Homology model of a novel thermostable xylanase from Bacillus subtilis-AK1

Xylanases are glycosyl hydrolases that catalyze the hydrolysis of internal beta-1,4 glycosidic bonds of xylan, the major hemi-cellulose component of the plant cell wall. Enzymes such as xylanases are used considerably in industries. Their industrial usage is especially attractive since they can replace some of the environmental pollutants. We have earlier isolated a family 11-xylanase gene from Bacillus subtilis-AK1, which is active at high temperature as well as at alkaline pH. In order to understand the factors liable for the adaptation of this enzyme, three dimensional model of B. subtilis-AK1 xylanase has now been obtained by homology modeling. Modeling was carried out using Molecular Operating Environment (MOE) software developed by Chemical Computing Group Inc., running on Pentium IV workstation. The model showed that B. subtilis-AK1 xylanase having molecular weight around 20 kDa contains in its fold an alpha-helix and two beta-sheets packed against each other forming a beta-sandwich. The conserved active site amino acids E78R, Y80L were mutated in this novel B. subtilis-AK1 strain, but the protein folding and the function was maintained with high thermal stability. Several minor modifications appeared to be responsible for the increased thermo stability of AK1. Docking studies of the substrate xylan with -AK1 shows the possibility of the Arg 78 acting as the nucleophile instead of Glu 78.     

Purification and partial characterization of serine protease from thermostable alkalophilic <Emphasis Type="Italic">Bacillus laterosporus</Emphasis>-AK1

An extracellular thermostable alkaline protease isolated from Bacillus laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced by the metal ions Ca²⁺ and Mg²⁺ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain completely when used with Wheel detergent.     

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k _cat/K _m determined for ABTS and syringaldazine were 9.2×10⁵ and 8.7×10⁵, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.     

Ganoderma australe from southern India

The genetic diversity of Ganoderma australe (Fr.) Pat. from southern India was investigated by using ITS1/2 rDNA. The phylogenetic analysis showed that six isolates clustered into two groups viz. biological species I and II. The four strains of BS I (YER03, MYC5, MYC2 and KE) clustered with G. australe TAI-01 and the two other strains of BS II (KMK3 and K39) were grouped with G. australe TAI-05 from Taiwan. The two strains namely TAI-01 and TAI-05 were described as G. australe intersterile Group 1 and 2 from Taiwan, respectively. The higher level nucleotide divergence among BS I and BS II and the high bootstrapping support clearly represent the presence of two biological species of G. australe in southern India which are genetically isolated.     

Protoplast Fusion in Streptomyces sp. for Increased Production of Laccase and Associated Ligninolytic Enzymes

Biodegradation of lignin by Streptomyces spp. results in the production of value-added chemicals such as Acid Precipitable Polymeric Lignins (APPLs), low molecular weight phenols, etc. To hasten the conversion metabolism through genetic manipulation, a preliminary attempt was made to standardize the methodology for isolation and regeneration of protoplasts. Protoplast fusion recombinants were developed and assayed for their ligninolytic activity, production of ligninolytic enzymes viz., peroxidase, laccase, polyphenol oxidase and crude protein. In comparison with the mutants and wild strain, fusion recombinant F₄ showed higher laccase activity and lower peroxidase activity. This attribute can be positively used for polymerization of free phenolics to polycondensates related to humic acids in soil or composting environments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anaerobic biodegradation of aromatic compounds

Many aromatic compounds and their monomers are existing in nature. Besides they are introduced into the environment by human activity. The conversion of these aromatic compounds is mainly an aerobic process because of the involvement of molecular oxygen in ring fission and as an electron acceptor. Recent literatures indicated that ring fission of monomers and obligomers mainly occurs in anaerobic environments through anaerobic respiration with nitrate, sulphate, carbon dioxide or carbonate as electron acceptors. These anaerobic processes will help to work out the better situation for bioremediation of contaminated environments. While there are plenty of efforts to reduce the release of these chemicals to the environment, already contaminated sites need to be remediated not only to restore the sites but to prevent the leachates spreading to nearby environment. Basically microorganisms are better candidates for breakdown of these compounds because of their wider catalytic mechanisms and the ability to act even in the absence of oxygen. These microbes can be grouped based on their energy mechanisms. Normally, the aerobic counterparts employ the enzymes like mono-and-dioxygenases. The end product is basically catechol, which further may be metabolised to CO2 by means of quinones reductases cycles. In the absense of reductases compounds, the reduced catechols tend to become oxidised to form many quinone compounds. The quinone products are more recalcitrant and lead to other aesthetic problems like colour in water, unpleasant odour, etc. On the contrary, in the reducing environment this process is prevented and in a cascade of pathways, the cleaved products are converted to acetyl co-A to be integrated into other central metabolite paths. The central metabolite of anaerobic degradation is invariably co-A thio-esters of benzoic acid or hydroxy benzoic acid. The benzene ring undergoes various substitution and addition reactions to form chloro-, nitro-, methyl- compounds. For complete degradation the side chains must be removed first and then the benzene ring is activated by carboxylation or hydroxylation or co-A thioester formation. In the next step the activated ring is converted to a form that can be collected in the central pool of metabolism. The third step is the channeling reaction in which the products of the catalysis are directed into central metabolite pool. The enzymes involved in these mechanisms are mostly benzyl co-A ligase, benzyl alcohol dehydrogenase. Other enzymes involved in this path are yet to be purified though many of the reactions products that have been theoretically postulated have been identified. This is mainly due to the instability of intermediate compounds as well as the association of the enzyme substrate is femoral and experimental conditions need to be sophisticated further for isolation of these enzymes. The first structural genes of benzoate and hydroxy benzoate ligases were isolated from Rhodopseudomonas palustris. This gene cluster of 30 kb size found in Rhodopseudomonas palustris coded for the Bad A protein. Similarly, some of the bph A,B,C and D cluster of genes coding for the degradation of pentachlorobenzenes were located in Pseudomonas pseudoalgaligenesKF 707.     

Diversity of probiotic adhesion genes in the gastrointestinal tract of goats

Gastrointestinal (GI) microflora is an important system in the host, as it has both pathogenic and probiotic bacteria. Most of the studies were focused on the human gut microflora and the available information on the intestinal microflora of goats was limited. This urged the need to inspect the impacts of the goat's gut microflora. Metagenomic investigation of probiotic bacteria in the GI tract of goat is one of the challenging streams because of the less available data of the uncultivable bacteria. In our report, comparative analysis of metagenomic and enrichment samples of goat intestinal content was done and this approach will be helpful in analyzing the identification of uncultivable and cultivable probiotic bacteria. This study mainly focused on three key probiotic adhesion genes, such as EF-Tu, mapA, and mub. The GI of four different goats were investigated for these genes. The data from this study showed that there is a wide diversity of these genes among goat intestinal samples.     

Biosynthesis of silver and gold nanoparticles using thermophilic bacterium Geobacillus stearothermophilus

Nanomaterials have assumed a great deal of importance as they often display unique and considerably modified physical, chemical and biological properties as compared to their counterparts of the macroscale. In this study, biogenic synthesis of silver and gold nanoparticles by Geobacillus stearothermophilus has been attempted. The exposure of G. stearothermophilus cell free extract to the metal salts leads to the formation of stable silver and gold nanoparticles in the solution. These nanoparticles were characterized by UV–Vis spectra, FTIR, TEM, and XRD. The silver and gold nanoparticles have absorption maxima at 423 nm and 522 nm respectively. The TEM micrograph revealed the formation of polydispersed particles in the case of silver nanoparticles and monodispersed particles with respect to the gold nanoparticles. High stability of the nanoparticle solution could be attributed to the secretion of certain capping proteins by the bacterium in the reaction mixture. The involvement of these proteins was confirmed by FTIR and SDS PAGE.     

Effect of biologically synthesized silver nanoparticles on Bacopa monnieri (Linn.) Wettst. plant growth metabolism

In this present study, interactions of biologically synthesized silver nanoparticles on hydroponically grown Bacopa monnieri (Linn.) Wettst. plant growth metabolism were documented. Estimates of protein, carbohydrate, total phenols, in addition antioxidant enzymes, catalase and peroxidise were assayed in various parts of the plants grown in hydroponic solution. The silver nanoparticles used in this study were synthesized by treating AgNO₃ with aqueous leaves extracts of Acalypha indica Linn., a medicinal herb as a source of reductants. Enhanced peroxidase and catalase activity, simulated the stress conditions induced by the silver nitrate treatment. No severe toxic effects were observed in silver nanoparticles treated plants in the morphological studies under scanning electron microscopy (SEM) while structural aberrations were observed in the light microscopic evaluation of root and stem anatomy. Further, the uptake of silver in the root and stem tissues of B. monnieri (Linn.) Wettst. was confirmed using atomic absorption spectrophotometer (AAS).