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

INTRODUCTION

Introduction

Agriculture plays a crucial role in the global economy, but its reliance on chemical pesticides poses significant environmental challenges The excessive use of these chemicals adversely affects human health, disrupts ecosystem balance, and leads to pollution of air, water, and soil Consequently, there is a growing demand for sustainable and eco-friendly agricultural practices that protect crops while minimizing environmental harm.

Lecanicillium lecanii, previously known as Verticillium lecanii, is an entomopathogenic fungus recognized as a promising alternative to chemical pesticides This biocontrol agent is effective against a wide variety of insect hosts, including those from the orders Homoptera, Coleoptera, Orthoptera, and Lepidoptera.

Lecanicillium lecanii has been extensively studied for its ability to produce extracellular enzymes such as protease, chitinase, and cellulase, which are effective in breaking down host defenses This allows the fungus to penetrate host bodies, form spores, and ultimately kill pests The application of L lecanii in agriculture serves as a biological control method to manage pest populations, thereby enhancing crop yields while minimizing environmental impact.

2018), (Hanan, Basit, Nazir, Majeed, & Qiu, 2020)

Viet Nam is currently on the direction to develop green agriculture with desiring to export agricultural products to potential markets such as Europe and the

Countries like the US enforce strict quality standards for imported products, particularly concerning pesticide residues As a result, the adoption of Good Agricultural Practices (GAP) and organic farming methods is rapidly increasing To comply with these standards, the use of organic fertilizers and biological pesticides is essential However, a report by the National Agency for Science and Technology Information indicates that domestic biopesticides account for only 37.3% of the total pesticides used, highlighting a significant gap in the market.

Vietnam faces a significant gap in techniques and research related to the production and application of biopesticides To address this issue, we are conducting a study focused on the ability of entomopathogenic fungi to produce extracellular enzymes, aiming to identify bio-compounds and enhance their activity for biopesticide development.

Purposes

- Studying on producing extracellular enzymes of Lecanicillium lecanii

- Evaluating effects to the activity of exoenzymes of HNL20

- Creeating the optimum medium to culture Lecanicillium lecanii HNL20

Requirements

- Determining the ability to produce extracellular enzymes of L lecanii

- Evaluating elements (Carbon sources, nitrogen sources, metals ion, pH, temperature and petroleum oil) affect to the activity of extracellular enzymes produced by Lecanicillium lecanii HNL20

- Building optimized medium for enhancing and determining exactly the activity of extracellular enzymes

LITERATURE REVIEW

Overview of Lecanicillium lecanii

Lecanicillium lecanii is an entomopathogenic fungus belonging to the division Ascomycota and the class Sordariomycetes, within the order Hypocreales and family Cordycipitaceae Previously classified as Verticillium lecanii, this species was reclassified to the genus Lecanicillium based on rDNA sequencing studies conducted by R Zare and Gams in 2001 and 2003 The genus Lecanicillium includes several species, such as L attenuatum, L longisporum, L muscarium, and L nodulosum.

Lecanicillium lecanii has the ability to against a number of pests such as

Homoptera, Coleoptera, Orthoptera, and Lepidoptera are affected by L lecanii, which functions as a microparasite by attaching to host bodies Once attached, conidia develop into hyphae that produce enzymes like chitinase and protease, enabling the hyphae to penetrate and eliminate pathogens Under suitable conditions, conidia are generated outside of the deceased pathogen, facilitating their spread to other hosts.

Depending on working as natural pesticides, researches of L lecanii direct to form biopesticides from conidia of L lecanii and study on mechanisms of enzymes

(chitinase, protease, cellulase and lipase) or toxic compounds produced by this fungus

Figure 2.1: Activating L leacnii HNL20 on PDA medium after 6 days at 28 o C and shaking 15rpm

Colonies develop to a size of 15–25 mm within 10 days at 24°C on PDA, appearing relatively compact and yellowish-white, with a deep yellow reverse The conidiogenous cells, known as phialides, are relatively short, measuring 11–20(–30) µm in length and 1.4–1.8 µm in width These cells are aculeate and taper strongly, produced either singly or in whorls of up to 6 directly on prostrate hyphae or on short, erect conidiophores, and may also be produced secondarily on existing phialides.

Figure 1.2: Conidiophores and conidia of L lecanii (Rasoul Zare & Gams, 2001)

Conidia formed in heads at apex of phialides, typically short-ellipsoidal, 2.5–3.5(–4.2) × 1–1.5 àm, homogeneous in size and shape Crystals present, octahedral Growth temperature optimum 21–24°C, no growth at 33°C (Rasoul Zare & Gams, 2001)

I don't know!

The life cycle of L lecanii begins with conidia that adhere to the insect's protective outer layer, known as the epicuticle Once attached, the conidia germinate and develop elongated germ tubes that penetrate this tough layer This penetration is facilitated by the secretion of various enzymes, including aminopeptidase, lipase, proteases, esterase, chitinases, and cellulase, which break down the epicuticle, allowing the germ to invade and grow within the host insect.

The hyphal growth of L lecanii leads to insect mortality through mechanical pressure and the action of secreted enzymes The fungus utilizes nutrients from the host insect to develop hyphae, which eventually differentiate into conidiophores that produce conidia on the insect's surface When conidia land on another host insect's epicuticle, L lecanii can initiate a new life cycle Currently, there is insufficient evidence to assess the toxicity of L lecanii to humans.

Figure 2.3: The distribution of L lecanii in the world (According to website: https://www.cabi.org/isc/datasheet/56281 accessed at 8:46 AM (GMT+7), Jan

L lecanii has a widespread geographical distribution across most continents, including Asia and the Americas However, it is notably absent in Africa due to the region's high-temperature climate Additionally, while Vietnam is part of Asia, it also faces unique environmental conditions that may affect the presence of L lecanii.

There is currently no distribution of L lecanii in Vietnam due to the country's tropical climate, characterized by high temperatures and humidity, which may hinder the natural growth of this species.

Application of L lecanii

Myzus persicae, commonly known as the green peach aphid, is a small green insect from the Aphididae family that significantly impacts plant health by stunting growth, causing leaf shriveling, and leading to tissue death This aphid is also a known vector for several plant viruses, including cucumber mosaic virus (CMV), potato virus Y (PVY), and tobacco etch virus (TEV) It infests a wide range of host plants, such as cabbage, dandelion, endive, mustard greens, parsley, turnip, tomato, cucumber, tobacco, potato, spinach, pepper, beet, celery, lettuce, and chard.

The cold winter significantly influences the life cycle of Myzus persicae, with a rapid development time of approximately 10-12 days for a new generation During unfavorable conditions, these aphids target Prunus spp in their egg stage As spring arrives and plants begin to grow, the eggs hatch, and the aphids feed on flowers, young foliage, and stems Following spring, winged insects disperse, sending nymphs to summer hosts, where adults mature and mate during the winter.

Figure 2.4: The nymphs (left) and adults (right) of Myzus persicae Photograph by

Lyle J Buss, University of Florida Website: http://entnemdept.ufl.edu/creatures/veg/aphid/green_peach_aphid.htm accessed at

Myzus persicae targets young plant tissues, leading to water stress, wilting, and reduced growth rates Prolonged infestations can significantly decrease yields in root and foliage crops Additionally, the green peach aphid serves as a vector for various viral diseases, including potato leafroll virus and potato virus Y affecting Solanaceae, beet western yellows and beet yellows viruses impacting Chenopodiaceae, lettuce mosaic virus on Compositae, and cauliflower mosaic and turnip mosaic viruses affecting Cruciferae Notably, some potato varieties may exhibit net necrosis in tubers due to the transmission of potato leafroll virus.

Solutions containing conidia of L lecanii are applied directly to eliminate adult Myzus persicae and inhibit nymph development This fungus effectively causes high mortality rates in Myzus persicae when cucumber plants serve as hosts.

Aphis gossypii, commonly referred to as the cotton aphid or melon aphid, is a small insect within the superfamily Aphidoidea of the order Hemiptera This pest primarily targets plants from the families Cucurbitaceae, Rutaceae, and Malvaceae, affecting crops such as watermelons, cucumbers, cantaloupes, squash, and pumpkins Additionally, citrus, cotton, and hibiscus plants are also susceptible to damage from the cotton aphid.

The adult body measures approximately 2 mm in length, featuring a black head and thorax Its body color ranges from yellow-green to dark green, occasionally appearing nearly black, with brown or dusky siphuncle, cauda, and the tips of the antennae and rostrum The apterate adult also exhibits variable coloration.

Each forewing features an elongated dark spot, while the nymphs exhibit a body color that ranges from very pale yellow to pale green and are smaller in size compared to the adults.

Figure 2.5: The nymphs and adults of Aphis gossypii (According to website: https://www.koppert.com/challenges/aphids/cotton-aphid/ accessed at 8:47 AM

The initial sign of Aphis gossypii infestation is the appearance of yellow leaves As aphid populations increase, leaves become puckered and curled Once their numbers are high, cotton aphids migrate to young leaves, flowers, and stems, leading to the development of a black sooty mold that thrives on the honeydew they secrete Ultimately, Aphis gossypii can cause severe twisting and death of the plants.

Solutions containing conidia of L lecanii are applied directly to eliminate adult Aphis gossypii and inhibit nymph development Research by Hall (1982) confirms the effectiveness of this method in controlling these pests.

Research on L.lecanii highlights its potential in controlling various pests, including whiteflies (Hall, 1982) and cereal aphids (Aqueel & Leather, 2013) Additionally, there is a strong emphasis on optimizing culture media and extracting biocompounds from L.lecanii (Shi, Xu, & Zhu, 2009; Yu et al., 2015) In Vietnam, the applications of entomopathogenic fungi, particularly L.lecanii, are gaining significant interest among researchers.

The purification of chitinase from the L lecanii strain 43H was carried out by Nguyen et al (2015) Additionally, Vũ Văn Hạnh (2012) enhanced the virulence and toxicity of L lecanii through mutations induced by UV light and N-methyl-N’-nitro-N-nitrosoguanidine.

Bioactive compounds

Figure 2.6: The structures of chitin and chitosan (Younes & Rinaudo, 2015)

Chitin, a linear polymer of β-1,4-N-acetylglucosamine (GlcNAC), is the second most abundant biopolymer on Earth, serving as the primary component of the exoskeletons of insects, fungi, and crustaceans, as well as in the internal structures of various invertebrates This white, hard substance is insoluble in water Chitosan, a polysaccharide derived from chitin through deacetylation with alkaline hydroxide, consists of randomly distributed β-(1→4)-linked D-glucosamine and N-acetyl-D-glucosamine units The degree of deacetylation can be analyzed using Nuclear Magnetic Resonance (NMR) spectroscopy Both chitin and chitosan are susceptible to degradation by chitinase.

Chitinases are glycosyl hydrolases that vary in size from 20 kDa to 90 kDa They are classified into glycosyl hydrolase families 18, 19, and 20 based on their amino acid sequences and domain architectures.

Chitinases, specifically enzymes 18 and 19, play a crucial role in the degradation of chitin Additionally, family 20 includes chitobiase and β-N-acetylhexosaminidase, which facilitate the breakdown of dimeric units such as N-acetylglucosamine (chitobiose) and terminal N-acetylgalactosamine or glucosamine from glyco-conjugates These chitinase enzymes are produced by various bacteria and fungi, including Aspergillus niger, Lecanicillium lecanii, and Beauveria bassiana.

Proteases are enzymes that catalyze the breakdown of proteins through proteolysis, converting protein molecules into smaller polypeptides or amino acids and significantly increasing the rate of these reactions They are categorized into seven groups based on their catalytic residues: serine, cysteine, threonine, aspartic, glutamic, metalloproteases, and asparagine peptide lyases Additionally, proteases are classified according to their optimal pH levels, which include acid, neutral, and basic (alkaline) proteases.

Entomopathogenic fungi possess proteases that facilitate the breakdown of insect cuticles Two key proteolytic enzymes identified are the subtilisin-like serine-protease Pr1 and the trypsin-like protease Pr2, whose activities have been thoroughly analyzed.

B bassiana, M anisopliae, Lecanicillium lecanni, Nomuraea rileyi and Metarhizium flavoviride (Liu, Meng, Yang, Fu, & Yang, 2007) These proteases are secreted during the first cuticle degradation stage, and they stimulate the signal transduction mechanism by activating protein kinase A mediated by AMPc (Cyclic adenosine monophosphate) (Fang et al., 2009) It has been validated that the extracellular involvement of protease Pr1 in cuticle penetration is initialized by the infection of the cuticle

Entomopathogenic fungi produce chitinases and proteases to dismantle protective barriers, facilitating their invasion of host insects Additionally, cellulases play a crucial role by providing degraded nutrients essential for fungal growth and development.

MATERIALS AND METHODS

Materials and equipment

Lecanicillium lecanii HNL20 strain was provided by Functional Bio- compounds Laboratory, Institute of Biotechnology, Vietnam Academy of Science and Technology

The instruments and equipment were used in Functional Bio-compounds Laboratory, Institute of Biotechnology, Vietnam Academy of Science and Technology:

Vortex shaker Italia pH titration machine Switzerland

Table 3.1: The instruments and equipment were used in the research

Table 3.2: Chemicals were used in the research

PDA medium (g/L) consisted of potato 200g; D-Glucose 20g; Agar 20g and 1 liter of water at pH=6

PDB medium (g/L) consisted of potato 200g; D-Glucose 20g and 1 liter of water at pH=6

CMC diffusion agar plate composed of 1% (w/v) CMC; 2% (w/v) Agar and water

Chitosan diffusion agar plate composed of 1% (w/v) Chitosan; 2% (w/v) Agar and water

Casein diffusion agar plate composed of 2% (w/v) Caseinand 2% (w/v) Agar and water

The components of Medium 1 are potato 200g; Glucose 20g; 1 liter of water; 2% (w/v) molasses; 1% (w/v) yeast extract; 1.5% (w/v) (NH4)2SO4; 0.25% (w/v) Urea; KH2PO4 10mM and MgSO4 10mM at pH=6

The components of Medium 2 are potato 200g; Glucose 20g; 1 liter of water; 0.02% (w/v) MgSO4; 0.02% (w/v) KH2PO4 and 0.01% (w/v) NH4NO3 at pH=6

Location: This subject was conducted in the Functional Bio-compounds Laboratory, Institute of Biotechnology, Vietnam Academy of Science and Technology

Research methods

3.2.1 Screening the morphology, the density of conidia and studying on the activity of extracellular enzymes of HNL20

HNL20 culture method: L lecanii HNL20 strain was stored in Glycerol 30%

A solution of L lecanii HNL20 was prepared at a concentration of (w/v) at -21 °C Subsequently, 100 µl of this solution was pipetted into PDA medium disks and incubated at 28 °C with shaking at 150 rpm for 5 days After incubation, the activated HNL20 disks were cut into 1x1 cm pieces and transferred into 250 ml medium flasks, which were then cultured at 28 °C and 150 rpm.

Screening morphology and the density of conidia after 5, 6, 7 and 8 culture days: After culturing 5, 6, 7 and 8 days, medium of culturing L lecanii HNL20

On the final day of culture, 14 samples were pipetted to assess morphology and determine the conidia density under a microscope The medium was then collected and centrifuged at 4000 rpm for 5 minutes to remove cells, and the crude extracellular enzymes were stored at a low temperature.

The activity of extracellular enzymes was assessed using the agar diffusion plate method (Tendencia, 2004) with substrates including 0.1% (w/v) CMC, 0.1% (w/v) chitosan, and 0.2% (w/v) casein A volume of 50 µL from each enzyme sample was pipetted into the wells of the plate and incubated at 30°C for 48 hours Following incubation, the substrate plates were stained with 0.5% (v/v) Lugol's solution and 20% (w/v) TCA Each experiment was conducted twice to ensure reliability of the results.

Note: The activity of enzymes was determined by the following the formula:

D-d (while D: Diameter of degradation round, d: diameter of well on plate)

3.2.2 Studying on the elements affecting to the activity of L lecanii HNL20 extracellular

3.2.2.1 Effects of carbon sources to the activity of L lecanii HNL20 extracellular enzymes

The examination for the activity of medium added carbon sources: 2% (w/v) glucose, 2% (w/v) sucrose and 2% (w/v) molasses were respectively added into PDB medium to culture L lecanii HNL20 at 28 o C, 150rpm, pH=6 L lecanii

HNL20 was cultured in PDB medium as a control sample After 6 days, the medium was collected and centrifuged at 4000 rpm for 5 minutes to obtain the supernatant The enzyme activity of all samples was assessed using the agar diffusion plate method (Tendencia, 2004) with substrates of 0.1% (w/v) CMC, 0.1% (w/v) chitosan, and 0.2% (w/v) casein A volume of 50 µL from each sample was pipetted into the wells of the plate and incubated at 30°C for 48 hours Following incubation, the substrate plates were stained with 0.5% (v/v) Lugol and 20% (w/v) TCA The testing experiments were conducted twice.

3.2.2.2 Effects of nitrogen sources to the activity of L lecanii HNL20 extracellular enzymes

The study investigated the effects of various nitrogen sources, including yeast extract, urea, and ammonium sulfate, on the activity of HNL20 cultured in PDB medium at concentrations ranging from 0.25% to 1.5% (w/v) under controlled conditions of 28°C, 150 rpm, and pH 6 A control sample of L lecanii HNL20 was also maintained in PDB medium After six days of cultivation, the medium was centrifuged at 4000 rpm for five minutes to collect the supernatant, which was then tested for enzyme activity using an agar diffusion plate method on substrates of 0.1% CMC, 0.1% chitosan, and 0.2% casein Each sample was pipetted into wells on the plate and incubated at 30°C for 48 hours Following incubation, the substrate plates were stained with 0.5% lugol and 20% TCA, and the experiments were conducted in duplicate.

3.2.2.3 Effects of petroleum oil to the activity of L lecanii HNL20 extracellular enzymes

The study investigated the effects of adding 1% (v/v) petroleum oil to PDB medium for culturing L lecanii HNL20 at 28°C and 150 rpm, with a pH of 6 A control sample of L lecanii HNL20 was also cultured in PDB medium without the oil After six days, the medium was collected and centrifuged at 4000 rpm for five minutes to obtain the supernatant Subsequently, 1 ml of the centrifuged supernatant from the L lecanii HNL20 culture in PDB medium was pipetted into tubes for further analysis.

The enzyme activity was assessed using an agar diffusion plate method (Tendencia, 2004) with a substrate consisting of 0.1% (w/v) carboxymethyl cellulose (CMC), 0.1% (w/v) chitosan, and 0.2% (w/v) casein, all in a 2% (v/v) petroleum solution A volume of 50 µL from each enzyme sample was pipetted into the wells of the plate and incubated at 30°C for 48 hours Following incubation, the substrate plates were stained with 0.5% (v/v) Lugol's solution and 20% (w/v) trichloroacetic acid (TCA) Each experiment was conducted in duplicate.

3.2.2.4 Effects of temperature to the activity of L lecanii HNL20 extracellular enzymes

The activity of HNL20 extracellular enzymes was assessed by incubating 1 ml of centrifuged supernatant from L lecanii HNL20 cultured in PDB medium at temperatures of 50 °C and 60 °C for 10 minutes.

Crude enzymes from L lecanii HNL20 cultured in PDB medium were used as a control sample, with temperatures set at 70 °C and 80 °C After a 10-minute incubation, enzyme activity was assessed using an agar diffusion plate method on substrates of 0.1% (w/v) CMC, 0.1% (w/v) chitosan, and 0.2% (w/v) casein A volume of 50 µL from each sample was pipetted into the wells of the plate and incubated at 30 °C for 48 hours Following incubation, the substrate plates were stained with 0.5% (v/v) Lugol's solution and 20% (w/v) TCA The testing experiments were conducted twice for accuracy.

3.2.2.5 Effects of pH to the activity of L lecanii HNL20 extracellular enzymes

The activity of L lecanii HNL20 extracellular enzymes was examined at various pH levels (3, 4, 5, 6, 7, and 8) by titrating collected crude enzymes and pipetting 1 ml into tubes Crude enzymes cultured in PDB medium served as the control sample The enzyme activity was assessed using an agar diffusion plate method on substrates of 0.1% (w/v) CMC, 0.1% (w/v) chitosan, and 0.2% (w/v) casein, with 50 µl of each sample pipetted into the wells and incubated at 30°C for 48 hours After incubation, the substrate plates were stained with 0.5% (v/v) Lugol and 20% (w/v) TCA, and the experiments were conducted twice for accuracy.

3.2.2.6 Effects of metal ion to the activity of L lecanii HNL20 extracellular enzymes

The study investigated the impact of various metal ions, including K\(^+\), Na\(^+\), Ca\(^{2+}\), Mg\(^{2+}\), Zn\(^{2+}\), and Cu\(^{2+}\), on the extracellular enzyme activity of L lecanii HNL20 These metal ions were introduced into PDB medium at concentrations of 5mM, 10mM, and 15mM The cultures of L lecanii HNL20 were maintained at 28°C, with shaking at 150 rpm and a pH of 6 to assess the crude enzyme production.

HNL20 cultured in PDB medium were used to be control sample After that, all samples of enzymes were tested the activity by agar diffusion plate (Tendencia,

2004) on the substrate of 0.1% (w/v) CMC, 0.1% (w/v) chitosan and 0.2% (w/v) casein 50àl of each sample was pipetted into the well of plate and incubated at

30 o C in 48 hours After incubating, the substrate plates were stained with lugol 0.5% (v/v) and TCA 20% (w/v) The experiments of testing are conducted 2 times

3.2.3 Determination the activity of chitinase through spectrophotometry

Principles: Enzyme chitinase degrades chitin to release N–acetyl–D– glucosamine which can be screened by DNS (3,5-dinitrosalisylic acid) (Miller,

1959) and absorbed at 535nm (Hà, 2012) Based on the standard curve of glucosamine and OD (optical density), the activity of chitinase can be calculated following the formula:

mol glucosamine V v.t.v’ (Unit/ml sample) While:

V: Total volume of method v: Volume of used enzyme t: Reaction time v’: Volume of spectrophotometry

The standard curve of D-glucosamine

Shaking and boiling in 5 minutes Distilled water

Shaking, resting 5 minutes and absorbed at 535nm

Table 3.3:The standard curve of D-glucosamine

Determining the activity of chitinase: Chitosan is a deacetylated derivative of chitin which was used as the substrate i) Sample:

- Mixing well 1ml crude enzyme and 1ml colloidal chitosan then incubating at

- Adding 1ml NaOH 1N and boiling in 5 minutes to stop reactions

- Centrifuging 4000rpm in 5 minutes and collecting the supernatant

- Preparing the mixture of 1ml supernatant and 1ml DNS 1% (w/v), shaking and boiling in 5 minutes after that immediately cooling

- Absorbing at 535nm ii) Control:

- Mixing well 1ml enzyme and 1 ml NaOH 1N, adding 1ml colloidal chitosan then boiling in 5 minutes

- Centrifuging 4000rpm in 5 minutes and collecting the supernatant

- Preparing the mixture of 1ml supernatant and 1ml DNS 1% (w/v), shaking and boiling in 5 minutes after that immediately cooling

3.2.4 Determination the activity of cellulase through spectrophotometry

Cellulase breaks down carboxymethyl cellulose (CMC) into reducing sugars, which can be quantified using DNS (3,5-dinitrosalicylic acid) and measured at 540 nm The activity of cellulase is determined by calculating the optical density (OD) against a standard curve of reducing sugars.

mol reducing sugar V v.t.v’ (Unit/ml sample) While:

The standard curve of glucose:

Volume of glucose (10 àmol/ml) (ml)

Shaking, putting in 100 o C thermostatic tank in 10 minutes the cooling to room temperature

Total volume of method (ml) 2.5 2.5 2.5 2.5 2.5

Table 3.4: The standard curve of glucose

Determining the activity of cellulase: i) Sample:

- Mixing well 0.5 ml crude enzyme and 0.5 ml CMC 0.5% (w/v) then incubating at

- Adding 1ml DNS 1% (w/v), shaking and boiling in 5 minutes after that immediately cooling

- Absorbing at 540nm ii) Control:

- Adding 0.5 ml crude enzyme to control tube then boiling in 5 minutes

- Continuing to add 0.5m ml CMC 0.5% (w/v) and 1ml DNS 1% (w/v) to tube, shaking well and incubated at 37 o C in 20 minutes

- Boiling control tube in 5 minutes and absorbing at 535nm

3.2.5 Determination the activity of protease through spectrophotometry

Protease activity leads to the degradation of proteins, resulting in the release of tyrosine along with other amino acids and short peptide chains The Folin-Ciocalteu reagent reacts with free tyrosine to form complexes that can be measured using spectrophotometry at a wavelength of 660 nm (Cupp-Enyard, 2008) The calculation of protease activity can be performed using a specific formula.

mol tyrosine V v.t.v’ (Unit/ml sample) While:

The standard curve of tyrosine:

Adding to all tubes 1.25ml Na2CO3 and 0.25ml Folin’s reagent, shaking and incubating at 37 o C in 30 minutes

Table 3.5: The standard curve of tyrosine

Determining the activity of protease:

Shaking and incubated at 37 o C in 10 minutes

Incubating at 37 o C in30 minutes then centrifuging to collect the supernatant

Table 3.6: The processes of determining the protease activity

Shaking and incubating at 37 o C in30 minutes

Filtrating before absorbing at 660nm

Table 3.7: The processes of spectrophotometric reaction

RESULTS

Evaluating effects (Carbon sources, nitrogen sources, metals ion, pH, temperature and petroleum oil) affect to the activity of

4.1.1 The morphology of hyphae, conidia and the exoenzyme activity of L lecanii HNL20

Figure 4.1: The morphology of L lecanii HL20 hyphae after culturing 5, 6, 7, 8 days in PDB medium at PH=6, 28 o C and shaking 150rpm

Figure 4.2: The degradation round of exoenzymes after culturing 5, 6,7 and 8 days (in PDB medium at pH=6, 28 o C, shaking 150rpm) on substrates: (A) 0.1% (w/v)

Table 4.1: The results of degradation round of L lecanii HNL20 extracellular enzymes cultured in PDB medium after 5, 6,7 and 8 days (pH=6, 28 o C, shaking

150rpm) on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C)

Table 4.2: The density of L lecanii HNL20’s conidia cultured in PDB medium after 5, 6,7 and 8 days (pH=6, 28 o C, shaking 150rpm)

After culturing 5, 6, 7 and 8 days in PDB medium at pH=6, 28 o C, shaking 150rpm, the results were recorded that:

In terms of morphology, L lecanii HNL20 had white long branching hyphae

The conidia of L lecanii HNL20 are small and elliptical in shape Significant hyphal development was observed on the 5th day, with maximum hyphal density reached on the 6th day, followed by a moderate decline on the 7th and 8th days Additionally, large quantities of conidia were produced on the 5th and 6th days, before decreasing in subsequent days.

The highest activity of exoenzymes was observed after 6 days of culture, indicating that the activity of extracellular enzymes is influenced by the development of hyphae and the density of conidia All enzymes used in the experiments were collected from flasks after this 6-day cultivation period.

4.1.2 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in PDB medium and PDB medium added Carbon sources (2% (w/v) molasses, 2% (w/v) glucose and 2% (w/v) sucrose)

Figure 4.3: 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,

28 o C, shaking 150rpm) on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein

Table 4.3 presents the degradation results of L lecanii HNL20 extracellular enzymes cultured in PDB medium, with additional carbon sources of 2% (w/v) molasses, glucose, and sucrose The experiments were conducted at pH 6 and a temperature of 28°C, with shaking at 150 rpm, using substrates of 0.1% (w/v) CMC and 0.1% (w/v) chitosan.

Adding 2% (w/v) molasses to the culture medium significantly increased the activity of extracellular enzymes on CMC, chitosan, and casein substrates, achieving measurements of 10mm, 14mm, and 9mm, respectively This enhancement was comparable to the enzyme activity observed in L lecanii HNL20 cultured in PDB medium, which recorded 10mm, 13mm, and 7mm Therefore, incorporating molasses into the culture medium for L lecanii HNL20 can effectively boost exoenzyme activity.

4.1.3 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in PDB medium and PDB medium added yeast extract

Figure 4.4: 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: 0.25% (w/v), 0.5% (w/v), 0.75% (w/v), 1%

(w/v), 1.25% (w/v) and 1.5% (w/v) (at pH=6, 28 o C, shaking 150rpm) on substrates: (A)

1: PDB medium (Control) 5: PDB medium + 1% (w/v) yeast extract

2: PDB medium + 0.25% (w/v) yeast extract 6: PDB medium + 1.25% (w/v) yeast extract

3: PDB medium + 0.5% (w/v) yeast extract 7: PDB medium + 1.5% (w/v) yeast extract 4: PDB medium + 0.75% (w/v) yeast extract

Table 4.4 presents the degradation results of L lecanii HNL20 extracellular enzymes cultured in PDB medium, with varying concentrations of yeast extract (0.25% to 1.5% w/v) at pH 6 and 28°C, shaken at 150 rpm The substrates tested include 0.1% (w/v) carboxymethyl cellulose (CMC), 0.1% (w/v) chitosan, and 0.2% (w/v) casein.

The results indicate that for the CMC substrate, the addition of 1.5% (w/v) yeast extract resulted in the largest degradation zone of 16mm, while other concentrations yielded approximately 14mm In the case of the chitosan substrate, increasing the yeast extract concentration to over 1% (w/v) led to a twofold increase in chitinase activity compared to the control, with degradation zones measuring 17mm and 8mm, respectively For the casein substrate, the optimal concentration of 0.25% (w/v) yeast extract achieved the highest degradation zone of 13mm Although the degradation diameters gradually decreased, they remained 1.5 times greater than those of the original medium Overall, the data suggest that adding 1-1.5% (w/v) yeast extract to the culture medium enhances the activity of three extracellular enzymes by 2 to 2.5 times compared to the initial medium.

4.1.4 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in PDB medium and PDB medium added (NH 4 ) 2 SO 4

The degradation round of L lecanii HNL20's exoenzymes was analyzed after culturing for 6 days in PDB medium, with varying concentrations of nitrogen sources, specifically ammonium sulfate \((NH_4)_2SO_4\) at 0.25%, 0.5%, 0.75%, 1%, 1.25%, and 1.5% (w/v) The experiments were conducted at pH 6, 28°C, and shaking at 150 rpm, using substrates including 0.1% (w/v) carboxymethyl cellulose (CMC), 0.1% (w/v) chitosan, and 0.2% (w/v) casein.

1: PDB medium (Control) 5: PDB medium + 1% (w/v)

Table 4.5 presents the degradation results of L lecanii HNL20 extracellular enzymes cultured in PDB medium, with varying concentrations of nitrogen sources, specifically ammonium sulfate \((NH_4)_2SO_4\) at 0.25%, 0.5%, 0.75%, 1%, 1.25%, and 1.5% (w/v) The experiments were conducted at a pH of 6, a temperature of 28°C, and a shaking speed of 150 rpm, utilizing substrates including 0.1% (w/v) carboxymethyl cellulose (CMC), 0.1% (w/v) chitosan, and 0.2% (w/v) casein.

The addition of more than 0.75% (w/v) concentration of (NH4)2SO4 to the culture medium significantly enhanced cellulase activity, recording a 2.6-fold increase compared to the medium without (NH4)2SO4, with measurements of 8mm versus 3mm In contrast, the activities of chitinase and protease experienced a slight decrease of about 1mm at the same concentrations Therefore, a 1.5% (w/v) concentration of (NH4)2SO4 is recommended for the culture medium of L lecanii HNL20 to boost cellulase activity while minimally impacting the activity of other exoenzymes.

4.1.5 The results of testing exoenzyme’s activity of L lecanii HNL20 cultured in PDB medium and PDB medium added Urea

The degradation of exoenzymes from L lecanii HNL20 was analyzed after culturing for 6 days in PDB medium, with the addition of varying concentrations of urea as a nitrogen source (0.25% to 1.5% w/v) The experiments were conducted at pH 6, 28°C, and shaking at 150 rpm, using substrates including 0.1% (w/v) CMC, 0.1% (w/v) chitosan, and 0.2% (w/v) casein.

1: PDB medium (Control) 5: PDB medium + 1% (w/v) urea

2: PDB medium + 0.25% (w/v) urea 6: PDB medium + 1.25% (w/v) urea

3: PDB medium + 0.5% (w/v) urea 7: PDB medium + 1.5% (w/v) urea

Table 4.6 presents the degradation results of L lecanii HNL20 extracellular enzymes cultured in PDB medium, with varying concentrations of urea as a nitrogen source (0.25% to 1.5% w/v) at pH 6 and 28°C, shaken at 150 rpm The substrates tested include 0.1% (w/v) carboxymethyl cellulose (CMC), 0.1% (w/v) chitosan, and 0.2% (w/v) casein.

The addition of 0.25% (w/v) urea to PDB medium for culturing L lecanii HNL20 resulted in a moderate increase in cellulase and chitinase activity, measuring 10mm and 13mm, respectively, compared to the control medium, which showed 7mm for both enzymes However, protease activity decreased to 8mm from 10mm in the PDB medium Notably, when urea concentration exceeded 0.25% (w/v), cellulase activity surged dramatically, increasing by 1.7-2.1 times, while the activity of other exoenzymes was inhibited Thus, incorporating 0.25% (w/v) urea in the culture medium enhances cellulase and chitinase activity, with higher urea concentrations yielding significantly increased cellulase activity.

4.1.6 The effects of temperature (50 o C, 60 o C, 70 o C and 80 o C) to the activity of

Figure 4.7: The degradation round of L lecanii HNL20’s exoenzymes after testing at 50 o C, 60 o C, 70 o C and 80 o C in 10 minutes on substrates: (A) 0.1% (w/v) CMC,

2: Crude enzyme at 50 o C in 10 minutes

Table 4.7: The results of degradation round of L lecanii HNL20’s exoenzymes after testing at 50 o C, 60 o C, 70 o C and 80 o C in 10 minutes on substrates: (A) 0.1% (w/v)

Graph 1: The degradation round of L lecanii HNL20’s exoenzymes after testing at

50 o C, 60 o C, 70 o C and 80 o C in 10 minutes on substrates: (A) 0.1% (w/v) CMC, (B)

The activity of crude enzymes from L lecanii HNL20 is significantly affected by temperature Notably, at temperatures ranging from 50 °C to 70 °C, there is a slight decrease in the activity of all extracellular enzymes, approximately 1-2 mm However, at an elevated temperature of 80 °C, the activity of cellulase, chitinase, and protease declines sharply, with reductions of 2 mm, 6 mm, and 7 mm, respectively, compared to the control enzyme measurements of 11 mm, 8 mm, and 10 mm To minimize enzyme inactivation, it is recommended that the crude enzyme of L lecanii HNL20 be utilized at temperatures not exceeding 70 °C.

The degradation round of L lecanii HNL20’s exoenzymes after testing at 50 o C, 60 o C, 70 o C and 80 o C

Control 50oC 60oC 70oC 80oC

4.1.7 The effects of pH (pH=3,4,5,5,7 and 8) to the activity of L lecanii HLN20 crude enzyme

Figure 4.8: The degradation round of L lecanii HNL20’s exoenzymes after testing at pH=3,4,5,5,7 and 8 on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein

1: Initial crude enzyme (control) 5: Crude enzyme at pH=6

2: Crude enzyme at pH=3 6: Crude enzyme at pH=7

3: Crude enzyme at pH=4 7: Crude enzyme at pH=8

Table 4.8: 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) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein

The incubation of crude enzyme from L lecanii HNL20 at various pH levels revealed that pH 5 significantly enhanced the activity of cellulase, chitinase, and protease, with measurements of 9 mm, 7 mm, and 5 mm, respectively, compared to control samples measuring 7 mm, 8 mm, and 5 mm In contrast, other pH levels led to a steady decline in exoenzyme activity, with most showing only 1 mm, while protease was inactivated at pH levels above 6 Therefore, pH 5 is identified as the optimal level for maximizing the activity of L lecanii HNL20 exoenzymes.

4.1.8 The effects of petroleum oil (SK Enspray 99EC) at 0.2% (v/v) to the activity of L lecanii HLN20 crude enzyme

Figure 4.9: The degradation round of L lecanii HNL20’s exoenzymes after incubating with 0.2% (v/v) petroleum oil on substrates: (A) 0.1% (w/v) CMC, (B)

Control (D-d) mm pH= 3 (D-d) mm pH=4 (D-d) mm pH= 5 (D-d) mm pH= 6 (D-d) mm pH= 7 (D-d) mm pH= 8 (D-d) mm

(Control) 2: Crude enzyme incubated with 0.2% (w/v) petroleum oil

Conc of Petroleum oil Substrate

Table 4.9: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating with 0.2% (v/v) petroleum oil on substrates: (A) 0.1% (w/v) CMC, (B)

Although 0.2% (v/v) petroleum oil (SK Enspray 99EC) was added to culture medium to improve the activity of extracellular enzymes, the results were not as expected There was no change in the diameters of degradation round (Table 4.9) so it was unnecessary to add petroleum oil to culture medium

4.1.9 The effects of metal ion (K + , Na + , Ca 2+ , Mg 2+ , Zn 2+ and Cu 2+ ) to the activity of L lecanii HLN20 crude enzyme

Figure 4.10: The degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Na + at 5mM, 10mM and 15mM on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein

1: Initial crude enzyme (control) 3: Initial crude enzyme incubated with

2: Initial crude enzyme incubated with Na + 5mM 4: Initial crude enzyme incubated with

Table 4.10: The results of degradation round of L lecanii HNL20’s exoenzymes after incubating respectively with Na + at 5mM, 10mM and 15mM on substrates: (A) 0.1%

The results of testing exoenzyme’s activity of L lecanii HNL20

4.2.1 Determination the activity of extracellular enzymes through agar plate diffusion

Figure 4.16: The degradation round of L lecanii HNL20’s exoenzymes after culturing 6 days in improved medium at pH=6, 28 o C, shaking 150rpm on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein

Table 4.17: The results of degradation round of L lecanii HNL20’s exoenzymes after culturing 6 days in improved medium at pH=6, 28 o C, shaking 150rpm on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein

1: Crude enzyme cultured in PDB medium (control) 3: Crude enzyme cultured in Medium

Graph 2: The degradation round of L lecanii HNL20’s exoenzymes after culturing 6 days in improved medium at pH=6, 28 o C, shaking 150rpm on substrates: (A) 0.1%

Initial testing on substrate plates revealed that the activity of extracellular enzymes in medium 1 was nearly twice as high as in the control medium Specifically, the measurements for medium 1 on CMC, chitosan, and casein substrates were 11mm, 16mm, and 13mm, respectively, compared to the control medium's 6mm, 6mm, and 7mm In medium 2, the degradation rounds for CMC, chitosan, and casein were slightly higher at 8mm, 8mm, and 7mm, respectively, compared to the control medium's 6mm, 6mm, and 7mm To accurately determine the activity of exoenzymes in the improved medium, spectrophotometry was utilized for calculations.

4.2.2 Determination the activity of extracellular enzymes through spectrophotometry

Table 4.18: OD values and activity of extracellular enzymes cultured in improved medium through spectrophotometry

The results of degradation round of L lecanii HNL20’s exoenzymes in PDB medium, Medium 1 and medium 2

Spectrophotometry was utilized to determine OD values for calculating the activity of exoenzymes cultured in different media Results indicated that medium 1 significantly enhanced the activity of cellulase, chitinase, and protease, yielding values of 70.3 U/ml, 0.057 U/ml, and 0.237 U/ml, respectively, compared to PDB medium, which showed 60.204 U/ml, 0.03 U/ml, and 0.213 U/ml In contrast, medium 2 exhibited lower enzyme activities than PDB medium, with values of 57.488 U/ml for cellulase, 0.034 U/ml for chitinase, and 0.139 U/ml for protease Notably, the chitinase activity was significantly lower than reported by Nguyen et al (2015), with only 0.057 U/ml compared to 0.528 U/ml In conclusion, while the optimized medium improved enzyme activity, the results fell short of expectations, indicating a need for further research to refine the medium.

CONCLUSSIONS AND SUGGESTTIONS

- Successfully determined L lecanii HNL20 had ability to produce extracellular enzymes such as cellulase, chitinase and protease

- The activity of extracellular enzymes can be went up when adding nutrients and metal ions at suitable concentrations

- Two optimized medium helped to enhance the activity of enzymes but results did not meet expectations

- Continue to research practically at higher scales and executed more studies to verify other bio compounds for enhancement of countering more pests

- Finding optimal culturing conditions for Lecanicillium lecanii HNL20 to produce the optimal amount and activity of enzymes and further production of biopesticides

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Graph 3: The tyrosine standard curve y = 5.1509x + 0.054 R² = 0.9036

Graph 4: The glucose standard curve

days in improved medium at pH=6, 28 o C, shaking

Table 4.17: The results of degradation round of L lecanii HNL20’s exoenzymes after culturing 6 days in improved medium at pH=6, 28 o C, shaking 150rpm on substrates: (A) 0.1% (w/v) CMC, (B) 0.1% (w/v) chitosan and (C) 0.2% (w/v) casein

1: Crude enzyme cultured in PDB medium (control) 3: Crude enzyme cultured in Medium

Graph 2: The degradation round of L lecanii HNL20’s exoenzymes after culturing 6 days in improved medium at pH=6, 28 o C, shaking 150rpm on substrates: (A) 0.1%

Initial testing results indicate that the activity of extracellular enzymes in medium 1 was nearly twice as high as in the control medium, with measurements of 11mm, 16mm, and 13mm for CMC, chitosan, and casein substrates, respectively, compared to 6mm, 6mm, and 7mm in the control Additionally, enzyme degradation on CMC, chitosan, and casein in medium 2 showed slight increases (8mm, 8mm, and 7mm) over the control medium (6mm, 6mm, and 7mm) To accurately assess the activity of exoenzymes in the improved medium, spectrophotometric analysis was performed.

4.2.2 Determination the activity of extracellular enzymes through spectrophotometry

Table 4.18: OD values and activity of extracellular enzymes cultured in improved medium through spectrophotometry

The results of degradation round of L lecanii HNL20’s exoenzymes in PDB medium, Medium 1 and medium 2

Spectrophotometry was utilized to determine OD values for calculating the activity of exoenzymes cultured in different media Results indicated that medium 1 significantly enhanced the activity of cellulase, chitinase, and protease, yielding values of 70.3 U/ml, 0.057 U/ml, and 0.237 U/ml, respectively, compared to PDB medium, which showed 60.204 U/ml, 0.03 U/ml, and 0.213 U/ml In contrast, medium 2 exhibited lower enzyme activities than PDB medium, with values of 57.488 U/ml for cellulase, 0.034 U/ml for chitinase, and 0.139 U/ml for protease Notably, the chitinase activity was significantly lower than reported by Nguyen et al (2015), which was 0.528 U/ml In conclusion, while the optimized medium improved enzyme activity, the results fell short of expectations, indicating a need for further research to refine the medium.