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Molecular Plant Breeding 2012, Vol.3, No.7, 63
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subtilisin-like protease isoforms (Pr1A, Pr1B, Pr1C)
from isolates of
M. anisopliae
and an extracellular
protease from
B. bassiana
(BBP) has also been
purified and characterized by Urtiz and Rice (2000).
The isoelectric point of BBP was 7.5 and it is 0.5 kDa
smaller than Pr1. Fang et al (2002) reported a cuticle-
degrading protease (CDEP
-
1) from
B. bassiana
pre-
dicting a protein of 377 amino acids (Mr 38 616, pI
8.302) with 1 134 bp. Southern analysis indicated that
CDEP
-
1 is a single-copy gene. St Leger et al (1996)
constructed an engineered mycoinsecticides based on
M. anisopliae
by over-expressing the toxic protease
Pr1 from
M. anisopliae
genome to accelerate the
killing speed of
M. anisopliae
. The over-expression of
Pr1 in the hemolymph of
M. sexta
activates the
phenoloxidase system which causes 25% reduction
in the time of death and 40% reduction in food
consumption.
4.2 Chitinases
The major component of insect cuticle is chitin,
therefore both endo and exo-chitinases play critical
roles in the cleavage of
N
-Acetylglucosamine (NAGA)
polymer of the insect cuticle into smaller units or
monomers. Khachatourians (1991) demonstrated that
the extracellular chitinases are virulence determinant
factors. Chitinolytic enzymes (
N
-acetyl
-
β
-
D-glucosa-
minidases and endochitinases were present in the
broth culture supplemented with insect cuticles from
M. anisopliae
,
M. flavoviride
, and
B. bassiana
(St
Leger et al., 1996). The chitinase from
M. anisopliae
consists of acidic (pI 4.8) proteins with molecular
masses 43.5 kDa and 45 kDa. The identified N-terminal
sequences of both bands were similar to an endochi-
tinase from
Trichoderma harzianum
. Valadares-Inglis
and Peberdy (1997) located chitinolytic enzymes in
enzymatically produced protoplasts and whole cells
(mycelia) of
M. anisopliae
. No significant induction
was observed from mycelia, yet protoplasts were
found to induce these enzymes significantly. The
majority of chitinolytic activity was cell-bound in both
whole cells and protoplast preparations, and the
activity was mainly located in the membrane fraction.
Kang et al (1998, 1999) reported a chitinase with
molecular mass of 60 kD from
M. anisopliae
grown in
a medium containing chitin as the sole carbon source
with an optimum pH of 5.0, which is different from
the chitinases values previously reported by St Leger
et al (1996) for endo-chitinases of 33.0, 43.5, and 45
kDa and exo-chitinases of 110 kDa. Screen et al (2001)
cloned the chitinase gene (
Chit1
) from
M. anisopliae
sf.
acridum
ARSEF strain 324 and
M. anisopliae
sf.
anisopliae
ARSEF strain 2575 (
Chit1
) using the pro-
moter of
Aspergillus
(
gpd
) for constitutive expression.
A 42 kD chitinase of
M. anisopliae
was expressed and
characterized in
Escherichia coli
by Baratto et al
(2003) using a bacteriophage T7- based promoter
expression vector. Baratto et al (2006) performed
transcriptional analysis of the chitinase
chi2
gene of
M.
anisopliae
var.
anisopliae
and showed that it has 1
542 bp encoding for a deduced 419 amino acids.
Nahar et al (2004) reported that the extracellular
constitutive chitin deacetylase (CDA) secreted by
M.
anisopliae
converts chitin (a β
-
1, 4
-
linked N-acetyl-
glucosamine polymer) into its deacetylated form
chitosan (a glucosamine polymer). This CDA was not
inhibited by solubilized melanin.
Fang et al (2005) purified an endochitinase from
liquid cultures of
B. bassiana
supplemented with
chitin. Bbchit1 was 33 kD (pI 5.4) and the encoding
gene,
Bbchit1
, and its upstream regulatory sequences
were cloned based on N-terminal amino acid sequence.
Bbchit1
contains no introns and it is present as a
single copy in the
B. bassiana
genome. The amino
acid sequence of Bbchit1 is similar to that of the
endochitinase of
Streptomyces avermitilis
,
S. coelicolor
and
T. harzianum
(Chit36Y), but not to EPFs that
reflect novel chitinases. Fang and co-worker (2005)
constructed a
B. bassiana
transformants (
gpd
-
Bbchit1
),
which overproduced
Bbchit1
and had enhanced
virulence.
4.3 Lipases
Although the major bulk components of the insect
cuticle are protein and chitin, the outermost epicuticular
surface layer are made up of a complex mixture of
non-polar lipids. Epicuticular lipids play a role in
chemical communication events (Blomquist and Vogt,
2003), and in keeping the cuticular surface dry which
affects insecticide and chemicals penetration (Hadley,
1981; Blomquist et al., 1987; Juárez, 1994). They