Study on the ability to produce extracellular enzymes of lecanicillium lecanii hnl20 and factors affecting the exo enzymatic activity (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -oOo - GRADUATION THESIS STUDY ON THE ABILITY TO PRODUCE EXTRACELLULAR ENZYMES OF LECANICILLIUM LECANII HNL20 AND FACTORS AFFECTING THE EXO-ENZYMATIC ACTIVITY Student : Pham Manh Hung Department : Microbial Biotechnology Supervisor : Vu Van Hanh, Assoc Prof : Nguyen Van Giang, Assoc Prof HANOI, 01/2021 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, January 2021 Pham Manh Hung i ACKNOWLEGDEMENT It is with great pride that I hereby present my graduation thesis During this six- month long process, I have gained a lot of knowledge of this research and it has been beneficial to my development in furthering my career and my studies in the future First and foremost, I would like to express absolute gratitude to teachers in the Faculty of Biotechnology and also the Department of Microbial Biology who have given me advantageous and valuable knowledge during time learning, practising and thesis I had a lot of chances to develop professionally my skills in both academical and social environment I would like to express my appreciation to Dr Nguyen Van Giang – Lecturer in the Faculty of Biotechnology He not only has oriented themes, dedicated guide throughout the process of the thesis but also given me the in-time supporting to help me get many chances in my future career I sincerely thank the staffs in the Functional Bio-compounds Laboratory, Institute of Biotechnology, Vietnam Academy of Science and Technology for helping and providing the most favorable conditions for me during my thesis Especially, I extend my gratitude to Dr Vu Van Hanh – Head of Functional Biocompounds Laboratory for his valuable guidance and support on completion of this thesis Finally, I would like to express profound thanks to my family and numerous friends who endured this long process with me, always offering support and love during the process of learning and research Thank you Sincerely! ii CONTENTS COMMITMENT i ACKNOWLEGDEMENT ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES ix ABBREVIATION LIST .xiii ABSTRACT xiv TÓM TẮT xv PART I: INTRODUCTION 1.1 Introduction 1.2 Purposes 1.3 Requirements PART II LITERATURE REVIEW 2.1 Overview of Lecanicillium lecanii 2.1.1 Introduction 2.1.2 Morphology characteristics of L lecanii 2.1.3 Life cycle of L lecanii 2.1.4 Geographical distribution of L lecanii 2.2 Application of L lecanii 2.2.1 Myzus persicae 2.2.2 Aphis gossypii 2.3 Bioactive compounds 2.3.1 Chitinase 2.3.2 Protease 10 PART III: MATERIALS AND METHODS 11 3.1 Materials and equipment 11 iii 3.1.1 Materials 11 3.1.2 Equipment 11 3.1.3 Chemicals 12 3.1.4 Medium 13 3.1.5 Location and time: 13 3.2 Research methods 13 3.2.1 Screening the morphology, the density of conidia and studying on the activity of extracellular enzymes of HNL20 13 3.2.2 Studying on the elements affecting to the activity of L lecanii HNL20 extracellular 14 3.2.3 Determination the activity of chitinase through spectrophotometry 17 3.2.4 Determination the activity of cellulase through spectrophotometry 19 3.2.5 Determination the activity of protease through spectrophotometry 21 PART IV: RESULTS 23 4.1 Evaluating effects (Carbon sources, nitrogen sources, metals ion, pH, temperature and petroleum oil) affect to the activity of exoenzymes 23 4.1.1 The morphology of hyphae, conidia and the exoenzyme activity of L lecanii HNL20 23 4.1.2 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in PDB medium and PDB medium added Carbon sources (2% (w/v) molasses, 2% (w/v) glucose and 2% (w/v) sucrose) 25 4.1.3 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in PDB medium and PDB medium added yeast extract 27 4.1.4 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in PDB medium and PDB medium added (NH4)2SO4 29 4.1.5 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in PDB medium and PDB medium added Urea 31 iv 4.1.6 The effects of temperature (50oC, 60oC, 70oC and 80oC) to the activity of L lecanii HLN20 crude enzyme 32 4.1.7 The effects of pH (pH=3,4,5,5,7 and 8) to the activity of L lecanii HLN20 crude enzyme 35 4.1.8 The effects of petroleum oil (SK Enspray 99EC) at 0.2% (v/v) to the activity of L lecanii HLN20 crude enzyme 36 4.1.9 The effects of metal ion (K+, Na+, Ca2+, Mg2+, Zn2+ and Cu2+) to the activity of L lecanii HLN20 crude enzyme 37 4.2 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in improved medium 44 4.2.1 Determination the activity of extracellular enzymes through agar plate diffusion 44 4.2.2 Determination the activity of extracellular enzymes through spectrophotometry 45 V CONCLUSSIONS AND SUGGESTTIONS 46 5.1 Conclusions 47 5.2 Suggestions 47 REFERENCES 47 APPENDIXES 51 v LIST OF TABLES Table 3.1: The instruments and equipment were used in the research 11 Table 3.2: Chemicals were used in the research 12 Table 3.3:The standard curve of D-glucosamine 18 Table 3.4: The standard curve of glucose 20 Table 3.5: The standard curve of tyrosine 21 Table 3.6: The processes of determining the protease activity 22 Table 3.7: The processes of spectrophotometric reaction 22 Table 4.1: The results of degradation round of L lecanii HNL20 extracellular enzymes cultured in PDB medium after 5, 6,7 and days (pH=6, 28oC, shaking 150rpm) on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 24 Table 4.2: The density of L lecanii HNL20’s conidia cultured in PDB medium after 5, 6,7 and days (pH=6, 28oC, shaking 150rpm) 24 Table 4.3: The results of degradation round of L lecanii HNL20 extracellular enzymes cultured in PDB medium and PDB medium added respectively Carbon sources: 2% (w/v) molasses, 2% (w/v) glucose and 2% (w/v) sucrose (at pH=6, 28oC, shaking 150rpm) on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 26 Table 4.4: The results of degradation round of L lecanii HNL20 extracellular enzymes cultured in PDB medium and PDB medium added respectively Carbon sources: (yeast extract) at concentrations: 0.25% (w/v), 0.5% (w/v), 0.75% (w/v), 1% (w/v), 1.25% (w/v) and 1.5% (w/v) (at pH=6, 28oC, shaking 150rpm) on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 28 vi Table 4.5: The results of degradation round of L lecanii HNL20 extracellular enzymes cultured in PDB medium and PDB medium added respectively Nitrogen sources: (NH4)2SO4 at concentrations: 0.25% (w/v), 0.5% (w/v), 0.75% (w/v), 1% (w/v), 1.25% (w/v) and 1.5% (w/v) (at pH=6, 28oC, shaking 150rpm) on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 30 Table 4.6: The results of degradation round of L lecanii HNL20 extracellular enzymes cultured in PDB medium and PDB medium added respectively Nitrogen sources: Urea at concentrations: 0.25% (w/v), 0.5% (w/v), 0.75% (w/v), 1% (w/v), 1.25% (w/v) and 1.5% (w/v) (at pH=6, 28oC, shaking 150rpm) on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 32 Table 4.7: The results of degradation round of L lecanii HNL20’s exoenzymes after testing at 50oC, 60oC, 70oC and 80oC in 10 minutes on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 33 Table 4.8: The results of degradation round of L lecanii HNL20’s exoenzymes after testing at pH=3,4,5,5,7 and on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 36 Table 4.9: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating with 0.2% (v/v) petroleum oil on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 37 Table 4.10: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Na+at 5mM, 10mM vii and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 38 Table 4.11: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with K+at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 39 Table 4.12: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Ca2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 40 Table 4.13: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Mg2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 41 Table 4.14: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Zn2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 42 Table 4.15: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Cu2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 43 Table 4.17: The results of degradation round of L lecanii HNL20’s exoenzymes after culturing days in improved medium at pH=6, 28oC, shaking 150rpm on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 44 Table 4.18: OD values and activity of extracellular enzymes cultured in improved medium through spectrophotometry 45 viii LIST OF FIGURES Figure 2.1: Activating L leacnii HNL20 on PDA medium after days at 28oC and shaking 15rpm Figure 2.2: Conidiophores and conidia of L lecanii (Rasoul Zare & Gams, 2001) Figure 2.3: Dead insects caused by L lecanii Figure 2.4: The distribution of L lecanii in the world (According to website: https://www.cabi.org/isc/datasheet/56281 accessed at 8:46 AM (GMT+7), Jan 12th2021 Figure 2.5: The nymphs (left) and adults (right) of Myzus persicae Photograph by Lyle J Buss, University of Florida Figure 2.6: The nymphs and adults of Aphis gossypii Figure 2.7: The structures of chitin and chitosan (Younes & Rinaudo, 2015) Figure 4.1: The morphology of L lecanii HL20 hyphae after culturing 5, 6, 7, days in PDB medium at PH=6, 28oC and shaking 150rpm 23 Figure 4.2: The degradation round of exoenzymes after culturing 5, 6,7 and days (in PDB medium at pH=6, 28oC, shaking 150rpm) on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 24 Figure 4.3: The degradation round of L lecanii HNL20’s exoenzymes after culturing days in PDB medium and PDB medium added respectively Carbon sources: 2% (w/v) molasses, 2% (w/v) glucose and 2% (w/v) sucrose (at pH=6, 28oC, shaking 150rpm) on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 25 Figure 4.4: The degradation round of L lecanii HNL20’s exoenzymes after culturing days in PDB medium and PDB medium added respectively Nitrogen sources (yeast extract) at concentrations: ix Conc of Na+ Substrates Control (D-d) mm 5mM (D-d) mm 10mM (D-d) mm 15mM (D-d) mm CMC 11± 0.5 9± 0.6 9± 0.1 9± 0.1 Chitosan 8± 0.8 8± 0.7 6± 0.6 Casein 10± 0.4 10± 0.1 10± 0.1 9± 0.5 Table 4.10: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Na+at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein Through testing the activity of exoenzymes on substrates, the results were recognized that there was a moderate decrease in the substrates degradation of extracellular enzymes which was incubated with Na+ at other concentrations The ranges of decrease ranged from 1mm to 2mm (Table 15) 4.1.9.2 Effects of K+ A B C Figure 4.11: The degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with K+at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein Note: 1: Initial crude enzyme (control) 3: Initial crude enzyme incubated with K+ 10mM 2: Initial crude enzyme incubated with K+ 5mM 4: Initial crude enzyme incubated with K+ 15mM 38 Conc of K+ Substrates CMC Chitosan Casein Control (D-d) mm 5mM (D-d) mm 10mM (D-d) mm 15mM (D-d) mm 5± 0.5 7± 0.9 4± 0.7 5± 0.6 7± 0.8 4± 0.6 5± 0.4 6± 5± 0.7 6± 0.6 6± 0.6 5± 0.6 Table 4.11: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with K+at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein For incubating with K+, enzyme cellulase and protease increased slightly in terms of diameters of degradation round on substrates, about 1mm at more than 10mM concentrations of K+ Nevertheless, the activity of chitinase incubated with K+ (6mm) was found to be a decrease against the control sample (7mm) 4.1.9.3 Effects of Ca2+ A B C Figure 4.12: The degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Ca2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein Note: 1: Initial crude enzyme (control) 3: Initial crude enzyme incubated with Ca2+ 10mM 2: Initial crude enzyme incubated with Ca2+ 5mM 4: Initial crude enzyme incubated with Ca2+ 15mM 39 Conc of Ca2+ Substrates CMC Chitosan Casein Control (D-d) mm 5mM (D-d) mm 10mM (D-d) mm 15mM (D-d) mm 6± 0.6 6± 6± 0.6 5± 0.6 7± 0.5 6± 0.7 6± 0.7 5± 0.6 6± 0.8 5± 0.5 5± 0.5 3± 0.5 Table 4.12: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Ca2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein In terms of Ca2+, on three substrates (CMC, chitosan and casein), the same result was observed that there were reductions of exoenzymes activity Especially, when incubating with Ca2+at 15mM, the degradation rounds of enzyme protease declined about times (3mm versus 6mm) Effects of Mg2+ 4.1.9.4 A B C Figure 4.13: The degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Mg2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein Note: 1: Initial crude enzyme (control) 3: Initial crude enzyme incubated with Mg2+ 10mM 2: Initial crude enzyme incubated with Mg2+ 5mM 4: Initial crude enzyme incubated with Mg2+ 15mM 40 Conc of Mg2+ Substrates CMC Chitosan Casein Control (D-d) mm 5mM (D-d) mm 10mM (D-d) mm 15mM (D-d) mm 6± 0.5 8± 4± 0.5 7± 0.9 8± 0.5 4± 0.8 6± 0.8 8± 1.2 8± 6± 0.6 7± 0.9 4± 0.4 Table 4.13: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Mg2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein When incubated crude enzymes with Mg2+ at 5mM and 10mM, the activity of cellulase, chitinase and protease were determined to increase in comparison with the initial enzymes Conversely, when increasing the concentrations of Mg2+ to 15mM, there was an unchangeable diameters of degradation rounds 4.1.9.5 Effects of Zn2+ A B C Figure 4.14: The degradation round of HNL20’s exoenzymes after incubating respectively with Zn2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein Note: 1: Initial crude enzyme (control) 3: Initial crude enzyme incubated with Mg2+ 10mM 2: Initial crude enzyme incubated with Mg2+ 5mM 4: Initial crude enzyme incubated with Mg2+ 15mM 41 Conc of Zn2+ Substrates CMC Chitosan Casein Control (D-d) mm 5mM (D-d) mm 10mM (D-d) mm 15mM (D-d) mm 7± 0.5 7± 0.5 7± 0.8 7± 0.9 6± 0.5 6± 0.5 7± 0.7 6± 0.5 3± 0.5 6± 0.5 5± 0.9 3± 0.4 Table 4.14: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Zn2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein Based on the results of Table 4.14, Zn2+ affected strongly to the activity of L lecanii HNL20 extracellular enzymes At concentration 5nM, the activity of enzymes decreased steadily on CMC, chitosan and protease substrates but when adding more than concentrations of Zn2+, exoenzymes lost significantly activity Specially, enzyme protease lost a half of activity at concentrations 10mM and 15mM of Zn2+ 4.1.9.6 Effects of Cu2+ A B C Figure 4.15: The degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Cu2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein Note: 1: Initial crude enzyme (control) 3: Initial crude enzyme incubated with Cu2+ 10mM 2: Initial crude enzyme incubated with Cu2+ 5mM 4: Initial crude enzyme incubated with Cu2+ 15mM 42 Conc of Control 5mM 10mM 15mM (D-d) (D-d) (D-d) (D-d) mm mm mm mm CMC 7± 0.5 5± 0.5 5± 0.4 6± 0.6 Chitosan 8± 1.1 7± 0.8 7± 1.2 6± 0.8 Casein 7± 0.4 3± 0.8 2± 0.4 Cu2+ Substrates Table 4.15: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Cu2+ at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein Through the collected data of Table 4.15, it was found to be that Cu2+ did not affect deeply to the activity of cellulase and chitinase Specially, when incubating at other concentrations of Cu2+, enzyme cellulase and chitinase decreased slightly degradation round on substrates, almost 1mm However, protease lost gradually the activity when Cu2+ was added to incubate; at concentrations of 15mM, protease lost all of the activity on casein substrate Overall, as reported by effects of metal ion, the vast majority of metal ion decreased the activity of extracellular enzymes, even lost all of activity like at concentration 15mM of Zn2+ However, when adding metal ions at suitable concentrations, the activity of exoenzymes can be went up such as 10-15mM K+, 510mM Mg2+ Therefore, two improved environments were established to check the enhancement of elements to the activity of extracellular enzymes: - The components of Medium are potato 200g; Glucose 20g; liter of water; 2% (w/v) molasses; 1% (w/v) yeast extract; 1.5% (w/v) (NH4)2SO4; 0.25% (w/v) Urea; KH2PO4 10mM and MgSO4 10mM - The components of Medium are potato 200g; Glucose 20g; liter of water; 0.02% (w/v) MgSO4; 0.02% (w/v) KH2PO4 and 0.01% (w/v) NH4NO3 - Both of medium and were cultured at pH=6, 28oC, shaking 150rpm in days 43 4.2 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in improved medium 4.2.1 Determination the activity of extracellular enzymes through agar plate diffusion A B C Figure 4.16: The degradation round of L lecanii HNL20’s exoenzymes after culturing days in improved medium at pH=6, 28oC, shaking 150rpm on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein Note: 1: Crude enzyme cultured in PDB medium (control) 2: Crude enzyme cultured in Medium Medium 3: Crude enzyme cultured in Medium Control Medium Medium (D-d) mm (D-d) mm (D-d) mm CMC 6± 0.5 11± 0.7 8± 0.4 Chitosan 6± 0.6 16± 1.4 8± 0.5 7± 13± 1.5 7± Substrates Casein Table 4.17: The results of degradation round of L lecanii HNL20’s exoenzymes after culturing days in improved medium at pH=6, 28oC, shaking 150rpm on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein 44 D- d (mm) The results of degradation round of L lecanii HNL20’s exoenzymes in PDB medium, Medium and medium 18 16 14 12 10 16 13 11 8 CMC Chitosan Casein Substrates Control Medium Medium Graph 2: The degradation round of L lecanii HNL20’s exoenzymes after culturing days in improved medium at pH=6, 28oC, shaking 150rpm on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein According to the initial results of testing on substrate plates, the activity of extracellular enzymes cultured in medium was nearly times higher than those of control medium Actually, the figures for medium on CMC, chitosan and casein substrate were 11mm, 16mm and 13mm, respectively against control medium, with 6mm, 6mm and 7mm While the degradation rounds on CMC, chitosan and casein of enzymes (8mm, 8mm and 7mm, in that order) cultured in medium were gently higher than those of control medium (6mm, 6mm and 7mm) To determined exactly the activity of exoenzymes in improved medium, spectrophotometry was conducted to calculate 4.2.2 Determination spectrophotometry Cellulase Chitinase Protease the activity PDB medium Activity OD (U/ml) 0.495 60.204 0.048 0.030 0.552 0.213 of extracellular Medium Activity OD (U/ml) 0.593 72.300 0.114 0.057 0.608 0.237 enzymes through Medium Activity OD (U/ml) 0.473 57.488 0.059 0.034 0.379 0.139 Table 4.18: OD values and activity of extracellular enzymes cultured in improved medium through spectrophotometry 45 Spectrophotometry was used to determine OD values which were applied to the formula to calculate the activity of exoenzymes cultured in medium Based on the results of Table 4.18, it was recognized that medium performed effectively to help enhance the activity of cellulase, chitinase and protease These figures were recorded respectively to be 70.3 U/ml, 0.057 U/ml and 0.237 U/ml in comparison with those of PDB medium, with 60.204 U/ml, 0.03 U/ml and 0.213 U/ml, in that order For medium 2, the activity of three enzymes (cellulase, chitinase and protease) were lower than those of PDB medium, with 57.488 U/ml, 0.034 U/ml and 0.139 U/ml, in that order Actually, the activity of chitinsae is much lower than that of (Nguyen et al., 2015) with only 0.057 U/ml against 0.528 U/ml In short, although optimized medium helped to enhance the activity of enzymes, results did not meet expectations so more and more studies should be researched to carry out the optimized medium 46 V CONCLUSSIONS AND SUGGESTTIONS 5.1 Conclusions - Successfully determined L lecanii HNL20 had ability to produce extracellular enzymes such as cellulase, chitinase and protease - The activity of extracellular enzymes can be went up when adding nutrients and metal ions at suitable concentrations - Two optimized medium helped to enhance the activity of enzymes but results did not meet expectations 5.2 Suggestions - Continue to research practically at higher scales and executed more studies to verify other bio compounds for enhancement of countering more pests - Finding optimal culturing conditions for Lecanicillium lecanii HNL20 to produce the optimal amount and activity of enzymes and further production of biopesticides 47 REFERENCES Abbas, W., & Mohammed, A (2019) Efficacy of entomopathogenic fungi Verticillium Lecanii and Isaria Fumosorosea against myzus persicae under laboratory conditions Plant Archives, 19, 1416-1419 Aqueel, M A., & Leather, S R (2013) Virulence of Verticillium lecanii (Z.) against cereal aphids; does timing of infection affect the 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