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Biomedical subjects

M N Gupta

Publications and source records attributed to M N Gupta.

At least 55 records · Page 3Linked to original sources
PubMed1998

Alginate beads as an affinity material for alpha amylases.

Calcium-alginate beads were found to bind a variety of enzymes in a nonspecific fashion. However, alpha amylases from porcine pancreas, Bacillus subtilis (BAN 240L) and wheat germ bound at a significant level and B. subtilis and wheat germ amylases could be eluted with 1M maltose. The wheat germ alpha amylase could be purified 45 fold with 70% recovery. The SDS-PAGE pattern showed significant purification by this single step strategy.

Alginates↗
PubMed1998

Chemical modification and chemical cross-linking for protein/enzyme stabilization.

The protein function as well as its stability is governed by the amino acid sequence which in turn defines the collective noncovalent interactions leading to its specific conformation. Hence, it is not surprising that chemical modification with monofunctional and bifunctional reagents (the latter is called chemical cross-linking) causes structural changes (sometimes even subtle) which can result in significant changes in the stability. This review, while recapitulating the early lessons, analyses recent work (including work from authors' laboratory) involving these twin approaches for protein stabilization. In the case of chemical modification, both surface hydrophilization and enhancing surface hydrophobicity are reported to have enhanced protein stability in different cases. For cross-linking, the nature, span, and position of the cross-link are important factors in the stabilization achieved. It is also pointed out that in the case of aqueous-organic cosolvent mixtures, protein stability may depend upon the nature of the organic solvents. In the case of polyphenol oxidase and trypsin (at least), it is possible to choose "good" solvents on the basis of the Polarity index of the solvent.

Cross-Linking Reagents↗
PubMed1998

Carbodi-imide coupling of enzymes to the reversibly soluble insoluble polymer Eudragit S-100.

The coupling of proteins and enzymes to soluble-insoluble polymers by carbodi-imide can be performed by using numerous variations of the protocol. This protocol has been investigated for the coupling of five different enzymes, namely wheatgerm acid phosphatase, beta-glucosidase, beta-galactosidase, trypsin and xylanase, to an enteric methacrylate polymer Eudragit S-100. The following results were found. (1) The activity of the bioconjugate was critically dependent on the physical state of the polymer and the pH of the coupling reaction. For example, in the case of wheatgerm acid phosphatase, the activity of the bioconjugate was 49% when coupling was performed at pH 7.2 and 67% when coupling was performed at pH 4. 5. With beta-galactosidase the corresponding values were 57% and 23% and with beta-glucosidase they were 57% and 52% respectively. (2) In some cases, such as beta-glucosidase and beta-galactosidase, it might be necessary to remove excess carbodi-imide before the addition of the enzyme to the activated matrix. (3) In most of the cases investigated, a sig-nificant amount of the enzyme (more than 90%) could be bound to the matrix merely by adsorption. (4) More importantly, after the carbodi-imide coupling procedure, a sufficient fraction of the bound enzyme could be eluted off the matrix, indicating that this was merely adsorbed and not covalently coupled.

Acid Phosphatase↗
PubMed1996

Sequential precipitation with reversibly soluble insoluble polymers as a bioseparation strategy: purification of beta-glucosidase from Trichoderma longibrachiatum.

Precipitation of proteins/enzymes with reversibly soluble-insoluble polymers is a simple approach which can be easily scaled up. It is shown here that considerable purification may be achieved if more than one polymer (chosen judiciously) is used in sequence for purification. The principle is illustrated with purification of beta-glucosidase from a commercial preparation of Trichoderma longibrachiatum. Precipitation with chitosan removed cellulase activity from the preparation with 4-fold purification and 99% recovery of beta-glucosidase activity in the supernatant. The treatment of the supernatant with Eudragit S-100 precipitated 98% of the enzyme activity. The two different elution procedures gave 82% recovery (with 14-fold purification) and 86% recovery (with 12-fold purification) of the enzyme activity from the precipitate. A low molecular weight protein still present in these preparations could be removed by gel filtration. This finally yielded an electrophoretically homogeneous enzyme with 74% recovery and 29-fold purification.

Cellulase↗
PubMed1996

An assessment of nonspecific adsorption to Eudragit S-100 during affinity precipitation.

The problem of nonspecific adsorption to the reversibly soluble-insoluble polymers is of considerable importance in the design of an affinity precipitation protocol. It was seen that activation and coupling of the affinity ligand to the polymer changes the nature of the polymer surface in a significant fashion. The results with pure trypsin, partially purified trypsin preparation, and crude protein extract-containing protein inhibitors of trypsin and alpha-amylase and the reversibly soluble-insoluble polymer Eudragit S-100, show that nonspecific adsorption may not be a severe limitation in such systems.

Acrylic Resins↗
PubMed1995

Immobilization of Aspergillus niger xylanase on magnetic latex beads.

Xylanase from Pectinex 3XL was purified 70-fold by precipitation with an enteric polymer, Eudragit S-100. The purified xylanase was immobilized on magnetic latex beads via carbodi-imide coupling. The immobilized preparation showed 80% of the total activity bound to the beads. The pH optimum remained unchanged at 6.0 and the Km increased from 0.25 g/100 ml (free enzyme) to 0.39 g/100 ml on immobilization. Immobilization resulted in significant thermal stability at 60 degrees C. The time course of hydrolysis of xylan at 60 degrees C by free enzyme as well as immobilized enzyme was also studied.

Aspergillus niger↗
PubMed1995

Purification and characterization of an acid phosphatase from Arachis hypogaea.

An acid phosphatase from Arachis hypogaea (peanuts) has been purified. The electrophoretically homogeneous enzyme preparation is free of any phophodiesterase activity. The enzyme has a molecular weight of 120,000. Among the various phosphomonoesters tested, p-nitrophenylphosphate was found to be its most effective substrate. The Km for p-nitrophenylphosphate was 1.21 mM at pH 5.0 and 25 degrees C. The enzyme was thermostable and did not loose activity after 1 hr at 50 degrees C.

Acid Phosphatase↗
PubMed1994

Affinity precipitation of trypsin with soybean trypsin inhibitor linked Eudragit S-100.

Soybean trypsin inhibitor linked to Eudragit S-100 was used for the affinity precipitation of trypsin. Polymer and ligand concentrations used in conjugate preparation showed remarkable effect on the trypsin recovery. Trypsin precipitation efficiency amounted to 89% and recovery was 74%. The final purification of relatively crude commercial trypsin resulted in 1.85-fold purification. The SDS-PAGE analysis indicated significant purification. The precipitated enzyme activity was around 96% and recovered enzyme activity was 83%.

Acrylic Resins↗
PubMed1994

An insoluble aggregate of commercially available bovine serum albumin shows antiproteolytic activity.

Chemical aggregation of bovine serum albumin by extensive chemical crosslinking with glutaraldehyde yielded an insoluble protein preparation with significant antiproteolytic activity. This was presumably due to the presence of alpha 2-macroglobulin in bovine serum albumin preparations. Chemical crosslinking of bovine serum albumin under optimum conditions was found to increase its antitryptic activity by about four times. The results indicate that enhanced rigidity of alpha 2-macroglobulin structure increases its antitryptic activity.

Animals↗
PubMed1993

Purification of endo-polygalacturonase by affinity precipitation using alginate.

The precipitation of alginate by Ca2+ at pH 3.8 was found to occur concomitantly with the precipitation of endo-polygalacturonase from Aspergillus niger. Under optimum conditions, 92% of the enzyme activity was precipitated. The enzyme could be recovered from the precipitate by washing with 0.5 M NaCl/0.2 M Ca2+. All the precipitated endo-polygalacturonase activity could be recovered in this way. The enzyme thus obtained was purified 10-fold. A comparison of SDS/PAGE gel patterns of the crude preparation and enzyme purified by the affinity precipitation also showed a significant purification of the enzyme.

Alginates↗
PubMed1992

Enzyme function in organic solvents.

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non-aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water less than 0.02% by vol.), perhaps reflects that the minimum necessity for water is for forming bonds with polar amino acids on the enzyme surface. The rigidity of enzyme structure at such low water content results in novel substrate specificities, pH memory and the possibility of techniques such as molecular imprinting. Limited data indicates that, while enhanced thermal stability invariably results, the optimum temperature for catalysis may not change. If true in general, this enhanced thermostability would have extremely limited benefits. Medium engineering and biocatalyst engineering are relevant techniques to improve the efficiency and stability of enzymes in such low water systems. Most promising, as part of the latter, is the technique of protein engineering. Finally, this review provides illustrations of applications of such systems in the diverse areas of organic synthesis, analysis and polymer chemistry.

Catalysis↗
PubMed1992

Effect of concanavalin A on the activity of acid and alkaline phosphatases.

The effect of Concanavalin A on the activity of acid and alkaline phosphatases has been investigated. Both the glycoproteins were inhibited by Con A (about 30-40% with 4 mg final concentration in the reaction mixture of Con A). In case of the acid phosphatase, the addition of 1.0 M NaCl, instead of 0.5 M, stimulated the enzyme activity at low concentrations of Con A. The significance of the lectin effect on the stability of glycoenzymes and its implications in bioseparation and enzyme immobilization is briefly discussed.

Acid Phosphatase↗
PubMed1991

Entrapment of proteins by aggregation within sephadex beads.

When a protein is aggregated by chemical crosslinking inside Sephadex beads of appropriate pore size, it gets trapped inside the beads. The above approach was used for immobilization of beta-galactosidase, acid phosphatase, trypsin, and concanavalin A. It was found to be a simple, convenient, and fast method for immobilization of proteins.

Acid Phosphatase↗
PubMed1988

Preparation of heteroenzyme conjugates: trypsin-chymotrypsin and trypsin-alkaline phosphatase.

Hybrid enzymes which have two different enzyme activities linked together covalently may be useful reagents for various applications, such as the determination of complex biological structures. The present paper describes the preparation and purification of two such enzyme-enzyme conjugates, namely, trypsin-chymotrypsin and trypsin-alkaline phosphatase. Whereas the former has been prepared by using the well-known bifunctional reagent glutaraldehyde, the latter exploited the Schiff base formation between the oxidized carbohydrate moiety of alkaline phosphatase and the free amino groups of trypsin.

Alkaline Phosphatase↗
PubMed1988

Crosslinking of concanavalin A with glutaraldehyde.

Crosslinking of Concanavalin A with low concentrations of glutaraldehyde gives a mixture of products. A specific product having about 66% of the biological activity of the native molecule was characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed the presence of monomers, dimers, trimers, tetramers, and a small amount of pentamers as products. The presence of alpha-methyl mannoside during crosslinking changed the nature of the products, yielding a product retaining 80% of the biological activity. The crosslinked products showed greater stability than the native molecule at alkaline pH. However, the greatest stability under alkaline conditions was shown by the native molecule itself where alpha-methyl mannoside was present.

Aldehydes↗