Study on characterization of chitinase from streptomyces

Effect of some parameters on chitinase activity of Streptomyces VN08-A0438 50 Figure 14.. Primary screening good Streptomyces strains for chitinase production Error!. ABSTRACT In this

Nguyen Thanh Huong

STUDY ON CHARACTERIZATION OF CHITINASE

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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

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ACKNOWLEDGEMENT

I would like to express my deep and sincere gratitude to my supervisor,

Dr Duong Van Hop, for ongoing support, encouragement, and guidance throughout my project Also for the invaluable learning experience and opportunity that was provided

I wish to express my warm and sincere thanks to PhD candidate, Ms Le Thi Hoang Yen for teaching me laboratory procedures and giving such helpful suggestions

I would also like to thank Dr Dao Thi Luong, Dr Nguyen Quynh Uyen and Ms Nguyen Thi Van for help with data analysis

I am grateful to all the lecturers of Vietnam National University, Hanoi (Vietnam) and University of Liege (Belgium) for their helpful scientific lectures and all of my classmates for their friendship and kind help during the mas ter course

Last but not least, I would like to thank my family: my parents and my husband Without their encouragement and understanding, it would have been impossible for me to finish this work

Hanoi, June 2011

Nguyen Thanh Huong

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

LIST OF FIGURES

Figure 4 Phylogenetic relationships of family 19 chitinases 17

Figure 5 Amino acid sequence of a chitinase from Streptomyces

erythraeus

18

Figure 7 Production of recombinant chitinase from Trichoderma

virens UKM-1 in E.coli

23

Figure 9 Clear zones’ diameters illustrated Chitinase activity of

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 16 Effect of temperature and pH on chitinase activity 54

Figure 17 TLC analyzing of final chitinase reaction products 55

LIST OF TABLES

Chapter 1

Table 1.2 Comparison of the characteristics of purified chitinase from

others reported Enterobacter sp

Table 3.2 Summary chitinase activities of 500 Streptomyces strains 42

Table 3.3 Chitinase avtivity of 60 selected Streptomyces strains 43

TABLE OF CONTENTS

ABSTRACT 1

CHAPTER 1 INTRODUCTION 4

1.1 Chitin and application of chitin and chitinoligosaccharides 4

1.1.1 Application of chitin in Agriculture and Environment 6

1.1.2 Application of chitin in Medicine 8

1.1.3 Application of chitin in cosmetic and industry 9

1.2 Compositions and me thods 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 assay Error! 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 medium Error! 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 11

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 (NH4)2SO4 - 2 g, at

35oC, 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 55oC 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

TÓM TẮT Tên luận văn: Nghiên cứu đặc tính chitinase từ xạ khuẩn

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)2SO4 (2 g/l) được sử dụng là nguồn cung cấp nitơ, điều kiện nhiệt độ 350C, 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 550C Kết quả phân tích TLC cho thấy sản phẩm tinh sạch có chứa toàn bộ mono-; di- và oligomers

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”

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 (C8H13O5N)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 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/cm3) 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

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