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

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

FACULTY OF BIOTECHNOLOGY
-------oOo-------

GRADUATION THESIS
STUDY ON CAPACITY FOR PRODUCING
EXTRACELLULAR ENZYMES OF BEAUVERIA BASSIANA
HNB20 AND FACTORS AFFECTING THOSE ENZYMES
ACTIVITY
Student

:

Nguyen Van Hieu

Class

:

K61CNSHE
Vu Van Hanh Assoc. Prof.

Supervisor

:
Nguyen Van Giang Assoc. Prof.

Hanoi 2/2021

COMMITMENT
I with this, declare that all the data and results that I have provided in
this study are true, accurate, and not used in any other reports.
I also assure that the literature cited in the thesis indicated the origin and
all help is thankful.

Hanoi February 2021

Nguyen Van Hieu

i


ACKNOWLEDGEMENT
In fact, there are no successes without associated with support or assistance,
whether more or less, directly or indirectly by others. This project consumed huge
amount of work, enthusiasm and dedication. Still, implementation would not have
been possible if we did not have a support of many people and organizations.
Therefore, we would like to extend our sincere gratitude to all of them.
Firsts of all, I would like expressing sincere thanks to the School Board, the
Dean of Biotechnology Faculty, and all teachers have imparted to me the knowledge is
advantageous and valuable during time learning, training, and implementation thesis.
Through working, I did not only gain much knowledge but more importantly, I also
had a great chance to sharpen my skills in a professional working environment. I have
developed myself both academically, professionally, and socially.
I would like to express our deep and sincere gratitude to my supervisors, Assoc.
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
ii


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
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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 Ca2+.................................................................................................. 34
4.4.7.Effect of Mg2+ ................................................................................................ 35
4.4.8.Effect of Zn2+................................................................................................. 36
4.4.9.Effect of Cu2+ ................................................................................................ 37
PART V. CONCLUSION AND SUGGESTION .................................................. 39
5.1. Conclusion ........................................................................................................ 40
5.2. Suggestion ........................................................................................................ 40
REFERENCES ........................................................................................................ 40
APPENDIX ............................................................................................................. 43

iv


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 (NH4)2SO4 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

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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 2+ 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 Mg2+ 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 2+ 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 2+ 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

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LIST OF FIGURES
Figure 2. 1: Infection cycle of B.bassiana ................................................................. 4

Figure 4. 1. A: Growth of B. bassiana on PDA medium; B: Morphological
feature of B. bassiana spores ....................................................................... 22
Figure 4. 2. Screening hyphea of Beauveria bassiana HNb20 under
microscope (400x)....................................................................................... 23
Figure 4. 3. Test enzymes activity of HNb20 was cultivated in PDB after 5
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 (NH4)2SO4 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

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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 Ca2+ 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 Mg2+ 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 2+ 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 2+ 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

viii


ABBREVIATION LIST

B.bassiana

Beauveia bassiana

CMC

Carboxymethyl cellulose

DNS

3,5-Dinitrosalicylic acid

kDa

Kilodalton

OD

Optical density

PDA

Potato Detroxes Agar

PDB

Potato Detroxes Broth

ix


ABSTRACT
The capacity for producing extracellular enzymes protease, cellulase,
chitinase of Beauveria bassiana HNb20 were demonstrated by agar radial
diffusion. The best enzymes activity was cultured in 28℃, 150 rpm after 6
days. These enzymes still work at 80℃ , the best pH for enzyme activity is 5.
The highest protease activity value reached 0.431 U/ml in PDB + 2% glucose +
0.75% high yeast extract + 0.75% urea , cellulase activity value was 87 U/ml in
PDB + 0.75% urea , chitinase activity value was 0.071 U/ml in PDB + 0.75%
high yeast extract.

x


TÓM TẮT
Khả năng sản sinh ra ba loại enzyme ngoại bào là protease, cellulase và
chitinase của Beauveria bassiana HNb20 đã được chứng minh bằng phương
pháp khuếch tán đĩa thạch. Thời gian nuôi cấy để hoạt độ cả ba loại enzyme này
tốt nhất là sau 6 ngày ở 28℃ lắc 150 vịng/phút. Ba loại enzyme này vẫn cịn
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.

xi


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

1


of

producing protease, chitosanase, cellulase by Beauveria bassiana and factors

affecting enzymes activity in order to increase effective bioinsecticide derived from
that entomopathogenic fungus.
Purposes:
- Demonstrated the ability to generate three types of extracellular enzymes
including cellulase, protease, chitinase of Beauveria bassiana HNb20

- Evaluating the factors affecting three types of enzymes such as cellulase,
protease, chitinase, thereby giving the most suitable culture conditions for the highest
enzyme activity
Requirements:
- Demonstrated the ability to generate three types of extracellular enzymes
including cellulase, protease, chitinase of Beauveria bassiana HNb20
- Determine the culture date for the best enzyme activity
- Testing the effect of Nitrogen, Carbon sources to enzyme activity
- Testing the effect of pH, temperature, petroleum oil, metal ion to enzyme
activity
- Find the medium for the best enzymes activity

2


PART II. 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

Order

Susceptible hosts

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

Homoptera

Eulecanium spp.

Diptera

Leria serrata

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
3


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.

4

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 tripartite 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

5

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.

6

2.3.5. Extracellular enzymes produced by Beauveria bassiana
2.3.5.1. Lipases
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are serine hydrolases that
are widely studied for their physiological and industrial potential. These enzymes help
to catalyse the hydrolysis of glycerol and long-chain fatty acids with synthesis of ester
bonds. The activities of lipases are triggered only when adsorbed to an oilewater
interface. Since the 1980s, the charms of the biocatalyst properties of lipases have
gained increasing demand in industry. Lipases are also extremely versatile because
they can catalyse numerous different reactions that are widely applied in multiple
industries, such as in dairy and food manufacture, in the leather and detergent
industries, in the production of cosmetics and pharmaceuticals and in organic synthesis
reactions, especially reactions in non-aqueous media. For example, in Brazil, there are
large quantities of fat wastes from vegetable oil refineries that could be used as a
carbon source in commercial lipase production.
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

7


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

8


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)

9


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.

10


- 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 exocellobiohydrolases 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).
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.

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Part III. MATERIALS AND METHODS
3.1. Materials and equipments
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
Name of equipments

Country

Laminar flow cabinet

America

Oven

China

Microscope

Japan

Incubator

China

Refrigerator

Japan

Microwave

Vietnam

pH meter

Switzerland

Heat diffuser

China

Votex machine

Italia

Water tank thermostat

Korea

Spectrometer

Australia

Incubator shaking

Germany

Refrigerator -80℃

Korea

Centrifuge 5000 rpm

China

Balances

China

Analytical balances

China

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3.1.3. Chemical
Chemicals were used:
Table 3. 2. Chemicals were used in this thesis
Chemical

Country

High yeast extract
Saccarose
Glucose

Vietnam

Molasses
D-Glucosamine

Germany

Tyrosine

Germany

CMC

Japan

Chitosan

Germany

Agar
DNS (3,5 Dinitrosalicylic
acid)
Ethanol
TCA (Trichloroacetic acid)
Folin
Casein
Urea
NaCl
Na2CO3
NaOH

China

Acid acetic
HCl
CaCl2
KCl
KH2PO4
MgSO4.7H2O
CuSO4
ZnSO4.7H2O
(NH4)2SO4 .H2O
NH4NO3

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