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Biomedical subjects

M N Gupta

Publications and source records attributed to M N Gupta.

At least 19 recordsLinked to original sources
PubMed2005

Ultrasonic pre-irradiation effect upon aqueous enzymatic oil extraction from almond and apricot seeds.

The present work evaluates the benefit of using ultrasonic pre-irradiation before extracting oil from almond and apricot seeds by aqueous enzymatic oil extraction (AEOE) process. The use of a commercial preparation which is a mixture of three proteases in AEOE gave 75% w/w oil yield from almonds at pH 4.0 in 18 h at 40 degrees C. The ultrasonic pre-irradiation at 70 W for 2 min increased the yield to 95%, w/w and reduced the extraction time to 6 h. The effect of ultrasonic pre-irradiation on meal morphology could be visually seen by scanning electron micrographs. It indicates development of of microfractures and disruption of cell walls in almond powder. With apricot, also, ultrasonic pre-irradiation also marginally increased the oil yield obtained by AEOE to 77% w/w and reduced the extraction time to 6 h. Thus, ultrasonic pre-irradiation step may reduce time required to extract oil from edible oils from plant sources and hence can improve through put in commercial oil production process.

Hydrogen-Ion Concentration↗
PubMed2005

One-step purification and characterization of an alkaline protease from haloalkaliphilic Bacillus sp.

An alkaline protease producer haloalkaliphilic bacteria (isolate Vel) was isolated from west coast of India. It was related to Bacillus pseudofirmus on the basis of 16S r RNA gene sequencing, lipid profile and other biochemical properties. The protease secreted by this bacteria was purified 10-fold with 82% yield by a single step method on Phenyl Sepharose 6 Fast Flow column. The apparent molecular mass based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was estimated to be 29 000 Da. The Km and Vmax towards caseinolytic activity were found to be 2 mg ml(-1) and 289.8 microg min(-1), respectively. The enzyme was active over the pH range of 8.5-12.0, the optimum being 10-11.0. The purified enzyme when kept at 45 degrees C and 50 degrees C for 40 min retained 92% and 85% protease activity, respectively. Effect of NaCl concentration on protease activity showed that the enzyme was slightly inhibited with high concentration of salt. The proteolytic activity was inhibited by PMSF, suggesting that the enzyme may belong to serine type protease. Interestingly, the activity was slightly enhanced with SDS (0.1%) and Triton X-100 (0.1%) but remained unaffected by Tween 80 (0.1%). The activity was affected by metal ions to varying extent. While Mn2+, Zn2+ and Mg2+ had no significant effect on protease activity, the enzyme was activated with Ca2+ (1 mM) and Cu2+ (5 mM). The stability of the enzyme in the presence of detergent components and surfactants is particularly attractive for its application in detergent industries.

Bacillus↗
PubMed2005

A solvent tolerant isolate of Enterobacter aerogenes.

A solvent tolerant strain of Enterobacter aerogenes was isolated from soil by cyclohexane enrichment. Presence of cyclohexane (20%) in culture media prolonged the lag phase and caused reduction in biomass. Transmission electron micrographs showed convoluted cell membrane and accumulation of solvent in case of the cells grown in cyclohexane. The Enterobacter isolate was able to grow in the range of organic solvents having log P above 3.2 and also in presence of mercury, thus showing potential for treatment of solvent rich wastes.

Cyclohexanes↗
PubMed2005

Purification and characterization of a solvent stable protease from Pseudomonas aeruginosa PseA.

A solvent tolerant Pseudomonas aeruginosa PseA strain was isolated from soil. It secreted a novel alkaline protease, which was stable and active in the presence of range of organic solvents, thus potentially useful for catalysis in non-aqueous media. The protease was purified 11.6-fold with 60% recovery by combination of ion exchange and hydrophobic interaction chromatography using Q-Sepharose and Phenyl Sepharose 6 Fast Flow matrix, respectively. The apparent molecular mass based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was estimated to be 35,000 Da. The enzyme was stable in the pH range of 6.0-9.0, the optimum being 8.0. The Km and Vmax towards caseinolytic activity were found to be 2.7 mg/ml and 3 micromol/min, respectively. The protease was most active at 60 degrees C and characterized as a metalloprotease because of its sensitivity to EDTA and 1,10-phenanthroline. It was tested positive for elastase activity towards elastin-orcein, thus appears to be an elastase, which is known as pseudolysin in other strains of P. aeruginosa. The protease withstands range of detergents, surfactants and solvents. It is stable and active in all the solvents having log P above 3.2, at least up to 72 h. These two properties make it an ideal choice for applications in detergent formulations and enzymatic peptide synthesis.

Bacterial Proteins↗
PubMed2005

Extraction of oil from Jatropha curcas L. seed kernels by combination of ultrasonication and aqueous enzymatic oil extraction.

Use of ultrasonication as a pretreatment before aqueous oil extraction and aqueous enzymatic oil extraction was found to be useful in the case of extraction of oil from the seeds of Jatropha curcas L. The use of ultrasonication for 10 min at pH 9.0 followed by aqueous oil extraction gave a yield of 67%. However, the maximum yield of 74% was obtained by ultrasonication for 5 min followed by aqueous enzymatic oil extraction using an alkaline protease at pH 9.0. Use of ultrasonication also resulted in reducing the process time from 18 to 6 h.

Enzymes↗
PubMed2005

An integrated process for separation of major and minor proteins from goat serum.

Analysis of minor proteins in animal sera is of considerable clinical significance. To be able to detect these proteins, depletion of major proteins (albumin and immunoglobulin G [IgG]) is necessary. Many of these proteins are also required in pure form for a variety of biochemical applications. The present work uses goat serum as the system and describes the separation and purification of both major and several minor proteins. This was carried out by judicious adaptation and combination of separation technologies such as immobilized metal ion affinity chromatography (on a somewhat novel matrix), dye affinity chromatography, and lectin affinity chromatography. Albumin, IgG, alpha2-macroglobulin, alpha1-proteinase inhibitor, and transferrin were obtained from the serum. The purified preparations were found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Animals↗
PubMed2004

Chitosan as a macroaffinity ligand purification of chitinases by affinity precipitation and aqueous two-phase extractions.

(1) Chitosan was found to be a suitable macroaffinity ligand for affinity precipitation of chitinase from Neurospora crassa, cabbage and puffballs. (2) The activity recoveries of 85, 82 and 90% with concomitant fold purifications in terms of specific activities were 27, 15 and 30 with N. crassa, cabbage and puffballs and were obtained with affinity precipitation. These results were obtained with clarified extracts/homogenates as the starting materials. (3) The incorporation of chitosan in poly(ethylene glycol) (PEG)-salt aqueous two-phase system allowed purification of chitinases from these sources directly from unclarified extracts/homogenates. (4) The 96% (w/v) chitosan (of initially introduced into the aqueous two-phase system) partitioned into PEG-phase and this enhanced the partitioning of chitinases into PEG-phase. The chitosan, free as well as bound to chitinases, could be separated from PEG-phase by increasing the pH to 7. (5) By the process of desorption with 2.0 M MgCl2, 86, 80 and 88% activity recoveries (% expressed in terms of total units of enzyme activities in the crude extract) were obtained in the case of N. crassa, cabbage and puffballs, respectively. The corresponding fold purifications in terms of specific activities were 34, 20 and 38. (6) The purified preparations gave single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the estimated molecular masses agreed with the reported values in the literature.

Chitinases↗
PubMed2004

Purification of recombinant green fluorescent protein by three-phase partitioning.

The technique of three-phase partitioning (TPP) was used to purify the green fluorescent protein (GFP) in a single step. TPP uses a combination of ammonium sulphate and tert-butanol to precipitate proteins from their crude extracts. In the first round of TPP with 20% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:1 (v/v), most of the GFP remains in the lower aqueous phase. When subjected to a second round of TPP with 60% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:2 (v/v) gives 78% recovery of GFP with a 20-fold purification. The sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of purified preparation shows single band. The fluorescence excitation and emission spectra agreed with values reported in literature.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Purification of phospholipase D by two-phase affinity extraction.

An aqueous two-phase system of polyethylene glycol (PEG)-salt was used for purification of phospholipase D (PLD) from peanuts and carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using 0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.

Arachis↗
PubMed2004

Smart biocatalysts: design and applications.

Smart materials respond to chemical or physical changes in their environment in a predictable fashion. One class of such materials are smart polymers which can be used to design reversibly soluble-insoluble biocatalysts. One important advantage of such soluble polymer enzyme conjugates is in bioconversion of macromolecular or insoluble substrates. In addition, they share the advantage of reusability with conventional immobilized enzymes. Stimuli that are used to "recover" smart polymer - enzyme conjugates for reuse include changes in pH, temperature, ionic strength and addition of chemical species like calcium. In addition to these, enzymes linked to photoresponsive polymers have also been described in the literature. Both adsorption and covalent coupling have been used to create such polymer conjugates. End-group conjugation and site-specific conjugation are recently described strategies to obtain biocatalysts with better designs for solving mass transfer constraints. Some important applications of such smart biocatalysts are hydrolysis of starch, cellulose and proteins. Work has also been carried out on hydrolysis of pectins and xylans. All the above applications involve hydrolysis and are hence carried out in aqueous media. For synthetic applications such as synthesis of peptides, some photoresponsive polymers linked to proteases have recently been described.

Biological Products↗
PubMed2004

A smart bioconjugate of trypsin with alginate.

Alginate is a polymer of guluronic acid and mannuronic acid residues and is an inexpensive, nontoxic polysaccharide of marine origin. Trypsin was immobilized noncovalently on alginate with 100% retention of activity. The enzyme did not leach off the polymer even in the presence of 0.01 M HCl and Triton X-100 (0.2% vv(-1)). The V(max)/K(m) values did not change significantly on immobilization. There was 22% loss of activity in first cycle of pH change and after that the conjugate could be reused upto 4 precipitation cycles without any further loss of activity. This smart bioconjugate was also found to have better operational stability in the presence of casein than free enzyme. Fluorescence studies were carried out to probe structural changes upon immobilization.

Alginates↗
PubMed2003

Magnetic alginate microparticles for purification of alpha-amylases.

Spherical magnetic alginate microparticles (25-60 microm in diameter) were prepared using the microemulsion system, with water-saturated 1-pentanol as the organic phase. The limited solubility of 1-pentanol in water enabled simple removal of the organic solvent from the prepared beads with water solution. The prepared alginate microparticles were used as magnetic affinity adsorbents for specific purification of alpha-amylases. Enzyme activity was eluted by 1.0 M maltose. alpha-Amylases from Bacillus amyloliquefaciens and porcine pancreatic acetone powder were purified 9- and 12-fold with 88 and 96% activity recovery, respectively.

Alginates↗
PubMed2003

Immobilization of xylan-degrading enzymes from Melanocarpus albomyces IIS 68 on the smart polymer Eudragit L-100.

Xylanase of Melanocarpus albomyces IIS 68 was immobilized on Eudragit L-100. The latter is a copolymer of methacrylic acid and methyl methacrylate and is a pH-sensitive smart polymer. The immobilization was carried out by gentle adsorption and an immobilization efficiency of 0.82 was obtained. The enzyme did not leach off the polymer even in the presence of 1 M NaCl and 50% ethylene glycol. The K(m) of the enzyme changed from 5.9 mg ml(-1) to 9.1 mg ml(-1) upon immobilization. The V(max) of the immobilized enzyme showed an increase from 90.9 micro mol ml(-1) min(-1) (for the free enzyme) to 111.1 micro mol ml(-1) min(-1). The immobilized enzyme could be reused up to ten times without impairment of the xylanolytic activity. The immobilized enzyme was also evaluated for its application in pre-bleaching of eucalyptus kraft pulp.

Enzymes, Immobilized↗
PubMed2003

pH-responsive polymer-assisted refolding of urea- and organic solvent-denatured alpha-chymotrypsin.

A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of alpha-chymotrypsin denatured with 8 M urea and 100 mM dithiothreitol at pH 8.2. The complete activity could be regained within 10 min during refolding. Both native and refolded enzymes showed emission of intrinsic fluorescence with lambda(max) of 342 nm. Gel electrophoresis showed that the presence of Eudragit S-100 led to dissociation of multimers followed by the appearance of a band at the monomer position. The unfolding (by 8 M urea) and folding (assisted by the polymer) also led to complete renaturation of alpha-chymotrypsin initially denatured by 90% dioxane. The implications of the data in recovery of enzyme activity from inclusion bodies and the interesting possibility in the in vivo context of reversing protein aggregation in amyloid-based diseases have been discussed.

Chymotrypsin↗
PubMed2003

Prospective comparative study of patients with culture proven and high suspicion of adult onset septic arthritis.

OBJECTIVE: To investigate whether patients with acute septic arthritis (SA) diagnosed by positive synovial fluid (SF) culture (Newman grade A) have different clinical and serological features from those with sterile SF in whom there is nonetheless a high suspicion of SA (Newman grades B and C). PATIENTS AND METHODS: A prospective 12 month multicentre hospital based study of adult patients with SA recruited 47 patients with culture positive SA and 35 patients with clinically suspected SA but sterile SF. RESULTS: Patient demography, clinical and laboratory features at presentation were similar irrespective of the underlying diagnosis, SF culture, and the presence of prosthetic joints. Medical and surgical treatment and outcome were comparable in the two patient groups. Patients with both suspected and proven SA were more likely to be from the more socially deprived areas of our community (p<0.0001). CONCLUSION: Patients in whom there is a high clinical suspicion of SA are comparable to those patients with SA with a positive SF culture and have similar morbidity and mortality on follow up. Therefore, if clinical suspicion of SA is high then it is correct to treat as SA in the absence of bacterial proof.

Acute Disease↗
PubMed2002

Macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) for purification of xylanase.

It is shown that eudragit S-100, a copolymer of methylacrylic acid and methylmethacrylate, undergoes three-phase partitioning. It was found that 95% eudragit S-100 could be recovered as the interfacial precipitate by using 30% (w/v) ammonium sulfate, 1:1 ratio of t-butanol to polymer solution at 40 degrees C. Three-phase partitioning of proteins uses simultaneous addition of ammonium sulfate and t-butanol to precipitate proteins in an interfacial layer separating the aqueous phase and organic solvent. Exploiting the affinity of xylanases towards eudragit S-100, it was possible to purify xylanase from Aspergillus niger; 60% recovery of activity with 95-fold purification could be obtained by this process. The purified enzyme showed A single band on SDS-PAGE. The technique shows promise to develop into a general method that could be termed "macroaffinity ligand-facilitated three-phase partitioning (MLFTPP).

Aspergillus niger↗