VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE SAMLEY MAM SCREENING AND CHARACTERIZATION OF CELLULASE IN BACILLUS sp Student: Samley MAM Student code: 24181065 Supervisor: Dr Nguyen Thi Thanh Thuy Department: Food safety and Quality management - VNUA AGRICULTURAL UNIVERSITY PRESS - 2017 DECLARATION This thesis contains no material that has been accepted for the award of any other degree or diploma in any educational institution and, to the best of my knowledge and belief, it contains no material previously published or written by other person, except where due to reference is made in the text of the thesis Hanoi, May 10th, 2017 Master candidate Samley MAM i ACKNOWLEDGEMENT First of all, I would like to sincerely thank the Vietnam National University of Agriculture (VNUA) in Vietnam, in particular the Faculty of Food Science and Technology, for providing all the conditions leading to this thesis and allowing me to follow the program Food Technology that financially supported by Académie de Recherche et d’Enseignement Supérieur – Commission de la Cooperation au Dévelopement (ARES-CCD) I express my gratitude to my supervisor Dr Nguyen Thi Thanh Thuy, for being my mentor during my thesis research on “Screening and characterization of cellulase in Bacillus sp.” Especially, I would like to acknowledge her precious advices, the very helpful discussions, and good explanation with her friendly encouragement I am very grateful to Ms Trinh Thi Thu Thuy for being my co-supervisor an offering me the great opportunity to perform my master work in her laboratory On the other hand, I really sincerely thank for her great support not only the idea but also her value time to solve the problem that incidentally faced during this research I particularly thank her for sharing her well-guided expertise in the field of characterization of cellulolytic enzyme, and for giving assistance supported I would like to offer a big thank to Dr Nguyen Hoang Anh, head of Central Laboratory, gave me a lot of advice and suggestion for my methodology Last but not least, I am deeply grateful to my beloved parents, namely, Mr SVAY KOU, Mrs SOUT HUN, and my beloved siblings This successful result could not be obtained if without their encouragement and support Hanoi, May 10th, 2017 Master candidate Samley MAM ii TABLE OF CONTENTS DECLARATION i ACKNOWLEDGEMENT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi THESIS ABSTRACT vii PART I – INTRODUCTION 1.1 Introduction 1.2 Objective 1.2.1 General objective 1.2.2 Specific objectives PART II –LITERATURE REVIEW 2.1 Cellulase 2.1.1 General information of cellulase 2.1.2 Classification of cellulase 2.1.3 Applications of cellulases in the industries 2.1.4 Microbial sources of cellulases 2.2 Factors affect for cellulase production by Bacilli and characterization of enzyme 13 2.2.1 General information of Bacillus sp and their cellulase production ability 13 2.2.2 Factors affect the cellulase production ability of Bacillus sp 16 2.2.3 Characterization of cellulase produced by Bacillus sp 18 PART III - MATERIALS AND METHODS 19 3.1 Materials 19 3.1.1 Bacteria strain 19 3.1.2 Medium 19 3.2 Research content 19 3.3 Methods 20 iii 3.3.1 Bacterial screening and characterization 20 3.3.2 Experiment design 21 3.3.3 Analytical method 22 3.3.4 Data analysis 24 PART IV RESULTS AND DISCUSSIONS 25 4.1 Screening Bacillus sp producing cellulase bacteria 25 4.2 Identification of selected Bacillus sp strains 28 4.2.1 Morphological characterization 28 4.2.2 Sequencing of 16S rDNA gene 28 4.2.3 Cellulase production of Bacillus pumilus B6.4 30 4.3 Characterization of cellulase activityof Bacillus pumilus B6.4 31 4.3.1 Effect of optimal temperature for cellulase activity 31 4.3.2 Effect of optimal pH for cellulase activity 32 4.3.3 Thermal stability on cellulase activity 33 4.3.4 pH stability 34 PART V CONCLUSIONS 36 REFERENCES 37 APPENDIX 47 iv LIST OF TABLES Table 2.1 Cellulase producing fungal strains 10 Table 2.2 Cellulase producing by bacterial strains 12 Table 2.3 Optimum temperature and pH of enzyme produced by Bacillus sp 14 Table 4.1 Clear zone of cellulase produced by 100 strains of Bacillus sp 26 Table 4.2 Ratio of clear zone category 27 Table 4.3.Thermal stability in enzyme activity (U/ml) 33 Table 4.4 pH stability in enzyme activity (U/ml) 35 v LIST OF FIGURES Figure 2.1 Efficiency in cooperation of members of cellulase enzyme system Figure 3.1 Diagram for bacterial characterization 20 Figure 3.2 Screening cellulase production by the agar-well diffusion method 20 Figure 4.1 Biggest clear zone of collection strains of A1.2, A1.8 and B 6.4 28 Figure 4.2 Cell morphology of strains A1.2, A1.8, and B6.4 28 Figure 4.3 Phylogenetic tree of Bacillus cereus A1.2 29 Figure 4.4 Phylogenetic tree Bacillus cereus A1.8 29 Figure 4.5 Phylogenetic tree Bacillus pumilus B 6.4 30 Figure 4.6 Effect of optimal temperature on enzyme production 31 Figure 4.7 Effect of optimal temperature on enzyme production 32 Figure 4.8 Thermal stability on enzyme production 34 Figure 4.9 pH stability on enzyme production 35 vi THESIS ABSTRACT Master candidate: Samley Mam Thesis title: Screening and characterization of cellulase in Bacillus sp Major: Food Technology Code:24181065 Educational Organization: Vietnam National University of Agriculture (VNUA) General objectives: Screening and characterization of cellulase in Bacillus sp and determine some characteristic of this enzyme Specific objectives - Screening Bacillus sp producing cellulase from the collection of Bacillus sp.; - Identification of selected strains by 16S rDNA gene sequencing; - Characterization of cellulase produced by selected strain (including optimal temperature, optimal pH, thermal stability and pH stability) Materials and Methods Materials A hundred strains of Bacillus sp were supplied by Faculty of Food Science and Technology, Vietnam National University of Agriculture There are two different sources of collection strains, one from the Muong Khuong chili sauce and the other from cow rumen Methods Microorganisms with cellulolytic activity were incubated in MT1 media and determined by the formation of clear zone around colony through the lugol overlay method; Cellulolytic bacteria were identified by using 16S rDNA gene sequencing; the neighbor - joining phylogenetic analysis was carried out with Tree view programme to show evolution relationship between selected strains and some others in the database; Cellulase was measured indirectly by spectrometric determination of reducing sugars by DNS method Optimal temperature was determined by incubating the enzyme at the various temperatures ranging from 40, 45, 50, 55, 60, 65, 70, 75, and 80°C; Thermal stability was first investigated by pre-incubating the enzyme at the various temperature including: 45, 55, 65, 75, and 85 °C for 30, 60, 90, 120, 150, 180, and 120 minutes; Residual enzyme activity was determined with % CMC in a 50 mM sodium acetate buffer with pH 5.0, at 37°C Optimal pH was determined with different buffer at various pH : 5.0, 5,5, 6.0, 6,5, 7.0, 7,5, and The pH stability was tested by pre-incubating the enzyme in variable pH buffer 5.5, 6.5, 7.5, and 8.5 at 37 °C for 30, 60, 90, 120, 150, 180 and 240 minutes vii Main findings and conclusion: Three among of 100 isolates collection strains were cultured on CMC agar plate for screening cellulase producing bacteria In which, isolates coded A1.2, A1.8 and B 6.4 having the highest diameter of clear zone as 24 mm were chosen for further studies Those strains were identified as Bacillus cereus A1.2, Bacillus cereus A1.8, and Bacillus pumilus B6.4 According to the Bacillus pumilus notified as a GRAS (FDA, 2015), it was chosen for further studies The result showed that the crude and purification enzyme activity of Bacillus pumilus B6.4 were found at 3.007 U/ml and 3.874 U/ml, respectively The optimal temperature and pH for cellulolytic cellulase of Bacillus pumilus B6.4 were found at 55 oC and 6.5, respectively On the other hand, the enzyme was maintained more than 58 % stability at 55 to 65 oC after 150 whereas the pH stability was maintained more than 56 % at 5.5 to 6.5 after 120 viii PART I INTRODUCTION 1.1 INTRODUCTION The last two decades, the using of enzyme in industrial process has significantly increased and remained a constant effort (Lima et al., 2005) According to the BBC Research (2011), the number usages of the total market for industrial enzyme reached to USD 4.4 billion in 2015 Some enzymes are commonly used may be mentioned as amylase, protease, lipase, xylanase, cellulase and so on (Bhat, 2000) The cellulase has its significance due to key role in biotechnology and industrial applications (Bhat, 2000) It has been widely utilized for bioremediation (Zahangir et al., 2005), food processing (Chandara et al., 2005), paper, pulp industry, supplement in animal feed industry (Chandara et al., 2005), textile industry (Ali and Saad, 2008), alcoholic beverage, malting and brewing (Sreeja et al., 2013), formulation of washing powders, extraction of fruit and vegetable juices, and starch processing (Camassola and Dillon, 2007) Bacillus sp is a Gram-positive aerobic or facultative endorspero-forming bacterium, rod-shaped bacterium (Shneath et al., 1986) which has been widely used in large-scale commercial enzyme application (Schallmey et al., 2004) Bacillus sp can produce a variety of extracellular cellulolytic enzymes that extremely express high cellulose degradation activities (Rastogi et al., 2010) The production of extracellular cellulase in microorganisms is significantly affected by a number of factors such as temperature, pH, aeration (Immanuel et al., 2006), agitation and medium constituents (Prasertsan and Doelle, 1987) Previously, the number of researchers in the Faculty of Food Science and Technology were investigated about some beneficial of Bacillus strains which can produce antibacteria, glucanase, protease, lactase and so on In this study, 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0,1 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 Glucose (mg/ml) Figure Glucose standard curve for the determination of the quantity of reducing sugar produced from enzyme assays and for determination of cellulolytic activity Absorbances’s were read at 540 nm Analyzing result of 16S sequence of selected strains Strain A1.2 >1st_BASE_2543389_A1_2_16sF AATAGCATTCGGGTCTATAATGCAGTCGAGCGAATGGATTAAGAGCTTGCTCTTAT GAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGG ATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGA AATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAG TTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGA TCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGG GAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAG GCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGC TGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCG CGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCA GGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGG AAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGA AATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAAC TGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTC CACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGGTTTCCGCCCCTTTAGTGCTG AAGGTTAACGCATTTAAGCACTCCCCCCTGGGGGAGTACGGCCCGCCAGGGCTTGA AACTCCAAAGGAAATTGAACGCGCGGCCCGCCACAAACCTGTCGGAGCCATGTTGG 47 TTTTAATTTCAAAACCAACCCCCCAAGAAACATTACCCATGGGCGTGGGAAGTGCC TCTTAAAAAACCCCAATCATATAAGTGGCTTTCTCCCTTCTCCGTAACCGGAATGTG ACCGAGTTTCAGTGCAAGTGTATAGTGGTGTCTCTCTCCGTGTCCCGAGAAGTGAGT GTGTGATGTAGGTCTCCCATTCCTCCTAGTCCAAAAACCCGCTAGTTGCTCTAAGTT TCGTCCGTCTCGTGTAATCCTTGTGGAGCAATCTCGGTAAGATCTGAAATTGACCAC TGGTGACTCCTACCTCGCCGGGAGAGAGACGGTGGTGCGTCGGGCCAGGTGCCGCC AACACGCACTCGCACGACCGCGCTTTCGGCTTCGCCACGCTGCGCCTGTAACAGAA CCCGCGCGCGTGGCACAC >1st_BASE_2543393_A1_2_16sR CAATTTGTCACTTAGCGGCTGGCTCCAAAAGGTTACCCCACCGACTTCGGGTGTTAC AAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCG GCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCT ACAATCCGAACTGAGAACGGTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCAGC TCTTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGAT TTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCA ACTTAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAAC ATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCTCCCGAAG GAGAAGCCCTATCTCTAGGGTTTTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCG CGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCT TTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAACTTC AGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGAC TACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGTGTCAGTTAC AGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCATATCTCTACGCATTTCACCG CTACACATGGAATTCCACTTTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGAC CCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCACCTGCGCGCG CTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTG GCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCAGCTTATTCAA CTAGCACTTGGTTCTTCCCTAACAACAGAATTTTAACGACCCGAAAGCCGTTCATCA CTTCAGGCGGGGGTTGGCTCCGTCCAGAACTTTCGGTCCATTGGCGAAAAGATTCC CTAACTGCTTGCCTTCCCGTAAGGAATCTGGGACCGGGGTTCGAGTCCCAGTTGTG GACCAAATCACCCTTCTCAAGGTCGGCTTACCCTTCGGTTGCCCTTGGTGGAGCCTG TTACCTCCACCAACTAACTTAAATGGCAAACGGGGGTCCCTTTCCCTAAAGGGAAA TTACTTAAACCCGGCCTTTTCAATTTTTGAAACCCTGGCGGGTTCCAAAATTGTTTA CCCGGGTATTAAATACGCCGGGGTTTCCCCGGGATATTTTCCCCCGTTATTTAATGG GGCGAGGGTAACCCACGTGGT A1-2 16S align GTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGT TGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGC CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAA GCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAG GGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTG TAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCT GGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATAC CCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGGTTTCCGCCCC TTTAGTGCTGAAGGTTAACGCATTTAAGCACTCCCCCCTGGGGGAGTACGGCCC - Result: Strain A1.2 is classified Bacillus cereus with identity 97% 48 Strain A1-8 >1st_BASE_2543390_A1_8_16sF ATTGGCTGGCGGCAGCTATAATGCAGTCGAGCGAATGGATTAAGAGCTTGCTCTTA TGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGG GATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCG AAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTA GTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTG ATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAG GGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAA GGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAG CTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCC GCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGC AGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTG GAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTG AAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAA CTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGT CCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGA AGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAA GGAATTGACCGGGGGCCCGCACAATCGGTGGGAGCATGTGGGTTTAATTCAAAGCC AACGCCCAAGAAACCTTACCAGGGTCTTGGACATCCTTCTGAACAACCCCTAGAAG ATAAGGGCTTCTGCCTTCCGGTAGCAGAAGTGAACAGGTAAGTGGCATGGTTAGTT CGTCGACCTCCTCTGCCGTGGAAAGGGTGAGGATTTAAGTGCCCGATCAACCTACA CGACAAGCCCTTGGATCCTTGAGTTGCCCAGCCTTGAAACTTTGGGCAAATTGTGA AGGTCGAATTGGCCTGTCAGTCCCAGCCGCAGAATAAAGGGAGGGGGGAATGGTC CGCTGATATCAATTCGCTGTGCCCATTTAGCCAACCTCTTGGTGATATCCCTCAGTG GTGCACTCACATTGGGCAGAGGGTCTAACAATGAGCGTGAGTCAAATATCTCCCGA ATTTACTGAGGCACTTACGATACCGGTTCAGCGCACACCTTCCTTTCTCTAAAATGT TTAAAGTGTGCTTAACTAACTATTAAATTAACCTTCTTCCTGAACCTGTGTAGACGT CCGGTTGGAGATCCAGATGGTGAACGTAAATCATAAC >1st_BASE_2543394_A1_8_16sR CACTCTGTCACCTTAGGCGGCTGGCTCCAAAAGGTTACCCCACCGACTTCGGGTGTT ACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCG CGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGC CTACAATCCGAACTGAGAACGGTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCA GCTCTTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGAT GATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGC CCAACTTAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCC AACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCTCCCG AAGGAGAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCT TCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATT CCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAAC TTCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTG GACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGTGTCAGT TACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCATATCTCTACGCATTTCA CCGCTACACATGGAATTCCACTTTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAAT GACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCACCTGCGC GCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTG CTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCAGCTTATT CAACTAGCACTTGTTCTTCCCTAACAACAGAATTTTACGACCCGAAAGCCTTCATCA 49 CTCAGCCGGCGTTGCTTCGTCAGACTTTCGTCCATTGCCGAAAGATTCCCTACTGCT GGCTTCCCGTAGGAGTCTGGGGCCGTGTCTCAGTCCCAGTGTGGGCCAATCACCCTT TCAAGGTCGGGTAACGCATCGGTTGCCCTTGGGGAAGGCCGTTACCTCACCCAACT AAACTAAATGCGAACGCGGGGTTCATTCCTTAAATTGAAAATCCAAAAACCCGCCT TTTCAATTTCCAAAACCTGGCGGGTTCCAAAAATGTTAACCCGGGATATAAACCCC CGGGTTTCTCCGGGAATTTTTCCCCGTCTTTTTAGGGGGGAGGGTTACCCACCGGGG TTTAACACACCTTGTCCTCCCGA >A1-8 align CGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAAC CGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCT TCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGG CTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGA CTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATG GACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAA AACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGT ACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT GGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGT CTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTG AGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATAT GGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCG AAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT GAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGC ACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACCGGGGG CCCGCACAATCGGTGGGAGCATGTGGGTTTAATTCAAAGCCAACGCCCAAGAAACC TTACCAGGGTCTTGGACATCCT Result: Strain A1.8 is classified Bacillus cereus with identity 99% Strain B6.4 >1st_BASE_2543391_B6_4_16sF TGCATGCGGCAGCTATACATGCAGTCGAGCGGACAGAAGGGAGCTTGCTCCCGGAT GTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATA ACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGA TGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTG GTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCG GCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAA TCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTT TCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAGTAACTGCTTGCA CCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTA ATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGG TTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACT GGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGC GTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACG CTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGC CGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAA CGCATTAAGCACTCCGCCTGGGGGAGTACGGTCGCCAGGACTGAAACTCAAAGGA ATTAACGAGGGGCCCGCACAAGCGGTGGGAGCATGTGGTTTTAATTTCGAAGCAAC 50 GCGCAAAAACCTTACCAGGGCCTGGACATCCCTCTGAAAAACCCTAGAAGATTAGG GCTTGTCCCTTTCCTGGGACAAGAATTGAACAGGTTGTTGTCATGGTTGTGTCTTTC TTTCTCCTTGTTCTTTGAGATGGTTGGTGTTAAAGTCCCCGCCAACCCTAGCGCCAA CTCCTTTGGATTCCTATGTACGTCCAAGGAATGTCA >1st_BASE_2543395_B6_4_16sR AAATCTGACCACCTTCGGCGGCTGGCTCATAAAGGTTACCTCACCGACTTCGGGTG TTGCAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCAC CGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCA GACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTAAACCTTGCGGTCTCG CAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGA TGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTG CCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACC CAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCC GAAGGGAAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTC TTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAAT TCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAG CTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGT GGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAG TTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTC ACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCAA TGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCG AGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCT GCCTGGCACGTATTTAGCCCGGGCCTTTCTGGTTAGGGTACCGTCAAGGTTGCAAG CAAGTTACTCTTGGCACTTGGTTCTTTCCCTAACCAACAGAAGCTTTTACGAATCCG AAAAACCTTTCATCCACTCAAGGCGGCGGTTGGCTCCGGTCAGAACTTTTCGTCCCA ATGGCGGAAAGAATTCCCTAACTGGCTGCCCTTCCCGTAAGGAATCTTGGGGCCGG GGTCCCAGGTCCCCAGTGGGGGGCCGAATCACCCTACTCCAGGGTCGGGCTTACCC AATCGGTCCCCCCTTGGTGTAAGCCCGTTAACCTCCACCCAACTAAGGCTAAATGG GCCCCCCGGGGGTCCCATTCTGGAAAATGCTACACCCGAAAAAACGGGTTTTTTAA TTCCCTTGGGACCCCTGGGGGGTGTTTAAAGGGGCATATTTTCTCTGGTAATTAATT GTTCTCGGTTTTACTCTCGGGGTGATTCTACCATTGTATTTTTCACTGCGAAGG >B6.4 Align CAAGTGCAAGAGTAACTGCTTGCACCTTGACGGTACCTAACCAGAAAGCCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTAT TGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTC AACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGA ATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAG GCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAG GATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGT TTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGGAGTACGG TCGCCAGGACTGAAACTCAAAGGAATTAACGAGGGGCCCGCACAAGCGGTGGGAG CATGTGGTTTTAATTTCGAAGCAACGCGCAAAAACCTTACCAGGG Result: B6.4 is clasified Bacillus pumilus with identity 97% 51 ... General objectives: Screening and characterization of cellulase in Bacillus sp and determine some characteristic of this enzyme Specific objectives - Screening Bacillus sp producing cellulase from... screen and characterization of cellulase in Bacillus sp and determines some characteristic of this enzyme 1.2.2 Specific objectives - Screening Bacillus sp producing cellulase from the collection of. .. CONTENT - Screening Bacillus sp produced cellulase from the collection of isolated of Bacillus sp. ; - Identification of selected Bacillus sp producing cellulase; - Characterization of cellulase