The oomycete pathogen, Phytophthora meadii, causes various diseases in Hevea brasiliensis at different stages of its life cycle. The study reports the structural characterization of the active principle from the culture filtrate of Alcaligenes sp. EIL-2 (GenBank ID: HQ641257) offering antagonistic activity against P. meadii. Gas Chromatography Mass Spectroscopy (GC-MS) analysis showed the similarity of the compound with phenazine derivatives. The specific representations of FT-IR spectrum such as 3200 cm(-1) (OH stretching, NH stretching and presence of aromatic ring), 1737 cm(-1) (carboxylic acid), 2200-2400 cm(-1) (conjugated dienes) and 1467 cm(-1), and 1422 cm(-1) (CN bonds) were an indicative of phenazine-1-carboxylic acid (PCA). The structure of the compound was further confirmed by (1)H NMR/(13)C NMR spectroscopy, DEPT experiments, and two-dimensional NMR spectral studies, including (1)H-(1)H COSY and (1)H-(13)C HSQC as PCA with the molecular formula of C₁₃H₈N₂O₂. P. meadii was sensitive to purified PCA extract from the endophyte and a concentration of 5 μg/ml completely inhibited the mycelia growth. PCA also showed zoosporicidal activity against P. meadii zoospores. This is the first study of this kind where PCA from an endophyte of H. brasiliensis is being confirmed to carry antagonistic activity against P. meadii.
We report the molecular characterization of β-1,3-glucanase-producing Bacillus amyloliquefaciens-an endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii. After cloning and sequencing, the β-1,3-glucanase gene was found to be 747 bp in length. A homology model of the β-1,3-glucanase protein was built from the amino acid sequence obtained upon translation of the gene. The target β-1,3-glucanase protein and the template protein, endo β-1,3-1,4-glucanase protein (PDB ID: 3o5s), were found to share 94% sequence identity and to have similar secondary and tertiary structures. In the modeled structure, three residues in the active site region of the template-Asn52, Ile157 and Val158-were substituted with Asp, Leu and Ala, respectively. Computer-aided docking studies of the substrate disaccharide (β-1, 3-glucan) with the target as well as with the template proteins showed that the two protein-substrate complexes were stabilized by three hydrogen bonds and by many van der Waals interactions. Although the binding energies and the number of hydrogen bonds were the same in both complexes, the orientations of the substrate in the active sites of the two proteins were different. These variations might be due to the change in the three amino acids in the active site region of the two proteins. The difference in substrate orientation in the active site could also affect the catalytic potential of the β-1,3 glucanase enzyme.
α-1,4-Amylase is one of the most important industrial enzymes and there is enormous interest in isolating α-1,4-amylase with better properties. The α-1,4-amylase producing endophytic Bacillus amyloliquefaciens was isolated and characterized from Hevea brasiliensis. The α-1,4-amylase gene after cloning and sequencing contained 1542 base pairs. A homology model of the α-1,4-amylase enzyme was built from the deduced amino acid sequence. The modelled and template α-1,4-amylase enzyme (PDB ID:3bh4) showed 97.7% sequence identity with similar secondary and tertiary structures. Computer aided docking studies of the substrate (maltotetraose) with the modelled as well as the template enzymes showed that although the binding energies were almost the same in both the complexes, the number of hydrogen bonds and van der Waals interactions in the active sites of the two enzymes were different. These variations might be due to the change in the amino acid residues of the active site regions of two enzymes. The mutated polar amino acids in the active site of modelled α-1,4-amylase favoured more hydrogen bond formation with the substrate. The difference in the active site interactions may improve the specificity of the enzyme and affect the catalytic potential of α-1,4-amylase.
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