Punit Srivastava

Characterization of gelatin‐immobilized pigeonpea urease and preparation of a new urea biosensor

Urease purified from pigeonpea seeds was immobilized on gelatin beads via cross‐linking with glutaraldehyde. The maximum immobilization (75%) was observed at 30 mg/ml gelatin, 0·414 mg of enzyme/bead, 1% (v/v) glutaraldehyde and 4 °C. Beads stored in 50 mM Tris/acetate buffer (pH 7·3) at 4 °C showed a half‐life of 240 days and there was practically no leaching of enzyme (less than 2%) over a period of 30 days. These beads can be reused more than 30 times (with 24 h intervals) without much loss of enzyme activity (i.e. less than 11%). The immobilized urease showed a shift in its optimum pH from 7·3 to 6·5 in Tris/acetate buffer. Optimum temperature also shifted from 47 to 65 °C compared with the soluble enzyme. Gelatin‐immobilized pigeonpea urease had a higher Km (8·3 mM) than that of the soluble enzyme (3·0 mM). The time‐dependent temperature inactivation pattern was also found to change from biphasic to monophasic kinetics. The immobilized beads were used for the preparation of a new urea biosensor with a response time of less than 2 min. At least 14 samples of urea can be measured with this biosensor within an hour. The beads, as well as the biosensor, were used to analyse the urea content in clinical samples from the local clinical pathology laboratories. The results obtained with the biosensor were strikingly similar to those obtained with the various commonly employed biochemical/autoanalyzer® methods used. These immobilization studies also have a potential role in haemodialysis machines that maintain the urea level in kidney patients and in the construction of a portable/wearable kidney. The easy availability of the pigeonpea urease, the ease of its immobilization on gelatin and a significantly lower cost of the urease described in the present study makes it a suitable product for future applications in therapeutics and diagnostics.

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L.) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.

Pigeonpea (Cajanus cajan L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications

Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05 M Trisacetate buffer, pH 7.3, at 4°C had a t1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations.

Immobilization of pigeonpea (Cajanus cajan) urease on DEAE-cellulose paper strips for urea estimation

Pigeonpea (Cajanus cajan) urease was immobilized on 1 cm×1 cm DEAE‐cellulose paper strips. The optimum immobilization (51% activity) was observed at 4 °C, with a protein concentration of 1.0 mg/strip. The apparent optimum pH shifted from 7.3 to 6.8. Immobilized urease showed an optimal stability temperature of 67 °C, compared with 47 °C for the soluble urease. Time‐dependent kinetics of the thermal inactivation of the immobilized urease were examined and found to be monophasic as compared with the soluble enzyme, which was biphasic. The Michaelis constant (Km) for the DEAE‐cellulose‐immobilized urease was found to be 4.75 mM, 1.5 times higher than the soluble enzyme. Immobilized strips stored at 4 °C showed an increased half‐life (t1/2=150 days). There was practically no leaching of the enzyme from the immobilized strips over a period of 2 weeks. These strips were used for estimating the urea content of blood samples; the results obtained matched well with those obtained in a clinical laboratory through an Autoanalyzer® (Zydus Co., Rome, Italy). The easy availability of pigeonpea urease, the ease of its immobilization on DEAE‐cellulose strips and the significantly lower cost of urease described in the present study makes it a suitable product for future applications in diagnostics.

Noncommunicating isolated enteric duplication cyst in childhood

Duplications of the alimentary tract are spherical or tubular structures lined by epithelium similar to intestine that are firmly attached to or share the wall of the alimentary tract and have a common blood supply with the adjacent segment of the bowel. Completely isolated duplication cysts are an extremely rare variety of gastrointestinal duplications with their own exclusive blood supply and do not communicate with the intestine. There are only 4 cases reported in the English literature-3 in childhood and 1 in an adult, and all are male. We report a case of noncommunicating isolated ileal duplication cyst in a 3-year-old female child that presented as a mass in the right iliac fossa, mimicking an intussusception. The duplication had its own blood supply arising from the terminal ileal mesentery. This report describes the first female patient with this condition and reviews the English literature.

Isolation and characterization of isocitrate lyase of castor endosperm

Abstract Isocitrate lyase (threo-DS-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: A t = A 0 2 · e −k 1 t + A 0 2 · e −k 2 t , where A0 and At are activities at time zero at t, and k1 and k2 are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg2+ (5.0 m m ) to a solution of this protein. Addition of Ba2+ or Mn2+ brings about partial recovery. Other metal ions are not effective.

Response of Rhizobium leguminosarum to nickel stress

Rhizobium leguminosarum strain P-5 biovar viciae was sensitive to Ni2+ (MIC, 75 μM) and showed concentration-dependent Ni2+ uptake in a wide concentration range (50–500 μM). Ni2+ uptake up to a certain threshold limit also increased thiol content (66 nmol mg−1 protein), proline content (10.85 nmol mg−1 protein) and urease specific activity (500 nmol min−1 mg−1 protein) maximum corresponding to 100 μM Ni2+ as the external concentration or 151 nmol Ni2+ mg−1 protein as the intracellular buildup. Proline synthesis was stimulated most even at much lower Ni2+ concentration (25 μM). Higher intracellular Ni2+ load neither favoured thiol nor proline biosynthesis nor urease activity. Ni2+ requirement of urease was ascertained by using EDTA-grown cells and the addition of bicarbonate (NaHCO3, 100 mM) to the crude extract. The induction of thiol or proline by Ni2+, therefore, reflects the possible strategies adopted by bacterial cells to overcome the environmental stress.

Unique activity of ureases immobilized on poly(styrene-co-acrolein) microspheres

Poly(styrene-co-acrolein) (P(S/A)) microspheres were synthesized by emulsifier-free emulsion-dispersion radical copolymerization of styrene and acrolein. Particles with a number average diameter (øD n) of 373 nm, a narrow diameter distribution (øD w/øD n) of 1.008 and a surface concentration of aldehyde groups from polyacrolein units of 2.51 · 10−6 mol/m2 were obtained. Ureases from jack beans (Urs-JB) and from pigeonpea (Urs-PP) were immobilized onto these microspheres. Activity of free and immobilized urease was determined using a standard phenol method. Surface concentration of attached enzymes was varied in the range from 0.06 to 1.6 mg/m2. Specific activity of Urs-JB in solution was 1.9 times higher than that of Urs-PP, however, upon immobilization of enzymes onto P(S/A) microspheres the reverse was true. Due to very high denaturation activity of immobilized Urs-JB was 10 times lower than in solution. In the case of Urs-PP, denaturation of the enzyme upon immobilization was much lower and possibly due to a cooperative effect resulting from a local high concentration of immobilized enzyme, its specific activity was from 3 to 9 times higher than that of immobilized Urs-JB.

Kinetics of inhibition and molecular asymmetry in pigeonpea (Cajanus cajan) urease.

Urease from seeds of pigeonpea showed a time-dependent and irreversible inactivation at very low concentrations of heavy metal ions. Concentration of Cu(2+), Hg(2+) and Ag(+) required for 50% inactivation, on 10 min of incubation, were found to be 2.2 x 10(-6), 2.9 x 10(-8) and 6.3 x 10(-12) M, respectively. The kinetics of inactivation with each of these metal ions was found to be biphasic, with half of the activity being lost in a fast phase and remaining in a slow phase. Acetohydroxamate (AHA) inhibits pigeonpea urease competitively and reversibly with a K(i) of 0.041 mM at pH 7.3. This inhibition was found to be pH dependent. A reversible and time-dependent inhibition was observed with AHA. AHA inhibition revealed biphasic kinetics as observed with the heavy metal ions. Pigeonpea urease was also inhibited by fluoride ions competitively with a K(i) value of 1.23 mM. These inhibition studies suggest the possible interaction of these inhibitors with active site thiol groups and Ni (II) ion. A mechanism has been proposed for each of these inhibitors and compared with inhibition studies reported for other ureases.

Duplication cyst of pyloroduodenal canal: a rare cause of neonatal gastric outlet obstruction: a case report

BackgroundA 21 day old male child presented with non bilious vomiting and abdominal mass.Case presentationThis case is reported because pyloroduodenal duplication cysts are an extremely rare congenital anomaly, whose clinical presentation often mimics those of hypertrophic pyloric stenosis. Ultrasound examination showed cystic mass at pyloric region and barium study was suggestive of extrinsic mass compressing the pyloric region. A laparotomy, a tense cystic mass was present at the pyloroduodenal junction (PDC) which was resected and end to end anastomosis was done. Patients followed an uneventful recovery and doing well.ConclusionThe clinical and radiological analysis can reveal configurational changes consistent with a large extrinsic mass rather than muscular hypertrophy and can lead to accurate preoperative diagnosis.