Study on capacity for producing exitracellular enzymes of beauveria bassiana hnb20 and factors affecting those enzymes activity (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -oOo - GRADUATION THESIS STUDY ON CAPACITY FOR PRODUCING EXTRACELLULAR ENZYMES OF BEAUVERIA BASSIANA HNB20 AND FACTORS AFFECTING THOSE ENZYMES ACTIVITY Student : Nguyen Van Hieu Class : K61CNSHE Vu Van Hanh Assoc Prof Supervisor : Nguyen Van Giang Assoc Prof Hanoi 2/2021 COMMITMENT I with this, declare that all the data and results that I have provided in this study are true, accurate, and not used in any other reports I also assure that the literature cited in the thesis indicated the origin and all help is thankful Hanoi February 2021 Nguyen Van Hieu i ACKNOWLEDGEMENT In fact, there are no successes without associated with support or assistance, whether more or less, directly or indirectly by others This project consumed huge amount of work, enthusiasm and dedication Still, implementation would not have been possible if we did not have a support of many people and organizations Therefore, we would like to extend our sincere gratitude to all of them Firsts of all, I would like expressing sincere thanks to the School Board, the Dean of Biotechnology Faculty, and all teachers have imparted to me the knowledge is advantageous and valuable during time learning, training, and implementation thesis Through working, I did not only gain much knowledge but more importantly, I also had a great chance to sharpen my skills in a professional working environment I have developed myself both academically, professionally, and socially I would like to express our deep and sincere gratitude to my supervisors, Assoc Prof Vu Van Hanh, PhD, Head of Functional Bio-compounds Labratory, Institute of Biotechnology, VietNam Academy of Science and Technology, Assoc Prof Nguyen Van Giang, PhD, Lecturer of Microbiology Department, Faculty of Biotechnology, Viet Nam National University of Agriculture for giving me the opportunity to complete this thesis and providing invaluable guidance through this thesis His dynamism, vision, sincerity and motivation have deeply inspired us He has taught us methodology to contribute a thesis and to present that as much as possible It was a great privilege and honor to work and study under his guidance We would also like to thank him for his friendship, empathy, and great sense of humor We are extending our heartfelt thanks to his wife, family for their acceptance for him to inspect our project Beside our instructor, I express my special thanks all of all members in Functional Bio-compounds Labratory, Institute of Biotechnology, VietNam Academy of Science and Technology for their effort during this thesis as much as they could We are extremely grateful to my family for their love, prayers, caring and sacrifices for educating and preparing us for our future Finally, our thanks go to all the people who have supported our group to complete the project directly or indirectly Hanoi, February, 2021 Sincerely, Nguyen Van Hieu ii CONTENTS COMMITMENT i ACKNOWLEDGEMENT ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES vii ABBREVIATION LIST viii ABSTRACT ix TÓM TẮT xi PART I INTRODUCTION PART II LITERATURE REVIEW 2.1 Brief of Beauveria bassiana 2.2 Mode of action of Beauveria bassiana 2.3 Effects of Beauveria bassiana to non-target organisms 2.3.1 Effects on plants 2.3.2 Effects on honey bees, earthworms, pollinators and other beneficial arthropods 2.3.3 Effects on aquatic organisms 2.3.4 Effects on mammals and human health 2.3.5 Extracellular enzymes produced by Beauveria bassiana 2.3.5.1 Lipases 2.3.5.2 Protease 2.3.5.3 Chitinases 2.3.5.4 Cellulase 10 Part III MATERIALS AND METHODS 12 3.1 Materials and equiments 12 3.1.1 Materials 12 3.1.2 Equiments 12 3.1.3 Chemical 12 3.2 Media 14 iii 3.3 Location and time study 14 3.4 Research methods 14 3.4.1 The capacity for producing extracellular enzyme and optimal time culture 14 3.4.2 Effects of Carbon soures to extracellular enzyme activity of HNb20 15 3.4.3 Effects of Nitrogen soures to extracellular enzyme activity of HNb20 15 3.4.4 Effects of pH, temperature, petroleum oil, metal ion to extracellular enzyme activity of HNb20 15 3.4.5 Optimal medium for extracellular enzyme activity of HNb20 16 PART IV RESULTS AND DISCUSSION 22 4.1.Screening mycelium of Beauveria bassiana HNb20 under microscope and optimal time for cultured 22 4.2.Effect of Carbon sources 24 4.3.Effect of Nitrogen sources 25 4.4.Effects of other factors : petroleum oil, pH, temperature and metal ion 29 4.4.1.Effect of petroleum oil 29 4.4.2.Effect of temperature 30 4.4.3.Effect of pH 31 4.4.4.Effect of Na+ 32 4.4.5.Effect of K+ 33 4.4.6.Effect of Ca2+ 34 4.4.7.Effect of Mg2+ 35 4.4.8.Effect of Zn2+ 36 4.4.9.Effect of Cu2+ 37 PART V CONCLUSION AND SUGGESTION 39 5.1 Conclusion 40 5.2 Suggestion 40 REFERENCES 40 APPENDIX 43 iv LIST OF TABLES Table Susceptible hosts of B bassiana from various insect orders Table List of equipments 12 Table Chemicals were used in this thesis 13 Table 3 Construct Tyrosine standard graph 17 Table Protease reaction process 17 Table Protease color reaction 18 Table Construct D-Glucosamine standard graph 19 Table Construct Glucose standard graph 20 Table Spore concentration of Beauveria bassiana 23 Table Zone clearance enzymes of HNb20 after cultivated in PDB at fifth day, sixth day, seventh day, eighth day on A: 0.1% CMC, B: 0.2% Casein,C: 0.1 % Chitosan (D-d, mm) 24 Table Zone clearance enzymes of HNb20 which cultivated in media that added (Molasses, saccarose, glucose) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 25 Table 4 Zone clearance enzymes of HNb20 which cultivated in media that added high yeast extract on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 26 Table Zone clearance enzymes of HNb20 which cultivated in media that added urea on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 27 Table Zone clearance enzymes of HNb20 which cultivated in media that added (NH4)2SO4 on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 29 Table Zone clearance enzymes of HNb20 under action of petroleum oil on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 30 v Table Zone clearance enzymes of HNb20 under action of temperatures (50℃, 60℃, 70℃, 80 ℃) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 30 Table Zone clearance enzymes of HNb20 after changed by different pH levels (3, 4, 5, 6, 7, 8) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 32 Table 10 Zone clearance enzymes of HNb20 under action of Na + on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 33 Table 11 Zone clearance enzymes of HNb20 under action of K + on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 34 Table 12 Zone clearance enzymes of HNb20 under action of Ca 2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 35 Table 13 Zone clearance enzymes of HNb20 under action of Mg2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 36 Table 14 Zone clearance enzymes of HNb20 under action of Zn 2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 36 Table 15 Zone clearance enzymes of HNb20 under action of Cu 2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 37 Table 16 Zone clearance enzymes of HNb20 were cultured in MT1, MT2, PDB on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 39 Table A OD and enzyme activity values 44 vi LIST OF FIGURES Figure 1: Infection cycle of B.bassiana Figure A: Growth of B bassiana on PDA medium; B: Morphological feature of B bassiana spores 22 Figure Screening hyphea of Beauveria bassiana HNb20 under microscope (400x) 23 Figure Test enzymes activity of HNb20 was cultivated in PDB after days, days, days, days on A: 0.1% CMC, B: 0.2% Casein,C: 0.1 % Chitosan 23 Figure 4.Test enzymes activity of HNb20 cultured in media that added (Molasses, saccarose, glucose) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 24 Figure Test enzymes activity of HNb20 cultured in media that added high yeast extract on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 25 Figure Test enzymes activity of HNb20 cultured in media that added urea on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 27 Figure Test enzymes activity activity of HNb20 cultured in media that added (NH4)2SO4 on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 28 Figure Test enzymes activity of HNb20 under action of petroleum oil on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 29 Figure Test enzymes activity of HNb20 under action of temperatures (50℃, 60℃, 70℃, 80 ℃) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 30 vii Figure 10 Test enzymes activity of HNb20 after changed by different pH levels (3, 4, 5, 6, 7, 8) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 31 Figure 11 Test enzymes activity of HNb20 under action of Na + on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 32 Figure 12 Test enzymes activity of HNb20 under action of K + on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 33 Figure 13 Test enzymes activity of HNb20 under action of Ca2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 34 Figure 14 Test enzymes activity of HNb20 under action of Mg2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 35 Figure 15 Test enzymes activity of HNb20 under action of Zn 2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 36 Figure 16 Test enzymes activity of HNb20 under action of Zn 2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 37 Figure 17 Test enzymes activity of HNb20 were cultured in MT1, MT2, PDB on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 38 viii ABBREVIATION LIST B.bassiana Beauveia bassiana CMC Carboxymethyl cellulose DNS 3,5-Dinitrosalicylic acid kDa Kilodalton OD Optical density PDA Potato Detroxes Agar PDB Potato Detroxes Broth ix Table Zone clearance enzymes of HNb20 under action of petroleum oil on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 20 ± 0.3 23 ± 0.4 17 ± 0.2 0.2% petroleum oil 18 ± 0.3 20 ± 0.4 15 ± 0.2 After added petroleum oil to raw enzyme we obtained the reduce of enzymes activity 11.76% on Chitosan substrate, 13.04% on Casein substrate, 10% on CMC substrate 4.4.2 Effect of temperature Figure Test enzymes activity of HNb20 under action of temperatures (50℃, 60℃, 70℃, 80 ℃) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan With PDB was control sample 50, 60, 70, 80 enzymes were affected at corresponding temperatures 50℃, 60℃, 70℃, 80 ℃ 30 Table Zone clearance enzymes of HNb20 under action of temperatures (50℃, 60℃, 70℃, 80 ℃) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 18 ± 0.3 21 ± 0.4 19 ± 0.2 50℃ 17 ± 0.3 21 ± 0.4 18 ± 0.2 60℃ 14 ± 0.3 21 ± 0.4 18 ± 0.2 70℃ 17 ± 0.3 14 ± 0.4 18 ± 0.2 80℃ 11 ± 0.3 ± 0.4 10 ± 0.2 4.4.3 Effect of pH A B C Figure 10 Test enzymes activity of HNb20 after changed by different pH levels (3, 4, 5, 6, 7, 8) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 31 Table Zone clearance enzymes of HNb20 after changed by different pH levels (3, 4, 5, 6, 7, 8) on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 14 ± 0.3 17 , (24*) ± 0.4 15 (11*) ± 0.2 pH = 13 ± 0.4 ± 0.2 pH = 15 ± 0.4 ± 0.2 pH = 14 ± 0.3 17 ± 0.4 15 ± 0.2 pH = ± 0.3 17* ± 0.4 6* ± 0.2 pH = ± 0.3 18* ± 0.4 7* ± 0.2 pH = 8 ± 0.3 19* ± 0.4 7* ± 0.2 Note : Compare ( value* ) in the same column because of different results of control sample on two tested dishes After cultivated pH of raw enzyme solution equal 5, so zone clearance at pH=5 was same size with zone clearance of control sample The highest enzymes activity at pH=5, and lowest enzymes activity at pH=3 4.4.4 Effect of Na+ C B A Figure 11 Test enzymes activity of HNb20 under action of Na+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan With PDB was control sample, Na+ (5 mM, 10 mM, 15 mM) were Na+ concentrations in raw enzyme solution 32 Table 10 Zone clearance enzymes of HNb20 under action of Na+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 14 ± 0.3 23 ± 0.4 12 ± 0.2 Na+ mM 12 ± 0.3 22 ± 0.4 10 ± 0.2 Na+ 10 mM 10 ± 0.3 20 ± 0.4 10 ± 0.2 Na+ 15 mM ± 0.3 18 ± 0.4 10 ± 0.2 Na+ concentration at mM; 10 mM; 15 mM on CMC substrate made enzyme activity declined respectively: 14.29%; 28.57%; 35.71% Similarly, it also decreased on Casein substrate: 4.35%; 13.04%; 21.74% On Chitosan substrate chitinase activity reduced 16.67% at the concentration of Na+ = 5mM, and maintained at next concentrations 4.4.5 Effect of K+ A B C Figure 12 Test enzymes activity of HNb20 under action of K+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan With PDB was control sample, K+ (5 mM, 10 mM, 15 mM) were K+ concentrations in raw enzyme solution 33 Table 11 Zone clearance enzymes of HNb20 under action of K+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 11 ± 0.3 18 ± 0.4 12 ± 0.2 K+ mM 10 ± 0.3 16 ± 0.4 11 ± 0.2 K+ 10 mM 10 ± 0.3 16 ± 0.4 10 ± 0.2 K+ 15 mM 10 ± 0.3 15 ± 0.4 ± 0.2 On CMC substrate, cellulase activity reduced 9.09% at the concentration of K+ = 5mM, and maintained at the next concentrations K+ concentration at mM; 10 mM; 15 mM on chitosan substrate made enzyme activity declined respectively: 8.33%; 16.67%; 25% Similarly, it also decreased on Casein substrate: 11.11%; 11.11%; 16.67% 4.4.6 Effect of Ca2+ A B C Figure 13 Test enzymes activity of HNb20 under action of Ca2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan With PDB was control sample, Ca2+ (5 mM, 10 mM, 15 mM) were Ca2+ concentrations in raw enzyme solution 34 Table 12 Zone clearance enzymes of HNb20 under action of Ca2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 15 ± 0.3 23 ± 0.4 11 ± 0.2 Ca2+ mM 12 ± 0.3 21 ± 0.4 11 ± 0.2 Ca2+ 10 mM 11 ± 0.3 21 ± 0.4 10 ± 0.2 Ca2+ 15 mM 10 ± 0.3 19 ± 0.4 10 ± 0.2 Ca2+ concentration at mM; 10 mM; 15 mM on CMC substrate made enzyme activity declined respectively: 20 %; 26.67%; 33.33% Similarly, it also decreased on Casein substrate: 8.70%; 8.7%; 17.39% On Chitosan substrate, chitinase activity maintained at the concentration of Ca+ = mM, reduced 9.09% at Ca+ = 10 mM, and maintained at Ca+ = 15 mM (compared to Ca+ = 10 mM) 4.4.7 Effect of Mg2+ A B C Figure 14 Test enzymes activity of HNb20 under action of Mg2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan With PDB was control sample, Mg2+ (5 mM, 10 mM, 15 mM) were Ca2+ concentrations in raw enzyme solution 35 Table 13 Zone clearance enzymes of HNb20 under action of Mg2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 10 ± 0.3 17 ± 0.4 12 ± 0.2 Mg2+ mM ± 0.3 15 ± 0.4 11 ± 0.2 Mg2+ 10 mM ± 0.3 13 ± 0.4 10 ± 0.2 Mg2+ 15 mM ± 0.3 11 ± 0.4 10 ± 0.2 Mg2+ concentration at mM; 10 mM; 15 mM on CMC substrate made enzyme activity declined respectively: 20%; 20%; 30% Similarly, it also decreased on Casein substrate: 11.76%; 23.53%; 35.29%, on chitosan substrate reduced 8.3%; 16.67%; 16.67% 4.4.8 Effect of Zn2+ A B C Figure 15 Test enzymes activity of HNb20 under action of Zn2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan With PDB was control sample, Zn2+ (5 mM, 10 mM, 15 mM) were Ca2+ concentrations in raw enzyme solution 36 Table 14 Zone clearance enzymes of HNb20 under action of Zn2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 10 ± 0.3 17 ± 0.4 13 ± 0.2 Zn2+ mM ± 0.3 15 ± 0.4 10 ± 0.2 Zn2+ 10 mM ± 0.3 13 ± 0.4 ± 0.2 Zn2+ 15 mM ± 0.3 11 ± 0.4 ± 0.2 Zn2+ concentration at mM; 10 mM; 15 mM on CMC substrate made enzyme activity declined respectively: 20%; 20%; 30% Similarly, it also decreased on Casein substrate: 11.76%; 23.53%; 35.29%, on chitosan substrate reduced 23.98%; 38.46%; 38.46% 4.4.9 Effect of Cu2+ A B C Figure 16 Test enzymes activity of HNb20 under action of Zn2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan With PDB was control sample, Cu2+ (5 mM, 10 mM, 15 mM) were Ca2+ concentrations in raw enzyme solution 37 Table 15 Zone clearance enzymes of HNb20 under action of Cu2+ on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 11 ± 0.3 17 ± 0.4 12 ± 0.2 Cu2+ mM ± 0.3 13 ± 0.4 ± 0.2 Cu 2+ 10 mM ± 0.3 ± 0.4 ± 0.2 Cu 2+ 15 mM ± 0.3 ± 0.2 On CMC substrate, cellulase activity fell sharply at Cu2+ centration equal mM 81.82%, reduced to 90.91% at next concentration ( compared to control sample) On Casein substrate, protease activity also decreased 23.53% at mM, 76.47% at 10mM, and no active at 15mM On Chitosan substrate made enzyme activity declined respectively: 50%; 66.67%; 91.67% From all results above the medium for highest protease was added 2% glucose (Eliana Tiemi Ito, March 2007), 0.75% urea for best cellulase and 0.75% high yeast extract for chitinase Consequently, we combined that in media called MT1, MT1: PDB + 2% glucose + 0.75% high yeast extract + 0.75% urea MT2: PDB + 0.02% MgSO4.7H2O + 0.02% KH2PO4 + 0.01% NH4NO3 Compared enzymes activity which culivated in three kind of media MT1, MT2 and PDB A B C Figure 17 Test enzymes activity of HNb20 were cultured in MT1, MT2, PDB on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 38 Table 16 Zone clearance enzymes of HNb20 were cultured in MT1, MT2, PDB on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) CMC Casein Chitosan PDB 22 ± 0.3 20 ± 0.4 13 ± 0.2 MT1 42 ± 0.4 ± 0.2 MT2 22 ± 0.3 20 ± 0.4 15 ± 0.2 MT1 for highest protease activity (0.431 U/ml), but it also reduced cellulase and chitinase activity MT2 was quite similar to PDB, so we could used PDB for save experiment cost (Appendix) 39 PART V CONCLUSION AND SUGGESTION 5.1 Conclusion - After days of cultured Beauveria bassiana HNb20 for the highest enzyme activity, the enzymes were heat resistant 80 ℃, adapted pH from to (cellulase to 8), preferably at pH of - Petroleum oil and salt of studied metals both reduce the activity of three enzymes PDB medium supplemented with 2% glucose + 0.75% yeast extract + 0.75% urea for highly reactive protease - PDB added 0.75% yeast extract for highly active chitinase - PDB adds 0.75% urea for highly active cellulase 5.2 Suggestion - Study on properties for improving quantitative of extracellular enzyme that Beauveria bassiana HNb20 can produce - Testing the ability to control pests and plant diseases by biological plant proteaction which use Beauveria bassiana HNb20 40 REFERENCES Vietnamese publication Cao Cường, Nguyễn Đức Lượng (2003), Khảo sát trình cảm ứng enzyme chitinase cellulase Trichoderma harzianum ảnh hưởng hai enzyme lên nấm bệnh Sclerotium rolfsii, Báo cáo khoa học, Hội nghị Cơng nghệ Sinh học tồn quốc, NXB Khoa học Kỹ thuật, Hà Nội: 321-324 Nguyễn Thị Hà (2012:),Tối ưu hóa điều kiện ni cấy chủng Aspergillus Protuberus sinh tổng hợp enzyme chitinase phân lập từ rừng ngập mặn Cần Giờ Tạp chí Khoa học, 2012:22b, 26-35 Foreign publication Austwick PKC 1980 The pathogenic aspects of the use of fungi: The need for risk analysis and registration of fungi Ecological Bulletin (Stockholm) 31:91_102 Burges HD 1981 Safety, safety testing and quality control 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35_62 25 Yoon CS, Bae SH, Song HH, Park HS, Lee C 2003a Effect of synnemata of Beauveria bassiana on the properties of noodle and the baking qualities of bread Institute of Food Technologists, Annual Meeting, Chicago, Abstract 60D-16 26 Yoon CS, Yu KW, Bae SH, Song HH, Park HS, Lee C 2003b Chemical properties and physiological activities of synnemata of Beauveria bassiana Journal of Microbiology and Biotechnology 13:125_133 42 APPENDIX Standard curve and results of enzymes activity TYROSINE STANDARD CURVE 1.4 y = 5.1509x + 0.054 R² = 0.9036 1.2 OD660 0.8 0.6 0.4 0.2 0 0.05 0.1 0.15 0.2 0.25 TYROSINE CONCENTRATION OD540 GLUCOSE STANDARD CURVE 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 y = 0.8101x + 0.0729 R² = 0.9394 0.2 0.4 0.6 0.8 GLUCOSE CONCENTRATION (µM) 43 1.2 D-GLUCOSAMINE STANDARD CURVE 0.5 y = 0.1208x - 0.0244 R² = 0.9591 0.4 OD535 0.3 0.2 0.1 0 -0.1 D-GLUCOSAMINE CONCENTRATION (µM) Table A OD and enzyme activity values Cellulase Sample OD540 Protease Enzyme activity Sample (U/ml) OD660 Chitinase Enzyme activity Sample (U/ml) OD535 Enzyme activity (U/ml) PDB 0.673 74 PDB 0.862 0.345 PDB 0.116 0.058 MT1 0.203 16 MT1 1.063 0.431 MT1 0.067 0.038 MT2 0.656 72 MT2 0.784 0.316 MT2 0.124 0.061 0.75% Urea 0.778 87 2% Glucose 0.353 0.75% High yeast extract 0.147 0.071 0.879 44