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Biomedical subjects

Munishwar N Gupta

Publications and source records attributed to Munishwar N Gupta.

At least 19 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

Kinetic resolution of (+/-)-1-phenylethanol in [Bmim][PF6] using high activity preparations of lipases.

Lipases from two different sources Candida rugosa (CRL) and Burkholderia cepacia (BCL) were formulated as enzyme precipitated and rinsed with organic solvents, organic solvent rinsed enzyme preparation, cross-linked enzyme aggregates (CLEAs) and protein coated micro-crystals (PCMCs). These various enzyme formulates were evaluated for the kinetic resolution of (+/-)-1-phenylethanol in ionic liquid [Bmim][PF(6)] by transesterification with vinyl acetate. Of all the enzyme forms evaluated EPRP and PCMC in the case of CRL showed the best results with 26 % (E value=153) and 53% (E value=79) conversion, respectively, at 35 degrees C in 24h. Carrying out this conversion with PCMC at lower temperature of 25 degrees C further improved the E value to 453 (with 44% conversion in 12h). For BCL the acetone-rinsed enzyme preparation (AREP), CLEA and PCMC performed equally well with % conversion of 50 and 99 ee(p) (%) (E value >1000) in just 2h, whereas, the free lipase gave only 8% conversion.

Benzyl Alcohols↗
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

Obtaining high transesterification activity for subtilisin in ionic liquids.

It is known that subtilisin shows poor transesterification activity in ionic liquids (ILs). The present work, taking subtilisin as the system, explores approaches for biocatalyst preparations, which are capable of yielding higher/adequate transesterification activity in these solvents. Of all the approaches tried, enzyme precipitated and rinsed with n-propanol (EPRP) gave the best results (about 10,000 times increase in initial rates in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF(6)]) over what is obtained with pH tuned lyophilized powders). In case of water soluble ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]), pH tuned lyophilized subtilisin did not show any transesterification activity. EPRP, however, gave an initial rate (for transesterification) of 2.78 mmol mg(-1) h(-1).

Chromatography, High Pressure Liquid↗
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

Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder.

Addition of bovine serum albumin (BSA) as a proteic feeder facilitates obtaining cross-linked enzyme aggregates (CLEAs) in cases where the protein concentration in the enzyme preparation is low and/or the enzyme activity is vulnerable to the high concentration of glutaraldehyde required to obtain aggregates. CLEAs of Pseudomonas cepacia lipase and penicillin acylase were prepared. CLEA of lipase prepared in the presence of BSA retained 100% activity whereas CLEA prepared without BSA retained only 0.4% activity of the starting enzyme preparation. Lipase CLEA showed 12-fold increase in activity over free enzyme powder when the CLEA was used in transesterification of tributyrin. For the transesterification of Jatropha oil, while free enzyme powder required 8 h and 50 mg lipase to obtain 77% conversion, CLEA required only 6 h and 6.25 mg lipase to obtain 90% conversion. In the case of penicillin acylase, 86% activity could be retained in CLEA prepared with BSA whereas CLEA made without BSA retained only 50% activity. CLEA prepared without BSA lost 20% activity after 8 h at 45 degrees C whereas CLEA with BSA retained full activity. CLEA prepared with BSA showed Vmax/Km of 36.3 min-1 whereas CLEA prepared without BSA had Vmax/Km of 17.4 min-1 only. Scanning electron microscopy analysis showed that CLEAs prepared in the presence of BSA were less amorphous and closer in morphology to CLEAs of other enzymes described in the literature.

Burkholderia cepacia↗
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↗
PubMed2005

Detection of native peptides as potent inhibitors of enzymes. Crystal structure of the complex formed between treated bovine alpha-chymotrypsin and an autocatalytically produced fragment, IIe-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp, at 2.2 angstroms resolution.

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in alpha-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 +/- 0.3 x 10(-11) M and thus works as a highly potent competitive inhibitor. The commercially available alpha-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated alpha-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mM sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 M ammonium sulfate. The rectangular crystals of small dimensions of 0.24 x 0.15 x 0.10 mm(3) were obtained. The X-ray intensity data were collected at 2.2 angstroms resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of alpha-chymotrypsin, which was readily interpreted as a 14 residue fragment of alpha-chymotrypsin corresponding to Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 sigma between the active site Ser195 Ogamma and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 angstroms between Ser195 Ogamma and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16-Trp29. In addition to the O-C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of alpha-chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of alpha-chymotrypsin.

Amino Acid Sequence↗
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

Obtaining higher transesterification rates with subtilisin Carlsberg in nonaqueous media.

Three phase partitioning (protein precipitate obtained as an interfacial layer between lower aqueous and upper t-butanol phases, formed by the addition of ammonium sulphate and t-butanol to the aqueous solution of protein) followed by lyophilization in the presence of two-component excipient resulted in 400-480x increases in transesterification activity of lyophilized powders of subtilisin Carlsberg, depending on the solvent. The three phase partitioned enzyme, 'dried' by washing with butanol, gave 3-4x higher rates (depending on the solvent used) than the enzyme preparation dried by lyophilization in the presence of two-component excipient system.

Catalysis↗