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”. [...]... 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... 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... strains of Streptomyces are plant pathogens, and family 19 chitinase from Streptomyces can be acquired from plants by horizontal gene transfer [34] 22 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 ≤... chemically A comparison table of the characteristics of purified chitinase from Enterobacter sp was established (table 2) [2] There are many methods for chitinase purification, depend on the sources where chitinase was purified from 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,... Recombinant chitinase Chitinase is known as a material for control of phytophathogenic fungi and insect pests Because of this reason, chitinase has attracted a lot of attention However, chitinase is uneconomic for commercialized due to the high cost in its productions The development in recombinant DNA technology has helped to reduce chitinase production cost and enhance its production Bacterial expression... 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... the consumption of exogenous foods In Japanese eel, chitinase was found in the stomach However, the digestive tract of eel contained also chitindecomposing bacteria [4] 25 * Role of chitinase in humans Chitinases in humans play a role in defense against pathogens, which have chitinous structures [12] 1.3.3 Purification of chitinase Chitinase is difficult to purify and modify chemically A comparison table... 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... 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... 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 screening of chitinase 2.3 Media * Agar medium (yeast extract - starch YS) was used for cultivation and maintenance of Streptomyces: (g/l) soluble starch: 10; Yeast extract: 4; MgSO4.7H2O: . 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. 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. Thanh Huong STUDY ON CHARACTERIZATION OF CHITINASE FROM STREPTOMYCES MASTER THESIS MAJOR BIOTECHNOLOGY HANOI – 2011 UNIVERSITY OF LIEGE *** VIETNAM NATIONAL UNIVERSITY,