.4.3. Identification of Streptomyces strain Error! Bookmark not defined.
2.4.4. Effect of culture conditions (temperature, pH, aeration, carbon, nitro sources) for chitinase
fermentation from Streptomyces Error! Bookmark not defined.
2.4.5. Purification of chitinase Error! Bookmark not defined.
2.4.6. SDS-PAGE and activity gel (zymogram) Error! Bookmark not defined.
2.4.7. Characterization of the partly purified chitinase Error! Bookmark not defined.

which is one of the natural polysaccharides is very 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. Chitin and its deacetylated product named chitosan have great benefits in agriculture and
environment, for instance, act as fertilizers to help plants develop (chitin) or decompose toxic
compounds (chitosan). Besides, chitin also plays important parts in other fields such as medicine
(possible pathway in human allergic disease, component of skin dressing), cosmetic and industry
(additive to thicken and stabilized foods, substance improving paper’s size and strength)…
Streptomyces species are important soil microorganisms. Some studies have been done on chitinase
from several streptomyces strains, nevertheless, these studies did not fully concentrate on
characteristics of chitinase. This study will be the one that completely solve that problems including
identifying streptomyces strains, purifying chitinase and determining chitinase characterization from
streptomyces strains in Hoang Lien Son national park, Vietnam.
The objectives of this study:
The aim of study was screened strains that are capable of producing chitinase. Since then, isolated
strains were identified and determined the characterization of their chitinase.
Content of study:
To discover the streptomyces strain having the highest chitinase activity from 500 strains of isolated
Streptomyces, the highest chitinase-producing streptomyces strain will be determined by primary
screening on agar Petri dish and P.V. Suresh and M. Chandrasekaran method.
To Identify of Streptomyces strain based on the morphology, biological criteria, chemotaxonomic
study together with 16S rDNA sequencing.
To purified the enzyme received from the target streptomyces strain.
To characterize the purified enzyme produced by the target streptomyces strain including pH and
temperature stability, TLC test.
Practical applicability: In the future, we intend to apply these isolated strains in different field
scales.
Contribution of the study:
Based on the morphology, biological criteria, chemotaxonomic study together with 16S rDNA
sequencing, strain VN08-A0438 which showed the highest ability in producing chitinase was identified as
Streptomyces chromofuscus.

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 [26]. 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.
It is said that the role of chitin in industry is of great value. 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. Chitosan also has been widely
used in food production, preservation and in diet diagrams.
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
skin-care creams, shampoos and hair spray.
1.2. Methods for producing chitin
Heterologous genes from viral, fungal, insect or other orsanisms can be achieved by many methods
to increase the amounts of chitin or to directly produce chitosan without the chemical modified chitin.
the compositions include polynucleotides encoding enzymes or polypeptides, a coding sequence for one
or more polypeptides, transformed fungi, bacteria, plants, plant cells, tissues and seeds.
It is almost easy and quick to produce chitin from shrimp waste by chemical methods. However,
people believed that this process may not be considered as a good recovery option because of
expensive cost and non - environmental friendly. It can be stated that physical methods are of great
value in producing chitin from seafood.
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

glucosamine (GlcNAc) monomers or diacetylchitobiose by b-(1,4)-N-acetylglucosaminidase activity (EC
3.2.1.30) or 1,4-b-chitobiosidase activity (EC 3.2.1.29), respectively.

1.4. Potential of chitin product application in Vietnam
Crustacean is an abundant aquatic product source accounting for one - third of total number of
fisheries production in Vietnam. The production of chitosan from shrimp shells can bring high economic
benefit. With the chitin and chitosan’s abilities in widen its applications, many countries including
Vietnam have studied to produce these products.
1.5. All domestic related studies
Scientists from Vietnam Academy of Science and Technology have found out 3 bio-procedures based
on the use of enzyme proteolytic to extract chitin from shrimp’s head and shell.
(i) Method that used enzyme bromelain in pineapple extracted solution.
(ii) Fermentation of the bacteria producing proteinase;
(iii) Natural fermentation
Doctors from Vietnam National Cancer Hospital have taken a research in 2003 on 60 patients from
ages 35 to 76 and found out that chitosan supported effectively in cancer treatments.
In medical-pharmaceutical, Vietnamese scientists produced Glusivac – a specific medicine for
osteoarthritis treatment. Besides, there are some weight loss pills made from Chitozan.
Vietnamese scientists have gained success in constructing chitin and chitosan production
techonology from seafood’s shell (shrimp, crab, shell, squid) for health and food sectors.

CHAPTER II. MATERIALS AND METHODS
2.1. Analytical instruments
The stuffs used in this research are standard ones belonging to the institute of Microbiology and
Biotechnology - Vietnam National University, Hanoi.
2.2. Microbes
The soil samples used for this experiment were collected from Hoang Lien Son national park. A
number of 500 strains of Streptomyces isolated from these samples were kept in VTCC and used for the
study on characteristics of chitinase.
2.3. Media

activity and weak chitinase activity belong to remaining 186 strains (Table 2)
Table 1. Summary chitinase activities of 500 strains Streptomyces.
Chitinase
Activity
No observation
Weak
Medium
Strong
Total
Number of
strains
186
108
146
60
500
Ratio (%)
37.2
21.6
29.2
12.0
100
Note: Diameter of growth zones D (mm); No activity: D ≤ 2; Weak: 2 < D ≤ 10; Medium: 10 < D < 20;
Strong: D ≥ 20.
All 60 strains with high chitinase activity will be used for next secondary screeing for chitinase
activity on liquid medium.
3.1.2. Chitinase activities of 60 strains Streptomyces in liquid medium
Sixty selected strains of streptomyces were grown on the medium (as described in the
methodology). The broths were taken for chitinase assay and measure as number of unit/ml. Almost
tested strains possess chitinase activities in the rank from 10 to 20 U/ml. There are 3 strains (VN10A-

-
3
Meso-Erythritol
-
4
D-Fructose
+
5
D-Glucose
+
6
Myo-Inositol
-
7
D-Raffinose
-
8
L-Rhamnose
+
9
Ribose
-
10
D-salicin
-
11
D-Sorbitol
-
12
Sucrose

The strain grow well at temperature (30-35
o
C), pH (6-7). It only could grow in medium with 1% - 2% NaCl
and could not grow at higher concentration of NaCl.
3.2.4. 16S rDNA sequencing of Streptomyces VN08-A0438
+ Extract total DNA and amplification of 16S rDNA.
Fig 3. Extraction of the total DNA
(A) and amplification of
16S rDNA (B) from strain
VN08A-438.

The value of optical (260/280) was about 1.95, the extracted DNA is good enough for processing PCR
amplification. The result of PCR amplification (Figure 3B) showed that we amplified 16S rDNA product
with the size of 15,000 bps. The product was then purified for determining DNA sequence.
+The 16S rDNA sequencing.
The sequence of Streptomyces VN08-A0438 was determined using ABI 3110 Avant Appied
Biosystems sequencer.
The result showed that a number of 1481 bps of the 16S rADN was sequenced. This sequence was

precipitation,
chitinase enzyme was precipitated in 60-80-100% ammonium sulfate saturation as described in the
methodology section. The precipitate was collected and dialyzed for the removal of ammonium sulfate.
The fractions collected from ammonium sulfate at 3 concentration 60, 80, 100% saturation were
determined the specific activity. The results showed that fraction of 80 % saturation gave higher specific
activity (40U/mg protein) in comparing to the others (22 U/mg and 31 U/mg for 60% and 100 %
saturation respectively). About of 3 ml of the chitinese was applied on a Sephadex G100 for further
purification.
Table 3. Summary of partly purification.

Steps of
purification
Total
activity
(U)
Protein
(mg)
Specific
activity
Purity
(fold)
Recovery
(%)
The
supernatant
6000
320 mg
18
1
100


Fig 7. Zymogram and SDS-PAGE
of chitinase.
3.5. Characterization of the
partly purified chitinase
From fig 8. A, B, C, D, it was clear
that chitinase shows good activity at
pH 5-5.5 and 55
o
C. This enzyme was
not stable at high temperature (60-
90) and pH (6-9).
We also checked the final product of
the enzyme reaction by TLC, using
colloidol chitin as substrate. The
results were showed on fig 9. Fig 8. Effect of temperature and pH on chitinase activity.
It was found that when using various subtrates
(N2:dimmer, trimer :N3 and hexamer: NG) the products containing ranks of mono-, di-, tri-, tetra- ,

percent- 12%) which have chitinase activities from 15 to 39 U/ml were selected. Further screening
showed that strain VN08-A0438 showed the highest chitinase activity (39.9 U/ml), followed by strains
VN10A-0046 (37.7 U/ml) and VN10A-0450 (36.8 U/ml).
2. Based on the results of morphology, physiological criteria, chemotaxonomy together with 16S
rDNA sequencing, the strain VN08-A0438 which has the highest chitinase activity was identified as
Streptomyces chromofuscus
3. The enzyme produced by VN08-A0438 was partly purified and characterized (including pH and
temperature stability, TLC test). The results showed that the enzyme sample be the mixture of
chitobiose, endo- and exochitinase.
FURTHER STUDY
The next studies should concentrate the work to completely purify and characterize of chitinase in
laboratory and apply the Streptomyces chromofuscus strain into the environment scale to test its
chitinase activity.