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UNIVERSITY OF SCIENCE AND TECHNOLOGY OF HANOI INSTITUTE OF BIOTECHNOLOGY MASTER REPORT MINING AMYLASE GENES THROUGH METAGENOMIC DATABASE OF BACTERIA MICROFLORA IN GUT OF TERMITE Coptotermes gestroi By BUI Thi Thuy Nga Biotechnology- Pharmacology Department Promoter: Professor Truong Nam Hai Co-promoter: Dr Do Thi Huyen Home institution: Institute of Biotechnology (IBT) Hanoi, September 2016 i ACKNOWLEDGMENTS First off, I would like to wholeheartedly thank my promoter: Professor Truong Nam Hai, whom I am also very grateful for his constant support and gave me the freedom to work in his department Next up is the wonderful person at the laboratory, my co-promoter: Dr Do Thi Huyen, who helped me lots in both academic knowledge and scientific research methods She always followed my process to aid as well as orienting the right direction for my working Especially I would like also performing my greatest pleasure to Le Ngoc Giang and Dao Trong Khoa who was a guidance to recover when my steps faltered, her patience and support helped me overcome many crisis situations and finish this dissertation Beside that I would like to thank to all staffs in the laboratory of Genetic Engineering, for being friendly, their supporting, giving me not only take care of the best conditions for me and gave me the very important advises, but also always answer all most my questions, taught and guided me about science during the time of my studying Indispensably, many thanks for all staffs and boards of managers of University of Science and technology of Hanoi (USTH); they equipped robustness knowledge for me Last but in no way the least, no word could describe all encouragements of my family and my friends to me, those made me have more motivations to keep living with the best efficient Thank you with my thankfulness! By Bui Thi Thuy Nga ii ABBREVIATIONS BLAST Basic Local Alignment Search Tool C.gestroi Coptotermes gestroi CAZy Carbohydrate-Active enZYmes EC number Enzyme Commission number GH Glycosyl hydrolase family ORF Open reading frame RDP Ribosomal Database Project rRNA Acid ribonucleic ribosome iii ABSTRACT The goal of this study is mining gene may be related to starch degradation through 5,6 Gb metagenome database of free-living microbial flora in the gut of the lower termite Coptotermes gestroi, the samples were harvested (6 nests) in Vietnam, sequenced and analyzed by using metagenomic and bioinformatics Of more than 12,000 ORFs with predicted functions related to carbohydrate metabolism, 587 encoding hydrolytic enzymes for cellulose, hemicellulose, and pectin were identified, of which 80% are bacterial genes Interestingly, 85 generated sequences were predicted related to coding enzyme hydrolysis starch Nevertheless, one sequence was predicted that encoding alpha-amylase was exploited and the estimated function will be used via bioinformatics tools suggesting adoption alpha-amylase would be synthesized and expressed in vitro and apply for further experiments With the progress in metagenomics and biotechnology, it will be successful application of relies on their partnership with a diverse community of bacteria in termite that create the ability to degrade starch gives termites an important place in the carbon cycle and this diverse array of microbial capabilities that could jumpstart a new biofuel industry promising models for the industrial conversion of raw material into microbial products and the production of biofuels Key words: Free-living gut bacterial community; Coptotermes gestroi (C gestroi); Amylase; Starch degradation; Metagenome iv VIETNAMESE ABSTRACT Mục tiêu nghiên cứu khai thác gen liên quan đến phân huỷ tinh bột thông qua 5,6 Gb sở liệu metagenome phân lập từ hệ vi sinh vật sống tự ruột mối cấp thấp Coptotermes gestroi, tất mẫu mối thu hoạch tổ Việt Nam, giải trình tự phân tích kỹ thuật metagenomic tin sinh học Có 12.000 ORF dự đốn có đầy đủ chức liên quan đến chu trình carbon, 587 ORF xác định với vai trị mã hóa cho enzyme thủy phân cellulose, hemicellulose pectin, số hệ gen vi khuẩn chiếm 80% Điều thú vị 85 trình tự phân lập dự đốn có liên quan đến việc mã hóa enzyme thủy phân tinh bột Tuy nhiên, trình tự dự đốn mã hóa alpha-amylase khai thác để nghiên cứu sâu chức thông qua việc sử dụng công cụ tin sinh việc tổng hợp biểu alphaamylase ống nghiệm cho thí nghiệm tương lai Với tiến metagenome công nghệ sinh học, biểu thành cơng gen mã hóa alpha-amylase thành công việc nghiên cứu đa dạng cộng đồng vi sinh vật ruột mối Đồng thời, khả phân hủy tinh bột cung cấp cho mối vị trí quan trọng chu trình cacbon, đồng thời đa dạng hệ thống vi sinh vật tạo tiền đề hình thành mơ hình cho việc chuyển đổi ngun liệu thơ thành sản phẩm vi sinh vật sản xuất nhiên liệu sinh học ngành công nghiệp nhiên liệu sinh học Từ khóa: cộng đồng vi khuẩn sống tự đường ruột; Coptotermes gestroi (C gestroi); amylaza; phân huỷ tinh bột; Metagenome v LIST OF TABLES Table Classification of Amylase Table Uses of amylases in various sectors of industry Table 3: Glycoside hydrolases (GHs) for the degradation of starch in C gestroi termite free-living gut microbiota 10 vi TABLE OF CONTENTS ABBREVIATIONS iii ABSTRACT iv VIETNAMESE ABSTRACT v LIST OF TABLES vi TABLE OF CONTENTS vii INTRODUCTION I Starch properties and amylolytic enzymes 1 Starch properties Enzymatic hydrolysis of starch 2.2 α-Amylase: 1,4-α-D-glucan 4-glucanohydorolase (EC 3.2.1.1) 2.3 Molecular structure of α-amylase 3 Application of amylolytic enzyme II Termites and community of microbiota in Gut’s Termite Outline of termites Microflora in the gut of termites Digestion in the gut of Lower termites III Metagenomic IV Objectives MATERIALS AND METHODS I Materials II Methods Functional Prediction and bacterial diversity prediction Conserved domains and active sites determining Three dimensional structure prediction Mining potential sequences by consensus probe RESULTS AND DISCUSSIONS I Functional annotation by BLAST2GO for amylase II Prediction of amylase properties based on the encoding ORFs 10 III Bacterial diversity of amylase producer in the termite gut 11 IV Protein conserved domains with Amylase activity 11 V Indentification of the consensus sequence 11 CONCLUSIONS 14 REFERENCES 15 APPENDIXS 17 vii INTRODUCTION I Starch properties and amylolytic enzymes Starch properties Starch is essentially a carbohydrate which is formed by the plants with the aid of glucose polymerization which storage carbohydrate in many plants It is the most common carbohydrate in human diets and is contained in large amounts in such staple foods as potatoes, wheat, corn, rice, and cassava Starch makes for two high molecular weight components, which consists of two types of molecules: the linear and helical amylose and the branched amylopectin (Figure 1) Depending on the plant, starch generally contains 20 to 25% amylose and 75 to 80% amylopectin by weight Amylose is a polymer approximately consisting of 500-1,000 glucose units that made of α-D-glucose units, bound to each other through α (1→4) glycoside bonds Moreover, Amylose consists of short α-1, linked linear chains of 10–60 glucose units and α-1, linked side chains composed of 15–45 glucose units An average number of branching points (α-1, linkage) in amylopectin are about 5%, and branches occur every 12 to 23 glucose units in the linear chain, depending on the botanical origin The amylose content varies between almost and 75%, with a common value of 15–25% Amylopectin is a major fraction of starch (75-85%) and is more complex than amylose because of its highly branched structure (Leveque et al, 2000) One amylopectin molecule can contain more than 2,000,000 glucose units, thereby representing the largest molecules in nature Amylose is hardly dissolved in water, whereas amylopectin is reasonably water- soluble Enzymatic hydrolysis of starch In 1815, Kirchhoff showed that gluten had the capacity to convert a larger quantity of starch into sugar Thus, Kirchhoff laid the foundation for the discovery of amylase In 1833, further describe and isolate amylase in powder form from barley malt and amylase was purified Amylases (EC 3.2.1.1) are starch-degrading enzymes They are widely distributed in microbial, plant and animal kingdoms Amylase hydrolyzes starch molecules to give diverse products including dextrins, and progressively smaller polymers composed of glucose units (Windish et al, 1965) Because of the complex structure of starch, cells usually require a combination of various types of hydrolyzing enzymes for the depolymerization of starch and related polysaccharides to diverse products including dextrin, smaller polymers or oligosaccharides (Windish et al,1965) In general, amylases or amylolytic enzymes and belong to the Glycoside Hydrolase groups (GH) Amylolytic enzymes catalyze the hydrolysis or transfer of glycosidic bonds between two carbohydrate monomers or between a carbohydrate monomer and a non-carbohydrate moiety in the starch or related polysaccharides Enzymes involved in the breakdown of starch chains, basically, amylases can be divided into three groups of the enzymes (Table 1): endoamylases, exoamylases and debranching The process of enzymatic starch conversion is displayed in Figure Table Classification of Amylase Endoamylase Amylase Exo-amylase Debranching Enzyme Α-amylase Classification EC 3.2.1.1 Cleavage Internal α-1,4 Product Dextrins Β-amylase Or Maltase Glucoamylase α-glucosidase Pullulanases Isoamylases EC 3.2.1.2 Outer regions of α-1,4 Maltose EC 3.2.1.3 EC 3.2.1.20 EC 3.2.1.41 EC 3.2.1.68 α-1,6 linkages Pullulan Dextrinases EC 3.2.1.142 α-1,6 linkages β-cyclodextrin and glucose Maltotriose Maltooligosaccharides Maltose 2.1 -Amylase family (glycoside hydrolase family 13, GH13) Most of the enzymes acting on starch belong to one major family They are classified into different GH families: GH13 amylases, GH14 amylases, and GH15-glucoamylases (Henrissat 1991) The amylase family or glycosyl hydrolases family 13 (GH13 enzyme) that is the major glycoside hydrolase family acting on substrates containing α-glucoside linkages GH13 contains hydrolases, transglycosidases, and isomerases; noticeably animal amino acid transporters, which have no glycosidase activity, are also GH13 members Among thousands of sequences and ~30 different enzymes specificities many are closely related to each other, GH13, therefore, has officially been subdivided into almost 40 subfamilies; several subfamilies, including exo/endo specificity, preference for hydrolysis or transglycosylation, -1,1, -1,4 or -1,6-glycosidic bond specificity and glucan synthesizing activity (Maarel et al., 2002) The amylase (EC 3.2.1.1), amylase (EC 3.2.1.2) and glucoamylase (EC 3.2.1.3), they differ in their primary and tertiary structures as well as in their catalytic machineries and reaction mechanisms 2.2 α-Amylase: 1,4-α-D-glucan 4-glucanohydorolase (EC 3.2.1.1) α-Amylase is one of three type of important enzyme belong to amylase group which catalyzes the hydrolysis of the α-1, 4-glucosidic linkages of starch, is widespread among microbes It is an Endoamylase, which liberates poly and oligosaccharide chains of varying lengths Besides, a few αamylases can degrade some α-l, 6-glucosidic bonds as well, but the rates of these reactions are much lower than those for 1,4-bonds (Fukumoto et al., 1963) Molecular weights of α-amylases vary from about 10,000 Da to 139,000 Da The lowest value, 10,000 Da, for B caldolyticus, is an estimate obtained by ultrafiltration, a technique that is not intended for quantitative molecular weight determination (Grootegoed et al., 1973) Molecular weights of microbial α-amylases are usually 50,000 Da to 60,000 Da, and yeast α-amylases are 38,000 Da to 62,000 Da, as shown directly by analysis of cloned α-amylase genes and deduced amino acid sequences ` 2.3 Molecular structure of α-amylase The GH13 enzymes are multi-domain proteins with a common (α/)8 or TIM-barrel catalytic domain, known as domain A folds symmetrically with eight inner, parallel β-strands surrounded by eight helices Domain B shows the highest variations in the length, sequence and secondary structure of domain B may be directly related to functional diversity and enzyme specificity Domain C, just after the catalytic TIM (β/α) 8-barrel (Figure 3) which could stabilize the catalytic domain (domain A) by protecting the hydrophobic residues of the of the (β/)8 -barrel from solvent (Katsuya et al 1998) Typical amylases (EC 3.2.1.1) harboring the domains A, B and C are monomeric, calcium-containing enzymes However, some amylases may have additional domains such as domain D (just after the TIM-barrel), and E (contain additional β-strand) following the C domain The enzymes possessing the catalytic (α/)8 barrel (domain A) and four domains B, C, D and E are thus five-domain proteins Application of amylolytic enzyme Amylases are among the most important enzymes and are of great significance for biotechnology and industrial applications They are used in industrial scales for a variety of different applications like starch liquefaction, textile resizing, bread improvement, alcohol production, and as an additive to detergent formulations (Table 2) Minor applications of these enzymes are found in the pulp and paper industry (Souza et al, 2010) Commercially, the heat stable amylase from B licheniformis or B stearothermophilus is currently used in hydrolyzing processes of starch (Maarel et al 2002) In detergent application, the demand for these enzymes is growing, both for laundering and dishwashing Amylases with high activity and stability under alkaline and oxidizing conditions (pH as high as 10.5) are of choice Most of the liquid detergents (account for 90%) contain amylase Table Uses of amylases in various sectors of industry (Sivaramakrishnan et al 2006) Sector Food industry Detergent industry Paper industry Textile industry Pharmaceutical industry Uses - Production of glucose syrups, maltose syrups crystalline glucose and high fructose corn syrups - Reduction of viscosity of sugar syrups - Reduction of haze formation in juices - Solubilisation and saccharification of starch for alcohol fermentation in brewing industry - Retardation of staling in baking industry Used as an additive to remove starch based dirt Reduction of viscosity of starch for appropriate coating of paper Warp sizing of textile fibbers Used as a digestive aid II Termites and community of microbiota in Gut’s Termite Outline of termites The fossil fuels are now considered a well settled and essential the world's energy source However, some difficulties and constraints still occur, such as greenhouse gasses emission, polluting In this context, renewable energy sources are getting to replace petroleum and its derivatives that are the important key actions (Franco et al, 2011) Some materials sources are available, which has been REFERENCES Bignell DE (2011) Morphology, physiology, biochemistry and functional design of the termite gut Biology of Termites: A Modern Synthesis pp 375–412 Springer-Verlag, Berlin Brune A Termite guts: the world's smallest bioreactors Trends Biotechnol 1998; 16:16–21 Do, T H., Nguyen, T N., Le, Q G., Nguyen, C., Kimura, K., and Troung, N H (2014) Mining biomass-degrading genes through Illumina-based de novo sequencing and metagenomics analysis of free-living bacteria in the gut of lower termite Coptotermes gestroi harvested in Vietnam J Biosci Bioeng 118, 665–671 Franco Cairo, J P L., Leonardo, F C., Alvarez, T M., Ribeiro, D A., Buchli, F., Costa-Leonardo, A M., Carazzolle, M F., Costa, F F., Paes Leme, A F., Pereira, G A., and Squina, F M.: Functional characterization and target dis- covery of glycoside hydrolases from the digestome of the lower termite Cop- totermes gestroi, Biotechnol 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