Correction to: Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study.

In the original version of our paper entitled "Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study" (2005) 27:415-424, some references to already published articles were inadvertently left out.

Rheological properties and microstructure of xylanase containing whole wheat bread dough.

The present research work was undertaken to investigate the effect of xylanase, produced by Penicillium citrinum, on rheological behavior of whole wheat bread dough at large and small deformation respectively. Dough attributes including textural properties (penetration) and structure related characteristics (oscillatory tests) were evaluated. Change in visco-elastic properties of xylanase containing dough was evaluated by oscillatory and creep measurements. The flow experiments were conducted under steady-state condition with shear rate ranging from 0.01 to 100 s-1. Frequency sweep experiments were performed between 0.01 and 10 Hz. It revealed that in both control and xylanase containing dough formulation, the elastic modulus was higher than viscous modulus in the entire range of frequency. Our results represent the adequacy of fitting of dynamic moduli in Power law model and week gel model. Peleg model as well as six element Kelvin model described well the creep behaviour of control and xylanase-containing dough. Uniaxial extensibility was assessed by Kieffer dough and gluten extensibility rig. Lyophilized powder of untreated and xylanase treated doughs were tested under scanning electron microscope. FTIR spectra of lyophilized powder of untreated and xylanase treated dough were recorded in the range of 600-4000 cm-1.

Arginine dependence of tumor cells: targeting a chink in cancer's armor.

Arginine, one among the 20 most common natural amino acids, has a pivotal role in cellular physiology as it is being involved in numerous cellular metabolic and signaling pathways. Dependence on arginine is diverse for both tumor and normal cells. Because of decreased expression of argininosuccinate synthetase and/or ornithine transcarbamoylase, several types of tumor are auxotrophic for arginine. Deprivation of arginine exploits a significant vulnerability of these tumor cells and leads to their rapid demise. Hence, enzyme-mediated arginine depletion is a potential strategy for the selective destruction of tumor cells. Arginase, arginine deiminase and arginine decarboxylase are potential enzymes that may be used for arginine deprivation therapy. These arginine catabolizing enzymes not only reduce tumor growth but also make them susceptible to concomitantly administered anti-cancer therapeutics. Most of these enzymes are currently under clinical investigations and if successful will potentially be advanced as anti-cancer modalities.

Demonstration of redox potential of Metschnikowia koreensis for stereoinversion of secondary alcohols/1,2-diols.

The present work reports the Metschnikowia koreensis-catalyzed one-pot deracemization of secondary alcohols/1,2-diols and their derivatives with in vivo cofactor regeneration. Reaction is stereoselective and proceeds with sequential oxidation of (R)-secondary alcohols to the corresponding ketones and the reduction of the ketones to (S)-secondary alcohols. Method is applicable to a repertoire of racemic aryl secondary alcohols and 1,2-diols establishing a wide range of substrate specificity of M. koreensis. This ecofriendly method afforded the product in high yield (88%) and excellent optical purity (>98% ee), minimizing the requirement of multistep reaction and expensive cofactor.

Nitrile hydratase of Rhodococcus erythropolis: characterization of the enzyme and the use of whole cells for biotransformation of nitriles.

The intracellular cobalt-type nitrile hydratase was purified from the bacterium Rhodococcuserythropolis. The pure enzyme consisted of two subunits of 29 and 30 kDa. The molecular weight of the native enzyme was estimated to be 65 kDa. At 25 °C the enzyme had a half-life of 25 h. The Michaelis-Menten constants Km and vmax for the enzyme were 0.624 mM and 5.12 µmol/min/mg, respectively, using 3-cyanopyridine as the substrate. The enzyme-containing freely-suspended bacterial cells and the cells immobilized within alginate beads were evaluated for converting the various nitriles to amides. In a packed bed reactor, alginate beads (2 % alginate; 3 mm bead diameter) containing 200 mg/mL of cells, achieved a conversion of >90 % for benzonitrile and 4-cyanopyridine in 38 h (25 °C, pH 7.0) at a feed substrate concentration of 100 mM. The beads could be reused for up to six reaction cycles.

Xanthine oxidoreductase: a journey from purine metabolism to cardiovascular excitation-contraction coupling.

Xanthine oxidoreductase (XOR) is a ubiquitous complex cytosolic molybdoflavoprotein which controls the rate limiting step of purine catabolism by converting xanthine to uric acid. It is known that optimum concentrations of uric acid (UA) and reactive oxygen species (ROS) are necessary for normal functioning of the body. The ability of XOR to perform detoxification reactions, and to synthesize UA and reactive oxygen species (ROS) makes it a versatile intra- and extra-cellular protective "housekeeping enzyme". It is also an important component of the innate immune system. The enzyme is a target of drugs against gout and hyperuricemia and the protein is of major interest as it is associated with ischemia reperfusion (I/R) injury, vascular disorders in diabetes, cardiovascular disorders, adipogenesis, metabolic syndrome, cancer, and many other disease conditions. Xanthine oxidoreductase in conjugation with antibodies has been shown to have an anti-tumor effect due to its ability to produce ROS, which in turn reduces the growth of cancer tissues. Apart from this, XOR in association with nitric oxide synthase also participates in myocardial excitation-contraction coupling. Although XOR was discovered over 100 years ago, its physiological and pathophysiological roles are still not clearly elucidated. In this review, various physiological and pathophysiological functional aspects of XOR and its association with various forms of cancer are discussed in detail.

Green pigment from Bacillus cereus M(1)(16) (MTCC 5521): production parameters and antibacterial activity.

A bacterial strain, Bacillus cereus M(1)(16) (MTCC 5521), isolated and identified in our laboratory produces a green pigment when grown in nutrient broth at stationary condition. Optimum fermentation parameters for maximum pigment production are pH 7.0, temperature 30°C, time of incubation 72 h and inoculum volume 1% from 20 h grown cell suspension. Magnesium ion enhances pigment production whereas calcium and zinc ions inhibit the process. The pigment is better extracted from the fermented broth with chloroform in comparison with diethyl ether, ethyl acetate, and butanol. The extracted crude pigment consists of three fractions as revealed from thin layer chromatogram on silica gel GF254 using ethyl acetate and hexane (1:1) solvent system. The major fraction C(3) shows antibacterial activity against different gram positive bacteria. The proposed structure of C(3) is 9-methyl-1,4,5,8-tetra-azaphenanthrene obtained by elemental analysis, GC-MS, and NMR spectra studies.

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor.

AIMS: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. METHODS AND RESULTS: Rhodococcus erythropolis MTCC 1526 was grown in 7-l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min(-1) , aeration 0.5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. CONCLUSION: Agitation and aeration rate affected the dissolved-oxygen concentration in the reactor which in turn affected the growth and enzyme production. SIGNIFICANCE AND IMPACT OF THE STUDY: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.

Immobilization of intracellular carbonyl reductase from Geotrichum candidum for the stereoselective reduction of 1-naphthyl ketone.

Different cell disintegration methods were used for the liberation of intracellular carbonyl reductase from Geotrichum candidum, in its active form. Solid shear (bead milling) was proved to be the best method for the extraction of the enzyme. Various solid supports were checked for the immobilization of the purified enzyme. Carbonyl reductase was immobilized on silica with an optimized protein loading of 4 mg/g support. Cross-linking with glutaraldehyde rendered the preparation more stable and suitable for use in consecutive batches. Carbonyl reductase of G. candidum immobilized on silica support and cross-linked by glutaraldehyde was found to be highly efficient biocatalyst formulation for the production of S(-)-1-(1'-naphthyl) ethanol.

Studies on the dephosphorylation of phytic acid in livestock feed using phytase from Aspergillus niger van Teighem.

Phytase from Aspergillus niger van Teighem efficiently hydrolyses phytate phosphorus present in various commercial live stock feeds and was not inactivated by various formulations and antibiotics present. The enzyme retained 90-95% phytase activity at 55 degrees C, pH 2.5 after 72 h of incubation with all the commercial feeds tested, thus indicating its suitability in feed application. The phytase hydrolysis increased with the increase in temperature and a significant release of 41 nmols P(i)/ml in phytase-treated feed over control sample was observed at 55 degrees C after 48 h. Besides this, the enzyme was maximally effective when used under acidic condition, releasing 21 and 42 nmols P(i)/ml at pH 1.5 and 2.5, respectively. As the pH shifted towards 5.5, significant decline in phosphorus release was observed. However, the enzyme was able to retain almost complete phytase activity in the presence of feed constituent even after 48 h over various pH tested. Thus it can be a potential candidate in animal nutrition where the ability of present phytase to retain activity over period of time in the presence of feed constituent is desired.

Enantioselective nitrilase from Pseudomonas putida: cloning, heterologous expression, and bioreactor studies.

Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(-)-mandelic acid.

Optimization of crucial reaction conditions for the production of nicotinamide by nitrile hydratase using response surface methodology.

The reaction conditions for the nicotinamide production by Rhodococcus erythropolis MTCC 1526 have been optimized by statistical experimental design. Application of this approach in the bioprocess can result in rallied product yield, reduced development time, and process variability. In this investigation, response surface methodology and central composite design were employed to predict the levels of variables such as reaction pH (6.5, 7, and 7.5), temperature (15, 20, and 25 degrees C), cell concentration (190, 200, and 210 mg/ml), and substrate concentration (18, 20, and 22 mM) on the production of nicotinamide. A total of 22 experiments were carried out in shake flasks, and a three-dimensional response surface was generated to determine the effect of crucial reaction parameters for the maximum conversion of 3-cynopyridine to nicotinamide. Using this methodology, the optimal values for the reaction conditions were reaction pH 6.85, temperature of 24.8 degrees C, cell concentration of 190.98 mg/ml, and substrate concentration of 21.98 mM. This statistical approach led to the increase of conversion of 3-cynopyridine (93%) as compared to the conversion obtained by one-factor-at-a-time approach (84%).

Predicting enzyme behavior in nonconventional media: correlating nitrilase function with solvent properties.

The insolubility of nitrile substrates in aqueous reaction mixture decreases the enzymatic reaction rate. We studied the interaction of fourteen water miscible organic solvents with immobilized nitrile hydrolyzing biocatalyst. Correlation of nitrilase function with physico-chemical properties of the solvents has allowed us to predict the enzyme behavior in such non-conventional media. Addition of organic solvent up to a critical concentration leads to an enhancement in reaction rate, however, any further increase beyond the critical concentration in the latter leads to the decrease in catalytic efficiency of the enzyme, probably due to protein denaturation. The solvent dielectric constant (epsilon) showed a linear correlation with the critical concentration of the solvent used and the extent of nitrile hydrolysis. Unlike alcohols, the reaction rate in case of aprotic solvents could be linearly correlated to solvent log P. Further, kinetic analysis confirmed that the affinity of the enzyme for its substrate (K (m)) was highly dependent upon the aprotic solvent used. Finally, the prospect of solvent engineering also permitted the control of enzyme enantioselectivity by regulating enantiomer traffic at the active site.

Development and validation of HPLC method for the resolution of drug intermediates: DL-3-Phenyllactic acid, DL-O-acetyl-3-phenyllactic acid and (+/-)-mexiletine acetamide enantiomers.

Sensitive and specific, high-performance liquid chromatography (HPLC) methods have been developed and validated for linearity, accuracy and precision for the quantification of dl-3-phenyllactic acid, dl-O-acetyl-3-phenyllactic acid and (+/-)-mexiletine acetamide enantiomers. Chromatographic separations were performed on a Chiralcel OJ-H column (0.46 mm x 250 mm, 5 microm, Daicel Chemical Industries, Japan) based on cellulose tris-(4-methyl benzoate) chiral stationary phase. The mobile phase consists of hexane and isopropanol (IPA) in the ratio of 90:10 containing 0.1% trifluoroacetic acid (in case of dl-3-phenyllactic acid and dl-O-acetyl-3-phenyllactic acid) and hexane and IPA (95:5) containing 0.1% triethylamine (in case of (+/-)-mexiletine acetamide) and the flow rate was set at 0.5 ml/min at 25 degrees C. The detection was carried out at 261 nm for dl-3-phenyllactic acid and dl-O-acetyl-3-phenyllactic acid and at 254 nm for (+/-)-mexiletine acetamide. The developed methods were utilized for monitoring the progress of lipase catalyzed enantioselective synthesis of O-acetyl-3-phenyllactic acid and mexiletine acetamide from dl-3-phenyllactic acid and (+/-)-mexiletine, respectively.

Studies on the production of enantioselective nitrilase in a stirred tank bioreactor by Pseudomonas putida MTCC 5110.

Nitrilases constitute an important class of hydrolases, having numerous industrial applications. The present work aims to address the production of nitrile hydrolyzing enzymes from Pseudomonas putida MTCC 5110 in a 6l bioreactor. Effect of various physico-chemical conditions and process parameters like pH, temperature, aeration and agitation rates and inducer concentration was studied. Further, the enzyme activity was enhanced by adopting the inducer feeding strategy. Various biochemical engineering parameters pertaining to the cultivation of P. putida in different physico-chemical conditions were reported. Finally, segregation of growth phase from the enzyme production phase allowed significant reduction in total fermentation time.

Improvement of carbonyl reductase production of Geotrichum candidum for the transformation of 1-acetonaphthone to S(-)-1-(1'-napthyl) ethanol.

The aim of this work was to study the influence of media components and physico-chemical parameters on the growth and carbonyl reductase production by Geotrichum candidum. Under optimized conditions, the conversion of the substrate increased to >93%, while the specific growth rate and enzyme activity were increased by 200% and 29%, respectively. The rate of conversion of the substrate was also very high in the cells grown in optimized medium. The volumetric productivity of the biotransformation process was much higher (0.27g/lh) with the cells grown in the optimized medium compared to that of grown in un-optimized medium (0.16g/lh). The cells were also highly stable in the operational condition, indicating the feasibility of their use in multiple batches of reaction.

Decolorization of triphenylmethane dye-bath effluent in an integrated two-stage anaerobic reactor.

In the present study, decolorization of a simulated dye waste containing three different triphenylmethane (TPM) dyes--Magenta, Malachite Green and Crystal Violet, was investigated in a laboratory scale, two-stage anaerobic high-rate reactor. The effect of various parameters (influent dye concentration, hydraulic and co-substrate loading rates) on color and COD removal efficiency of the reactor has been studied. It has been shown that the influent dye concentration had little effect on overall COD and color removal efficiency. More than 99% color removal and 96% COD removal efficiency were maintained even at a dye concentration of 500 mg/l and a dye loading rate of 1000 mg/l day. However, a minimum level of glucose as supplementary carbon source is required to maintain the maximum color removal efficiency and it drops appreciably when no glucose is added to the influent. The study also showed that the acidogenic phase of the reaction plays an important role in decolorization of the TPM dyes. In addition, the two-stage anaerobic reactor was observed to have distinct advantages over the single-stage system, as the drop in color and COD removal efficiency of stage 1 are adequately compensated by stage 2 of the reactor especially under high dye loading rates accompanied by low co-substrate loading and under reduced HRTs.

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis.

(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (phi). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.