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

22 4.1.Screening mycelium of Beauveria bassiana HNb20 under microscope and optimal time for cultured .... ABSTRACT The capacity for producing extracellular enzymes protease, cellulase,

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

ACTIVITY

Vu Van Hanh Assoc Prof

Nguyen Van Giang Assoc Prof

Hanoi 2/2021

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

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 1

PART II LITERATURE REVIEW 3

2.1 Brief of Beauveria bassiana 3

2.2 Mode of action of Beauveria bassiana 3

2.3 Effects of Beauveria bassiana to non-target organisms 5

2.3.1 Effects on plants 5

2.3.2 Effects on honey bees, earthworms, pollinators and other beneficial arthropods 5

2.3.3 Effects on aquatic organisms 6

2.3.4 Effects on mammals and human health 6

2.3.5 Extracellular enzymes produced by Beauveria bassiana 7

2.3.5.1 Lipases 7

2.3.5.2 Protease 8

2.3.5.3 Chitinases 9

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

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 Ca

34

4.4.7.Effect of Mg

35

4.4.8.Effect of Zn

36

4.4.9.Effect of Cu

37

PART V CONCLUSION AND SUGGESTION 39

5.1 Conclusion 40

5.2 Suggestion 40

REFERENCES 40

APPENDIX 43

LIST OF TABLES

Table 2 1 Susceptible hosts of B bassiana from various insect orders 3

Table 3 1 List of equipments 12

Table 3 2 Chemicals were used in this thesis 13

Table 3 3 Construct Tyrosine standard graph 17

Table 3 4 Protease reaction process 17

Table 3 5 Protease color reaction 18

Table 3 6 Construct D-Glucosamine standard graph 19

Table 3 7 Construct Glucose standard graph 20

Table 4 1 Spore concentration of Beauveria bassiana 23

Table 4 2 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 4 3 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 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 4 5 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 4 6 Zone clearance enzymes of HNb20 which cultivated in media that added (NH

)

SO

on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 29

Table 4 7 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

Table 4 8 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 4 9 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 4 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 4 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 4 12 Zone clearance enzymes of HNb20 under action of Ca

on A:

0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 35 Table 4 13 Zone clearance enzymes of HNb20 under action of Mg

on A:

0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 36 Table 4 14 Zone clearance enzymes of HNb20 under action of Zn

on A:

0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 36 Table 4 15 Zone clearance enzymes of HNb20 under action of Cu

on A:

0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan (D-d, mm) 37 Table 4 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 1 OD and enzyme activity values 44

days, 6 days, 7 days, 8 days on A: 0.1% CMC, B: 0.2% Casein,C:

0.1 % Chitosan 23 Figure 4 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 4 5 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 4 6 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 4 7 Test enzymes activity activity of HNb20 cultured in media that

added (NH

)

SO

on A: 0.1% CMC, B: 0.2% Casein, C: 0.1 %

Chitosan 28 Figure 4 8 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 4 9 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

Figure 4 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 4 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 4 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 4 13 Test enzymes activity of HNb20 under action of Ca

on A:

0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 34 Figure 4 14 Test enzymes activity of HNb20 under action of Mg

on A:

0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 35 Figure 4 15 Test enzymes activity of HNb20 under action of Zn

on A:

0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 36 Figure 4 16 Test enzymes activity of HNb20 under action of Zn

on A:

0.1% CMC, B: 0.2% Casein, C: 0.1 % Chitosan 37 Figure 4 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

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

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

PART I 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.//baonamdinh.com.vn) On average, Viet Nam used as much as 40% of the four most used pesticides in the world (According to http.//vusta.vn)

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

of producing protease, chitosanase, cellulase by Beauveria bassiana and factors

affecting enzymes activity in order to increase effective bioinsecticide derived from that entomopathogenic fungus

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

PART II LITERATURE REVIEW

2.1 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 This

observation led to establishment of the concept of biological control of

entomopathogens of various cash crops B.bassiana is a cosmopolitan fungi found on infected insects in both temperate and tropica regions Habitats for B bassiana range from desert soils to forests and cultivated soils Microbial

pesticides in India were included in the schedule to the Insecticide Act, 1968,

while B bassiana for commercial production and distribution was included in the Gazette of India on March 26, 1999 B bassiana has been isolated from insects of diverse orders Catalogued hosts of B bassiana are listed in Table 2.1

(Keswani, Singh, & Singh, 2013)

Table 2 1 Susceptible hosts of B bassiana from various insect orders

Coleoptera

Lathrobium brunnipes , Calvia quattuordecimguttata , Phytodectra olivacea, Otiorhynchus sulcatus , Sitona lineatus , S sulcifrons, S macularius , S hispidulus , Anthonomus pomorum, Hylaster ater

Hymenoptera Ichneumonidae, Lasius fuliginosus , Vespula spp., Bombus

pratorum

Lepidoptera Hepialus spp., Hypocrita jacobaea, Cydia nigricans

Heteroptera Picromerus bidens , Anthocoris nemorum

2.2 Mode of action of Beauveria bassiana

As in other entomopathogenic fungi, Beauveria species attack their host

insects percutaneously The infection pathway consists of the following steps: (1) attachment of the spore to the insect cuticle, (2) spore germination on

cuticle, (3) penetration through the cuticle, (4) overcoming the host immune response , (5) proliferation within the host, (6) saprophytic outgrowth from the dead host and production of new conidia

Figure 2 1: Infection cycle of B.bassiana

(Keswani et al 2013)

Spore germination and successful infection by B bassiana relies on

various factors, e.g susceptible host, host stage and certain environmental

factors, such as temperature and humidity Generally, germination of B bassiana conidia starts after about 10 hour and completed in 20 hour at 25 ℃

Afterwards, the germinated spore penetrates nonsclerotised areas of the cuticle like joints, mouthparts and between segments producing extracellular proteases and chitinases that degrade these proteinaceous and chitinous components, allowing hyphal penetration After successful penetration, the fungus invades other tissues of the host insect by extensive vegetative growth and the production of toxic secondary metabolites ultimately leading to host’s death

2.3 Effects of Beauveria bassiana to non-target organisms

2.3.1 Effects on plants

B bassiana is a typical soil dwelling fungus and has globally been used

for almost a century as an eco-friendly alternative for the control of leaf and root feeding insects Recent research has demonstrated that there are various tri-partite interactions between plant, pest insect and entomopathogenic fungi Most interesting interactions can be summarized as (a) Plants may affect the infection by the entomopathogen, (b) Plants may affect the persistence of the

entomopathogen (c) B bassiana can persist as an endophyte within plants

Another important aspect of this tritrophic interaction is the fact that toxic

metabolites of Beauveria spp may enter the plants, though such repots

validating the hazardous effects of its toxins on environmental health are available

2.3.2 Effects on honey bees, earthworms, pollinators and other beneficial arthropods

Owing to its wide host range B bassiana has been extensively used in

agricultural practices in various Asian countries since past cen tury, but an important issue raised by microbial ecologists is about the host specificity being

a strain-specific trait This is especially important when commercial products of these fungi are used on a larger scale Though there is a difference between physiological and ecological host range The physiological host range demonstrates the range of insect species that can be infected in the laboratory, while the ecological host range demonstrates which insects can be infected in nature or under field conditions Non-target insects which are infected under laboratory conditions may not necessarily be infected in nature Despite the

wide host range of B bassiana, evidence suggests that this fungus can be used

with minimal impact on beneficial organisms

2.3.3 Effects on aquatic organisms

No toxicity or pathogenicity was observed in Daphnia magna when

exposed to 1x109 conidia of Beauveria bassiana strain GHA per litre for 21

days (Goettel & Jaronski 1997) Strain GHA was also not infectious against the

grass shrimp, Palaemonetes pugio, after percutaneous and oral contamination (Genthner et al 1994b) In the mysid shrimp Americamysis bahia (formerly Mysidopsis bahia) Beauveria bassia conidia caused high mortalities, but these

were attributed to a high particulate density since heat-killed controls also proved lethal (Genthner et al 1994a) Beauvericin has been found to be highly

toxic towards Artemia salina larvae and murine cell lines and can induce apoptosis (Pascale et al 2002) In the mysid Americamysis bahia, beauvericin

was toxic at an LC50 of 0.56 mg L-1 (Genthner et al 1994)

2.3.4 Effects on mammals and human health

Safety of entomopathogenic fungi, especially B bassiana and B brongniartii,

to mammals and humans is of primary concern and has to be considered as one

of the main potential hazards of using fungi as biocontrol agents Therefore, it

is not unusual that allergic, pathogenic or toxic risks for humans and mammals have been stressed in many papers (Steinhaus 1957; Muller-Kogler 1967; Ignoffo 1973; Austwick 1980; Burges 1981; Saik et al 1990; Siegel & Shadduck 1990; Goettel et al 1997, 2001; Vestergaard et al 2003) Recently, some papers from South Korea on the addition of B bassiana to human food documents a totally new aspect of this fungus Yoon et al (2003b) reported that

extracts of B bassiana synnemata had anticoagulant and immune system

modulating activity, which could provide beneficial physiological activities for

humans In another paper, Yoon et al (2003a) found that B bassiana

synnemata could be used as an additive to wheat flour for the preparation of noodle and bread

2.3.5 Extracellular enzymes produced by Beauveria bassiana

The epicuticle, the external layer of the insect's cuticle, is hydrophobic in nature and acts as the first barrier against microbial attack A heterogeneous mix of lipids, long-chain alkenes, esters and fatty acids is the main constituent of insect cuticle Lipases are responsible for the hydrolysis of ester bonds of lipoproteins, fats and waxes found at the interior part of the insect integument They significantly penetrate the cuticle and initiate nutrient release by breaking down the integument The degradation of the epicuticle is followed by the production of fungal protease (Pr1), which degrades the proteinaceous material positioned in the procuticle A defence mechanism of insects has been identified and associated with the secretion of lactone

B, which is responsible for the inhibition of lypolytic activity, which impedes subsequent entomopathogenic infection Adhesion of the spores to the epicuticle with the help of lipase is a mandatory pre-step that initiates the degradation of fatty acids and alkenes in the cuticle waxy surface

There are two types of lipases (Lipases I and II) that have been purified to homogeneity, using column chromatography on DEAE-Toyopearl Lipase I consists of

two polypeptide chains [chain A is small peptide in size, and it conjoins with a sugar moiety, whereas chain B is a large peptide chain of 34 kDa molecular weight]; Lipase

II is a 30 kDa protein with a single polypeptide chain It was reported by Ohnishi et al., that an Aspergillus oryzae strain produced at least two kinds of extracellular lipolytic enzymes, L1 and L2 It was found that Lipase L1, a monomeric protein, has a molecular weight of 24 kDa, and it has the ability to cleave all ester bonds present in triolein

In B bassiana, the Bbcyp52x1 gene encoded the lipase activity with an

enzymatic complex known as CYP52X1 It has been proved that an additional combined activity of u hydroxylase, which is capable of adding terminal hydroxyl groups in fatty acids and epoxides, is shown by CYP52X1 CYP52 fungal enzymes were found to be flexible; the presenting isoforms showed different activities and specificities in relation to the kind of alkanes and/or fatty acids, offering a great advantage to the entomopathogenic fungi to use these substrates as nutrients Moreover, epicuticle degradation by the Bbcyp52x1 gene cluster of B bassiana has been found during the initial stages of the infection Nevertheless, after degradation of the cuticle, its role was no longer required For this reason, the breakdown of lipid substrates by the entomopathogenic fungi occurs just at the time of cuticle penetration (Mondal, Baksi, Koris, & Vatai, 2016)

2.3.5.2 Protease

Proteases build up a large group of hydrolytic enzymes that cleave the peptide bonds of proteins and break them into small peptides and amino acids Since proteases play a role in almost cellular functions, they are found in plants and animals, as well as

in microorganisms, including viruses However, proteases are extensively present in nature, and microbes also serve as a preferred source of these enzymes If we take a brief view of the industrial enzymes, 75% are hydrolases and proteases from plant, animal and microbe sources, and they account for approximately 60% of total enzyme sales Proteases are the enzymes that are considered as the most important for the

infective process, and these characteristics and the potential industrial demand for these enzymes promote the production of these enzymes

After the epicuticle has been broken down by lipases, the invading fungi produce great quantities of Pr1 (serine-protease), which degrades the proteinaceous material On the other hand, further degradation of solubilised proteins in to amino acids by amino peptidases and exopeptidases is done to provide nutrients for entomopathogenic fungi The most frequently studied proteolytic enzymes are the subtilisin-like serine-protease Pr1 and trypsin-like protease Pr2 The Pr1 gene is related to eleven isoforms that have been identified and cloned, including a metallo protease The molecular structure of subtilisin-like protease Pr1 consists of five cysteines forming two disulphide bridges, and the residual cysteine was found near the catalytic triad made of Asp39, His69 and Ser224 The activities of Pr1 and Pr2 have been determined in B bassiana These proteases are secreted during the first cuticle degradation stage, and they stimulate the signal transduction mechanism by activating protein kinase A (PKA) mediated by AMPc It has been validated that the extracellular involvement of protease Pr1 in cuticle penetration is initialised by the infection of the cuticle.(Mondal et al., 2016)

2.3.5.3 Chitinases

Chitin is a combined polymer of β-1,4 N-acetyl glucosamine and is one of the most abundant polymers in nature after cellulose It was considered the main structural component of fungal cellular walls and of exoskeletons of invertebrates Chitinases hydrolyse the β-1,4 bonds of chitin polymer, producing a predominant N, N’-diacetylchitobiose This is carried out by the break down of N-acetyl glucosamine (GlcNAc) monomer by chitobiose Chitinases are widely distributed in plants, bacteria, fungi, insects and vertebrates They collaborate with proteases to degrade the insect's cuticle and are associated with different stages of the life cycle (germination, hyphal growth, morphogenesis, nutrition and defence against competitors) of entomopathogenic fungi The genome of filamentous fungi contains chitinases responsible for various physiological functions including: a) chitin degradation in the fungal cellular walls or in the exoskeletons of arthropods used as nutrient sources; b)

remodelling of cell walls during hyphae growth, branching, hyphae fusion, autolysis and competence; c) also, protection from other fungi located in the same ecological niche

The Bbchit1 gene of B bassiana coded for a protein with a molecular weight of

33 kDa, and it was also homologous to T harzianum and Streptomyces avermitilis

MA-4680 chitinases Nonetheless, it was not similar to chitinases produced by other entomopathogenic fungi, and they indicated that there were many differences among chitinases produced by these fungi Meanwhile, the Bbchit1 gene was demonstrated to contain two ChBD binding sites; its chitinolytic activity increased by an evolution process directed by the construction of a series of variants The variants SHU-1 and SHU-2 showed maximum enzymatic activity as result of the amino acid mutations

outside of the catalytic and substrate binding regions The virulence of B bassiana improved for silkworm mouth Bombyx mori with production from a recombinant

Bbchit1 gene, constructed by fusing the Bbchit1 gene with the chitin binding domain (ChBD), under the regulation of the promoter with overexpression of chitinase and reducing the desiccation period of the infected insect Afterwards, a hybrid protein with the ability to increase the binding capability of protease to chitin if insect cuticle

has been obtained by recombination of the ChBD fragment from B mori with the CDEP-1 gene of B bassiana was shown to have serine protease activity This recombinant strain showed increased pathogenicity over Myzus persicae larvae due to

the solubilization of protein components during insect cuticle degradation (Mondal et al., 2016)

2.3.5.4 Cellulase

Cellulases break down the cellulose molecule into monosaccharides such as

beta-glucose, or shorter polysaccharides and oligosaccharides

There are three main cellulose enzymes:

- Cellobiohydrolase (CBH or 1,4-β-D-glucan cellobioydrolase, EC 3.2.1.91): This enzyme cuts the non-reducing end of the cellulose chain to form cellobiose The molecular weight of the enzymes of this group ranges from 53 to 75 kDa These enzymes are unable to break down crystalline cellulose but only change their physical and chemical properties

- Endo-β-1,4-cellulase (EG or endo-1,4-β-D-glucan 4-glucanohydrolase, EC 3.2.14) has a molecular weight of between 42 and 49 kDa They are active at relatively high temperatures and participate in the breakdown of the β-1,4 glucoside bonds in cellulose in lichenine and β-D-glucan Products of decomposition are cellodextrin, cellobiose, and glucose

- β-glucosidase (BG-EC 3.2.1.21): capable of operating at very wide pH (pH 4.4

- 4.8), molecular weight in the range of 50 - 98 kDa, pI = 8.4 and yes Can be operated

at high temperatures β-glucosidase participates in the breakdown of cellobiose, forming glucose (Nguyễn Đức Lượng, 2003)

Cellulose hydrolytic enzymes can be broken down into several components, such as microbial cellulase enzyme which may consist of one or more CBHs, one or more EGs and possibly β-glucosidase The complete system consists of CBH celulase,

EG and BG together to convert cellulose into glucose The enzymes cellobiohydrolases and endocellulases work together to hydrolyze cellulose into short segments of oligosaccharides The oligosaccharides (mainly cellobiose) are then hydrolyzed to glucose with β-glucosidase (Bguin P, Henrissat B, 1994)

exo-Celulase can be synthesized from a variety of natural sources, mainly from microorganisms like bacteria, bacteria, molds, and certain types of yeasts Because of the advantages of growth time, size and efficiency of enzyme production, microorganisms are often used to produce enzyme preparations

Several studies have been reported to detect extracellular cellulase activity

from B bassiana This is due to the enzyme-forming capacity of these microorganisms

that decompose all major plant biologically active substances: cellulose hemi-cellulose and xylan which is the main constituents of hemicelluloses Considering design strategies concerned with view of microbiological control of insects, the aim of this

study was to investigate the production of cellulase enzymes of insect fungus B

bassiana when grown on Substrates with the presence of glucose in the medium search

for use these strains to control insects

Part III MATERIALS AND METHODS

3.1.1 Materials

Beauveria bassiana HNb20 is provided by Functional Bio-compounds

Labratory, Institute of Biotechnology, VietNam Academy of Science and

Technology

3.1.2 Equipments

Equipments are provided by Functional Bio-compounds Labratory, Institute of Biotechnology, VietNam Academy of Science and Technology Equipments consist of:

Table 3 1 List of equipments

Laminar flow cabinet America

Water tank thermostat Korea

3.1.3 Chemical

Chemicals were used:

Table 3 2 Chemicals were used in this thesis

High yeast extract

Vietnam Saccarose

Glucose Molasses D-Glucosamine Germany

Ethanol TCA (Trichloroacetic acid) Folin

Casein Urea NaCl

Na2CO3

NaOH Acid acetic HCl

CaCl2

KCl

KH2PO4

MgSO4.7H2O CuSO4

ZnSO4.7H2O (NH4)2SO4 H2O

NH4NO3

- CMC substrate (Carboxymethyl cellulose) : 0.1%CMC; 2% agar

- Casein substrate : 0.2% Casein; 2% agar

- Chitosan substrate : 0.1% Chitosan; 2% agar (Ha, Tuyen, Nhue, & Binh, 2019)

3.3 Location and time study

Location: This subject was conducted in Functional Bio-compounds Labratory, Institute of Biotechnology, VietNam Academy of Science and Technology

Time : 08/2020 – 01/2021

3.4 Research methods

3.4.1 The capacity for producing extracellular enzyme and optimal time culture

Activated Beauveria bassiana HNb20 from original strain Pipeting 100

µl original strain stored in -80℃ then activated on PDA dish, after that put it in

incubator at 28℃ from 3 to 5 days

Cultured Beauveria bassiana HNb20 Cut 1cmx1cm square of PDA dish

containing HNb20 after 3 to 5 days Putting this piece into 250 ml volume flask had 50ml PDB (pH=6) , then that was put in incubator shaking at 28℃, 150 rpm, and record at fifth day, sixth day, seventh day, eighth day

Screening mycelia and count conidia of HNb20 After 5 days, 6 days, 7 days, 8 days cultivated, next pipeted 1 ml culture fluid for screening mycelia and count conidia of HNb20 under microscope

Determined enzyme activity by agar radial diffusion: Collected culture fluid at fifth day, sixth day, seventh day, eighth day cultured then centrifuged at 4000rpm in 5 minutes and got supernatant Used a steriled cork (d= 8 mm) drilled wells on three kind of substrate (Hà, 2012:): 0.1% CMC; 0.2% Casein; 0.1% Chitosan on petri plate Pipeted 50 μl raw enzyme in to wells were prepared on three kind of substrate: 0.1% CMC; 0.2% Casein; 0.1% Chitosan and set in incubator at 30℃ After 48 hours the plates were stained by TCA, lugol or both to mesure zone of clearance (D – d, mm), D was diameter of the clear zone From the results gave the day for greatest enzyme activity.(Davis, 1977)

3.4.2 Effects of Carbon soures to extracellular enzyme activity of HNb20

Supplied carbon soures by added: 2% Glucose; 2% Saccrose; 2% Molasses into PDB medim, pH=6 after addition Cultivated incubator shaking

at 28℃, 150rpm After that did the same process in 3.4.1 gave the substance for greatest enzyme activity

3.4.3 Effects of Nitrogen sources to extracellular enzyme activity of HNb20

Supplied nitrogen sources by adding: High yeast extract , urea , (NH4)2SO4 following by 0.25%; 0.5%; 0.75%; 1%; 1.25%; 1.5% concentration into PDB medim, pH=6 after addition (each supplement did six experiments correspond 6 different concentrations from 0.25% to 1.5%) Cultivated incubator shaking at 28℃, 150rpm After that did the same process in 3.4.1 gave the substance for greatest enzyme activity

3.4.4 Effects of pH, temperature, petroleum oil, metal ion to extracellular enzyme activity of HNb20

pH effect Collected culture liquid after cultivated in PDB medium, then

centrifuged at 4000rpm in 5 minutes and got supernatant Adjusted pH of supernatant following by pH=3, pH=4, pH=5 pH=6, pH= 7, pH=8 After that did the same process in 3.4.1 gave the most suitable pH for best enzyme activity

Temperature effect Collected culture liquid after cultivated in PDB

medium, then centrifuged at 4000rpm in 5 minutes and got supernatant Pipeted 1ml supernatant into 4 eppendorf were marked 50℃, 60℃, 70℃, 80℃ on surface of each tube After that put each tube in oven in corresponding temperature that were written on each tube in 10 minutes After that did the same process in 3.4.1 gave what temperature the enzyme still active

Petroleum oil effect Collected culture liquid after cultivated in PDB

medium, then centrifuged at 4000 rpm in 5 minutes and got supernatant Pipeted petroleum oil so as to reached 0.2% petroleum oil in solution that mixed oil with supernatant After that did the same process in 3.4.1 gave effect

of petroleum oil to enzyme activity

Metals ion effect.(Quan, Thi, Thanh, & Hanh, 2015) Substances were

consumed for this experiment: NaCl; KCl; CaCl2; MgSO4.7H2O; ZnSO4.7H2O; CuSO4 Collected culture liquid after cultivated in PDB medium, then centrifuged at 4000rpm in 5 minutes and got supernatant Added NaCl; KCl; CaCl2; MgSO4.7H2O; ZnSO4.7H2O; CuSO4 to concentration following by 5

mM, 10 mM, 15 mM (each metal ion carries out 3 experiments correspond 3

different concentrations 5 mM, 10 mM, 15 mM) After that did the same

process in 3.4.1 imparted effect of Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+ at difference concentrtion to enzyme activity

3.4.5 Optimal medium for extracellular enzyme activity of HNb20

From the above experiments, suitable factors have been selected for

cultivated Beauveria bassiana HNb20 and media contaned that factors called

MT1 medium MT2 medium consisted of : PDB + 0.02% MgSO4.7H2O + 0.02% KH2PO4 + 0.01% NH4NO3 Compartion enzymes activity was cultured

in MT1, MT2 and PDB by both qualitative and quantitative

Qualitative enzymes activity by agar radial diffusion in 3.4.1 Quantitative enzymes activity by spectroscopic measurement Finally gave media for the best enzymes activity