Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.     

Partial role of nitric oxide in infarct size limiting effect of quercetin and rutin against ischemia-reperfusion injury in normal and diabetic rats

Reperfusion injury is remarkable clinical issue that needs to be resolved as ischemia-reperfusion is a common phenomenon encountered in numerous clinical situations. The present communication report the involvement of nitric oxide (NO) in cardioprotection offered by flavonoids (rutin and quercetin) against myocardial ischemia reperfusion. Rutin produced better cardioprotection than quercetin in normal and diabetic rats. The observed cardioprotection offered with quercetin and rutin was partially abolished by prior administration of nitric oxide synthase inhibitor, L-NAME (N-nitro-L-arginine methyl ester) in both normal and diabetic rats. L-NAME abolished the cardioprotective actions of rutin more strongly than the cardioprotective actions of quercetin. However, mechanistic study with NOS inhibitor implied the possible partial role of nitric oxide in infarct size limiting effect of quercetin and rutin     

Gamma-radiation induced modifications in substrate specificity of glucose dehydrogenase and carbon source utilization pattern of phosphate-solubilizing <Emphasis Type="Italic">Pantoea</Emphasis> strains

Glucose, maltose, and mannose as sole carbon sources, induced synthesis of glucose dehydrogenase (GDH) in three strains of Pantoea with specific activities from 0.14 to 0.6 U/mg proteins. Utilization of lactose indicated that the enzyme belongs to GDH type B isozyme. Of mutant clones, developed through radiation mutagenesis, P2-M2 utilized ribose with GDH specific activity of 0.57 U/mg protein, P4-M3 grown on glucose gave 1.5 U/mg protein and P4-M5 had high activities, when grown on galactose, maltose, and lactose. Clones P3-M2 and P2-M5 had versatile utilization of sugars and released higher amounts of P from tri-calcium phosphate and can be efficiently used for biofertilization.     

Synthesis,characterization and biological evaluation of some novel 2,4-thiazolidinediones as potential cytotoxic,antimicrobial and antihyperglycemic agents

A series of some novel 2,4-thiazolidinediones (TZDs) (2a–x) have been synthesized and characterized by FTIR, ¹H NMR, ¹³C NMR and LC mass spectral analysis. All the synthesized compounds were evaluated for their cytotoxicity, antimicrobial and in vivo antihyperglycemic activities. Among the tested compounds for cytotoxicity using Brine Shrimp Lethality assay, compound 2t ((Z)-5-(4-((E)-3-oxo-3-(thiophen-2-yl)prop-1-enyl)benzylidene)-1,3-thiazolidine-2,4-dione) exhibited significant inhibitory activity at ED₅₀ value 4.00 ± 0.25 μg/mL and this level of activity was comparable to that of the reference drug podophyllotoxin with ED₅₀ value 3.61 ± 0.17 μg/mL. Antimicrobial activity was screened using agar well diffusion assay method against selected Gram-positive, Gram-negative and fungal strains and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. From the results of antimicrobial activity compound 2s ((Z)-5-(4-((E)-3-(3,5-bis(benzyloxy)phenyl)-3-oxoprop-1-enyl)benzylidene)-1,3-thiazolidine-2,4-dione) was found to be the most active against all the tested strains of microorganisms with MIC value 16 μg/mL. In vivo antihyperglycemic effect of twenty four TZDs (2a–x) at different doses 10, 30 and 50 mg/kg b.w (oral) were assessed using percentage reduction of plasma glucose (PG) levels in streptozotocin-induced type II diabetic rat models. From the results, the novel compound 2x ((Z)-5-(4-((E)-3-(9H-fluoren-2-yl)-3-oxoprop-1-enyl)benzylidene)-1,3-thiazolidine-2,4-dione) exhibited considerably potent blood glucose lowering activity than that of the standard drug rosiglitazone and it could be a remarkable starting point to evaluate structure–activity relationships and to develop new lead molecules with potential cytotoxicity, antimicrobial and antihyperglycemic activities. In addition molecular docking studies were carried out against PPARγ molecular target using Molegro Virtual Docker v 4.0 to accomplish preliminary confirmation of the observed in vivo antihyperglycemic activity.     

Comparative study of thermostabilty and ester synthesis ability of free and immobilized lipases on cross linked silica gel

A novel support has been utilized for immobilization of lipase, which was prepared by amination of silica with ethanolamine followed by cross linking with glutaraldehyde. Lipases from Rhizopus oryzae 3562 and Enterobacter aerogenes were immobilized on activated silica gel, where they retained 60 and 50% of respective original activity. The thermal stability of the immobilized lipases was significantly improved in comparison to the free forms while the pH stability remained unchanged. E. aerogenes and R. oryzae 3562 lipases retained 75 and 97% of respective initial activity on incubation at 90 degrees C, whereas both the free forms became inactive at this temperature. The conversion yield of isoamyl acetate was found to be higher with the immobilized fungal (90 vs. 21%) and bacterial lipases (64 vs. 18%) than the respective free forms. Immobilized R. oryzae 3562 lipases retained 50% activity for isoamyl acetate synthesis up to ten cycles whereas it was eight cycles for E. aerogenes.     

Isolation and Characterization of ACC Deaminase Gene from Two Plant Growth-Promoting Rhizobacteria

Lowering of plant ethylene by deamination of its immediate precursor 1-aminocyclopropane-1-carboxylate (ACC) is a key trait found in many rhizobacteria. We isolated and screened bacteria from the rhizosphere of wheat for their ACC-degrading ability. The ACC deaminase gene (acdS) isolated from two bacterial isolates through PCR amplification was cloned and sequenced. Nucleotide sequence alignment of these genes with previously reported genes of Pseudomonas sp. strain ACP and Enterobacter cloacae strain UW4 showed variation in their sequences. In the phylogenetic analysis, distinctness of these two genes was observed as a separate cluster. 16S rDNA sequencing of two isolates identified them to be Achromobacter sp. and Pseudomonas stutzeri.     

Thermal inactivation of glucose oxidase. Mechanism and stabilization using additives

Thermal inactivation of glucose oxidase (GOD; beta-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 degrees C. The activation energy increased from 60.3 kcal mol-1 to 72.9, 76.1, and 88.3 kcal mol-1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal x mol-1 x deg-1 in the presence of 7.1 x 10-5 m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25-90 degrees C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 degrees C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 degrees C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.     

Verification and Rapid Identification of Soybean Rhizobia in Indian Soils

Sixty root nodule isolates of soybean rhizobia indigenous to eight field sites in India were characterized using PCR-RFLP for repeated sequence RSα a 1195-bp DNA fragment, indole acetic acid production, and nitrogenase activity. Site-dependent variations were observed in terms of IAA production and nitrogenase activities. RSα was conserved in slow-growing soybean rhizobia across locations and sites and was absent in other Rhizobiaceae members and other bacterial genera. The results suggest that RSα can be a useful molecular marker for slow-growing soybean rhizobia. The study also showed the low presence of soybean nodulating fast growers in Indian soils.     

Genetic Diversity and Population Structure in Fragmented Natural Populations of Melia dubia Cav. Revealed by SSR Markers—Its Implications on Conservation

Melia dubia, a fast-growing tree species with multi-various uses, is suitable species for agro and farm forestry. In an extensive survey in eight districts of Karnataka, India, it was observed that the majority of M. dubia natural populations are fragmented and tree number is very less. Sixty trees were selected to assess the genetic variability using SSR markers. Analysis revealed a moderate level of genetic diversity (Ho?=?0.47; He?=?0.69) and inbreeding as evinced by overall positive fixation index of 0.30. Mean Fst observed was 0.16 indicating moderate genetic differentiation among populations. The analysis of molecular variance ascribed 99% of total genetic diversity to within population variation. Cluster analysis by unweighted pair group method and genetic differentiation through structure analysis did not differentiate 10 populations as per geographic location, showing that moderate genetic diversity was not due to geographic distance. As genetic diversity was more within population, selection of individual plants would be more effective for genetic improvement to capture the natural variation within the population. An understanding of genetic diversity and differentiation of M. dubia natural populations will help in exploiting the genetic resource for tree improvement and also in formulating management and conservation strategies for this species.