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. Supervisor : Nguyen Van Giang Assoc. 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.
Vu Van Hanh, PhD, Head of Functional Bio-compounds Labratory, Institute of Biotechnology, VietNam Academy of Science and Technology, Assoc. 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. iii LIST OF TABLES.
v LIST OF FIGURES. vii ABBREVIATION LIST. Brief of Beauveria bassiana. Mode of action of Beauveria bassiana.
Effects of Beauveria bassiana to non-target organisms. Effects on plants. Effects on honey bees, earthworms, pollinators and other beneficial arthropods. Effects on aquatic organisms.
Effects on mammals and human health. Extracellular enzymes produced by Beauveria bassiana. MATERIALS AND METHODS. Materials and equiments.
Location and time study. The capacity for producing extracellular enzyme and optimal time culture. Effects of Carbon soures to extracellular enzyme activity of HNb20. Effects of Nitrogen soures to extracellular enzyme activity of HNb20.
Effects of pH, temperature, petroleum oil, metal ion to extracellular enzyme activity of HNb20. Optimal medium for extracellular enzyme activity of HNb20. RESULTS AND DISCUSSION .Screening mycelium of Beauveria bassiana HNb20 under microscope and optimal time for cultured .Effect of Carbon sources .Effect of Nitrogen sources .Effects of other factors : petroleum oil, pH, temperature and metal ion .Effect of petroleum oil .Effect of temperature.Effect of pH .Effect of Na+ .Effect of Ca2+.Effect of Mg2+ .Effect of Zn2+.Effect of Cu2+. CONCLUSION AND SUGGESTION.
43 iv LIST OF TABLES Table 2. Susceptible hosts of B. bassiana from various insect orders. List of equipments.
Chemicals were used in this thesis. Construct Tyrosine standard graph. Protease reaction process. Protease color reaction.
Construct D-Glucosamine standard graph. Construct Glucose standard graph. Spore concentration of Beauveria bassiana. Zone clearance enzymes of HNb20 after cultivated in PDB at fifth day, sixth day, seventh day, eighth day on A: 0.
Zone clearance enzymes of HNb20 which cultivated in media that added (Molasses, saccarose, glucose) on A: 0. Zone clearance enzymes of HNb20 which cultivated in media that added high yeast extract on A: 0. Zone clearance enzymes of HNb20 which cultivated in media that added urea on A: 0. Zone clearance enzymes of HNb20 which cultivated in media that added (NH4)2SO4 on A: 0.
Zone clearance enzymes of HNb20 under action of petroleum oil on A: 0. Zone clearance enzymes of HNb20 under action of temperatures (50℃, 60℃, 70℃, 80 ℃) on A: 0. Zone clearance enzymes of HNb20 after changed by different pH levels (3, 4, 5, 6, 7, 8) on A: 0. Zone clearance enzymes of HNb20 under action of Na + on A: 0.
Zone clearance enzymes of HNb20 under action of K + on A: 0. Zone clearance enzymes of HNb20 under action of Ca 2+ on A: 0. Zone clearance enzymes of HNb20 under action of Mg2+ on A: 0. Zone clearance enzymes of HNb20 under action of Zn 2+ on A: 0.
Zone clearance enzymes of HNb20 under action of Cu 2+ on A: 0. Zone clearance enzymes of HNb20 were cultured in MT1, MT2, PDB on A: 0. OD and enzyme activity values. 44 vi LIST OF FIGURES Figure 2.
1: Infection cycle of B. bassiana on PDA medium; B: Morphological feature of B. Screening hyphea of Beauveria bassiana HNb20 under microscope (400x). Test enzymes activity of HNb20 was cultivated in PDB after 5 days, 6 days, 7 days, 8 days on A: 0.Test enzymes activity of HNb20 cultured in media that added (Molasses, saccarose, glucose) on A: 0.
Test enzymes activity of HNb20 cultured in media that added high yeast extract on A: 0. Test enzymes activity of HNb20 cultured in media that added urea on A: 0. Test enzymes activity activity of HNb20 cultured in media that added (NH4)2SO4 on A: 0. Test enzymes activity of HNb20 under action of petroleum oil on A: 0.
Test enzymes activity of HNb20 under action of temperatures (50℃, 60℃, 70℃, 80 ℃) on A: 0. Test enzymes activity of HNb20 after changed by different pH levels (3, 4, 5, 6, 7, 8) on A: 0. Test enzymes activity of HNb20 under action of Na + on A: 0. Test enzymes activity of HNb20 under action of K + on A: 0.
Test enzymes activity of HNb20 under action of Ca2+ on A: 0. Test enzymes activity of HNb20 under action of Mg2+ on A: 0. Test enzymes activity of HNb20 under action of Zn 2+ on A: 0. Test enzymes activity of HNb20 under action of Zn 2+ on A: 0.
Test enzymes activity of HNb20 were cultured in MT1, MT2, PDB on A: 0. 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 ABSTRACT The capacity for producing extracellular enzymes protease, cellulase, chitinase of Beauveria bassiana HNb20 were demonstrated by agar radial diffusion. The best enzymes activity was cultured in 28℃, 150 rpm after 6 days. These enzymes still work at 80℃ , the best pH for enzyme activity is 5.
The highest protease activity value reached 0.431 U/ml in PDB + 2% glucose + 0.75% high yeast extract + 0.75% urea , cellulase activity value was 87 U/ml in PDB + 0.75% urea , chitinase activity value was 0.071 U/ml in PDB + 0.75% high yeast extract. x TÓM TẮT Khả năng sản sinh ra ba loại enzyme ngoại bào là protease, cellulase và chitinase của Beauveria bassiana HNb20 đã được chứng minh bằng phương pháp khuếch tán đĩa thạch. Thời gian nuôi cấy để hoạt độ cả ba loại enzyme này tốt nhất là sau 6 ngày ở 28℃ lắc 150 vòng/phút. Ba loại enzyme này vẫn còn hoạt tính ở 80 ℃.
Hoạt tính của protease, chitinase, cellulase tốt nhất tại pH bằng 5. Hoạt tính protease cao nhất đạt được là 0.431 U/ml trong môi trường PDB + 2% glucose + 0.75% cao nấm men + 0.75% ure , hoạt độ cellulase là 87 U/ml trong môi trường PDB + 0.75% ure , hoạt độ chitinase là 0.071 U/ml trong môi trường PDB + 0.75% cao nấm men. INTRODUCTION In the recent few decades, Viet Nam has always been in the Pesticide Crisis, and the number of importing crop protection has not decreased. From 1981 to 1986, the amount of crop protection is around 9 thousand tons and up to 75.8 thousand tons imported until 2010.
In 5 years lately, Viet Nam spent 500- 700 million US dollars on crop protection. In these amounts, there are 48% of herbicides equal with 19 thousand tons, and the rest are 16 thousand tons of pesticides and 900 tons of growth stimuli. The active volume agent of crop protection per hectares of plant per year in Viet Nam is scaling up to 2kg while in other countries is at 0,2 to 1kg per hectares (According to http. On average, Viet Nam used as much as 40% of the four most used pesticides in the world (According to http.
This implication lead to destroy the environment, that harm the pest, but also human. Children and any young developing organisms are particularly vulnerable to them, even when exposed to very low level. The expose to pesticide can have several side effects such as memory loss, loss of coordination, reduce the speed of response to stimuli, reduced the visual ability, altered mood or behavior, reduced motor skills, asthma, allergies, and hypersensitivity. More serious conditions such as cancer, hormone disruption, problems with production and fetal development have also been linked to the consumption of pesticides.
The use of insecticides is broadly spread, not simply on agricultural field, but also in house, school,. The intensive use of pesticide bring not simply consequences to what you eat, the air you breathe, the water you drink. Therefore, the solution for problem is using bioinsecticides, that are friendly to human, animals and enviroment. One kind of bioinsecticide is mentioning derived from entomopathogenic fungus Beauveria bassiana.
As the conidia of Beauveria bassiana germinate and germ tube penetrates the cuticle, using a specific series of enzymes, which in degrade the lipids, protein and chitin in the insect cuticle. Consequently, ‘ Study on capacity for producing extracellular enzymes of Beauveria bassiana HNb20 and factors affecting those enzyme activity’ demonstrated the ability 1 of producing protease, chitosanase, cellulase by Beauveria bassiana and factors affecting enzymes activity in order to increase effective bioinsecticide derived from that entomopathogenic fungus. Purposes: - Demonstrated the ability to generate three types of extracellular enzymes including cellulase, protease, chitinase of Beauveria bassiana HNb20 - Evaluating the factors affecting three types of enzymes such as cellulase, protease, chitinase, thereby giving the most suitable culture conditions for the highest enzyme activity Requirements: - Demonstrated the ability to generate three types of extracellular enzymes including cellulase, protease, chitinase of Beauveria bassiana HNb20 - Determine the culture date for the best enzyme activity - Testing the effect of Nitrogen, Carbon sources to enzyme activity - Testing the effect of pH, temperature, petroleum oil, metal ion to enzyme activity - Find the medium for the best enzymes activity 2 PART II. Brief of Beauveria bassiana Initial investigation by Agostino Bassi di Lodi (1835) on the disease of silkworms (Bombyx mori) which he called ‘white muscardine’ verified for the first time that a fungus (Beauveria bassiana) can cause diseases in insects.