Production of biosurfactant can be substantially increased by the addition of precursors like vegetable oils, petroleum products, and other water-insoluble substances. Pseudomonas Ptm+ strain produces biosurfactant in the presence of hexachlorocyclohexane (HCH), which specifically emulsifies HCH, a recalcitrant organochlorine pesticide. Addition of previously produced crude biosurfactant by the same organism as a precursor instead of HCH increased production of biosurfactants with a decrease in the total fermentation time from 32 to 24 h. The main objective of this paper was to find alternatives for HCH as an inducer.

A polyclonal antibody-based enzyme-linked immunosorbent assay ( ELISA) method was developed for the N-methylcarbamate insecticide bendiocarb ( 2,2-dimethyl-1,3-benzodioxol-4-yl methylcarbamate). Two novel haptens having dimethylbenzodioxyl and dimethylbenzofuranyl groups connected to oxyacetyl-gamma-aminobutanoic acid and oxyacetyl-beta-alanine spacer arm respectively were synthesised. The first hapten was conjugated to carrier proteins to make antigens that were used to raise polyclonal antibodies in rabbits. The antibodies specifically recognised bendiocarb and its metabolite 2,2-dimethyl-1,3-benzodiox-4-ol with an IC50 value of 9 ppb ( ng ml(-1)). The assay was standardised using the competitive ELISA format at 0.0625 mg antibody concentration and at 1/10k pesticide-HRP dilution. Matrix effect studies were carried out in four vegetable and cereal food samples. Matrix effect elimination in cabbage, cauliflower and rice was achieved by simple dilution of the extract. Five different approaches were attempted to achieve matrix clean up in paddy rice. C-18 column and gel permeation column chromatography ( GPC) helped in the matrix removal. The spike and recovery studies for all the four food samples gave a recovery in the range of 75-95%, thus indicating the efficiency of the matrix elimination procedures developed.

Sensitive detection of pesticides is of utmost importance in environment and food analysis. Immunological methods are widely used to detect pesticides in agricultural and environmental samples wherein antibodies are employed against the target molecules. Accurate diagnosis depends on the affinity and specificity of the antibody preparation used, and high affinity antibodies are essential for the detection of very small amounts of pesticides. Enzyme linked immuno sorbent assay (ELISA) coupled with flow injection analysis (FIA) technique provides a very high sensitivity with high throughput of analyses. Automation of this analysis scheme ensures precise detection with high accuracy. The present development aims at providing a user-friendly system for achieving this objective. It employs a 8952 microcontroller for precise flow of reagents, samples, substrate and conjugates used for analysis to be passed through an immobilized antibody column at predetermined time. With the sequence and flow control of buffers used, it also provides the option for reuse of the immobilized antibody column. The system is flexible to accommodate multiple sequences up to a maximum of 99 steps. It is customizable for different flow ELISA applications. It can control up to eight solenoid valves (dc 24V) and two peristaltic pumps and has one 12bit analog channel for data acquisition. With the serial interface port, the system provides convenient means for data acquisition into the computer. The system has been successfully tested for immuno analysis of organophosphorous pesticide methyl parathion.

We have utilized a microbe, which can degrade caffeine to develop an Amperometric biosensor for determination of caffeine in solutions. Whole cells of Pseudomonas alcaligenes MTCC 5264 having the capability to degrade caffeine were immobilized on a cellophane membrane with a molecular weight cut off (MWCO) of 3000-6000 by covalent crosslinking method using glutaraledhyde as the bifunctional crosslinking agent and gelatin as the protein based stabilizing agent (PBSA). The biosensor system was able to detect caffeine in solution over a concentration range of 0.1 to 1 mg mL(-1). With read-times as short as 3 min, this caffeine biosensor acts as a rapid analysis system for caffeine in solutions. Interestingly, successful isolation and immobilization of caffeine degrading bacteria for the analysis of caffeine described here was enabled by a novel selection strategy that incorporated isolation of caffeine degrading bacteria capable of utilizing caffeine as the sole source of carbon and nitrogen from soils and induction of caffeine degrading capacity in bacteria for the development of the biosensor. This biosensor is highly specific for caffeine and response to interfering compounds such as theophylline, theobromine, paraxanthine, other methyl xanthines and sugars was found to be negligible.Although a few biosensing methods for caffeine are reported, they have limitations in application for commercial samples. The development and application of new caffeine detection methods remains an active area of investigation, particularly in food and clinical chemistry. The optimum pH and temperature of measurement were 6.8 and 30 +/- 2 degrees C, respectively. Interference in analysis of caffeine due to different substrates was observed but was not considerable. Caffeine content of commercial samples of instant tea and coffee was analyzed by the biosensor and the results compared well with HPLC analysis. (c) 2006 Elsevier B.V. All rights reserved.

The fermentation parameters of production of nigerloxin, an inhibitor of lipoxygenase, through solid state fermentation by Aspergillus niger CFR-W-105 and its recovery was studied. Inhibitor production was influenced by the nature of the solid substrate, moisture content, incubation temperature, carbon, nitrogen, lipid and mineral supplements. Nigerloxin production was the highest when wheat bran was supplemented with 5% (w/w) trisodium citrate. The maximum nigerloxin obtained was 5.06mg/g of dry wheat bran. Extraction of the fermented wheat bran using ethyl acetate under acidic pH range of 4–5.5 under agitated conditions resulted in maximum inhibitor recovery. Optimum time and temperature of extraction were determined to be 90min and 30°C, respectively.

Due to the known adverse effects of caffeine in the widely consumed beverages like coffee and tea, a caffeine free product is desirable. The commonly used solvents in decaffeination suffer from the disadvantage of being carcinogenic or increase the process costs tremendously. A biotechnological approach for decaffeination involving microbial cells or enzymes is viewed as a potential alternative approach.Present studies on the degradation of caffeine were carried out using a strain of Pseudomonas alcaligenes CFR 1708, isolated from coffee plantation soil. The enzymes responsible for caffeine degradation were found to be inducible. Pre-induction of the microbial cells in a medium containing caffeine as the sole source of carbon and nitrogen was carried out for 48 h. The induced bacterial cells were found to be capable of completely degrading caffeine (1 g/l) from solutions containing caffeine, within 4 to 6 h at 30 +/- 2 degrees C in the pH range of 7.0-8.0. To make the process of decaffeination application oriented immobilisation of cell debris were done and used in designed bioreactor for continuous decaffeination process. (c) 2005 Elsevier Inc. All rights reserved.

A novel method based on immuno-chemiluminescence and image analysis using charge coupled device (CCD) for the qualitative detection of methyl parathion (MP) with high sensitivity (up to 10ppt) is described. MP antibodies raised in poultry were used as a biological sensing element for the recognition of MP present in the sample. The immuno-reactor column was prepared by packing in a glass capillary column (150μl capacity) MP antibodies immobilized on Sepharose CL-4B through periodate oxidation method. Chemiluminescence principle was used for the detection of the pesticide. Light images generated during the chemiluminescence reaction were captured by a CCD camera and further processed for image intensity, which was correlated with pesticide concentrations. K3Fe(CN)6 was used as a light enhancer to obtain detectable light images. Different parameters including concentrations of K3Fe(CN)6, luminol, urea H2O2, antibody, addition sequence of reactants and incubation time to obtain best images were optimized. The results obtained by image analysis method showed very good correlation with that of competitive ELISA for methyl parathion detection. Competitive ELISA method was used as a reference to compare the results obtained by CCD imaging.

Stability of glucose oxidase (GOD) immobilized with lysozyme has been considerably enhanced by modification of free thiols generated by reducing disulfide bonds using β-mercaptoethanol and N-ethylmaleimide in conjunction with additives like antibiotics and salts. Thermal stability of immobilized GOD was quantified by means of the transition temperature, Tm and the operational stability by half-life t1/2 at 70 °C. Modification of the free thiols in the enzyme coupled with the presence of kanamycin, NaCl, and K2SO4, led to increase in Tm, to 80, 82 and 84 °C (compared to 75 °C in control) and t1/2 by 7.7-, 11- and 22-fold, respectively, indicating that this method can be effectively used for enhancing the stability of enzymes.

An important requirement of immobilized enzyme based biosensors is the thermal stability of the enzyme. Studies were carried out to increase thermal stability of glucose oxidase (GOD) for biosensor applications. Immobilization of the enzyme was carried out using glass beads as support and the effect of silane concentration (in the range 1–10%) during the silanization step on the thermal stability of GOD has been investigated. Upon incubation at 70°C for 3h, the activity retention with 1% silane was only 23%, which increased with silane concentration to reach a maximum up to 250% of the initial activity with 4% silane. Above this concentration the activity decreased. The increased stability of the enzyme in the presence of high silane concentrations may be attributed to the increase in the surface hydrophobicity of the support. The decrease in the enzyme stability for silane concentrations above 4% was apparently due to the uneven deposition of the silane layer on the glass bead support. Further work on thermal stability above 70°C was carried out by using 4% silane and it was found that the enzyme was stable up to 75°C with an increased activity of 180% after 3-h incubation. Although silanization has been used for the modification of the supports for immobilization of enzymes, the use of higher concentrations to stabilize immobilized enzymes is being reported for the first time.

Reliable analysis using an immunosensor strongly depends on the specificity, activity, and sensitivity of the antibody. Immobilization of antibody on the solid matrix enables its repeated use, for which it is required to dissociate the antigens and antigen-enzyme conjugate from the immobilized antibody matrix after each use and while doing so, a maximum retention of activity and specificity are crucial requirements. In the present investigation, on the development of an immunosensor for the organophosphorus pesticide ethyl parathion (EP) using EP antibodies, different dissociating agents such as organic solvents, detergents and acidic buffers, that is, dimethyl sulphoxide (DMSO), Tween-20, cetyl trimethylammonium bromide (CTAB), methanol, chloroform, guanidium chloride (GdmCl), glycine-HCl (Gly-HCl) buffer in the pH range of 1.5-3.0, pierce buffer and combination of DMSO and methanol in phosphate buffer and Gly-HCl buffer and salts like NaCl and MgCl2 were used. Generally about 50-60% dissociation was obtained with some degree of denaturation of the antibody immobilized on the sepharose matrix. However, 1% DMSO in combination with 0.2 M Gly-HCl buffer at a pH of 2.3 showed 97% dissociation and the immobilized antibody retained sufficient activity to carry out 14 reproducible assays for EP.

Solid state fermentation which involves growth of microorganisms on moist solid substrates in the absence of free flowing water, has gained considerable attention of late due its several advantages over submerged fermentation. Solid-state fermentation is also finding increased application in the production of enzymes, antibiotics, surfactants, biocides etc. as also for the production of value-added products from wastes. There have been significant additions to the science and engineering knowledge of solid-state fermentations in recent years. This paper aims to present an overview of these developments emphasizing important aspects such as mass and heat transfer, design, scale-up, monitoring and control.

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 60degreesC. 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.mol(-1).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 25-90degreesC 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 59degreesC, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62degreesC. 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.

Immunoassays for two groups of organochlorine insecticides, cyclodienes (endosulfan and heptachlor) and DDT were applied to the analysis of a diverse range of plant-derived foods. Water-miscible solvent extracts of high-moisture, low-fat foods such as cauliflower, cabbage, green and red blue grapes and tomato caused little or no interference with the assays, enabling methanol or acetonitrile extracts of the foods to be analysed directly by immunoassay, after dilution in assay buffer. Reasonable recoveries of spikes of these pesticides were obtained by direct analysis of extracts of spiked commodities, with reliable detection down to 0.025 mg kg(-1) heptachlor or endosulfan and 0.1 mg kg(-1) DDT in the commodities. Acetonitrile extracts of milk could also be analysed directly for DDT. In contrast, extracts of low moisture, non-fatty (rice) and fatty (cottonseed) food commodities interfered appreciably with the assays, reducing assay colour and detection sensitivity. Some simple cleanup methods were developed to remove interference and enable detection of spiked organochlorines in these foods. Extracts of coloured foods, such as tea, coffee and spinach caused similarly major interference in the assays, and a number of simple clean-up methods were ineffective in removing interference. However, use of an immunoaffinity chromatography method for cyclodienes enabled quantitative recoveries to be obtained in extracts of several of these foods when analysed by either ELISA or gas chromatography. Direct analysis was suited for screening purposes but immunoaffinity chromatography results were more quantitative. These results indicate that ELISAs can be applied under developing country conditions to a range of diverse foods, but that cleanup strategies need to be tailored to different types of foods.

Immunoassays, capable of detecting 0.05 mug l(-1) and 0.5 mug l(-1) , respectively, have been developed to detect the organophosphate pesticides, methyl-parathion and parathion. Using haptens based on derivatization of the phosphate ester of the methyl and ethyl forms in the target compounds, there was selectivity in detection of methyl-parathion and parathion, respectively, using the two assays. Antisera to methyl-parathion detected parathion with 25-20% cross-reaction, while the parathion antisera detected methyl-parathion with 30-40% cross-reaction in water. The only other commonly-used agrochemical that cross-reacted in the assays was fenitrothion, the 3-methyl derivative of methyl-parathion. The assays were applied to the analysis of residues of these pesticides in water and several food matrices representative of different classes following the extraction of residues using simple procedures. Methanol extracts of most fruits and vegetables tested (high-moisture, low fat foods), including green and blue grapes, cauliflower and cabbage, could be analysed directly in the methyl-parathion assay, as could rice and basmati rice (low-moisture, low fat foods). Methanol extracts of butter and milk (high-fat foods) provided interference, but this was overcome by either further dilution or using acetonitrile as the extractant. A coagulating reagent was used to remove matrix interference from strongly coloured foods (tea and spinach). The parathion assay was subject to greater matrix interference, so it was preferable to analyse parathion in samples on plates coated with methyl-parathion antibody. With these foods, and with water samples, near-quantitative recoveries of spiked methyl-parathion or parathion were usually obtained, while with high-fat foods (milk and butter) and strongly coloured foods recoveries were poorer.

Aldose reductase (EC 1.1.1.21) catalyzes the conversion of glucose to sorbitol and promotes the accumulation of sorbitol in various tissues under the condition of hyperglycemia such as diabetes mellitus. The accumulated intracellular sorbitol causes development of diabetic complications such as cataracts, neuropathy, retinopathy and nephropathy1). Inhibitors of aldose reductase have been shown to reverse these biochemical changes and have been proven effective in delaying and even preventing several diabetic pathologies. Thus, aldose reductase has become an attractive pharmacological target for the treatment of diabetic complications. It has been reported that inhibitors of aldose reductase reduce the tissue sorbitol content in diabetic animals and are useful as therapeutic agents for diabetic complications2). In our screening programme on bioactive molecules through the fermentation route3-7), we screened 15 different strains of Aspergillus sp. for potential inhibitors against rat lens aldose reductase (RLAR). We found that, Aspergillus niger CFR-1046 produced a compound, termed by us as asperaldin, which exhibits potent inhibitory activity against RLAR. The present paper describes the fermentation, isolation, physico-chemical properties and biological activities of asperaldin. The organism used in this study Aspergillus niger CFR 1046 was obtained from CFTRI culture collection. Submerged fermentation was carried out in a potato dextrose (soluble starch 0.4% and glucose 2%) medium. A portion of the mature agar slant was inoculated into 100ml of potato dextrose broth in a 500ml Erlenmeyer flask and incubated at 30°C on a rotary shaker at 250rpm for 8 days.