Citation
Kiong, Diana Siaw Boon
(2015)
Physical characterization of hydrophobin-like proteins from aerial conidia of entomopathogenic fungus (metarhizium anisopliae var.anisopliae) and their role in controlling termite (coptotermes curvignathus).
Masters thesis, Universiti Putra Malaysia.
Abstract
Hydrophobin-like protein (HLP) was found in the aerial conidia of this fungus
and play a role in attachment on the cuticle surface of the termite during
pathogenesis. The aim of this study was to isolate and physically characterized
HLP from the aerial conidia of two local isolates of M. anisopliae, also to
evaluate the interaction effect between HLP and M. anisopliae conidia
suspension on the mortality of C. curvignathus. The protein samples were
isolated based on their insolubility in hot sodium dodecyl sulfate (SDS). The
SDS-insoluble proteins were then purified by three purification methods namely
formic acid only, formic acid followed by performic acid, and trifluoroacetic acid.
The isolated and purified protein samples were further quantified and
characterized as HLP by studying the contact angle when applied on the
hydrophobic surface, wetting ability when applied on hydrophilic surface and
emulsifying properties. Virulence test of termite bioassay was performed. The
HLP samples were verified by their low molecular weight and unique
characteristics. Due to the stringent protocol of isolation and purification, the
formic acid followed by performic acid purified proteins showed four
conspicuous highly intensified bands (13.5 kDa, 15.0 kDa, 16.5 kDa and
17.0 kDa). This method of purification produced highest concentration of HLP
(17.82 μg mL-1 and 15.64 μg mL-1 for both isolates TA and LR2, respectively)
among the three purification methods. The contact angle measurement showed
that both protein isolates HLPTA (58.43° ± 0.25°) and HLPLR2 (57.06°± 0.38°)
have the contact angle of < 90°when applied on the hydrophobic surface as
compared to the water (108.37° ± 0.50°). When hydrophilic paper was coated
with a layer of HLP solution, the paper resisted wetting by water up to 439 ±
20.52 seconds. These results indicated that the HLP is able to convert
hydrophilic surface into hydrophobic property and vice versa. This was due to
the unique characteristic of HLP which can self-assemble themselves and
configure the adsorption at the hydrophobic-hydrophilic interfaces. The HLP also revealed good emulsification effect in the oil-water phase. Aerial conidia of
M. anisopliae showed good dispersion in aqueous solution with the application
of pre-coated HLP prior to the mixing of conidia suspension. Two application
methods were used to test the efficacy and pathogenicity of M. anisopliae with
HLP samples. The termite, C. curvignathus showed the fastest trend of
mortality (100%) by the pre-coated method with HLPTA prior to the conida
topical application on the Day 4. Similar trend of mortality was observed for the
same method of application for HLPLR2. HLP treatment alone on termite did
not revealed any toxic effect. In conclusion, the most applicable purifying agent
used for the purification process of hydrophobin-like proteins (HLP) samples
was 98% formic acid followed by perfomic acid. This study showed the strain
specificity of the HLP on its application and was found to have enhancing effect
but non-toxic when applied alone on C. curvignathus. These unique properties
of HLP from local isolates of M. anisopliae var. anisopliae are of great potential
to be used in wide range of industrial applications especially in enhancing the
formulation of the biological control agent in controlling insect pests.
Download File
Additional Metadata
Actions (login required)
![View Item View Item](/style/images/action_view.png) |
View Item |