Study on characterization of chitinase from streptomyces

Study on characterization of chitinase from streptomyces

Nguyen Thanh Huong

STUDY ON CHARACTERIZATION OF CHITINASE

FROM STREPTOMYCES

MASTER THESIS MAJOR BIOTECHNOLOGY

HANOI – 2011

UNIVERSITY OF LIEGE

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VIETNAM NATIONAL UNIVERSITY, HANOI

INSTITUTE OF MICROBIOLOGY AND BIOTECHNOLOGY

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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

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VIETNAM NATIONAL UNIVERSITY, HANOI

INSTITUTE OF MICROBIOLOGY AND BIOTECHNOLOGY

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TABLE OF CONTENTS

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

CHAPTER 3 RESULTS AND DISCUSSION Error! Bookmark not defined 3.1 Screening of chitinase-producing Streptomyces Error! Bookmark not defined.

3.1.1 Primary screening good Streptomyces strains for chitinase production Error! Bookmark not

defined

3.1.2 Chitinase activities of 60 Streptomyces strains in liquid mediumError! Bookmark not defined 3.2 Identification of Streptomyces strain VN08-A0438 Error! Bookmark not defined.

3.2.1 Morphology of strain VN08-A0438 Error! Bookmark not defined

3.2.2 Studying carbon sources assimilation of the culture Error! Bookmark not defined

3.2.3 Some physiological criteria of the culture Error! Bookmark not defined

3.2.4 16S rDNA sequencing of Streptomyces VN08-A0438 Error! Bookmark not defined

3.3 Selecting medium and conditions for chitinase production Error! Bookmark not defined 3.4 Purification of chitinase Error! Bookmark not defined 3.5 Characterization of the partly purified chitinase Error! Bookmark not defined CONCLUSION Error! Bookmark not defined FURTHER STUDIES Error! Bookmark not defined.

BIBLIOGRAPHY Error! Bookmark not defined

ABSTRACT

Chitin (C8H13O5N)n 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

This is the first time the enzyme produced by Streptomyces chromofuscus was partly purified and

characterized (including pH and temperature stability, TLC test) in Vietnam

Distribution of thesis: thesis contained 67 pages, 8 tables, 17 figures, and 43 references

Distribution of thesis concluded: Abstract (1 page), Foreword (1 page), Introduction (24 pages), materials and methods (11 pages), results and discussion (13 pages), conclusions and futher study (1 page), and reference (4 pages), appendix (6 pages)

CHAPER 1 INTRODUCTION 1.1 Chitin and application of chitin and chitinoligosaccharides

Chitin (C8H13O5N)n is one of the natural polysaccharides including a copolymer of N-acetyl-D-glucosamine and D-N-acetyl-D-glucosamine residues These two components are linked together by β-1,4 glycosidic bonds

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 Beside, in agriculture and horticulture, chitosan-a substance derived from chitin can be used as a bio-control elictor [26] 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 Chitosan - a substance derived from chitin can trigger plant defensive mechanisms as a vaccine in human, stimulate plant growth and induce unexpected effects of certain enzymes

Scientists believe that chitin is used for environmental treatment because of its features: natural origin and being biodegraded

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 [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

1000oC 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 1000oC for 1 hour → Chitin

Physical method in producing chitosan from chitin is more effective in comparison with chemical method because of its simple and fast features in degradation

In biological method, Proteases can be used for the deproteinization of crustacean shells for the

production of chitin or chitosan

1.3 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 Chitinase have been detected in a wide variety of

organisms including organismsthat do not contain chitin, such as bacteria, fungi, viruses, plants and insects The roles of chitinases in different organisms are diverse Chitinases from all organisms are classified into two phylogenetically distinct families 18 and 19 among the 87 families of glycosyl hydrolases based on amino acid sequence of their catalytic domains

Chitinase is difficult to purify and modify chemically Plant chitinase is specific example for chitinase purification Chitinases can be purified from a total homogenate, from the intercellular fruid or from latex Affinity chromatography, using chitin or colloidal chitin, is a successful method for the purification

of the chitinases from barley leaf intercellular fluid, soybean, bean, tobacco and wheat Chitinases have a very basic or very acid isoelectric points (and they are described as basic or acidic proteins)

The development in recombinant DNA technology has helped to reduce chitinase production cost and enhance its production Bacterial expression systems are the most common and preferred method for the overexpression of recombinant proteins because of fast cell growth

There are four forms of chitinase: endochitinase is defined as an enzyme splitting within the chitin polymer, exochitinase (EC 3.2.1.14) as an enzyme releasing chitobiose, β-N-acetylglucosaminidase releases N-acetylglucosamine monomers from chitin and chitobiase hydrolyses chitobiose Endochitinases (EC 3.2.1.14) cleave chitin polymers at randominternal sites, whereas exochitinases cleave chitin progressively starting at the non-reducing ends of the chains and release N-acetyl-D-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

* Agar medium (yeast extract - starch YS), Medium 1 (M1) (culture fermentation) and medium ISP4

2.4 Methodology

2.4.1 Screening of chitinase-producing Streptomyces and culture conditions

- Preparing culture broth of Streptomyces

- Primary screening on agar Petri dish

2.4.2 Selecting good chitinese producers by chitinase activity assay

2.4.3 Identification of Streptomyces strain

- Morphology and growth characterization

- Some biological criteria of Streptomyces target strain

- Chemotaxonomic Study of Streptomyces

- 16S rDNA sequencing

2.4.4 Effect of culture conditions (temperature, pH, aeration, carbon, nitro sources) for chitinase fermentation from Streptomyces

2.4.5 Purification of chitinase

2.4.6 SDS-PAGE and activity gel (zymogram)

2.4.7 Characterization of the partly purified chitinase

CHAPTER III RESULTS AND DISCUSSION

3.1 Screening of chitinase-producing Streptomyces

3.1.1.Primary screening good streptomyces strains for chitinase production:

Five hundred collected Streptomyces strains in Hoang Lien Son national park were isolated and

maintained at Vietnam Type Culture Collection - Institute of Microbiology and Biotechnology - Vietnam National University, Hanoi Agar Petri disc method was used in primary screening step Chitinase activity

was determined according to the clear zones’ diameters (fig 1) The zone diameters on plates varied in size (2 - 30 mm)

Fig 1 Clear zones’ diameters illustrated Chitinase activity

of Streptomyces strains

A number of 60 strains showing strong chitinase activities, 146 strains showing medium chitinase 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

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