Anaerobic degradation of pectin by mixed consortia and optimization of fermentation parameters for higher pectinase activity

Samples from biogas digesters, sewage ponds, animal house effluents and food processing wastes were used in enrichment systems seeking anaerobic bacteria producing pectinases. Among the 46 anaerobic consortia developed from various samples, four showed high pectinase activity under static anaerobic conditions. Investigation of fermentation variables showed the optimum conditions for pectinase activity were pH 7.0, 45°C and 72 h of growth with 0.5% pectin in the cultivation medium. A 1.4- to 1.6-fold increase in the pectinase activity was achieved under these conditions. The maximum yield of enzymes (62.72 U ml-1 of pectinase, 4.74 U ml^-1 of polygalacturonase, 113.30 U ml^-1 of pectin lyase, 2.10 U ml^-1 of pectinesterase, 0.75 U ml^-1 of total cellulase and 9.27 U ml^-1 of xylanase) was recorded with the consortia C-S2 developed from decomposed plant samples collected from a pond.     

Genomic relationships in the genus Glycine (Fabaceae: Phaseoleae): use of a monoclonal antibody to the soybean Bowman-Blrk inhibitor as a genome marker

We investigated the use of a monoclonal antibody (MAb 238) to the soybean Bowman-Birk inhibitor (BBI) to verify and understand the intergenomic relationships among the wild perennial Glycine species. Competitive enzyme linked immunosorbent assay and western blot screening studies revealed that the accessions of B-genome (G. latifolia, G. microphylla, and G. tabacina, 2n = 40) and C-genome (G. curvata and G. cyrtoloba) species did not contain the MAb 238 crossreactive proteins (BBI-nulls). By contrast, all the A-genome (G. argyrea, G. canescens, G. clandestina, and G. latrobeana), E-genome (G. tomentella, 2n = 38), and F-genome (G. falcata) species, G. arenaria (genome unknown), and the polyploid (2n = 78,80) G. tomentella accessions were BBI-positive. The D-genome G. tomentella (2n = 40) and tetraploid G. tabacina (2n = 80) contained both BBI-null and BBI-positive type accessions. Among the recently described species, G. hirticaulis (2n = 40), G. lactovirens, and G. pindanica contained the MAb 238 crossreactive proteins while G. albicans did not. Glycine hirticaulis, G. pindanica, and G. tomentella (2n = 38) displayed highly similar MAb 238 crossreactive isoelectric focusing banding patterns, indicating that they are genomically close to each other. Glycine hirticaulis was found to have both diploid (2n = 40) and tetraploid (2n = 80) cytotypes. We demonstrated that the MAb 238 was specific to the trypsin inhibitor domain of the BBI. The MAb 238 clearly reflected all the previously established relationships in the genus Glycine, validating its use as a genome marker.     

Characterization of crystals of the thermostable DNA polymerase I from thermus aquaticus

Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 Å resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermo stability in proteins. © 1995 Wiley-Liss, Inc.     

Movement and distribution pattern of the residues of granular insecticides in tropical soil and okra plants

Summary The increased downward mobility of phorate, quinalphos and carbofuran residues was detected in soil with increase in depth of soil column whereas aldicarb was found to remain localised mainly in 0–7.5 cm and 7.5–15.0 cm layers. Persistence of organophosphate insecticides was higher as compared to carbamates in all the soil layers. Residues of all the four insecticides got distributed in all parts of okra plant through uptake but accumulated in higher amounts in fruits only. Contribution No. 312/83 from I.I.H.R. Bangalore (India)     

Hepatic and extra-hepatic glutathione-S-transferase activity in wild pigeons (Columba livia)

Glutathione-S-transferase (EC 2.5.1.18) activity was assayed in hepatic and extra-hepatic tissues of pigeons using l-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Gluthathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene in pigeon was in the order: kidney > liver > testes > brain > lung> heart. The enzyme activity with 1-chloro-2,4-dinitrobenzene as substrate was 40–44 times higher in pigeon liver and kidney than that observed with 1,2-dichloro-4-dinitrobenzene as substrate.K _m values of hepatic and renal glutathione transferase with l-chloro-2,4-dinitrobenzene as substrate were 2.5 and 3 mM respectively. Double reciprocal plots with varying reduced gluthathione concentrations resulted in biphasic curves with twoK _m values (liver 0.31 mM and 4mM; kidney 0.36 mM and 1.3 mM). The enzyme activity was inhibited by oxidized gluthathione in a dose-dependent pattern. 3-Methylcholanthrene elicited about 50% induction of hepatic glutathione transferase activity whereas phénobarbital was ineffective.     

A general multistate model for the analysis of hydrogen-exchange kinetics

In proton nmr, the chemical exchange rates of slowly exchanging labile hydrogens (with lifetimes in the range ～ 10 msec – ～ 1 sec) of peptides, proteins, and nucleic acids can be measured in H₂O by a combination of two separate experiments: (1) the transfer of solvent saturation and (2) saturation-recovery experiments. When these molecules exist in a dynamic equilibrium among different conformations, the experiments cannot be analyzed in a straightforward manner to derive the intrinsic exchange rates. In the present study we have derived analytical expressions for the above two experiments on a biomolecule under certain limiting conditions: (1) the extreme low-motility limit, where each of the conformational transitions is much slower than the corresponding hydrogen exchange rate with the solvent; (2) the high-motility limit (EX₂ mechanism), which is the opposite extreme of the previous limit; and (3) the low-motility limit (EX₁ mechanism), which is a mixture of limits (1) and (2), i.e., for some of the conformations, the exchange rate with the solvent is much faster than their conformational transition rates, while for the remaining conformations the reverse situation is realized. The results may be considered as a generalization to an arbitrary number of states of the two-state model treated by Hvidt. Equations have also been derived that are applicable to the iostope exchange method of measuring very slow exchange rates (with life-times of the order of minutes and longer) in biomolecules. The saturation recovery experiments performed in H₂O on the active pentapeptide fragment of thymopoietin serve to illustrate the high-motility limit. The theoretical formulation presented in this study can be easily adapted to other double-resonance techniques and also to situations where the kinetics of an arbitrary system existing in a multistate equilibrium are of interest.     

The amino acid sequence of ferredoxin from the alga Mastigocladuslaminosus

Ferredoxin was purified from the thermophilic blue-green alga, Mastigocladuslaminosus. The physicochemical properties of this ferredoxin are similar to those of other [2Fe-2S] plant ferredoxins except for its unusual thermal stability. The primary structure of the protein was determined and consists of 98 amino acid residues, 5 of which are cysteines. The positions of 4 cysteines which bind the iron atoms of the active centre are identical to those in other ferredoxins. The primary structure of the ferredoxin does not reveal any special features to account for its high thermal stability.     

Hydroxylamine inhibition of the nitrate reductase complex from Amaranthus

Nitrate reductase from Amaranthus viridis is similar to nitrate reductase from other plant sources. NH₂OH inhibits nitrate reduction from NADH by the nitrate reductase complex, but it does not inhibit either the NADH-dehydrogenase activity or nitrate reduction from reduced flavin mononucleotides. The inhibition observed was non-competitive with nitrate when the enzyme was pre-incubated with NH₂OH and NADH, and competitive with nitrate without pre-incubation. The K_i values for NH₂OH were 5 μM and 30 μM with or without pre-incubation respectively.     

NMR studies of the variant-3 neurotoxin from Centruroides sculpturatus Ewing

We report a preliminary high-resolution proton nuclear magnetic resonance characterization of the variant-3 toxin from the scorpion Centruroides sculpturatus Ewing (range Southwestern USA). This toxin assumes a well defined folded conformation in aqueous solutions at room temperature and undergoes reversible thermal denaturation. A number of amide hydrogens exhibit exchange life times varying from several minutes to several hours. A few tentative assignments of the low field aromatic CH resonances has been made on the basis of 2D-COSY and NOE experiments. The upfield shifts exhibited by Trp-47 suggest a unique microenvironment for this residue. The NMR data suggest that there is some degree of correlation between the solution structure of the variant-3 toxin and its crystallographic structure. Our studies provide a basis for a detailed elucidation of the structure-function relationships of these interesting scorpion toxins which bind to the sodium channels of excitable membranes and delay sodium current inactivation.     

Synthesis of lysine-containing fragments of the Proteus mirabilis O27 O-specific polysaccharide and neoglycoconjugates therefrom.

Amide-linked lysine mono- and di-uronic acid fragments of the O-specific polysaccharide from P. mirabilis O27 have been synthesised. N epsilon-Boc-L-lysine tert-butyl ester was condensed with 2-azidoethyl glycosides of glucuronic acid and beta-D-GlcpNAc-(1----3)-beta-D-GlcpA. Transformation of the products into 2-acrylamidoethyl glycosides, followed by deprotection using trifluoroacetic acid, gave the target monomers that were converted into high-molecular-weight copolymer-type neoglycoconjugates.