Browsing by Author "Alka Dwevedi"

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A β-galactosidase from pea seeds (PsBGAL): purification, stabilization, catalytic energetics, conformational heterogeneity, and its significance
(2009) Alka Dwevedi; Arvind M. Kayastha
A basic glycosylated β-galactosidase (PsBGAL) has been purified from pea seeds by 910-fold with a specific activity of 77.33 μmol min-1 mg-1 protein. The purified enzyme is an electrophoretically homogeneous protein consisting of a single protein band with an apparent Mr of 55 kDa, while the deglycosylated enzyme has a Mr of 54.2 kDa on SDS-PAGE under reducing conditions. According to MALDI-TOF measurements of the 55 kDa band, the enzyme showed a homology with BGAL from other sources present in the SWISS-PROT database, while it showed no resemblance to any lectin. The N-terminal sequence of PsBGAL was determined as TIECK and showed a resemblance to BGAL from Arabidopsis thaliana (Q93Z24). The enzyme showed an unique property of multiple banding patterns on SDS-PAGE at 20 mA current, with tryptic digests of all bands having similar m/z values (using MALDI-TOF) while it showed only a single band at 10 mA current. PsBGAL is effectively compartmentalized during seed maturation inside vacuoles (pH ∼ 5). The enzyme is capable of hydrolyzing pea seed xyloglucan, and it may be involved in modifying the cell wall architecture during seedling growth and development. The enzyme has a protonated carboxyl group at its active site as observed by ionization constant, thermodynamics, and chemical modification studies. © 2009 American Chemical Society.
Biochemical and thermodynamic characterization of de novo synthesized β-amylase from fenugreek
(Elsevier B.V., 2019) Dinesh Chand Agrawal; Alka Dwevedi; Arvind M. Kayastha
β-Amylase has been de novo synthesized from germinating fenugreek seeds. Enzyme has been isolated and purified from 36 h germinated seeds with 226-fold purification and specific activity of 763 U/mg. Homogeneity of the purified β-amylase has been confirmed with size-exclusion chromatography, SDS-PAGE and MALDI MS/MS analysis. The isoelectric point, optimum pH and temperature of the enzyme were found to be pH 5.2, 5.7 and 57 °C, respectively. The enzyme was specific for soluble starch with Km and Vmax of 2.4 mg/mL and 833.3 U/mg, respectively. Maltose was found to be competitive inhibitor of the enzyme with inhibition constant (Ki) of 14 mM. However, metallic ions like Ag+ and Hg2+ were found to be non-competitive inhibitors of the enzyme. Thermodynamic parameters like Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes have further revealed that thermal denaturation of the enzyme has followed first-order with the enzyme unfolding rather an aggregation with the process being irreversible. The activation energy of β-amylase during thermal activation and denaturation were 27.5 kJ/mol and 145.23 kJ/mol, respectively at R2 > 0.92. Thus, the enzyme was stable even at higher temperature with ability of undergoing catalysis making it commercially exploitable, particularly in food and pharmaceutical industries. © 2019 Elsevier B.V.
Current and future trends on polymer-based enzyme immobilization
(Elsevier, 2021) Ranjana Das; Alka Dwevedi; Arvind M. Kayastha
Enzymes (nature’s catalysts) have been the crucial partner in various human activities, from daily chores to commercial market. It has been well validated now that enzymes have the solutions for every problem ranging from clinical, industrial, environmental, physiological, etc. The best alluring aspect of enzymes is that they can perform actions under mild conditions with a very high degree of substrate specificity with least generation of by-products. However, their utilization for various applications has been limited due to their relative instability and their high costs of isolation. Enzyme immobilization has found solution to this problem as it allows reusability of enzymes in addition to its stability, activity, inhibition by reaction products, and selectivity toward nonnatural substrates. Thorough studies have been going on across the world to look for excellent matrices for enzyme immobilization as well regular improvisation of immobilization techniques to obtain immobilized enzyme with excellent physicochemical properties. The parameters like matrix properties, including mean particle diameter, swelling behavior, mechanical strength, compression behavior, and surface area, have been most critical in determining the performance of immobilized enzymes. These parameters are useful in designing correct bioreactor for any given large-scale industrial processes. The present chapter has provided a summary on the wide range of polymeric matrices and their utilization for enzyme immobilization for various industrial applications. © 2021 Elsevier Inc. All rights reserved.
Enzyme immobilization: A breakthrough in enzyme technology and boon to enzyme based industries
(Nova Science Publishers, Inc., 2011) Alka Dwevedi; Arvind M. Kayastha
Enzyme technology is the application involving modification of enzyme's structure as well as its function. It is currently considered as a useful alternative to conventional process technology in enzyme based industrial as well as analytical fields. Enzyme immobilization is the foremost technique used in enzyme technology, which has gained popularity for the last few decades. It stabilizes structure of the enzymes, thereby allowing their applications under harsh environmental conditions (pH, temperature, organic solvents), and thus enable their uses in non-aqueous enzymology, and in the fabrication of biosensors. Furthermore, it allows repetitive use of enzyme as well as helpful in prevention of product contamination with the enzyme (especially useful in the food and pharmaceutical industries). Recently, the development of techniques for immobilization of multi-enzymes along with cofactor regeneration and retention system are gainfully exploited in developing biochemical processes involving complex chemical conversions. Various methods for enzyme immobilization used in bioreactors and biosensors include adsorption on or covalent attachment to a support, micro-encapsulation, and entrapment within a membrane, film or gel. The ideal immobilization method should employ mild chemical conditions, allow large quantities of enzyme to be immobilized, provide a large surface area for enzyme-substrate contact within a small total volume, minimize barriers to mass transport of substrate and product, and provide a chemically and mechanically robust system. Immobilization helps in the development of continuous processes allowing more economic organization of the operations,automation, decrease of labour and investment to capacity ratio. The present chapter delineates the current status and future potentials of immobilized enzymes in the emerging enzyme based industries with suitable examples. © 2011 by Nova Science Publishers, Inc. All rights reserved.
Enzyme immobilization: A breakthrough in enzyme technology and boon to enzyme based industries
(2012) Alka Dwevedi; Arvind M. Kayastha
Enzyme technology is the application involving modification of enzyme's structure as well as its function. It is currently considered as a useful alternative to conventional process technology in enzyme based industrial as well as analytical fields. Enzyme immobilization is the foremost technique used in enzyme technology, which has gained popularity for the last few decades. It stabilizes structure of the enzymes, thereby allowing their applications under harsh environmental conditions (pH, temperature, organic solvents), and thus enable their uses in non-aqueous enzymology, and in the fabrication of biosensors. Furthermore, it allows repetitive use of enzyme as well as helpful in prevention of product contamination with the enzyme (especially useful in the food and pharmaceutical industries). Recently, the development of techniques for immobilization of multi-enzymes along with cofactor regeneration and retention system are gainfully exploited in developing biochemical processes involving complex chemical conversions. Various methods for enzyme immobilization used in bioreactors and biosensors include adsorption on or covalent attachment to a support, micro-encapsulation, and entrapment within a membrane, film or gel. The ideal immobilization method should employ mild chemical conditions, allow large quantities of enzyme to be immobilized, provide a large surface area for enzyme-substrate contact within a small total volume, minimize barriers to mass transport of substrate and product, and provide a chemically and mechanically robust system. Immobilization helps in the development of continuous processes allowing more economic organization of the operations, automation, decrease of labour and investment to capacity ratio. The present chapter delineates the current status and future potentials of immobilized enzymes in the emerging enzyme based industries with suitable examples. © Nova Science Publishers Inc.
Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications
(2009) Sandeep Kumar; Alka Dwevedi; Arvind M. Kayastha
The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol-1, 5.02 kcal mol-1, 6.45 kcal mol-1 for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in Km value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of Vmax for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 102 μmol NH3 min-1 mg-1 protein, 2.65 × 102 μmol NH3 min-1 mg-1 protein and 2.85 × 102 μmol NH3 min-1 mg-1 protein, respectively. By contrast, reusability studies showed that chitosan-urease beads can be used almost 14 times with only 20% loss in original activity while alginate-urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer®. The immobilization studies have a potential role in haemodialysis machines. © 2008 Elsevier B.V. All rights reserved.
Insights into pH-induced conformational transition of β-galactosidase from pisum sativum leading to its multimerization
(2010) Alka Dwevedi; Vikash Kumar Dubey; Medicherla V. Jagannadham; Arvind M. Kayastha
Although β-galactosidases are physiologically a very important enzyme and have may therapeutics applications, very little is known about the stability and the folding aspects of the enzyme. We have used β-galactosidase from Pisum sativum (PsBGAL) as model system to investigate stability, folding, and function relationship of β-galactosidases. PsBGAL is a vacuolar protein which has a tendency to multimerize at acidic pH with protein concentration ≥100 μg mL-1 and dissociates into its subunits above neutral pH. It exhibits maximum activity as well as stability under acidic conditions. Further, it has different conformational orientations and core secondary structures at different pH. Substantial predominance of β-content and interfacial interactions through Trp residues play crucial role in pH-dependent multimerization of enzyme. Equilibrium unfolding of PsBGAL at acidic pH follows four-state model when monitored by changes in the secondary structure with two intermediates: one resembling to molten globule-like state while unfolding seen from activity and tertiary structure of PsBGAL fits to two-state model. Unfolding of PsBGAL at higher pH always follows two-state model. Furthermore, unfolding of PsBGAL reveals that it has at least two domains: α/β barrel containing catalytic site and the other is rich in β-content responsible for enzyme multimerization. © 2010 Springer Science+Business Media, LLC.
Lactose nano-probe optimized using response surface methodology
(2009) Alka Dwevedi; Ashwani Kumar Singh; Dinesh Pratap Singh; Onkar Nath Srivastava; Arvind M. Kayastha
A lactose nano-probe has been developed by immobilization of PsBGAL onto gold nanoparticles (AuNps). It is helpful for severe lactose intolerants for quality check of lactose hydrolyzed milk and estimation of hidden lactose present in variety of food products. Optimization of PsBGAL immobilization onto AuNps using spacer arm (cysteamine-glutaraldehyde) was carried out by response surface methodology (Box-Behnken design). The process has led to immobilization of enzyme onto AuNps with an efficiency of 140.81%. AuNp-PsBGAL was characterized using transmission electron microscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. Immobilized enzyme showed broad temperature and pH optima and a significant enhancement in catalytic efficiency (Vmax/Km) with respect to soluble PsBGAL. AuNp-PsBGAL was stable under dried conditions than wet conditions for 6 months with loss of 10.2% and 87.53%, respectively. It has reusability of over five batchwise uses, with almost no loss in activity. Hill's coefficient was found to be 1.71 corresponding to lactose concentration ranging from 0.1% to 2.0%. © 2009 Elsevier B.V. All rights reserved.
Optimal immobilization of β-galactosidase from Pea (PsBGAL) onto Sephadex and chitosan beads using response surface methodology and its applications
(2009) Alka Dwevedi; Arvind M. Kayastha
Response surface methodology (RSM) and centre composite design (CCD) were used to optimize immobilization of β-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 °C, respectively. Chitosan-PsBGAL showed higher rate of lactose hydrolysis present in milk and whey at room temperature and 4 °C than Sephadex-PsBGAL. In both cases, lactose of milk whey was hydrolyzed at higher rate than that of milk. © 2008 Elsevier Ltd. All rights reserved.
Plant β-galactosidases: Physiological significance and recent advances in technological applications
(Springer, 2010) Alka Dwevedi; Arvind M. Kayastha
The β-galactosidase (BGAL) is one of the oldest ubiquitous enzymes, known for more than 100 years. The enzyme is known to perform various functions in different organisms, however with similar mode of action. There is an immense literature available related to bacterial, fungal as well as animal BGAL compared to plant BGAL. Initially, it was believed that lactose is the only substrate for the enzyme. Later, it was observed that enzyme specificity is due to hydrolysable bond rather than the substrate. The present review is based on the role of BGAL in plants, the in vivo substrates and their physiological significances. Similarity as well as dissimilarity with BGAL from bacterial as well as fungal sources is also discussed. Plant BGAL would be best suited for industrial applications because of its easy availability, cost effectiveness and easy adaptability.
Response surface analysis of nano-ureases from canavalia ensiformis and cajanus cajan
(Elsevier B.V., 2011) Alka Dwevedi; Satya Brata Routh; Amit Singh Yadav; Ashwani Kumar Singh; Onkar Nath Srivastava; Arvind M. Kayastha
Ureases isolated from leguminous sources, Canavalia ensiformis and Cajanus cajan were immobilized onto gold nanoparticles (nano-ureases). Optimization of the urease immobilization was carried using response surface methodology based on Central Composite Design. Immobilization efficiency of nano-urease from C. ensiformis and C. cajan were found to be 215.10% and 255.92%, respectively. The methodology adopted has deviation of 2.56% and 3.01% with respect to experimental values in case of C. ensiformis and C. cajan, respectively. Nano-urease from C. cajan has broad physico-chemical parameters with pH optimum from 7.1 to 7.3 and temperature optimum from 50 to 70 °C. Nano-urease from C. ensiformis has sharp pH and temperature optima at 7.3 and 70 °C, respectively. Fourier transform infra-red spectroscopy has revealed involvement of groups viz. amino, glycosyl moiety, etc. in urease immobilization onto gold nano-particles. Transmission and scanning electron micrographs revealed that arrangement of urease onto gold nano-particles from C. ensiformis was uniform while it was localized in case of C. cajan. Nano-urease from C. ensiformis has higher specificity and catalysis toward urea as compared to nano-urease from C. cajan. Nano-ureases from both sources are equally stable for 6 months under dried conditions and can be used for 10 washes. © 2011 Elsevier B.V.
Soil sensors: detailed insight into research updates, significance, and future prospects
(Elsevier, 2017) Alka Dwevedi; Promod Kumar; Pravita Kumar; Yogendra Kumar; Yogesh K. Sharma; Arvind M. Kayastha
Soil is an important natural resource that requires crucial attention due to its significant role in crop yield. There is substantial ongoing research on soil health, particularly its water retention capability, moisture content, salinity, temperature, pH, concentration of dissolved gases, etc. Technologies have evolved that can remotely monitor soil and track its various parameters without being physically present in the field, using various probes (coaxial impedance dielectric reflectometry sensors, frequency domain reflectometry sensors, time domain reflectometry sensors, gypsum blocks, neutron probes, etc.). These probes are extremely accurate and provide information about the soil’s surface as well as its inner layers with respect to moisture content, salinity, and temperature. Thus, the probes are helpful for farmers in increasing crop yield by adopting an optimum level of irrigation and suitable fertilizers. The usage of soil probes would be helpful for the economical use of water, as well as of chemical fertilizers to subsequently reduce their toxicity to growing crops. Research scientists studying earth and its environmental processes are always keen to know the processes going on inside the soil. These soil sensors buried in different soil horizons are able to provide complete information to scientists’ lab desktops, even without going to the topographical location. These soil probes are kept intact within the soil with no gaps between the soil and probes to obtain an accurate picture of the soil environment. However, the most tedious aspect is to select the right sensor based on the type of soil and the precision required. This chapter details recent developments on soil sensors, data analysis obtained, and their applications in fields, as well as their shortcomings, if any. © 2017 Elsevier Inc. All rights reserved.
Stabilization of β-galactosidase (from peas) by immobilization onto Amberlite MB-150 beads and its application in lactose hydrolysis
(2009) Alka Dwevedi; Arvind M. Kayastha
The soluble PsBGAL (from Pisum sativum) is extremely unstable with loss of over 80% in enzyme activity within 24 h at 4 °C when the protein concentration was lower than 0.1 mg/mL Enzyme immobilization onto Amberlite MB-150 beads (diameter = 5 μm) greatly stabilized the enzyme preparation, with almost no loss for 12 months at room temperature (27 °C). Enzyme (21.9 μg) 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 Km 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. © 2009 American Chemical Society.
Wastewater remediation via combo-technology
(Elsevier, 2018) Alka Dwevedi; Arvind M. Kayastha
The availability of safe water has been an ongoing problem and is becoming worse with increasing urbanization and population density. Only about 30% of the fresh water total is available on earth, in groundwater and surface water, for drinking and other daily human activities as well as industrial and agricultural activities. Further, 20% of the available fresh water is not available to humans living in remote areas, 60% of fresh water cannot be captured, as it comes at inconvenient times and places (e.g., monsoons and floods), and 18% of fresh water is contaminated by human activities. Only about 2% of fresh water is available for usage. Developed countries have advanced technology for water treatment and are thus able to manage the problem of water scarcity to a large extent, but advanced technology is largely unprocurable in developing and undeveloped countries due to cost. Based on the current population growth rate, it has been estimated that over 3.5 billion people will be in a water scarcity condition by 2025. The problem has become exacerbated due to the introduction of various recalcitrant, nondegradable compounds by agricultural and industrial activities. These compounds cannot be removed completely by any current available technologies. Thus, there is an urgent need of easily procurable technology with excellent efficacy in wastewater treatment. © © 2019 Elsevier Inc. All rights reserved.