Dihydrofolate reductase from soybean seedlings: A fluorescence and circular dichroism study

The fluorescence emission spectrum of soybean dihydrofolate reductase suggests that the emitting tryptophan residues are situated in a hydrophobic microenvironment. The dissociation constants determined from fluorescence and circular dichroism data reveal that the soybean enzyme has a lower affinity for substrates and substrate analogs than that determined for dihydrofolate reductases isolated from other sources. The binding of methotrexate to the soybean enzyme does not affect the binding of NADPH. Similarly, the binding of NADPH has no effect on subsequent methotrexate binding. Polarimetric study indicates that the enzyme has a low (ca. 5%) α-helical content. Addition of dihydrofolate to the soybean enzyme results in the generation of a positive ellipticity band at 298 nm with a molar ellipticity, [θ], of 186,000, whereas the binding of folate induces a negative ellipticity band at 280 nm with [θ] of −181,000. The qualitative and quantitative differences in the circular dichroism of the enzyme-dihydrofolate and enzyme-folate complexes indicate that the mode of binding of these ligands may be different. The formation of an enzyme-NADPH complex is accompanied by a negative Cotton effect at 270 nm. These studies indicate that the binding of substrates or inhibitors causes significant conformational changes in the enzyme and also leads to the formation of a number of spectroscopically identifiable complexes.

Immobilization of Aspergillus oryzae alpha-galactosidase in gelatin and its application in removal of flatulence-inducing sugars in soymilk

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean-derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. In this study, alpha-galactosidase from Aspergillus oryzae was entrapped in gelatin using formaldehyde as the hardener. The immobilization yield was 64.3% under the optimum conditions of immobilization. The immobilized alpha-galactosidase showed a shift in optimum pH from 4.8 to 5.4 in acetate buffer. The optimum temperature also shifted from 50 degrees C to 57 degrees C compared with soluble enzyme. Immobilized alpha-galactosidase was used in batch, repeated batch and continuous mode to degrade RO present in soymilk. In the repeated batch, 45% reduction of RO was obtained in the fourth cycle. The performance of immobilized alpha-galactosidase was tested in a fluidized bed reactor at different flow rates and 86% reduction of RO in soymilk was obtained at 25 ml h(-1) flow rate. The study revealed that immobilized alpha-galactosidase in continuous mode is efficient in reduction of RO present in soymilk.

Unfolding studies on soybean agglutinin and concanavalin a tetramers: A comparative account

The unfolding pathway of two very similar tetrameric legume lectins soybean agglutinin (SBA) and Concanavalin A ( ConA) were determined using GdnCl-induced denaturation. Both proteins displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provided values for conformational stability of the two proteins. It was found that the DeltaG of unfolding of SBA was much higher than ConA at all the temperatures at which the experiments were done. ConA had a T-g 18 degreesC less than SBA. The higher conformational stability of SBA in comparison to ConA is largely due to substantial differences in their degrees of subunit interactions. Ionic interactions at the interface of the two proteins especially at the noncanonical interface seem to play a significant role in the observed stability differences between these two proteins. Furthermore, SBA is a glycoprotein with a GlcNac(2)Man(9) chain attached to Asn-75 of each subunit. The sugar chain in SBA lies at the noncanonical interface of the protein, and it is found to interact with the amino acid residues in the adjacent noncanonical interface. These interactions further stabilize SBA with respect to ConA, which is not glycosylated.

Enzymatic Degradation of Poly(soybean oil-g-methyl methacrylate)

This study discusses grafting of methyl methacrylate units from thepolymeric soybean oil peroxide to produce poly(soybean oil-graft-methyl methacrylate) (PSO-g-PMMA). The degradation of this copolymer in solution was evaluated in the presence of different lipases, viz Candida rugosa (CR), Lipolase 100T (LP), Novozym 435 (N435) and Porcine pancreas (PP), at different temperatures The copolymer degraded by specific chain end scission and the mass fraction of the specific product evolved was determined The degradation was modeled using continuous distribution kinetics to determine the rate coefficients ofmenzymatic chain end scission and deactivation of the enzyme The enzymes, CR. LP and N435 exhibited maximum activity for the degradation of PSO-g-PMMA at 60 degrees C, while PP was most active at 50 degrees C. The thermal degradability of the copolymer, assessed by thermo-gravimetry, indicated that the activation energy of degradation of the copolymer was 154 kJ mol(-1), which was lesser than that of the PMMA homopolymer.

Studies on the tryptophan residues of soybean agglutinin. Involvement in saccharide binding

Modification of tryptophan side chains of soybean agglutinin (SBA) with N-bromosuccinimide results in a loss of the hemagglutinating and carbohydrate binding activities of the protein. One residue/subunit is probably essential for the binding activity. Modification leads to a large decrease in the fluorescene of the protein accompained by a blue shift. Iodide ion quenching of the protein fluorescence shows that saccharide binding results in a decreased accessibility of some of the tryptophan side chains. These results strongly point towards the involvement of tryptophan residues in the active site of SBA.

An assessment of the current Indian scene in biotechnology

Biotechnology holds great promise, and is sure to make an impact in India in the nineties. The role of the government's Department of Biotechnology in focusing attention and resources on crucial problems and in supporting basic research is laudable.

Developments in Biotechnology for Environmentally Benign Iron Ore Beneficiation

Biomineralization and biogenesis of iron ore deposits are illustrated in relation to indigenous microorganisms inhabiting iron ore mines. Aerobic and anaerobic microorganisms indigenous to iron oxide mineralization are analyzed. Microbially-induced flotation and flocculation of iron ore minerals such as hematite, alumina, calcite and quartz are discussed with respect to use of four types of microorganisms, namely, Paenibacillus polymyxa, Bacillus subtilis, Saccharomyces cerevisiae and Desulfovibrio desulfuricans. The role of the above organisms in the removal of silica, alumina, clays and apatite from hematite is illustrated with respect to mineral-specific bioreagents, surface chemical changes and microbe-mineral interaction mechanisms. Silica and alumina removal from real iron ores through biobeneficiation is outlined. Environmental benefits of biobeneficiation are demonstrated with respect to biodegradation of toxic reagents and environmentally-safe waste disposal and processing.

Rethinking production of Taxol (R) (paclitaxel) using endophyte biotechnology

Taxol (R) (generic name paclitaxel) represents one of the most clinically valuable natural products known to mankind in the recent past. More than two decades have elapsed since the notable discovery of the first Taxol (R) producing endophytic fungus, which was followed by a plethora of reports on other endophytes possessing similar biosynthetic potential. However, industrial-scale Taxol (R) production using fungal endophytes, although seemingly promising, has not seen the light of the day. In this opinion article, we embark on the current state of knowledge on Taxol (R) biosynthesis focusing on the chemical ecology of its producers, and ask whether it is actually possible to produce Taxol (R) using endophyte biotechnology. The key problems that have prevented the exploitation of potent endophytic fungi by industrial bioprocesses for sustained production of Taxol (R) are discussed.

Copolyesters from Soybean Oil for Use as Resorbable Biomaterials

A family of soybean oil (SO) based biodegradable cross-linked copolyesters sourced from renewable resources was developed for use as resorbable biomaterials. The polyesters were prepared by a melt condensation of epoxidized soybean oil polyol and sebacic acid with citric acid (CA) as a cross-linker. D-Mannitol (M) was added as an additional reactant to improve mechanical properties. Differential scanning calorimetry revealed that the polyester synthesized using only CA as the cross-linker was semicrystalline and elastomeric at physiological temperature. The polymers were hydrophobic in nature. The water wettability, elongation at break and the degradation rate of the polyesters decreased with increase in M content or curing time. Modeling of release kinetics of dyes showed a diffusion controlled mechanism underlies the observed sustained release from these polymers. The polyesters supported attachment and proliferation of human stem cells and were thus cytocompatible. Porous scaffolds induced osteogenic differentiation of the stern cells suggesting that these polymers are well suited for bone tissue engineering. Thus, this family of polyesters offers a low cost and green alternative as biocompatible, bioresobable polymers for potential use as resorbable biomaterials for tissue engineering and controlled release.