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Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.

The activities of malate dehydrogenase in whole tissue extract (NAD(+)-MDH) as well as in mitochondrial (NAD(+)-MDH) and chloroplastic (NADP(+)-MDH) preparations of aminating (NADH-GDH) and deaminating(NAD(+)-GDH) glutamate dehydrogenases were studied in two sets of rice cultivars differing in salt tolerance grown under moderate (7 dS m(-1)) and high (14 dS m(-1)) NaCl salinity levels. A contrasting response to salinity on enzyme activities was found between the sensitive and tolerant cultivars during a 5-20-day growth period of study. NaCl salinity in situ caused increase in all three MDH activities in salt tolerant cvs. CSR-1 and CSR-3 whereas in salt sensitive cvs. Ratna and Jaya 16-100% inhibition in activities was noted. Chloroplastic MDH was extremely sensitive to NaCl. In seedlings of salt tolerant cultivars concomitant increase in both aminating and deaminating GDH activities was observed with increase in salinity level, whereas in sensitive cultivars under higher salinity level decrease in GDH activity was noted. Under in vitro conditions NaCl concentration in the range 1-1000 mM caused gradual inhibition in MDH activity. With 400 mM NaCl in vitro, complete loss of mitochondrial and chloroplastic MDH activities was observed. GDH activity increased with increasing concentration of NaCl up to 200 mM NaCl and other salts in vitro and was inhibited thereafter. However 800 mM NaCl caused complete loss of deaminating GDH activity from sensitive cultivar but not from tolerant cultivar. Results suggest varying behaviour of MDH and GDH in two sets of rice cultivars differing in salt tolerance and that inhibition in the activities of dehydrogenases in salt sensitive rice cultivars due to salinity may be one of the possible reasons for decreased growth of rice plants under saline conditions.

It has been shown that when two enzymes showing similar actions act in close proximity of each other they influence each other synergistically. The phenomenon of synergism is, however, not observed if the two enzymes are of dissimilar action type. The condition of closest proximity has been simulated by conducting the enzymic reactions inside the reversed micelles. In the present study we have experimented with alpha-amylase and invertase both hydrolysing enzymes and also with peroxidase and invertase which do not show similar actions.

Sequestering of Zn(II) ions at the surface of rice husk has been studied as a function of contact time, adsorptive concentration, temperature and pH, using the radiotracer technique employing a 65Zn radiotracer. A relatively slow uptake of metal ion increasing with bulk dilution (10(-2) to 10(-7) mol/dm3) was seen, which obeys the first-order rate law and agrees well with the classical Freundlich adsorption isotherm. The removal of metal ions from the bulk increases with the increase in pH (3.2-10.2) and temperature (293-323K) and evaluation of the thermodynamic data shows that the process involved is endothermic and apparently irreversible in nature, suggesting an ion exchange mechanism along with surface complexation. On the other hand Cd(II) was not adsorbed on the surface of the rice husk at pH 6.2.

Studies have been conducted on the enzymic activity of Baker's yeast and also of Brewer's yeast entrapped into the reversed micelles formed by cetyl pyridinium chloride (CPC1) in n-hexane. The activities of alpha-amylase and invertase enzymes in the entrapped cells have been estimated and compared with those in the control experiments where there was no entrapment. The following significant observations have been made: 1. except for invertase, enzymes in Brewer's yeast, the entrapped yeast cells showed enhanced enzymic activities; 2. when the yeast cells were entrapped inside the reversed micelles along with substrates of the two enzymes, alpha-amylase, and invertase, the activity of each of these enzymes showed a further enhancement in comparison to that showed in the experiments in which substrates of the individual enzymes alone were entrapped-the phenomenon of synergism; 3. when the yeast cells and the respective substrates were entrapped inside separate reversed micelles and the solutions containing entrapped cells and entrapped substrates were mixed, the activities of the individual enzymes, alpha-amylase and invertase, showed further enhancement in comparison to the case in which the cells and the substrates were entrapped inside the same reversed micelle (in this case also the phenomenon of synergism was observed); and (4) In the case of experiments in which there was no entrapment, it was observed that the presence of substrates induced more release of enzymes from the yeast cells. These observations on yeast cells, which to the best of our knowledge have not been reported before, should be biotechnologically relevant.

A rationale of arriving at the concentration of surfactant additives in the pesticidal formulations has been discovered. Fungitoxic activity of each of the two dithanes, Dithane M-45 (maneb) and Dithane Z-78 (zineb) has been estimated in the presence of various concentrations of three surfactants namely sodium lauryl sulphate, cetyl pyridinium chloride and Tween 80. Data reveal that the concentration of the surfactant additives in the pesticidal formulations should be at least equal to its critical micelle concentration (CMC) for enhanced biological activity. This rationale for the concentration of surfactant additives in the pesticidal formulation has so far not been clearly brought out in the literature. Since biological activity of the pesticides is enhanced in the presence of surfactant additives the present study is relevant to the reduction of environmental pollution due to pesticide residues.

Observations of the activity of two hydrolyzing enzymes-protease and alpha-amylase--entrapped inside the reversed micelles formed by surfactants in hexane, benzene, and cyclohexane are reported. The surfactants chosen for this study are: Tween 80, a nonionic surfactant, Cetyl pyridinium chloride, a cationic surfactant, and two anionic surfactants, sodium lauryl sulfate and Aerosol OT. Tween 80 enhances the activity of both protease and alpha-amylase. Sodium lauryl sulfate and Aerosol OT, which are ionic surfactants, enhance the activity of protease, but inhibit the activity of alpha-amylase. Cetyl pyridinium chloride, however, enhances the activity of alpha-amylase, but inhibits the activity of protease. Enhanced activity is generally severalfold greater in comparison to the activity observed in the usual aqueous system in the absence of reversed micelles. It has also been observed that the enhanced activity of the enzymes entrapped inside the reversed micelles remains preserved for a much longer period of time in comparison to the activity in the usual aqueous systems. These observations, which support the view that with proper choice of surfactant and the organic solvent, reversed micelles act like a microreactor that provides a favorable aqueous micro-environment for enzyme activity, have biotechnological overtones.

In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml-1 and 1 ng ml-1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 micrograms ml-1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.

A synthetic medium containing ammonium nitrate, urea, D-glucose, glycerol, and sodium glutamate was developed for growth of Xanthomonas oryzae and preparation of its phytotoxin. Culture filtrates and partially purified toxin preparations caused blight symptoms when inoculated in leaves of 30 days old rice plant cv. T(N)1. Toxin elicited quicker response than living cells. This activity was due to a non-dialysable, partial acid and heat labile glycopeptide. There was a sharp decrease in activity when culture filtrates and crude toxin preparations were heated at 100 degrees C for 30 min. Maximum activity was observed in pH range of 6.0-7.0 and a gradual loss at lower pH (5.0-3.0) and higher pH values (8.0-10.0). Unlike living cells which caused continuous lesion enlargement, toxin inoculation caused maximum attainment of lesion length within 4-6 weeks. Toxin preparation in concentration of 45 micrograms glucose/ml failed to evoke response. The study demonstrates that the purified toxin being easy in storage and causing quicker response than living cells when inoculated in rice leaves, may be used to screen rice varieties for bacterial blight resistance.

Culture filtrates and partially purified enterotoxins of 6 strains of Aeromonas hydrophila isolated from faeces of diarrhoeic and healthy persons, drinking water, sewage and faeces of domestic animals caused induration and increased capillary permeability in skin of adult albino rabbits. The activities were less in crude enterotoxins indicating partial loss during purification. Heat treatment for 30 min at 60 degrees C and 56 degrees C inactivated the induration and permeability effects of culture filtrates and crude enterotoxins respectively. The activities were most pronounced at pH 8.0 showing gradual loss of activities at lower pH values disappearing completely in culture filtrates at 3.0 and in crude toxins at 4.0. The induration and permeability factor activities of crude enterotoxin were neutralised proportionally in vivo with antitoxin raised against the same in rabbits. Suppression effect was more when antitoxin was injected prior to inoculation of toxin. This study demonstrates that the assay of the activity of skin permeability factor being an easier process than loop test, may be used for assay of enterotoxin of A. hydrophila.

Cell-free culture filtrates and crude enterotoxin preparations from six strains of Aeromonas hydrophila caused the accumulation of fluid in rabbit ileal loops. This activity was due to a non-dialysable, heat and acid-labile antigenic protein and was lost when culture filtrates and crude enterotoxin preparations were heated at 60 degrees C for 20 min. or 56 degrees C for 30 min. respectively. Maximum activity was observed at pH 8.0-10.0; there was a gradual loss at lower pH and activity was abolished in culture filtrates held at pH 3.0 and crude enterotoxin preparations held at pH 4.0. Titration of the crude enterotoxin preparations in rabbit ileal loops showed that the ED50 (equivalent to 1 unit of toxin) was contained in 25 microgram of protein; a logarithmic plot of the neutralisation coefficients against antiserum concentrations showed that one unit of antitoxin was contained in 42 x 10(-4) ml of the antiserum.