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Kalyani Mondal

Publications and source records attributed to Kalyani Mondal.

18 recordsLinked to original sources
PubMed2007

Role of stimuli-sensitive polymers in protein refolding: alpha-amylase and CcdB (controller of cell division or death B) as model proteins.

Alginate, a calcium-sensitive polymer, could carry out simultaneous purification and refolding of 8 M urea/100 mM dithiothreitol (DTT) denatured and thermally denatured alpha-amylase present in a commercial preparation. Activity recoveries of 80 and 70% in the former and the latter cases, respectively, were obtained. The fluorescence spectra showed refolding, and PAGE showed the absence of any aggregates in the refolded preparation. As another example, Eudragit S-100, a pH-sensitive poly(methyl methacrylate), was used to refold CcdB (controller of cell division or death B) protein. Initial experiments with wild-type (WT) CcdB showed that Eudragit bound and precipitated (upon lowering the pH to 4.0) CcdB quantitatively from the latter's aqueous solution. The bioconjugate showed DNA gyrase inhibition activity of CcdB and could be recycled. The inclusion bodies of CcdB mutant CcdB-17P were solubilized in 8 M urea/100 mM dithiothreitol. This preparation could be refolded by precipitation with Eudragit. The fluorescence and CD spectra showed that protein refolding has occurred.

Alginates↗
PubMed2006

Leveraging protein purification strategies in proteomics.

The proteomic studies, although, tend to be analytical in nature, yet many strategies of preparative protein purification can be usefully employed in such studies. This review points out the importance of purification techniques which are capable of dealing with samples which are suspensions rather than clear solution, e.g. aqueous two phase partitioning, three phase partitioning, expanded bed chromatography, etc. The review also outlines the potential of non-chromatographic techniques in dealing with fractionation of proteomes. Separation protocols which can deal with post-translationally modified (PTM) proteins are also considered.

Chromatography, Affinity↗
PubMed2006

Microwave-assisted preparation of affinity medium.

Microwave assistance was used for preparing polyethylene glycol (PEG)-Cibacron blue 3GA and Sepharose CL-4B-Cibacron blue 3GA affinity materials. The former was used as the affinity macroligand in a PEG-dextran aqueous two-phase system for purification of alcohol dehydrogenase and EcoRI. The Sepharose CL-4B-Cibacron blue 3GA was used for affinity chromatography of the above two enzymes. It was found that microwave assistance could reduce the time of PEG-dye preparation to 5 min (from 7h). Similarly, Sepharose CL-4B-Cibacron blue 3GA preparation time could be reduced to 21 min (from 3.5h). The performances of affinity macroligand PEG-dye and the affinity medium Sepharose-dye prepared by conventional methods and with microwave assistance were similar during purification of these enzymes.

Chromatography, Affinity↗
PubMed2006

A bioconjugate of Pseudomonas cepacia lipase with alginate with enhanced catalytic efficiency.

A bioconjugate of Pseudomonas cepacia lipase with alginate was prepared by simple adsorption. Atomic force microscope (AFM) images showed that this bioconjugate resulted from adsorption rather than entrapment of the enzyme as enzyme molecules were visible on the gel surface. The soluble bioconjugate exhibited increased enzyme activity in terms of high effectiveness factor (effectiveness factor was 3 for the immobilized preparation) and greater Vmax/Km value (Vmax/Km increased 25 times upon immobilization). This constitutes one of the less frequently observed instances of lipase activation by lid opening as a result of binding to a predominantly hydrophilic molecule. The bioconjugate was also more stable at 55 degrees C as compared to the free enzyme and could be reused for oil hydrolysis up to 4 cycles without any loss in activity. Fluorescence emission spectroscopy showed that the immobilized enzyme had undergone definite conformational changes.

Adsorption↗
PubMed2006

Alginate-chaperoned facile refolding of Chromobacterium viscosum lipase.

Urea denatured lipase from Chromobacterium viscosum lipase could be refolded by addition of alginate with high guluronic acid content. The refolded molecule could be recovered by affinity precipitation. This approach resulted in recovery of 80% (of original activity) as compared to classical dilution method which gave only 21% activity recovery. Dynamic light scattering showed that binding required about 45 min and activity data obtained from affinity precipitation experiments indicated that refolding was almost instantaneous after binding. Circular dichroism (CD) and fluorescence data showed that refolded molecule was identical to the native molecule. It also showed that refolding takes place at the binding stage and not at the precipitation stage. Preliminary studies showed that the refolding strategy worked equally well with lipases from wheat germ and porcine pancreas.

Alginates↗
PubMed2006

The affinity concept in bioseparation: evolving paradigms and expanding range of applications.

The meaning of the word affinity in the context of protein separation has undergone evolutionary changes over the years. The exploitation of molecular recognition phenomenon is no longer limited to affinity chromatography modes. Affinity based separations today include precipitation, membrane based purification and two-phase/three-phase extractions. Apart from the affinity ligands, which have biological relationship (in vivo) with the target protein, a variety of other ligands are now used in the affinity based separations. These include dyes, chelated metal ions, peptides obtained by phage display technology, combinatorial synthesis, ribosome display methods and by systematic evolution of ligands by exponential enrichment (SELEX). Molecular modeling techniques have also facilitated the designing of biomimetic ligands. Fusion proteins obtained by recombinatorial methods have emerged as a powerful approach in bioseparation. Overexpression in E. coli often result in inactive and insoluble inclusion bodies. A number of interesting approaches are used for simultaneous refolding and purification in such cases. Proteomics also needs affinity chromatography to reduce the complexity of the system before analysis by electrophoresis and mass spectrometry are made. At industrial level, validation, biosafety and process hygiene are also important aspects. This overview looks at these evolving paradigms and various strategies which utilize affinity phenomenon for protein separations.

Affinity Labels↗
PubMed2006

Enhancing the synthetic utility of aldolase antibody 38C2.

Three-phase partitioning (TPP) treated aldolase antibody 38C2 was evaluated for aldol reaction between p-nitrobenzaldehyde and acetone to give 4-(4'-nitrophenyl)-4-hydroxy-2-butanone. While TPP-treated 38C2 transformed 65% of p-nitrobenzaldehyde, the untreated 38C2 gave only 24% transformation in 18 h at 25 degrees C. However, since TPP-treated 38C2 also gave an additional (unidentified) product, its synthetic utility was limited. Crosslinked aggregate of 38C2, however, gave the biocatalyst which gave a single product and could be reused at 40 degrees C five times without loss of activity.

Acetone↗
PubMed2006

Applications of alginate in bioseparation of proteins.

Alginate is a polysaccharide that is a block polymer consisting of block units of guluronic acid and mannuronic acid. It shows inherent biological affinity for a variety of enzymes such as pectinase, lipase, phospholipase D, a and ss amylases and glucoamylase. Taking advantage of its precipitation with Ca2+ and the above-mentioned property, alginate has been used for purification of these enzymes by affinity precipitation, aqueous two phase separation, macroaffinity ligand facilitated three phase partitioning, immobilized metal affinity chromatography and expanded bed affinity chromatography. Thus, this versatile marine resource has tremendous potential in bioseparation of proteins.

Alginates↗
PubMed2006

Preparation and properties of thermoresponsive bioconjugates of trypsin.

Covalent attachment of enzymes and other proteins to the smart polymer, poly(N-isopropylacrylamide) [poly (NIPAAm)], has been widely used as a method for the preparation of thermosensitive protein conjugates. In the present study, reversible soluble-insoluble polymer-enzyme conjugates were prepared by conjugating a copolymer of NIPAAm with 5-mol % of 6-acrylaminohexanoic acid to trypsin by the carbodiimide-NHS (N-hydroxysuccinimide) coupling method. Four bioconjugates with different units of enzyme coupled to the matrix were prepared. Increased enzymatic activity in terms of high effectiveness factor (in the range of 3-5) was found in the conjugates. Kinetic parameters for the immobilized and free enzyme were determined. The Vmax/Km value of the enzyme significantly increased on immobilization by the factors in the range of 12-28. The immobilized enzyme also showed stability to autolysis at 50 degrees C.

Acrylic Resins↗
PubMed2004

Simultaneous refolding/purification of xylanase with a microwave treated smart polymer.

Affinity precipitation with a smart polymer, Eudragit S-100 (a methyl methacrylate polymer), was exploited for simultaneous refolding and purification of xylanase. Affinity precipitation consisted of this reversibly soluble-insoluble polymer-binding xylanase selectively. The complex was precipitated by lowering the pH and xylanase was eluted off the polymer using 1 M NaCl. For refolding experiments, the commercial preparation of Aspergillus niger xylanase was denatured with 8 M urea. Addition of microwave irradiated Eudragit S-100 and affinity precipitation led to recovery of 96% enzyme activity by refolding. Simultaneously, the enzyme was purified 45 times. Thermally inactivated preparation, when subjected to similar steps, led to 95% recovery of enzyme activity with 42-fold purification. The strategy has the potential for recovering pure proteins in active forms from overexpressed proteins, which generally form inclusion bodies in E. coli.

Affinity Labels↗
PubMed2004

Affinity precipitation of Aspergillus niger pectinase by microwave-treated alginate.

Affinity precipitation is a simple, single plate separation process in which the complex of a smart macroaffinity ligand with the target protein (from a crude broth) can be selectively precipitated by application of a suitable stimulus. Alginate is a copolymer of guluronic acid and mannuronic acid residues and precipitates with Ca(2+) ions. It was found to bind to pectinase present in a commercial preparation of Aspergillus niger, Pectinex Ultra-SPL. Microwave pretreatment of alginate at 75 degrees C was found to enhance the selectivity of the affinity precipitation. Using microwave-treated alginate, 83% of the enzyme activity with 20-fold purification could be recovered. SDS-PAGE upon silver staining confirmed the enhanced selectivity of affinity precipitation when microwave-treated alginate was used.

Alginates↗
PubMed2003

Separation of enzymes by sequential macroaffinity ligand-facilitated three-phase partitioning.

Pectinase and cellulase were separated from a commercial enzyme preparation called Pectinex Ultra SP-L. This was carried out using a process called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this method, a water-soluble polymer is floated as an interfacial precipitate by adding ammonium sulfate and tert.-butanol. The polymer (appropriately chosen) in the presence of an enzyme for which it shows affinity, selectively binds to the enzyme and floats as a polymer-enzyme complex. In the first step, pectinase was purified (with alginate as the polymer) 13-fold with 96% activity recovery. In the second MLFTPP step, using chitosan, cellulase was purified 16-fold with 92% activity recovery. Both preparations showed a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis. This illustrative example shows that the strategy of sequential MLFTPP can be used to separate important biological activities from a crude broth.

Affinity Labels↗
PubMed2003

Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate.

Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this process, a crude preparation of the enzyme was mixed with alginate. On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase. Enzyme activity from this interfacial precipitate was recovered using 1M maltose. Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively. Both the purified preparations showed a single band on SDS-PAGE.

Alginates↗
PubMed2003

kappa-Carrageenan as a carrier in affinity precipitation of yeast alcohol dehydrogenase.

kappa-Carrageenan is a non-toxic polymer from seaweeds, which becomes reversibly insoluble upon the addition of K(+). Its conjugate with the dye, Cibacron Blue 3GA, was used to purify alcohol dehydrogenase from crude yeast extract by affinity precipitation. Response surface methodology was used to optimize conditions for affinity precipitation of the enzyme with the polymer-dye conjugate. Recovery of 58% of the enzyme activity with 13.6-fold purification was obtained.

Alcohol Dehydrogenase↗
PubMedDate not supplied

Macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) of alpha-amylases using a modified alginate.

The crude extracts of alpha-amylases when mixed with alginate, tert-butyl alcohol, and ammonium sulfate resulted in an interfacial precipitate containing polymer-bound amylase. The precipitate was dissolved in 1 M maltose to recover alpha-amylase activity. The recovery of alpha-amylases were 74%, 77%, and 92% in the case of Bacillus amyloliquefaciens, wheat germ, and porcine pancreas, respectively. All purified preparations showed a single band on SDS-PAGE.

Adsorption↗
PubMedDate not supplied

Accelerating enzymatic hydrolysis of chitin by microwave pretreatment.

Response surface analysis was used to determine optimum conditions [2% (w/v) chitin, 57.5 degrees C, 38 min] for microwave irradiation of chitin to improve its enzymatic hydrolysis. V(max)/K(m) of cabbage chitinase toward untreated and microwave-irradiated chitin was found to be 21.1 and 31.7 nmol h(-1) mg(-2) mL, respectively. Similar improvement was observed in the case of pectinase in its unusual catalytic activity of chitin degradation. It was found that a greater extent of chitin hydrolysis by chitinase was possible after the substrate chitin was irradiated with microwaves.

Acetylglucosamine↗