Arvind M. Kayastha

Optimal immobilization of β-galactosidase from Pea (PsBGAL) onto Sephadex and chitosan beads using response surface methodology and its applications.

Response surface methodology (RSM) and centre composite design (CCD) were used to optimize immobilization of beta-galactosidase (BGAL) from Pisum sativum onto two matrices: Sephadex G-75 and chitosan beads. The immobilization efficiency of 75.66% and 75.19% were achieved with Sephadex G-75 and chitosan, respectively. There was broad divergence in physico-chemical properties of Sephadex-PsBGAL and chitosan-PsBGAL. Chitosan-PsBGAL was better suited for industrial application based on its broad pH and temperature optima, higher temperature stability, reusability etc. Sephadex-PsBGAL and chitosan-PsBGAL showed much variation in their catalytic properties with respect to soluble enzyme. About 50% loss in activity of Sephadex-PsBGAL and chitosan-PsBGAL were observed after 12 and 46 days at 4 degrees C, respectively. Chitosan-PsBGAL showed higher rate of lactose hydrolysis present in milk and whey at room temperature and 4 degrees C than Sephadex-PsBGAL. In both cases, lactose of milk whey was hydrolyzed at higher rate than that of milk.

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.

Stabilization of β-galactosidase (from peas) by immobilization onto amberlite MB-150 beads and its application in lactose hydrolysis.

The soluble PsBGAL (from Pisum sativum ) is extremely unstable with loss of over 80% in enzyme activity within 24 h at 4 degrees C when the protein concentration was lower than 0.1 mg/mL. Enzyme immobilization onto Amberlite MB-150 beads (diameter = 5 microm) greatly stabilized the enzyme preparation, with almost no loss for 12 months at room temperature (27 degrees C). Enzyme (21.9 microg) was immobilized by 62.56% onto activated 100 mg of Amberlite MB-150 beads using 4% glutaraldehyde, at pH 6.0 (50 mM, sodium phosphate buffer). Statistical analysis carried out by ANOVA revealed that all parameters used during immobilization were equally important at P < 0.05 (level of significance). An approach toward commercial exploitation of Amberlite-PsBGAL especially in lactose hydrolysis was anticipated due to improved physicochemical properties including broad optimum pH and temperature, with a K(m) of 4.11 +/- 0.21 mM for lactose. Amberlite-PsBGAL hydrolyzed 64.57 and 69.18% of lactose present in milk and milk whey, respectively, within 10 h (at room temperature). Immobilized enzyme has reusability of over 10 batchwise uses, with almost no loss in activity. The easy accessibility of enzyme source, ease of its immobilization on Amberlite, lower cost of Amberlite, enhanced stability of Amberlite-PsBGAL, and comparable lactose hydrolysis in milk and milk whey described here make it a suitable product for future applications at laboratory and industrial scale.

Thermal, Chemical and pH Induced Denaturation of a Multimeric β-Galactosidase Reveals Multiple Unfolding Pathways

Background In this case study, we analysed the properties of unfolded states and pathways leading to complete denaturation of a multimeric chick pea β-galactosidase (CpGAL), as obtained from treatment with guanidium hydrochloride, urea, elevated temperature and extreme pH. Methodology/Principal Findings CpGAL, a heterodimeric protein with native molecular mass of 85 kDa, belongs to α+β class of protein. The conformational stability and thermodynamic parameters of CpGAL unfolding in different states were estimated and interpreted using circular dichroism and fluorescence spectroscopic measurements. The enzyme was found to be structurally and functionally stable in the entire pH range and upto 50°C temperature. Further increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were irreversible, non-coincidental and sigmoidal. Free energy of protein unfolding (ΔG0) and unfolding constant (Kobs) were also calculated for chemically denatured CpGAL. Significance The protein seems to use different pathways for unfolding in different environments and is a classical example of how the environment dictates the path a protein might take to fold while its amino acid sequence only defines its final three-dimensional conformation. The knowledge accumulated could be of immense biotechnological significance as well.

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.

Cicer α-galactosidase immobilization onto functionalized graphene nanosheets using response surface method and its applications.

Cicer α-galactosidase was immobilized onto functionalized graphene with immobilization efficiency of 84% using response surface methodology (Box-Behnken design). The immobilized enzyme had higher thermal stability than the soluble one, attractive for industrial applications. Immobilization of the enzyme lowered the Km to 1/3rd compared to the soluble enzyme. Raffinose family oligosaccharides (RFOs) are mainly responsible for flatulence by taking soybean derived food products. The immobilized enzyme can be used effectively for the hydrolysis of RFOs. After ten successive runs, the immobilized enzyme still retained approximately 60% activity, with soybean RFOs. The easy availability of enzyme source, ease of its immobilization on matrices, non-toxicity, increased stability of immobilized enzyme and effective hydrolysis of RFOs increase the Cicer α-galactosidase application in food processing industries.

Immobilization of β-galactosidase onto functionalized graphene nano-sheets using response surface methodology and its analytical applications.

Background β-Galactosidase is a vital enzyme with diverse application in molecular biology and industries. It was covalently attached onto functionalized graphene nano-sheets for various analytical applications based on lactose reduction. Methodology/Principal Findings Response surface methodology based on Box-Behnken design of experiment was used for determination of optimal immobilization conditions, which resulted in 84.2% immobilization efficiency. Native and immobilized functionalized graphene was characterized with the help of transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Functionalized graphene sheets decorated with islands of immobilized enzyme were evidently visualized under both transmission and scanning electron microscopy after immobilization. FTIR spectra provided insight on various chemical interactions and bonding, involved during and after immobilization. Optimum temperature and energy of activation (Ea) remains unchanged whereas optimum pH and Km were changed after immobilization. Increased thermal stability of enzyme was observed after conjugating the enzyme with functionalized graphene. Significance Immobilized β-galactosidase showed excellent reusability with a retention of more than 92% enzymatic activity after 10 reuses and an ideal performance at broad ranges of industrial environment.

Optimisation of immobilisation conditions for chick pea β-galactosidase (CpGAL) to alkylamine glass using response surface methodology and its applications in lactose hydrolysis

Response surface methodology was advantageously used to optimally immobilise a β-galactosidase from chick pea onto alkylamine glass using Box-Behnken experimental design, resulting in an overall 91% immobilisation efficiency. Analysis of variance was performed to determine the adequacy and significance of the quadratic model. Immobilised enzyme showed a shift in the optimum pH; however, optimum temperature remained unaffected. Thermal denaturation kinetics demonstrated significant improvement in thermal stability of the enzyme after immobilisation. Galactose competitively inhibits the enzyme in both soluble and immobilised conditions. Lactose in milk whey was hydrolysed at comparatively higher rate than that of milk. Immobilised enzyme showed excellent reusability with retention of more than 82% enzymatic activity after 15 uses. The immobilised enzyme was found to be fairly stable in both dry and wet conditions for three months with retention of more than 80% residual activity.

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.