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

Nghiên cứu khả năng sản xuất enzyme ngoại bào của Lecanicillium lecanii HNL20 và các yếu tố ảnh hưởng đến hoạt động enzym ngoại bào.

• Tác giả: Pham Manh Hung
• Người hướng dẫn: Assoc. Prof. Vu Van Hanh
• Trường học: Vietnam National University of Agriculture
• Chuyên ngành: Microbial Biotechnology
• Đề tài: Study on the ability to produce extracellular enzymes of Lecanicillium lecanii HNL20 and factors affecting the exo-enzymatic activity
• Loại tài liệu: Graduation thesis
• Năm xuất bản: 2021
• Địa điểm: Hanoi

Mục lục chi tiết

• COMMITMENT
• ACKNOWLEGDEMENT
• 1. PART I: INTRODUCTION
• 1.1. Introduction
• 1.2. Purposes
• 1.3. Requirements
• 2. PART II: OVERVIEW OF LECANICILLIUM LECANII
• 2.1. Introduction
• 2.2. Morphology characteristics of L. leacnii HNL20 on PDA medium after 6 days at 28oC and shaking 15rpm
• 2.3. Life cycle of L.
• 2.4. Geographical distribution of L.
• 3. PART III: MATERIALS AND METHODS
• 3.1. Materials and equipment
• 3.2. Location and time
• 3.3. Screening the morphology, the density of conidia and studying on the activity of extracellular enzymes of HNL20
• 3.4. Studying on the elements affecting to the activity of L. lecanii HNL20 extracellular
• 3.5. Determination the activity of chitinase through spectrophotometry
• 3.6. Determination the activity of cellulase through spectrophotometry
• 3.7. Determination the activity of protease through spectrophotometry
• 4. PART IV: RESULTS
• 4.1. Evaluating effects (Carbon sources, nitrogen sources, metals ion, pH, temperature and petroleum oil) affect to the activity of exoenzymes
• 4.2. The morphology of hyphae, conidia and the exoenzyme activity of L.
• 4.3. 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)
• 4.4. The results of testing exoenzyme’s activity of L. lecanii HNL20 cultured in PDB medium and PDB medium added yeast extract
• 4.5. The results of testing exoenzyme’s activity of L. lecanii HNL20 cultured in PDB medium and PDB medium added (NH4)2SO4
• 4.6. The results of testing exoenzyme’s activity of L. lecanii HNL20 cultured in PDB medium and PDB medium added Urea
• 4.7. The effects of temperature (50oC, 60oC, 70oC and 80oC) to the activity of L. lecanii HLN20 crude enzyme
• 4.8. The effects of pH (pH=3,4,5,5,7 and 8) to the activity of L. lecanii HLN20 crude enzyme
• 4.9. The effects of petroleum oil (SK Enspray 99EC) at 0.2% (v/v) to the activity of L. lecanii HLN20 crude enzyme
• 4.10. The effects of metal ion (K+, Na+, Ca2+, Mg2+, Zn2+ and Cu2+) to the activity of L. lecanii HLN20 crude enzyme
• 4.11. The results of testing exoenzyme’s activity of L. lecanii HNL20 cultured in improved medium
• 4.12. Determination the activity of extracellular enzymes through agar plate diffusion
• 4.13. Determination the activity of extracellular enzymes through spectrophotometry
• CONCLUSSIONS AND SUGGESTTIONS

Tóm tắt

I. Giới thiệu về Lecanicillium lecanii HNL20

Lecanicillium lecanii HNL20 là một loại nấm ký sinh gây bệnh cho côn trùng, thuộc phân lớp Ascomycota, lớp Sordariomycetes. Nấm này được biết đến với khả năng sản xuất enzyme ngoại bào như cellulase, protease và chitinase. Những enzyme này có vai trò quan trọng trong việc phá vỡ hàng rào bảo vệ của côn trùng, cho phép nấm xâm nhập và tiêu diệt chúng. Nghiên cứu này nhằm mục đích xác định khả năng sản xuất enzyme của chủng HNL20 và các yếu tố ảnh hưởng đến hoạt tính enzyme. Theo nghiên cứu, Lecanicillium lecanii có thể được sử dụng như một biện pháp sinh học thay thế cho thuốc trừ sâu hóa học, giúp bảo vệ mùa màng mà không gây hại cho môi trường.

II. Khả năng sản xuất enzyme ngoại bào

Khả năng sản xuất enzyme ngoại bào của Lecanicillium lecanii HNL20 được đánh giá thông qua việc nuôi cấy trong môi trường PDB. Kết quả cho thấy, hoạt tính của các enzyme như cellulase, chitinase và protease phụ thuộc vào sự phát triển của hệ sợi và mật độ bào tử. Nghiên cứu đã chỉ ra rằng, việc tối ưu hóa môi trường nuôi cấy có thể nâng cao đáng kể hoạt tính enzyme. Cụ thể, môi trường nuôi cấy cải tiến với các thành phần như rỉ mật và chiết xuất nấm men đã cho thấy sự gia tăng hoạt tính enzyme lên đến 70.237 U/ml cho cellulase. Điều này chứng tỏ rằng việc điều chỉnh các yếu tố môi trường có thể ảnh hưởng tích cực đến khả năng sản xuất enzyme của nấm.

III. Các yếu tố ảnh hưởng đến hoạt tính enzyme

Nghiên cứu đã xác định nhiều yếu tố ảnh hưởng đến hoạt tính enzyme của Lecanicillium lecanii HNL20, bao gồm nguồn carbon, nguồn nitơ, pH, nhiệt độ và ion kim loại. Các thí nghiệm cho thấy, nguồn carbon như glucose và rỉ mật có tác động tích cực đến sự phát triển của nấm và hoạt tính enzyme. Nhiệt độ tối ưu cho sự phát triển của nấm là từ 21 đến 24°C, trong khi pH từ 6 đến 7 cho thấy hoạt tính enzyme cao nhất. Ngoài ra, các ion kim loại như K+, Na+, Ca2+ cũng có ảnh hưởng đến hoạt tính enzyme, với các nồng độ khác nhau cho kết quả khác nhau. Việc hiểu rõ các yếu tố này sẽ giúp tối ưu hóa quy trình sản xuất enzyme trong tương lai.

IV. Giá trị và ứng dụng thực tiễn

Nghiên cứu về khả năng sản xuất enzyme ngoại bào của Lecanicillium lecanii HNL20 không chỉ có giá trị trong lĩnh vực sinh học mà còn có ứng dụng thực tiễn trong nông nghiệp. Việc phát triển các biện pháp sinh học như nấm Lecanicillium có thể giúp giảm thiểu việc sử dụng thuốc trừ sâu hóa học, từ đó bảo vệ sức khỏe con người và môi trường. Hơn nữa, việc sản xuất enzyme từ nấm có thể được ứng dụng trong nhiều lĩnh vực khác nhau như chế biến thực phẩm, sản xuất sinh học và xử lý chất thải. Nghiên cứu này mở ra hướng đi mới cho việc phát triển các sản phẩm sinh học an toàn và hiệu quả trong nông nghiệp.

Trích đoạn nội dung tài liệu

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 Bio- compounds 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. Sincerely! ii CONTENTS COMMITMENT .iii LIST OF TABLES.

v LIST OF FIGURES. ix ABBREVIATION LIST. xv PART I: INTRODUCTION. Overview of Lecanicillium lecanii.

Morphology characteristics of L. Life cycle of L. Geographical distribution of L. 10 PART III: MATERIALS AND METHODS.

Materials and equipment. Location and time:. Screening the morphology, the density of conidia and studying on the activity of extracellular enzymes of HNL20. Studying on the elements affecting to the activity of L.

lecanii HNL20 extracellular. Determination the activity of chitinase through spectrophotometry. Determination the activity of cellulase through spectrophotometry. Determination the activity of protease through spectrophotometry.

21 PART IV: RESULTS. Evaluating effects (Carbon sources, nitrogen sources, metals ion, pH, temperature and petroleum oil) affect to the activity of exoenzymes. The morphology of hyphae, conidia and the exoenzyme activity of L. 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). The results of testing exoenzyme’s activity of L. lecanii HNL20 cultured in PDB medium and PDB medium added yeast extract. The results of testing exoenzyme’s activity of L.

lecanii HNL20 cultured in PDB medium and PDB medium added (NH4)2SO4. The results of testing exoenzyme’s activity of L. lecanii HNL20 cultured in PDB medium and PDB medium added Urea. The effects of temperature (50oC, 60oC, 70oC and 80oC) to the activity of L.

lecanii HLN20 crude enzyme. The effects of pH (pH=3,4,5,5,7 and 8) to the activity of L. lecanii HLN20 crude enzyme. The effects of petroleum oil (SK Enspray 99EC) at 0.2% (v/v) to the activity of L.

lecanii HLN20 crude enzyme. The effects of metal ion (K+, Na+, Ca2+, Mg2+, Zn2+ and Cu2+) to the activity of L. lecanii HLN20 crude enzyme. The results of testing exoenzyme’s activity of L.

lecanii HNL20 cultured in improved medium. Determination the activity of extracellular enzymes through agar plate diffusion. Determination the activity of extracellular enzymes through spectrophotometry. CONCLUSSIONS AND SUGGESTTIONS.

51 v LIST OF TABLES Table 3.1: The instruments and equipment were used in the research .2: Chemicals were used in the research .3:The standard curve of D-glucosamine .4: The standard curve of glucose .5: The standard curve of tyrosine.6: The processes of determining the protease activity .7: The processes of spectrophotometric reaction .1: The results of degradation round of L. lecanii HNL20 extracellular enzymes cultured in PDB medium after 5, 6,7 and 8 days (pH=6, 28oC, shaking 150rpm) on substrates: (A) 0.2: The density of L. lecanii HNL20’s conidia cultured in PDB medium after 5, 6,7 and 8 days (pH=6, 28oC, shaking 150rpm) .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) 0.4: 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: 0.5: 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: 0.6: 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: 0.7: 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) 0.8: The results of degradation round of L.

lecanii HNL20’s exoenzymes after testing at pH=3,4,5,5,7 and 8 on substrates: (A) 0.9: The results of degradation round of L. lecanii HNL20’s exoenzymes after incubating with 0.10: 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) 0.11: The results of degradation round of L. lecanii HNL20’s exoenzymes after incubating respectively with K+at 5mM, 10mM and 15mM on substrates: (A) 0.12: The results of degradation round of L.

lecanii HNL20’s exoenzymes after incubating respectively with Ca2+ at 5mM, 10mM and 15mM on substrates: (A) 0.13: The results of degradation round of L. lecanii HNL20’s exoenzymes after incubating respectively with Mg2+ at 5mM, 10mM and 15mM on substrates: (A) 0.14: The results of degradation round of L. lecanii HNL20’s exoenzymes after incubating respectively with Zn2+ at 5mM, 10mM and 15mM on substrates: (A) 0.15: The results of degradation round of L. lecanii HNL20’s exoenzymes after incubating respectively with Cu2+ at 5mM, 10mM and 15mM on substrates: (A) 0.17: The results of degradation round of L.

lecanii HNL20’s exoenzymes after culturing 6 days in improved medium at pH=6, 28oC, shaking 150rpm on substrates: (A) 0.18: OD values and activity of extracellular enzymes cultured in improved medium through spectrophotometry. 45 viii LIST OF FIGURES Figure 2. leacnii HNL20 on PDA medium after 6 days at 28oC and shaking 15rpm .2: Conidiophores and conidia of L.3: Dead insects caused by L.4: The distribution of L. lecanii in the world (According to website: https://www.org/isc/datasheet/56281 accessed at 8:46 AM (GMT+7), Jan 12th2021.5: The nymphs (left) and adults (right) of Myzus persicae.

Photograph by Lyle J. Buss, University of Florida.6: The nymphs and adults of Aphis gossypii .7: The structures of chitin and chitosan (Younes & Rinaudo, 2015) .1: The morphology of L. lecanii HL20 hyphae after culturing 5, 6, 7, 8 days in PDB medium at PH=6, 28oC and shaking 150rpm.2: The degradation round of exoenzymes after culturing 5, 6,7 and 8 days (in PDB medium at pH=6, 28oC, shaking 150rpm) on substrates: (A) 0.3: The degradation round of L. lecanii HNL20’s exoenzymes after culturing 6 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) 0.4: The degradation round of L.

lecanii HNL20’s exoenzymes after culturing 6 days in PDB medium and PDB medium added respectively Nitrogen sources (yeast extract) at concentrations: ix 0.5: The degradation round of L. lecanii HNL20’s exoenzymes after culturing 6 days in PDB medium and PDB medium added respectively Nitrogen sources (NH4)2SO4 at concentrations: 0.6: The degradation round of L. lecanii HNL20’s exoenzymes after culturing 6 days in PDB medium and PDB medium added respectively Nitrogen sources (Urea) at concentrations: 0.7: The degradation round of L. lecanii HNL20’s exoenzymes after testing at 50oC, 60oC, 70oC and 80oC in 10 minutes on substrates: (A) 0.8: The degradation round of L.

lecanii HNL20’s exoenzymes after testing at pH=3,4,5,5,7 and 8 on substrates: (A) 0.9: The degradation round of L. lecanii HNL20’s exoenzymes after incubating with 0.10: The degradation round of L. lecanii HNL20’s exoenzymes after incubating respectively with Na+at 5mM, 10mM and 15mM x on substrates: (A) 0.11: The degradation round of L. lecanii HNL20’s exoenzymes after incubating respectively with K+at 5mM, 10mM and 15mM on substrates: (A) 0.12: The degradation round of L.

lecanii HNL20’s exoenzymes after incubating respectively with Ca2+ at 5mM, 10mM and 15mM on substrates: (A) 0.13: The degradation round of L. lecanii HNL20’s exoenzymes after incubating respectively with Mg2+ at 5mM, 10mM and 15mM on substrates: (A) 0.14: The degradation round of HNL20’s exoenzymes after incubating respectively with Zn2+ at 5mM, 10mM and 15mM on substrates: (A) 0.15: The degradation round of L. lecanii HNL20’s exoenzymes after incubating respectively with Cu2+ at 5mM, 10mM and 15mM on substrates: (A) 0.16: The degradation round of L. lecanii HNL20’s exoenzymes after culturing 6 days in improved medium at pH=6, 28oC, shaking 150rpm on substrates: (A) 0.

44 xi LIST OF GRAPHS Graph 1: The degradation round of L. lecanii HNL20’s exoenzymes after testing at 50oC, 60oC, 70oC and 80oC in 10 minutes on substrates: (A) 0. 34 Graph 2: The degradation round of L. lecanii HNL20’s exoenzymes after culturing 6 days in improved medium at pH=6, 28oC, shaking 150rpm on substrates: (A) 0.

45 Graph 3: The tyrosine standard curve. 51 Graph 4: The glucose standard curve. 51 Graph 3: The D-glucosamine standard curve. 52 xii ABBREVIATION LIST L.

lecanii Lecanicillium lecanii PDA Potato Dextrose Agar PDB Potato dextrose agar CMC Carboxymethyl cellulose TCA Trichloroacetic acid OD Optical density DNS 3,5-Dinitrosalicylic acid w/w Weight/weight w/v Weight/volume xiii ABSTRACT Lecanicillium lecanii HNL20 was cultured in PDB medium and to determine the ability to produce extracellular enzymes (cellulase, protease and chitinase) and screened the morphology of hyphae and conidia. The activity of extracellular enzymes was determined to depend on the development of hyphae and density of conidia. Based on the study of effecting factors, two improved environments were established to check the enhancement of elements to the activity of extracellular enzymes. The components of Medium 1 are potato 200g; Glucose 20g; 1 liter of water; 2% (w/v) molasses; 1% (w/v) yeast extract; 1.25% (w/v) Urea; KH2PO4 10mM and MgSO4 10mM.The components of Medium 2 are potato 200g; Glucose 20g; 1 liter of water; 0.02% (w/v) KH2PO4 and 0.

Through spectrophotometry, the activity of cellulase, chitinase and protease was recorded respectively to be 70.237 U/ml for Medium 1. In terms of medium 2, the results were observed to be 57.139 U/ml, in that order. xiv TÓM TẮT Chủng Lecanicillium lecanii HNL20 được nuôi cấy trong môi trường PDB để xác định khả năng sản sinh các enzyme ngoại bào (cellulase, protease and chitinase) và quan sát hình thái của hệ sợi và bào tử. Theo đó, hoạt độ của các enzyme này phụ thuộc vào sự phát triển của hệ sợi và mật độ của bào tử.

Dựa vào sự nghiên cứu các yếu tố ảnh hưởng, hai môi trường cải tiến được tạo ra để kiểm tra các yếu tố giúp tăng hoạt tính của các enzyme ngoại bào.