Squalene hopene cyclases and oxido squalene cyclases: potential targets for regulating cyclisation reactions.

Squalene hopene cyclases (SHC) convert squalene, the linear triterpene to fused ring product hopanoid by the cationic cyclization mechanism. The main function of hopanoids, a class of pentacyclic triterpenoids in bacteria involves the maintenance of membrane fluidity and stability. 2, 3-oxido squalene cyclases are functional analogues of SHC in eukaryotes and both these enzymes have fascinated researchers for the high stereo selectivity, complexity, and efficiency they possess. The peculiar property of the enzyme squalene hopene cyclase to accommodate substrates other than its natural substrate can be exploited for the use of these enzymes in an industrial perspective. Here, we present an extensive overview of the enzyme squalene hopene cyclase with emphasis on the cloning and overexpression strategies. An attempt has been made to explore recent research trends around squalene cyclase mediated cyclization reactions of flavour and pharmaceutical significance by using non-natural molecules as substrates.

An Esterase with Increased Acetone Tolerance from Bacillus subtilis E9 over Expressed in E. coli BL21 Using pTac Bs-est Vector.

Bacillus subtilis E9 was identified as a potential strain producing esterase. The gene coding esterase from B. subtilis E9 was amplified using esterase-specific primers and the sequence was translated in silico. The presence of conserved catalytic triad amino acid residues (His-Ser-Asp/Glu) confirmed the functional nature of the esterase enzyme. Docking studies conducted with modeled protein and the ligand p-nitrophenyl acetate showed that the amino acid residues interacting with the ligand were Ser77, His76, and Gly103. The gene coding for esterase from B. subtilis E9 was cloned into an assembled vector having Tac promoter (pTac), pUC origin of replication, Ni-Histidine residues, ampicillin cassette, and T7 terminator using Golden gate DNA assembly method. The generated pTac Bs-est (4598 bp) recombinant plasmid was transformed and heterologously expressed in Escherichia coli BL21 (DE3) strain. The tagged recombinant protein was purified to yield 43.4% pure protein with specific activity of 772 U/mg. The purified recombinant protein was subjected to peptide sequencing and the identity was confirmed as esterase by peptide tandem mass fragmentation method using the LC-MS/MS analysis. The purified recombinant esterase was found to be organic solvent stable and tolerant up to 5 days retaining around 95% residual activity in 30-90% v/v Acetone. The recombinant esterase expressed in our study was found to exhibit better organic solvent stability and tolerance than compared to the original bacterial esterase from B. subtilis E9, a property which could be explored in the biocatalytic and synthetic transformation reactions for industrial applications.

Pineapple Peel Extract as an Effective Substrate for Esterase Production from Bacillus subtilis E9.

Esterase, belonging to hydrolase class of enzymes catalyzes the cleavage and formation of ester bonds. Esterase producing isolates E9 and E46, isolated from pineapple waste enriched soil were identified as Bacillus subtilis E9 and Bacillus sp. E46 respectively. Bacillus subtilis E9 with 10 U/mg esterase activity in basal media was further chosen for media optimization studies. Several factors including the effect of organic solvents and fruit peel extracts were studied by one factor at a time optimization method and statistical models. An enhanced enzyme production of 250.50 U/mg could be obtained under the optimal conditions of pH 6.5, incubation time 25 h and 1.8%v/v of acetone extract of pineapple peel. The four-stage purification improved the purity of the enzyme by 1.5-fold with 5.3% recovery and specific activity of 384 U/mg. The monomeric nature and the molecular weight (45 KDa) of the enzyme were determined by performing SDS PAGE and its activity was confirmed by zymogram analysis. The substrate specificity of the purified fraction exhibited a higher activity towards lower chain length esters, indicating the enzyme as esterase. The partially purified esterase showed an optimal temperature of 40 °C at an optimum pH of 7. Km and Vmax of the enzyme were 1.12 mM and 1.18 mM of released pNP · min-1 respectively.

Phenazine-1-carboxylic acid mediated anti-oomycete activity of the endophytic Alcaligenes sp. EIL-2 against Phytophthora meadii.

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.

In silico characterization of a novel ß-1,3-glucanase gene from Bacillus amyloliquefaciens--a bacterial endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii.

We report the molecular characterization of ß-1,3-glucanase-producing Bacillus amyloliquefaciens-an endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii. After cloning and sequencing, the ß-1,3-glucanase gene was found to be 747 bp in length. A homology model of the ß-1,3-glucanase protein was built from the amino acid sequence obtained upon translation of the gene. The target ß-1,3-glucanase protein and the template protein, endo ß-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 (ß-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 ß-1,3 glucanase enzyme.