Cuticle degrading enzyme production by some isolates of the entomopathogenic fungus, Metarhizium anisopliae (Metsch.)

Abstract

Context: Entomopathogenic fungi have been recognized as viable alternate options to chemicals in insect pest control. Unlike other potential biocontrol agents, fungi do not have to be ingested to infect their hosts but invade directly through the cuticle. Entry into the host involves both enzymic degradation of the cuticle barrier and mechanical pressure. Production of a range of cuticle degrading enzymes is an important event in the interaction of entomopathogenic fungi and host. Enzyme secretion is believed to be a key contributor for the virulence of a fungal isolate. Objectives: The potentiality of nine isolates of M. anisopliae were tested to produce to produce three important cuticle degrading enzymes, viz., chitinase, protease and lipase. Materials and Methods: Nine isolates of M. anisopliae were evaluated for chitinase, protease and lipase enzyme production by determining the enzyme index and activities. Results: Chitinase index of these isolates were ranged from 1.5 to 2.2 and chitinolytic activity from 0.525 to 1.560 U/ml. The isolates showed protease index in the range of 1.2 to 3.3 and the activity ranged from 0.020 to 0.114 U/ml. Lipase index ranged from 1.15 to 7.0 and the enzyme activity ranged from 0.153 to 0.500 U/ml. A strong relationship was observed between virulence of the isolates and cuticle degrading enzyme production as increased enzyme production was observed for virulent isolates. Conclusion: In the present study three isolates as (MIS2, MIS7 and MIS13) demonstrated cuticle degrading enzyme (CDE) that indicate higher virulence based on the bioassay conducted earlier by the authors as strongly substantiating the role of CDEs is considered the virulence of Metarhizium isolates. So, these isolates may be as ecofriendly insect-pest control agent in future.

Full text

J. bio-sci. 20: 25-32, 2012 ISSN 1023-8654
http://www.banglajol.info/index.php/JBS/index
CUTICLE DEGRADING ENZYME PRODUCTION BY SOME ISOLATES OF THE
ENTOMOPATHOGENIC FUNGUS, METARHIZIUM ANISOPLIAE (METSCH.)
N Sapna Bai1, O K Remadevi2 ∗, T O Sasidharan1, M Balachander2,
Priyadarsanan Dharmarajan1
1Ashoka Trust for Research in Ecology and the Environment, Royal Enclave, Srirampura,
Jakkur P.O., Bengaluru-560064, India
2Institute of Wood Science and Technology, 18th Cross, Malleswaram, Bengaluru-560003, India
Abstract
Context: Entomopathogenic fungi have been recognized as viable alternate options to chemicals in
insect pest control. Unlike other potential biocontrol agents, fungi do not have to be ingested to infect
their hosts but invade directly through the cuticle. Entry into the host involves both enzymic degradation
of the cuticle barrier and mechanical pressure. Production of a range of cuticle degrading enzymes is an
important event in the interaction of entomopathogenic fungi and host. Enzyme secretion is believed to
be a key contributor for the virulence of a fungal isolate.
Objectives: The potentiality of nine isolates of M. anisopliae were tested to produce to produce three
important cuticle degrading enzymes, viz., chitinase, protease and lipase.
Materials and Methods: Nine isolates of M. anisopliae were evaluated for chitinase, protease and
lipase enzyme production by determining the enzyme index and activities.
Results: Chitinase index of these isolates were ranged from 1.5 to 2.2 and chitinolytic activity from
0.525 to 1.560 U/ml. The isolates showed protease index in the range of 1.2 to 3.3 and the activity
ranged from 0.020 to 0.114 U/ml. Lipase index ranged from 1.15 to 7.0 and the enzyme activity ranged
from 0.153 to 0.500 U/ml. A strong relationship was observed between virulence of the isolates and
cuticle degrading enzyme production as increased enzyme production was observed for virulent
isolates.
Conclusion: In the present study three isolates as (MIS2, MIS7 and MIS13) demonstrated cuticle
degrading enzyme (CDE) that indicate higher virulence based on the bioassay conducted earlier by the
authors as strongly substantiating the role of CDEs is considered the virulence of Metarhizium isolates.
So, these isolates may be as ecofriendly insect-pest control agent in future.
Key words: Metarhizium, CDE, Chitinase, Protease, Lipase.
Introduction
The increasing use of chemical products has generated negative aspects for the biotic complex of nature,
affecting plants, animals and humans resulting in a growing demand for alternatives to chemical control.
Entomopathogenic fungi have already been recognized as a viable alternate control option for chemicals.
Myco-biocontrol is an environmentally sound and effective means of mitigating insect-pests. Past researches
have shown fungi being a potential biological control agent mainly due to their high reproductive capabilities,
target specific activity, short generation time and resting stage producing capabilities that can ensure their
survival for a longer time when no host is present (Sandhu et al. 2012). Entomopathogenic fungi need to
penetrate through the cuticle into the insect body to obtain nutrients for their growth and reproduction.
∗ Corresponding author E-mail: okremadevi@icfre.org, okremadevi@gmail.com

Sapna et al.
26
Penetration of the insect cuticle requires both mechanical pressure by penetrated hyphae and enzymic
degradation by a range of extracellular cuticle degrading enzymes, including chitinases, lipases and at least
four different classes of proteases (Hegedus and Khachatourions 1995, St. Leger et al. 1996). Proteases
play an important role in providing nutrients before and after the cuticle is penetrated. On the other hand
chitinase is required only for a brief period during penetration of host cuticle and is tightly regulated by chitin
degradation products. Chitinolytic enzymes usually act after the pathogen proteases have significantly
digested the cuticle protein and unmasked the chitin component of the cuticle (St. Leger et al. 1998).
Enzyme secretion is believed to be a key factor in determining the virulence of the isolate and this is
considered as a rationale for the enhance virulence of certain isolates (Mustafa and Kaur 2009). Highly
pathogenic strains show detectable amounts of extracellular chitinase, lipase, and protease activities
Samuels et al. (1989). In this study the potentiality of nine M. anisopliae were assessed to produce chitinase,
protease and lipase enzymes by determining of chitinase, protease and lipase index and activities.
Materials and Methods
Fungus
Among the nine fungal isolates (MIS1 to MIS25) used in this study, 6 were isolated either from soil or from
infected insects and 3 procured from different institutions.
Enzyme Assay
Preparation of Culture filtrate
Pure cultures of the isolates were prepared on potato dextrose agar yeast (PDAY) plates. After 4 days of
incubation, suspension of the different isolates with a concentration of 1x107 spores ml-1 were prepared and
inoculated (5 ml) into 500 ml of Potato Dextrose Broth (PDB) fortified with 1% Yeast extract and incubated for
7 days at 28±1°C and 90% RH in dark. The mycelia were filtered through Whatman No.1 filter paper and the
suspensions were further filtered through 0.22 µm filter (Millipore) before use.
Chitinase assay
Chitinase index was assessed (De Boer et al. 2004, Valadares-Inglis and Azevedo 1997) by measuring the
clear zone produced by degradation of chitin in Chitin yeast extract agar (CYEA) in combination with (2%
colloidal chitin, 0.05% yeast extract, 2% agar and 0.01% congo red). Ten mili meter well was cut in the
centre of the CYEA plate and 50 µl of crude culture filtrate was added to the well. The zone of clearance
around the well was measured after four days of incubation. Each plate served as a replicate with three
replications per treatment. Enzymatic index was calculated based on diameter of the halos with well, divided
by well diameter.
Chitinolytic activity was assayed according to the method of Valdimir et al. (2002) by measuring the release
of reducing saccharides from colloidal chitin: A reaction mixture containing 1 ml of crude culture filtrate, 0.3
ml of 1M sodium acetate buffer (pH 4.7) and 0.2 ml of colloidal chitin was incubated at 40º C for 6h and then
centrifuged at 12,225 g for 5 min at 6º C. After centrifugation, an aliquot of 0.75 ml of the supernatant, 0.25
ml of 1% solution of dinitrosalicylic acid in 0.7M NaOH and 0.1 ml of 10M NaOH were mixed in 1.5ml
eppendorf tubes and heated at 100º C for 5 min. Absorbance of the reaction mixture at 582 nm was
measured after cooling in room temperature. Each tube served as a replicate and three replications were
done for per treatment. A calibration curve with N-acetyl D-glucosamine as a standard was used to
determine the reducing sugar concentration. One unit of enzyme activity was defined as the amount of
enzyme that released 1µ mole of N-acetyl D-glucosamine per min under conditions described.

Cuticle degrading enzyme 27
Protease assay
Protease index in solid medium was tested (St. Leger et al. 1999, Valadares-Inglis and Azevedo 1997) by
measuring the clearing zone produced by degradation of milk protein in pH indicator medium (0.01% yeast
extract, 2% agar, 0.01% bromocresol purple adjusted to pH 5.2) containing 1% skimmed milk. Ten mili meter
well was cut in the centre of the medium and 50 µl of crude culture filtrate added to the well and the zone of
clearance around the well was measured after 24-48 h of incubation. Each plate served as a replicate and three
replications were done per treatment. Enzymatic index was calculated based on diameter of the halos with well
divided by well diameter.
Protease activity was assayed according to the method of (Hossain et al. 2006).The reaction mixture containing
3 ml of 1% (w/v) casein in 3 ml 0.1M citrate-phosphate buffer, pH 7.0 and 3 ml of crude culture filtrate was
incubated at 40 ± 1°C for 1 hr. The reaction was stopped by the addition of 5 ml 20 % (w/v) TCA and the
absorbance of the solution was measured at 650 nm in a spectrophotometer (SP3000 PLUS, CE OPTIMA,
Tokyo, Japan).Each tube served as replicate with three replications were done for per treatment. The amount of
amino acids released was calculated from a standard curve plotted against known concentrations of tyrosine.
One unit of enzyme was defined as the amount of enzyme that released 1µg of tyrosine ml-1 of substrate.
Lipase assay
Lipase index in solid medium was measured by the clearing zone produced by degradation of lipids in tributyrin
agar (0.5% peptone, 0.3% yeast extract, 2% agar, 0.01% methyl red, pH 7.5 with 1% tributyrin). One percent
sterile tributyrin was added to the media after cooling to 80°C and mixed thoroughly to emulsify the tributyrin
completely and poured to maintain uniform turbidity. Ten mili meter well was cut in the centre of tributyrin agar
plate and 50 µl of crude culture filtrate added to the well and the zone of clearance around the well measured
after 24-48 h of incubation. Each plate served as a replicate with three replications per treatment. Enzymatic
index was calculated based on diameter of the halos with well divided by well diameter.
Lipase activity was measured by a titrimetric assay with 0.05N NaOH using emulsified olive oil as substrate
(Kamimura et al. 1999). One ml of crude culture filtrate was added to 5 ml emulsion containing 25% (v/v) olive
oil and 75% (v/v) gum arabic and 2 ml 10 mM phosphate buffer at pH 7. The reaction mixture was incubated at
37°C for 30 min. The reaction was stopped by adding 15 ml acetone–ethanol (1:1v/v) and the amount of fatty
acids was then titrated. Each flask served as a replicate and three replications were done for per treatment. One
unit of lipase was defined as the amount of enzyme that released 1µ mole of fatty acids per min under these
conditions.
Results
Qualitative Assay
The nine isolates of M. anisopliae were tested for chitinase. All the isolates showed positive results and
chitinase index were ranged from 1.5 to 2.2 (Fig. 1). Maximum index of 2.2 was shown by the isolate MIS2
and next of index was exhibited by isolates MIS7, MIS13, MIS24, MIS20, MIS19 and MIS18. The lowest
index of 1.5 was recorded for the isolates MIS1 and MIS3.
Protease index varied from 1.2 to 3.3 for the nine isolates (Fig. 2). Isolate MIS7 showed highest protease
index of 3.33. Moderate index values were recorded in isolates MIS13, MIS2 and MIS19. Isolates MIS3 and
MIS1 showed intermediate index value of 1.83 and the lowest was recorded in MIS24.
Lipase index varied significantly between the tested isolates (Fig. 3) and the values ranged from 1.15 to 7.0.
The highest index of 7.0 was recorded in isolates MIS7 and MIS13. Moderate index values of 6 to 4.80 were
recorded for isolates MIS2, MIS24 and MIS18 respectively and the lowest index of 1.15 was exhibited for
isolate MIS1.

Sapna et al.
28
Chitinase index of Metarhizium isolates
0
0.5
1
1.5
2
2.5
MIS1 MIS2 MIS3 MIS7 MIS13 MIS18 MIS19 MIS20 MIS24
Isolates
Chitinase inde
x
Fig. 1. Chitinase index of Metarhizium isolates.
Protease index of Metarhizium isolates
0
0.5
1
1.5
2
2.5
3
3.5
4
MIS1 MIS2 MIS3 MIS7 MIS13 MIS18 MIS19 MIS20 MIS24
Isolates
Prote ase inde
x
Fig. 2. Protease index of Metarhizium isolates.
Lipas e inde x of Met arh izium isolates
0
1
2
3
4
5
6
7
8
MIS1 MIS2 MIS3 MIS7 MIS13 MIS18 MIS19 MIS20 MIS24
Isolates
Lipas e inde
x
Fig. 3. Lipase index of Metarhizium isolates.

Cuticle degrading enzyme 29
Quantitative Assay
All the isolates tested for chitinase production in submerged culture and the chitinolytic activity were ranged
from 0.525 to 1.560 U/ml (Table 1). The highest activity of 1.560 U/ml was shown by the isolate MIS2
followed by MIS7. Moderate activity was in the range of 1.001 U/ml to 1.152 U/ml for isolates MIS13, MIS1
and MIS18. and the lowest activity was exhibited in MIS20.
Table 1. Chitinase activity of Metarhizium isolates.
Isolates Chitinase Activity (U/ml)
MIS1 1.082d
MIS2 1.560a
MIS3 0.662g
MIS7 1.430b
MIS13 1.152c
MIS18 1.001e
MIS19 0.603h
MIS20 0.525i
MIS24 0.780f
SED CD(.05) CD(.01)
0.0252 0.0535 0.0737
Protease activity ranged from 0.020 to 0.114 U/ml (Table 2). The highest protease activity was recorded in
MIS7 (1.560 U/ml) followed by MIS20 (0.107 U/ml), MIS18(0.092 U/ml) MIS2(0.096 U/ml) MIS13(0.095U/ml).
The isolate MIS20 showed the lowest protease activity.
Table 2. Protease activity of Metarhizium isolates.
Isolates Protease Activity (U/ml)
MIS1 0.075e
MIS2 0.096c
MIS3 0.020f
MIS7 0.114a
MIS13 0.095c
MIS18 0.092c
MIS19 0.083d
MIS20 0.107b
MIS24 0.079de
SED CD(.05) CD(.01)
0.0027 0.0058 0.0080

Sapna et al.
30
The lipolytic activity for the nine isolates were recorded and ranged from 0.153 to 0.500 U/ml (Table 3).
Maximum lipase production was recorded for MIS7 with an activity of 0.50 U/ml followed by isolates MIS2
and MIS13 with activity of 0.492 U/ml. Moderate activity was observed in isolates MIS24 and MIS18. The
lowest lipase activity of 0.153 U/ml was recorded in MIS1.
Table 3. Lipase activity of Metarhizium isolates.
Isolates Lipase Activity (U/ml)
MIS1 0.153f
MIS2 0.492a
MIS3 0.200d
MIS7 0.500a
MIS13 0.492a
MIS18 0.364c
MIS19 0.196de
MIS20 0.190e
MIS24 0.400b
SED CD(.05) CD(.01)
0.0045 0.0096 0.0132
Discussion
From the result it was observed that all the nine isolates showed chitinase index ranged from 1.5 to 2.2 and
the chitinolytic activity ranged from 0.525 to 1.560 U/ml. in early works chitinase activity 0.01-0.0398U/ml was
recorded for various M. anisopliae isolates studied by Nahar et al. (2004). Braga et al. (1998) evaluated
chitinolytic activity of seventeen isolates of M. anisopliae isolates and reported the activity to vary from
0.0261 to 0.1340U/ml. Similarly St. Leger et al. (1986) reported chitinase activity of 0.027U/ml for M.
anisopliae. Wu (2010) evaluated the chitinase activity of M. anisopliae and recorded chitinase yield of
105.32mU/ml. Markedly higher chitinolytic activity (8.66mU/ml) was detected in the culture fluid when M.
anisopliae were grown in a medium containing colloidal chitin as a sole carbon source (Kang et al. 1999).
The nine isolates showed protease index in the range of 1.2 to 3.3 and protease activity ranged from 0.020 to
0.114 U/ml. This result support the study of Nahar et al. (2004) who reported protease activity of 0.01U/ml for
M. anisopliae in both YPG and chitin medium. Protease index of forty segregants of M. anisopliae were
ranged from 1.357-1.923 (Valadares-Inglis and Azevedo 1997). Protease index on mineral agar medium
amended with gelatin was 3.56 and 3.25 and in mineral agar medium amended with casein was 1.87 and 2.2
respectively at pH 6.8 and 8.5 for B. bassiana (Dias et al. 2008). M. anisopliae is the mainly targeted
entomopathogenic fungi for the study of cuticle-degrading protease as they are reported to produce a variety
of fungal proteases (Cole et al. 1993).
In the present study index was recorded 1.15 to 7.0 were recorded by the nine isolates in medium containing
tributyrin containing medium. Screening of lipase producers on agar plates is frequently done by using
tributyrin as a substrate (Cardenas et al. 2001) and clear zones around the colonies indicate production of
lipase (Sharma et al. 2001). The lipase activities of the isolates were ranged from 0.153 to 0.500 U/ml. Nahar
et al. (2004) showed lipolytic activity of 0.312 and 0.015U/ml in YPG and chitin medium respectively. Fungi
are widely recognized as preferable lipase sources.

Cuticle degrading enzyme 31
Braga et al. (1998) carried out chitinolytic activity together with estimates of the genetic parameters of such
activities and suggested that these parameters help in improving these traits in M. anisopliae. Mustafa and
Kaur (2009) studied in-vitro production of cuticle-degrading enzymes, such as chitinase, proteinase,
caseinase, lipase and amylase in fourteen isolates of M. anisopliae and suggested that the enzyme
production exhibited significant natural isolate variability. Two chymoelastases and three trypsinlike
proteases were separated from culture filtrates of the entomopathogen M. anisopliae by St. Leger et al.
(1987) who reported rapid production of proteases (Prl and Pr2) by Metarhizium anisopliae in culture media
and in situ on insect cuticle (St. Leger et al. 1987a). The production of the cuticle-degrading extracellular
proteases, chymoelastase (Pr1) and trypsin (Pr2) were reported by Pinto et al. (2002) by the isolates of M.
flavoviride.
In present study three isolates such as (MIS2, MIS7 and MIS13) demonstrated higher CDE production to
have higher virulence based on the bioassay conducted earlier by the authors (Remadevi et al. 2010). This
strongly substantiates the role of CDEs in deciding the virulence of Metarhizium isolates.
Conclusion
Variation in the overall enzyme production of each isolate parallels the differing virulence among isolates,
indicating that the whole cuticle-degrading enzyme machinery, rather than the individual enzymes,
determines virulence (Santiago and Gabriel 2000). The present investigation suggests that the isolates of M.
anisopliae are virulent as produced increased amount of chitinase, protease and lipase enzyme. Attempts to
enhance the enzyme production of the isolates by strain improvement, modification of culture conditions or
genetic manipulation may facilitate the development of a much proficient pest control strategy.
Acknowledgement
The authors are grateful to the Department of Biotechnology, New Delhi for providing financial support to
carry out this work. Thanks are due to the Director, ATREE and Director, IWST, Bangalore for providing
facilities to undertake the study. The permission granted by the PCCF Karnataka and PCCF Kerala to
undertake survey in the states is also acknowledged.
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