Abstract
Crystal (Cry) proteins produced by Bacillus thuringiensis (Bt) species are δ-endotoxins known
for their insecticidal activity against agricultural pests and disease vectors. These endotoxins
exert their effect by binding to specific receptors in the insect midgut, leading to pore formation
and cell lysis. The increasing demand for eco-friendly insecticidal agents has driven research
into novel Cry-producing strains with enhanced specificity and potency. The present study
aimed to profile the crystal proteins of Bacillus dicomae and investigate, through in silico
approaches, their potential biotechnological applications. Bacillus dicomae is a novel species
previously isolated from medicinal plant Dicoma anomala. The species was found to produce
Cry like proteins. Cry proteins were extracted using alkaline solubilization, yielding a crude
protein mixture enriched in parasporal inclusions. Scanning electron microscopy (SEM)
confirmed the presence of bipyramidal, cuboidal and spherical crystalline structures typical of
Cry proteins produced by Bt. Partial purification using the ÄKTA Go fast protein liquid
chromatography (FPLC) system revealed three Ultraviolet (UV) absorbance peaks during a salt
gradient, with Peak C (~53.7 mL) corresponding to the main Cry protein fraction. Sodium
deodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified
fractions showed protein bands within the 130–48 kDa range, consistent with Cry protein
molecular weights. Liquid chromatography mass spectrometry (LC-MS/MS) profiling
validated the expression of Cry4Da1, Cry1Hc1, and Cry2Ba1 through detection of proteotypic
peptides. Genomic interrogation via BLASTX identified Cry4Da1 gene fragments with
homology to conserved domains, including glycosyltransferases and DinB-like proteins.
Phylogenetic analysis further supported the evolutionary placement of B. dicomae within Cryproducing
clades. To evaluate the structural and functional potential of Cry4Da1, a threedimensional
model was generated using SWISS-MODEL, guided by a Cry protein template
with 38.49% sequence identity. Structural validation via PROCHECK, GMQE, QMEAN, and
RMSD (0.407 Å) confirmed the reliability of the predicted fold. Multiple sequence alignment
with Cry1Ac revealed conserved secondary structure motifs and a seven-domain protoxin
architecture. Superimposition analysis demonstrated strong structural overlap with the
reference template, supporting domain-level homology. Electrostatic surface potential
mapping identified charged regions within receptor-facing domains, suggesting functional
relevance for midgut receptor interaction. Molecular docking simulations were performed to
assess receptor-binding specificity between Cry4Da1 and two Aedes aegypti midgut receptors:
aminopeptidase N (APN) and alkaline phosphatase (ALP). Homology models of APN and ALP
iv
were constructed and validated prior to docking using ClusPro. Electrostatic surface analysis
and RING-based contact mapping revealed that the Cry4Da1–ALP complex exhibited a
compact, domain-integrated binding interface involving Domains II and III, supported by
strong electrostatic complementarity and a favourable docking score (−1061.8 kcal/mol). In
contrast, the Cry4Da1–APN complex displayed a more diffuse interaction pattern, lower
docking affinity (−940.8 kcal/mol), and fewer stabilizing contacts. These findings suggest that
ALP is a structurally and energetically preferred receptor for Cry4Da1, consistent with its
known role in pore formation. Based on literature review and results obtained from this study,
it is concluded that B. dicomae is a novel source of Cry proteins with mosquitocidal potential.
To the best of our knowledge, this is the first report characterizing Cry4Da1 from B.
dicomae and its interaction with Aedes aegypt midgut receptors. Cry4Da1, in particular,
demonstrates strong receptor-binding specificity and structural integrity, supporting its
candidacy for further development as a targeted bio-insecticide.