Study on improvement of the cellulase activity of trichoderma mutated by gamma irradiation

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ================== UNDERGRADUATE THESIS PROJECT: STUDY ON IMPROVEMENT OF THE CELLULASE ACTIVITY OF Trichoderma MUTATED BY GAMMA IRRADIATION HANOI - 2022/05 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ============== UNDERGRADUATE THESIS PROJECT: STUDY ON IMPROVEMENT OF THE CELLULASE ACTIVITY OF Trichoderma MUTATED BY GAMMA IRRADIATION Student’s name : Nguyen Thi Lan Anh Class : K62CNSHE Major : Biotechnology Instructor : Tran Bang Diep MSc Bui Thi Thu Huong Ph.D HANOI – 2022/05 COMMITMENT I hereby declare that this entire thesis was completed by my own knowledge, ability to learn, and scientific research, under the dedicated guidance of Tran Bang Diep MSc - Manager of Radiation Technology Department, Hanoi Irradiation Center, Vietnam Atomic Energy Institute, and Bui Thi Thu Huong Ph.D - Deputy Head of Biology Department, Faculty of Biotechnology, Vietnam National University of Agriculture, and colleagues in the radiation technology room The figures, images, and results presented are truthful and accurate The sources of information cited in this thesis are clearly indicated I accept all responsibility for my promises to the Council Hanoi, May 10th, 2022 Student Nguyen Thi Lan Anh i ACKNOWLEDGMENT During the time of studying, researching, and completing the thesis, I have received the guidance and dedicated guidance of teachers, the help and encouragement of friends, colleagues, and family With all my heart, I would like to express my deepest respect and gratitude to Tran Bang Diep MSc - Manager of Radiation Technology Department, Hanoi Irradiation Center, Vietnam Atomic Energy Institute and Bui Thi Thu Huong Dr — Deputy Head of Biology Department, Faculty of Biotechnology, Vietnam National University of Agriculture has constantly helped, guided me with enthusiasm, closeness, and created the best conditions during my thesis work I would like to express my deep gratitude to the members of the Radiation Technology Department, Hanoi Irradiation Center, Vietnam Atomic Energy Institute for their enthusiastic help and valuable comments as well as for making great efforts Their guidance and support supported me to carry out this thesis I would also like to thank the leadership of Hanoi Irradiation Center, Vietnam Atomic Energy Institute for creating conditions for me to have time to study during my time here I would like to express my sincere thanks to the Faculty of Biotechnology, Vietnam National University of Agriculture for creating all conditions for me to study and research in this field throughout the thesis Ultimately, I would like to express my deep gratitude to my family members and close friends who have always been by my side, encouraging and encouraging me throughout my study and research process HaNoi, May 10th, 2022 Student Nguyen Thi Lan Anh ii CONTENTS COMMITMENT I ACKNOWLEDGMENT II CONTENTS III LIST OF ABBREVIATION VI LIST OF FIGURES AND CHARTS VIII ABSTRACT IX INTRODUCTION 1 RATIONABLE TARGET RESEARCH CONTENTS CHAPTER LITERATUTURE REVIEW 1.1 CELLULASE 1.1.1 Overall 1.1.2 Classification 1.1.3 Mechanism of cellulase 1.1.4 Source of cellulase 1.1.5 Applications of Cellulases 10 1.1.5.1 Industrial applications 10 1.1.5.2 Environment treatment 12 1.1.5.5 Biofuel production 14 1.2 AN OVERVIEW OF THE Trichoderma AND THE DEGRADABILITY OF Trichoderma’s CELLULOSE 14 1.2.1 Trichoderma morphological and structural features 14 1.2.2 Reproduction of Trichoderma 16 1.2.3 The capacity of Trichoderma to decompose cellulose 17 1.3 APPLICATION OF IONIZING RADIATION IN MUTAGENESIS OF MICROORGANISMS – ACCELERATING CELLULASE PRODUCTION OF Trichoderma 19 1.3.1 Ionizing radiation 20 1.3.1.1 Definition 20 iii 1.3.2 Radioactive source 21 1.3.3 Biological effects of ionizing radiation 22 1.3.4 The biological effects caused by ionizing radiation 26 1.3.5 Advanced resistance irradiation in irradiation treatment 27 CHAPTER MATERIALS, CONTENTS, AND RESEARCH METHODS 31 2.1 MATERIALS AND EQUIPMENT 31 2.1.1 Materials 31 2.1.2 Chemicals 32 2.1.3 Equipment 32 2.2 RESEARCH LOCATION AND TIMES 33 2.3 RESEARCH METHOD 33 2.3.1 Seed storage method 33 2.3.2 Irradiation treatment method for spores’ solution 34 2.3.3 Determine spore quantity method 34 2.3.4 Cellulase qualitative and quantitative methods 35 2.3.4.1 Diffusion method on PDA agar plate 35 2.3.4.2 DNS method (axit 3,5 dinitrosalicylic) 36 2.3.5 Sellect to colonies radiation resistance with hight cellulase secreation method 38 2.3.6 Assessing the stability of the mutant Trichoderma strain method 38 2.3.7 Analysis data methods 39 3.1 SELECTION OF Trichoderma STRAIN WITH HIGH CELLULASE ACTIVITY 40 3.1.1 Speed of growth and colony morphology of Trichoderma strains 40 3.1.2 Selection of Trichoderma strain with high and stable cellulase activity 44 3.1.2.1 The cellulase ability of Trichoderma strains 44 3.1.2.2 T reesei VTCC 31572 strain’s characteristic 47 3.2.1 Effecting of irradiation on the viability of T reesei VTCC 31572 48 3.2.2 The effect of irradiation treatment on cellulase secretion of Trichoderma strains 49 iv 3.3 MUTATED Trichoderma STRAIN WITH HIGH CELLULASE SECRETION 51 3.3.1 Screening for radiation-resistant Trichoderma colonies with high cellulase secretion 51 3.4 SCREENING FOR HIGH CELLULASE SECRETION Trichoderma STRAIN MUTATED BY RADIATION 53 3.4.1 CMCase activity 53 3.4.2 Fpase activity 54 3.5 ASSESSING THE STABILITY OF THE MUTANT Trichoderma STRAIN 54 3.5.1 The morphology of resistance radiation lines with high cellulase secretion 54 3.5.2 The stability of the mutant Trichoderma strain 56 CONCLUSION AND RECOMMENDATIONS 58 CONCLUSION 58 RECOMMENDATIONS 58 REFERENCES 60 APPENDIX 66 PPENDIX 67 v LIST OF ABBREVIATION Abbreviation Definition RNA Axit ribonucleic CBD Cellulose binding domain CFU Colony Forming Units CMC Carboxymethyl cellulose DNA Axit deoxyribonucleic DNS Axit 3,5- dinitrosalicylic FPase Filter paper activity Gy Gray HC Hydrolysis capacity kDa Kilodalton LET Linear Energy Transfer PDA Potato dextrose agar vi LIST OF TABLES Table 1.1 Values of LET in water medium (source: ICRU 1995) 22 Table 2.1 Trichoderma strains fungi with the ability to synthesize cellulase 31 Table 3.1 Colonies morphology of Trichoderma strains cultured after days on PDA medium 40 Table 3.2 The ability of cellulase secretion of Trichoderma strains 45 Table 3.3 The HC value of T reesei VTCC 31572 strain after at 28 oC for 24 cultured and inculcation at for days 47 Table 3.4 Cellulose degradation in T reesei colonies treated with different doses of irradiation 50 Table 3.5 HC values of colonies mutated capable high cellulase secretion by irradiation 52 Table 3.6 CMCase activity of VTCC(r) I-1 radiation resistance line with high cellulase secretion capacity after times inoculation 56 vii LIST OF FIGURES AND CHARTS Figure 1.1 Synergistic action of Cellulose (Um Y and Kim K, 2013) Figure 1.2 Mechanism of hydrolysis of cellulose and cellulosome Figure 1.3 The morphology of Trichoderma colony on PDA medium (7 days), 15 Figure 1.4 Morphological characterization of T viride 17 Figure 1.5 The inventory of cellulolytic encoding genes in Trichoderma species Numbers on the plots indicate number of nucleotide sequences deposited in NCBI GenBank database (Strakowska et al., 2014) 19 Figure 1.6 Direct and indirect effects of ionizing radiation to DNA 23 Figure 1.7 Types of damage caused by the direct impact of ionizing radiation on DNA 25 Figure 3.1 Some Trichoderma strain’s CMC degradation ring after culture at 28oC for 24 hours and incubation at 37 oC for days 46 Figure 3.2 T reesei VTCC 31572 strain on PDA medium after days cultured (A- The surface of colonies, B- The backside of colonies, CReproductive organs) 48 Figure 3.3 The correlation between the spore number T reesei VTCC 31572 surviving in spore’s epidemic and radiation dose 49 Figure 3.4 Does-treated T reesei spores’ solution at 700 doses, diluted 105 times 51 Figure 3.5 The CMC degradation ring of T reesei strain after 24 hours cultured at 28 oC and incubated for days at 37 oC 52 Figure 3.6 CMCase and Fpase activity of survivals’ T reesei colonies with high cellulase secretion by irradiation 53 Figure 3.7 T reesei VTCC(r) I-1 wild type colony cultured on PDA medium at 28oC 55 (A- Colony morphology after 24 hours; B- The ability of pigment secretion after days; C- The morphology of spore after days) 55 viii 3.4 SCREENING FOR HIGH CELLULASE SECRETION Trichoderma STRAIN MUTATED BY RADIATION 3.4.1 CMCase activity The semi-quantitative (HC value determination) and quantitative methods in this study both used the same low-viscosity CMC substrate in medium culture However, CMC was recommended to be used as a preferred measure of endoglucanase activity and was not suitable for all cellulase types The advantage of the method using CMC substrates was that the endoglucanase activity can be evaluated through many parameters, such as the viscosity of liquid cellulose medium culture, the degree of Hydrolysis Capacity (HC value) or the amount of reducing sugars formed Figure 3.6 CMCase and Fpase activity of survivals’ T reesei VTCC colonies with high cellulase secretion by irradiation The obtained results show that the potential colonies have higher CMCase activity than wild type The CMCase activity of colonies VTCC(r) I-1 and VTCC(r) I-5 which were potential colonies obtained from irradiated strains of T reesei were both greater than 2.5U/ml While CMCase activity in VTCC(r) I1 reached 3,020 U/ml and this value was 2,557 U/ml in VTCC(r) I-5 This result was quite like the semi-quantitative results of determining the values of HC, 53 VTCC(k) I-1 and VTCC(k) I-3 as well as VTCC(r) I-1 and VTCC(r) I-5 were colonies whose HC values were superior to those of the remaining colonies (screened from the same wild type initially irradiated) 3.4.2 Fpase activity The cellulase activity that hydrolyzes the Fatman No.1 filter paper substrate (abbreviated as FPase) from T reesei wild type strain was 0.189 U/ml Using the same CMC substrate in liquid culture, but Miklaszewska et al found that the FPase activity in some Trichoderma strains was quite low, typically FPase activity of T koningii strain was 0.031 U/ml and T harzianum was 0.023 (Miklaszewska et al., 2016) While the FPase determined by Shahbazi et al was significantly higher, the FPase of T reesei was 5.77 U/ml (Shahbazi et al., 2014) Irradiation treatment not only increased the CMCase activity in T reesei VTCC 31572, but the FPase activity of these fungal strains also improved markedly All five potential colonies obtained in each strain after screening showed increased FPase activity compared with the original strain Colonies of VTCC(r) I-1 and VTCC(r) I-5 screened from irradiated T reesei strain had FPase activities of 0.434 and 0.355 U/ml, respectively (Figure 3.6) The results showed that the potential colonies in each Trichoderma strains (produced in the dose range 700-1500 Gy) had higher CMCase and FPase activities than wild type CMCase activity in potential colonies screened from irradiated T reesei strain had CMCase activity from 1.48 to 2.47 times higher than wild type, FPase activity was from 1.34 to 2.29 times higher than wild type 3.5 ASSESSING THE STABILITY OF THE MUTANT Trichoderma STRAIN 3.5.1 The morphology of resistance radiation lines with high cellulase secretion Potential colonies with superior CMCase and FPase activities were VTCC(r) I-1 screened from T reesei VTCC 31572 wild type cultured on PDA medium Their growth rates, colony morphology and spore were observed and compared with pure strains and were depicted in Figure 3.7 54 A B C T reesei (Wild type) VTCC (r) I-1 Figure 3.7 T reesei VTCC(r) I-1 wild type colony cultured on PDA medium at 28oC (A- Colony morphology after 24 hours; B- The ability of pigment secretion after days; C- The morphology of spore after days) After days of cultured on PDA medium, I noticed a difference in growth rate between the T reesei wild type and the VTCC(r) I potential mutant, reflected in their colony sizes (Figure 3.7A) The larger the colony diameter, the faster the growth rate of the strain The wild type grew more slowly but produced significantly more yellow pigment than the post-irradiation screened strain Although there were differences in growth rate and pigment secretion during culture between wild type and screened strains after irradiation, their spore morphology was not significantly different when observed with a digital microscope with 40x magnification (Figure 3.7 C) Reproductive organs emantled with phialides, conidiophore and conidiophore fully appeared in both wild type and strains after irradiation In the irradiation strain, I observed double 55 or triple branching at the same position; the new branches have a longer length than the arising branch Cylindrical body, the conidiospores were elongated and elliptical, and the peduncle can be easily observed 3.5.2 The stability of the mutant Trichoderma strain VTCC(r) I-1 colonies were preserved by inoculation on inclined agar tubes containing PDA medium After switching to liquid medium culture and collecting crude enzyme extracts, CMCase and FPase activities were quantified in each generation after each inoculation (Table 3.6) Table 3.6 CMCase activity of VTCC(r) I-1 radiation resistance line with high cellulase secretion capacity after times inoculation Number of CMCase activity (U/ml) Fpase activity (U/ml) 3,020±0,035a 0,434±0,008ab 3,152±0,075a 0,408±0,006a 2,982±0,121a 0,445±0,015b 3,114±0,104a 0,427±0,012ab inoculation times Note: The different characteristics at the same column are significant for the differentiation which statistical significance between the average values with reliability about 95 percent T reesei VTCC 31572 wild type had CMCase and FPase activities of 1.220 and 0.189 U/ml, respectively The quantitative results showed that strain VTCC(r) I-1 screened from strain T reesei had a CMCase activity of 3.02 (greater than 2.47 times), FPase activity of 0.434 U/ml (greater than the 2.29 times) VTCC(r) I-1 strain activity was stable at least after consecutive generations (4 subcultures, month apart) The CMCase and FPase activities of the first and fourth generation mutant lines were also tested and confirmed by the Department of Microbial Biotechnology-Institute of Food Industry 56 In addition, decoding and analyzing differences in the endoglucanaseencoding gene of the radiation-resistant Trichoderma lines with high cellulase capacity revealed a mutation leading to the substitution of the amino acid alanine radical to threonine at position 325 and an additional one arginine triad at position 413 on the endoglucanase-encoding gene of mutant line VTCC(r)-1 compared with wild type These changes may have increased the cellulase biosynthetic level of VTCC(r) I-1 (the results were not presented in the thesis) 57 CONCLUSION AND RECOMMENDATIONS CONCLUSION The T reesei VTCC 31572 strain with high and stable cellular secretion were selected and used as metagenic agent from eleven (11) wild type strains The T reesei VTCC 31572 strain spores were radiated at dose range from to 2500 Gy on Co-60 source at Hanoi Irradiation Center showed that the survival rate of spores gradually decreased with increasing dose D10 of T reesei VTCC 31572 strain was about 400 Gy The survival rate at 500 Gy was 0.65%, respectively, and non-viable T reesei spores were detected at 2500 Gy Irradiation treatments were capable high cellulase-secret mutant colonies The highest mutation rate was 12.57-15.87 % at the dose range from 700 to 1500 Gy when the number of surviving cells after irradiation decreased from 4-6 Log units compared with the wild type without irradiation From radiation resist-colonies (dose range from 700 to 1500 kGy) and high cellulase-secret colonies screened on PDA medium supplemented with CMC and red Congo, five potential mutant lines have superior cellular capacity and stable was selected In particular, the VTCC(r)-1 line had a CMCase activity of 3.02 U/ml (2.47 times higher than the wild type) and a FPase activity of 0.434 U/ml (2.29 times higher than the wild type) RECOMMENDATIONS To the results of the research are quickly application in the field, I proposal to continues some next studies bellow: - Study to determine the optimal fermentation parameters of mutant strains by radiation 58 - Study on creating products to degrading rice straw and agricultural byproducts from mutated Trichoderma strains with predominance cellulase production ability - Identification of rice straw degrading of probiotics produced at the laboratory and field scale 59 REFERENCES ENGLISH Van Hung N., Maguyon-Detras M.C., Migo M.V., et al., 2020, Rice straw overview: Availability, properties, and management practices Sustainable rice straw management Springer international publishing, Cham, pp 1–13 Xu F., Wang J., Chen S., Qin W., Yu Z., Zhao H., Xing X., H., 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