UNIVERSITY OF LIEGE *** VIETNAM NATIONAL UNIVERSITY, HANOI INSTITUTE OF MICROBIOLOGY AND BIOTECHNOLOGY *** Nguyen Thanh Huong STUDY ON CHARACTERIZATION OF CHITINASE FROM STREPTOMYCES MASTER THESIS MAJOR BIOTECHNOLOGY HANOI - 2011 i VIETNAM NATIONAL UNIVERSITY, HANOI LIEGE UNIVERSITY INSTITUTE OF MICROBIOLOGY *** AND BIOTECHNOLOGY *** 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 ii 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 iii ABBREVIATIONS DNA - Deoxyribonucleic acid DNS - Dinitrosalicylic acid E.coli - Escherichia coli GlcNAc - N-acetyl-D-glucosamine M1 - Medium 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 Natural sources of chitin Figure Chemical structures of cellulose and chitin Figure Structure of chitin and chitosan 13 Figure Phylogenetic relationships of family 19 chitinases 17 Figure Amino acid sequence of a chitinase from Streptomyces erythraeus 18 Figure Purification of the pea antifungal hydrolases 21 Figure Production of recombinant chitinase from Trichoderma virens UKM-1 in E.coli 23 Figure Calibration curve of N - acetyl – Glucosamine 33 Figure 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 Table 1.1 Chitin content of some organisms Table 1.2 Comparison of the characteristics of purified chitinase from others reported Enterobacter sp 22 List of instruments 29 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 Chapter Table 2.1 Chapter vi TABLE OF CONTENTS ABSTRACT CHAPTER INTRODUCTION 1.1 Chitin and application of chitin and chitinoligosaccharides 1.1.1 Application of chitin in Agriculture and Environment .6 1.1.2 Application of chitin in Medicine 1.1.3 Application of chitin in cosmetic and industry 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 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 v 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 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 vi 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 - g, colloidol chitin g and nitrogen source as (NH4)2SO4 - g, at 35oC, pH 6.5 with shacking rate 200 rpm for 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 Người hướng dẫn: TS Dương Văn Hợp Viện Vi sinh vật 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 phân lập từ Vườn Quốc gia Hoàng Liên Sơn tiến hành tuyển chọn xác định hoạt tính chitinase Thông qua trình sàng lọc bản, chủng xạ khuẩn mang kí hiệu VN08-A0438 chủng có hoạt tính sinh chitinase cao nhất, chủng lựa chọn để phục vụ cho mục đích nghiên cứu Cũng nghiên cứu này, chủng VN08-A0438 tiến hành định loại dựa đặc điểm hình thái, hóa sinh giải trình tự 16S rDNA, kết cho thấy xạ khuẩn phân lập chủng Streptomyces chromofuscus Bên cạnh đó, tiến hành thí nghiệm xác định điều kiện tối ưu cho phát triển sinh chitinase chủng Streptomyces chromofuscus VN08-A0438 bao gồm thiết lập nhiệt độ, pH, chế độ thoáng khí, thời gian lên men, thử nghiệm nguồn cácbon nitơ khác Kết phân tích cho thấy chủng phát triển tốt môi trường có nguồn cacbon glucoza (5g/l), colloidol chitin (5g/l) (NH4)2SO4 (2 g/l) sử dụng nguồn cung cấp nitơ, điều kiện nhiệt độ 350C, pH = 6.5 lắc 200 vòng/phút ngày Chitinase chủng Streptomyces VN08-A0438 sau tinh sơ kết tủa amôn sunphat Chitinase nghiên cứu đặc tính độ bền nhiệt, pH sắc ký mỏng (TLC) Kết phân tích cho thấy chitinase bền pH 5.5 nhiệt độ 550C Kết phân tích TLC cho thấy sản phẩm tinh có chứa toàn mono-; di- oligomers (synthesis of phytoalexins, chitinases, pectinases, glucanases, and lignin) The approach using chitosan - an organic compound opens a real promising of biocontrol 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 physicochemicaltype 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] 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 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” 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 10 BIBLIOGRAPHY Aye K N., Stevens W F (2000), “Improved chitin production by pretreatment of shrimp shells”, Journal of Chemical Technology 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28.http://cdtvn.net/index.php?option=com_content&view=article&id=435:chitin -chitosan-produce&catid=40:life&Itemid=255 (site accessed: 26/02/2011) 29 http://www.cyberchemvn.com/cong-nghe-va-ung-dung/chitosan-tong-quan- nghien-cuu-ung-dung.html (site accessed: 12/02/2011) 30 http://en.wikipedia.org/wiki/Chitin (site accessed: 12/02/2011) 31 http://www.evven.netfirms.com/chitin,novel,application.html (site accessed: 12/02/2011) 32.http://www.ibt.ac.vn/index.php?option=com_content&task=view&id=438&It emid=702 (site accessed: 03/03/2011) 14 33 http://www.mhhe.com (site accessed: 14/02/2011) 34 http://mic.sgmjournals.org/cgi/content/abstract/145/12/3353 (site accessed: 03/03/2011) 35 http://www.nanobiosaccharides.org/Pages/project_summary.htm (site accessed: 26/02/2011) 36 http://www.newwealthchallenge.com (site accessed: 14/02/2011) 37 http://www.niabi.in (site accessed: 14/02/2011) 38.http://www.phuongduy.com.vn/index.php?page=detailContent&content_id=5 (site accessed: 12/02/2011) 39.http://www.plasticstrends.net/index2.php?option=com_content&do_pdf=1&i d=12 (site accessed: 14/02/2011) 40 http://www.redsquarepools.wordpress.com (site accessed: 14/02/2011) 41 http://www.scribd.com/doc/24129694/%E1%BB%A9ng-d%E1%BB%A5ngkhang-khu%E1%BA%A9n-c%E1%BB%A7a-chitosan (12/02/2011) 15 APPENDIX Table 3.1 Primarily screening chitinase activities of 500 Streptomyces strains No 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Strain VN10-A0001 VN10-A0002 VN10-A0003 VN10-A0004 VN10-A0005 VN10-A0006 VN10-A0007 VN10-A0008 VN10-A0009 VN10-A0010 VN10-A0011 VN10-A0012 VN10-A0013 VN10-A0014 VN10-A0015 VN10-A0016 VN10-A0017 VN10-A0018 VN10-A0019 VN10-A0020 VN10-A0021 VN10-A0022 VN10-A0023 VN10-A0024 VN10-A0025 VN10-A0026 VN10-A0027 VN10-A0028 VN10-A0029 VN10-A0030 VN10-A0031 VN10-A0032 VN10-A0033 VN10-A0034 VN10-A0035 VN10-A0036 VN10-A0037 VN10-A0038 VN10-A0039 VN10-A0040 VN10-A0041 VN10-A0042 VN10-A0043 VN10-A0044 Chitinase activity (mm) 10 10 0 13 18 25 0 10 10 5 25 0 15 22 25 20 24 0 0 0 0 0 21 0 10 20 16 No 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 Strain VN10-A0045 VN10-A0046 VN10-A0047 VN10-A0048 VN10-A0049 VN10-A0050 VN10-A0051 VN10-A0052 VN10-A0053 VN10-A0054 VN10-A0055 VN10-A0056 VN10-A0057 VN10-A0058 VN10-A0059 VN10-A0060 VN10-A0061 VN10-A0062 VN10-A0063 VN10-A0064 VN10-A0065 VN10-A0066 VN10-A0067 VN10-A0068 VN10-A0069 VN10-A0070 VN10-A0071 VN10-A0072 VN10-A0073 VN10-A0074 VN10-A0075 VN10-A0076 VN10-A0077 VN10-A0078 VN10-A0079 VN10-A0080 VN10-A0081 VN10-A0082 VN10-A0083 VN10-A0084 VN10-A0085 VN10-A0086 VN10-A0087 VN10-A0088 Chitinase activity (mm) 26 20 20 22 26 15 10 12 13 17 10 14 23 18 16 16 16 24 14 16 10 14 20 13 12 10 10 10 10 10 10 14 18 10 0 12 No 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 Strain VN10-A0089 VN10-A0090 VN10-A0091 VN10-A0092 VN10-A0093 VN10-A0094 VN10-A0095 VN10-A0096 VN10-A0097 VN10-A0098 VN10-A0099 VN10-A0100 VN10-A0101 VN10-A0102 VN10-A0103 VN10-A0104 VN10-A0105 VN10-A0106 VN10-A0107 VN10-A0108 VN10-A0109 VN10-A0110 VN10-A0111 VN10-A0112 VN10-A0113 VN10-A0114 VN10-A0115 VN10-A0116 VN10-A0117 VN10-A0118 VN10-A0119 VN10-A0120 VN10-A0121 VN10-A0122 VN10-A0123 VN10-A0124 VN10-A0125 VN10-A0126 VN10-A0127 VN10-A0128 VN10-A0129 VN10-A0130 VN10-A0131 VN10-A0132 VN10-A0133 Chitinase activity (mm) 10 11 10 13 0 10 13 13 17 0 15 5 0 15 10 23 15 13 0 0 10 5 10 0 10 10 17 No 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 Strain VN10-A0134 VN10-A0135 VN10-A0136 VN10-A0137 VN10-A0138 VN10-A0139 VN10-A0140 VN10-A0141 VN10-A0142 VN10-A0143 VN10-A0144 VN10-A0145 VN10-A0146 VN10-A0147 VN10-A0148 VN10-A0149 VN10-A0150 VN10-A0151 VN10-A0152 VN10-A0153 VN10-A0154 VN10-A0155 VN10-A0156 VN10-A0157 VN10-A0158 VN10-A0159 VN10-A0160 VN10-A0161 VN10-A0162 VN10-A0163 VN10-A0164 VN10-A0165 VN10-A0166 VN10-A0167 VN10-A0168 VN10-A0169 VN10-A0170 VN10-A0171 VN10-A0172 VN10-A0173 VN10-A0174 VN10-A0175 VN10-A0176 VN10-A0177 VN10-A0178 Chitinase activity (mm) 0 10 12 12 22 0 0 0 15 15 5 16 0 0 0 0 20 18 15 10 10 0 0 10 25 25 10 No 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 Strain VN10-A0179 VN10-A0180 VN10-A0181 VN10-A0182 VN10-A0183 VN10-A0184 VN10-A0185 VN10-A0186 VN10-A0187 VN10-A0188 VN10-A0189 VN10-A0190 VN10-A0191 VN10-A0192 VN10-A0193 VN10-A0194 VN10-A0195 VN10-A0196 VN10-A0197 VN10-A0198 VN10-A0201 VN10-A0202 VN10-A0203 VN10-A0204 VN10-A0205 VN10-A0206 VN10-A0207 VN10-A0208 VN10-A0209 VN10-A0210 VN10-A0211 VN10-A0212 VN10-A0213 VN10-A0214 VN10-A0215 VN10-A0216 VN10-A0217 VN10-A0218 VN10-A0219 VN10-A0220 VN10-A0221 VN10-A0222 VN10-A0223 VN10-A0224 VN10-A0225 Chitinase activity (mm) 20 0 13 0 19 12 0 0 14 25 13 0 0 10 10 10 10 0 10 12 19 10 10 17 15 18 14 15 10 23 12 18 No 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 Strain VN10-A0226 VN10-A0227 VN10-A0228 VN10-A0229 VN10-A0230 VN10-A0231 VN10-A0232 VN10-A0233 VN10-A0234 VN10-A0235 VN10-A0236 VN10-A0237 VN10-A0238 VN10-A0239 VN10-A0240 VN10-A0241 VN10-A0242 VN10-A0243 VN10-A0244 VN10-A0245 VN10-A0246 VN10-A0247 VN10-A0248 VN10-A0249 VN10-A0250 VN10-A0251 VN10-A0252 VN10-A0253 VN10-A0254 VN10-A0255 VN10-A0256 VN10-A0257 VN10-A0258 VN10-A0259 VN10-A0260 VN10-A0261 VN10-A0262 VN10-A0263 VN10-A0264 VN10-A0265 VN10-A0266 VN10-A0267 VN10-A0268 VN10-A0269 VN10-A0270 Chitinase activity (mm) 12 12 20 15 15 0 20 0 0 21 19 15 0 19 24 0 0 15 15 17 18 15 19 10 0 18 15 12 17 19 15 30 20 No 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 Strain VN10-A0271 VN10-A0272 VN10-A0273 VN10-A0274 VN10-A0275 VN10-A0276 VN10-A0277 VN10-A0278 VN10-A0279 VN10-A0280 VN10-A0281 VN10-A0282 VN10-A0283 VN10-A0284 VN10-A0285 VN10-A0286 VN10-A0287 VN10-A0288 VN10-A0289 VN10-A0290 VN10-A0291 VN10-A0292 VN10-A0293 VN10-A0294 VN10-A0295 VN10-A0296 VN10-A0297 VN10-A0298 VN10-A0299 VN10-A0300 VN10-A0301 VN10-A0302 VN10-A0303 VN10-A0304 VN10-A0305 VN10-A0306 VN10-A0307 VN10-A0308 VN10-A0309 VN10-A0310 VN10-A0311 VN10-A0312 VN10-A0313 VN10-A0314 VN10-A0315 Chitinase activity (mm) 16 0 0 19 13 20 24 14 10 17 14 10 20 15 10 13 15 20 10 17 18 14 24 17 15 15 20 17 18 18 16 15 30 20 20 16 18 30 19 No 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 Strain VN10-A0316 VN10-A0317 VN10-A0318 VN10-A0319 VN10-A0320 VN10-A0321 VN10-A0322 VN10-A0323 VN10-A0324 VN10-A0325 VN10-A0326 VN10-A0327 VN10-A0328 VN10-A0329 VN10-A0330 VN10-A0331 VN10-A0332 VN10-A0333 VN10-A0334 VN10-A0335 VN10-A0336 VN10-A0337 VN10-A0338 VN10-A0339 VN10-A0340 VN10-A0341 VN10-A0342 VN10-A0343 VN10-A0344 VN10-A0345 VN10-A0346 VN10-A0347 VN10-A0348 VN10-A0349 VN10-A0350 VN10-A0351 VN10-A0352 VN10-A0353 VN10-A0354 VN10-A0355 VN10-A0356 VN10-A0357 VN10-A0358 VN10-A0359 VN10-A0360 Chitinase activity (mm) 17 18 19 25 0 0 14 10 35 0 13 10 0 0 0 0 20 0 0 12 10 16 10 0 0 10 No 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 Strain VN10-A0361 VN10-A0362 VN10-A0363 VN10-A0364 VN10-A0365 VN10-A0366 VN10-A0367 VN10-A0368 VN10-A0369 VN10-A0370 VN10-A0371 VN10-A0372 VN10-A0373 VN10-A0374 VN10-A0375 VN10-A0376 VN10-A0377 VN10-A0378 VN10-A0379 VN10-A0380 VN10-A0381 VN10-A0382 VN10-A0383 VN10-A0384 VN10-A0385 VN10-A0386 VN10-A0387 VN10-A0388 VN10-A0389 VN10-A0390 VN10-A0391 VN10-A0392 VN10-A0393 VN10-A0394 VN10-A0395 VN10-A0396 VN10-A0397 VN10-A0398 VN10-A0399 VN10-A0400 VN10-A0401 VN10-A0402 VN10-A0403 VN10-A0404 VN10-A0405 Chitinase activity (mm) 12 16 5 14 13 26 0 16 18 5 0 22 14 22 11 10 16 13 10 15 14 20 10 11 20 12 10 22 24 13 13 10 20 No 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 Strain VN10-A0406 VN10-A0407 VN10-A0408 VN10-A0409 VN10-A0410 VN10-A0411 VN10-A0412 VN10-A0413 VN10-A0414 VN10-A0415 VN10-A0416 VN10-A0417 VN10-A0418 VN10-A0419 VN10-A0420 VN10-A0421 VN10-A0422 VN10-A0423 VN10-A0424 VN10-A0425 VN10-A0426 VN10-A0427 VN10-A0428 VN10-A0429 VN10-A0430 VN10-A0431 VN10-A0432 VN10-A0433 VN10-A0434 VN10-A0435 VN10-A0436 VN10-A0437 VN10-A0438 VN10-A0439 VN10-A0440 VN10-A0441 VN10-A0442 VN10-A0443 VN10-A0444 VN10-A0445 VN10-A0446 VN10-A0447 VN10-A0448 VN10-A0449 VN10-A0450 Chitinase activity (mm) 0 0 10 0 12 12 10 0 10 12 12 10 10 27 17 18 10 0 18 0 23 15 10 15 20 15 14 20 15 17 17 17 20 No 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 Strain VN10-A0451 VN10-A0452 VN10-A0453 VN10-A0454 VN10-A0455 VN10-A0456 VN10-A0457 VN10-A0458 VN10-A0459 VN10-A0460 VN10-A0461 VN10-A0462 VN10-A0463 VN10-A0464 VN10-A0465 VN10-A0466 VN10-A0467 VN10-A0468 VN10-A0469 VN10-A0470 VN10-A0471 VN10-A0472 VN10-A0473 VN10-A0474 VN10-A0475 Chitinase activity (mm) 10 10 25 19 17 23 22 28 15 10 14 19 15 25 15 25 11 18 15 15 No 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 Strain VN10-A0476 VN10-A0477 VN10-A0478 VN10-A0479 VN10-A0480 VN10-A0481 VN10-A0482 VN10-A0483 VN10-A0484 VN10-A0485 VN10-A0486 VN10-A0487 VN10-A0488 VN10-A0489 VN10-A0490 VN10-A0491 VN10-A0492 VN10-A0493 VN10-A0494 VN10-A0495 VN10-A0496 VN10-A0497 VN10-A0498 VN10-A0499 VN08-A0438 Chitinase activity (mm) 15 23 16 0 5 21 17 14 18 14 15 14 8 10 0 11 30 Note: Streptomyces strains having diameters of clear zone more than 20 cm were marked as bold 21 [...]... 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 3 CHAPTER 1 INTRODUCTION 1.1 Chitin and application of chitin and chitinoligosaccharides... desalting and group separation”, Nature, 183, pp.1657-9 17a Nikolova1 S A., Stefanova1 V., Yocheva L (2006-2007), “Taxonomy study of Streptomyces sp strain 34-1”, Journal of culture collections, 5, pp 1015 18 Saito A., Fujii T and Miyashita K (2003), “Distribution and evolution of chitinase genes in Streptomyces species: involvement of gene-duplication and domain-deletion”, Antonie van Leeuwenhoek, 84(1),... Tewari R., Tiwari R P., Hoondal G S (2005), Chitinase from Enterobacter sp NRG4: Its purification, characterization and reaction pattern”, Electronic Journal of Biotechnology, 8(2), pp 0717-3458 3 Delic I., Robbins P., and Westpheling J (1992), “Direct repeat sequences are implicated in the regulation of two Streptomyces chitinase promoters that are subject to carbon catabolite control”, Proc Natl Acad... Lapboonrueng S., Pechsrichuang P., Pesatcha P., Yamabhai M (2010), “Expression and characterization of Bacillus licheniformis chitinase (ChiA), suitable for bioconversion of chitin waste”, Bioresource Technology, 101, pp 4096–4103 23 Suresh P V and Chandrasekaran M (1998), “Utilization of prawn waste for chitinase production by marine Beauveria bassiana under solid state fermentation”, World Journal of. .. isolated from plant litter in Vietnam”, International Journal of Systematic and Evolutionary Microbiology, 59, pp 550–554 20 Sarkar S., et al (2010), “Bioprocessing Data for the Production of Marine Enzymes”, Mar Drugs, 8, pp 1323-1372 21 Shirling, E B & Gottlieb, D (1966), “Methods for characterization of Streptomyces species, International Journal Systematic Bacteriology, 16, pp 313–340 22 Songsiriritthigul... chitin and chitosan”, International Bureau, WO/2006/124779 8 Hamsah N B (2009), Bachelor thesis: “Optimization of inducer on express of recombinant chitinase in Escherichia coli using respone surface”, University Malaysia Pahang, Malaysia 11 9 International atomic energy agency (2002), “Radiation processing application for agrowaste (subproject: radiation processing of chitin/chitosan)”, Project Coordinators’... (2005), “Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi) gene and characterization of its protein”, Genetics and Molecular Biology, 28(4), pp 821-826 27 Zhu X F., Zhou Y., Feng J L (2007), “Analysis of both chitinase and chitosanase produced by Sphingomonas sp CJ-5”, Journal of Zhejiang University SCIENCE B, 8(11), pp 831-838 Websites: 28.http://cdtvn.net/index.php?option=com_content&view=article&id=435:chitin... 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... 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 5 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... 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