Virendra K. Rathod

Arsenic and fluoride contaminated groundwaters: A review of current technologies for contaminants removal.

Chronic contamination of groundwaters by both arsenic (As) and fluoride (F) is frequently observed around the world, which has severely affected millions of people. Fluoride and As are introduced into groundwaters by several sources such as water-rock interactions, anthropogenic activities, and groundwater recharge. Coexistence of these pollutants can have adverse effects due to synergistic and/or antagonistic mechanisms leading to uncertain and complicated health effects, including cancer. Many developing countries are beset with the problem of F and As laden waters, with no affordable technologies to provide clean water supply. The technologies available for the simultaneous removal are akin to chemical treatment, adsorption and membrane processes. However, the presence of competing ions such as phosphate, silicate, nitrate, chloride, carbonate, and sulfate affect the removal efficiency. Highly efficient, low-cost and sustainable technology which could be used by rural populations is of utmost importance for simultaneous removal of both pollutants. This can be realized by using readily available low cost materials coupled with proper disposal units. Synthesis of inexpensive and highly selective nanoadsorbents or nanofunctionalized membranes is required along with encapsulation units to isolate the toxicant loaded materials to avoid their re-entry in aquifers. A vast number of reviews have been published periodically on removal of As or F alone. However, there is a dearth of literature on the simultaneous removal of both. This review critically analyzes this important issue and considers strategies for their removal and safe disposal.

A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification

The major complications in fruit juice quality improvement are the presence of polysaccharides components in the form of disrupted fruit cell wall and cell materials. Hence, breakdown of cellulose along with pectin and starch is important for the juice processing. In this context, magnetic tri-enzyme nanobiocatalyst was prepared by simultaneously co-immobilizing three enzymes; α-amylase, pectinase and cellulase onto amino-functionalized magnetic nanoparticle by 60mM glutaraldehyde concentration with 10h cross-linking time for one pot juice clarification. The prepared nanobiocatalyst was characterized by FT-IR, SEM and XRD. The thermal (50-70°C) and pH (3-6) stability studies indicated more than two folds increment in half-life and enhanced tolerance to lower pH. The immobilized enzymes retained up to 75% of residual activity even after eight consecutive cycles of reuse. Finally, the clarification of apple, grapes and pineapple juices using magnetic tri-enzyme showed 41%, 46% and 53% respective reduction in turbidity till 150min treatment.

Self-assembled organicinorganic hybrid glucoamylase nanoflowers with enhanced activity and stability

An organic-inorganic hybrid glucoamylase nanoflower was prepared in single pot by simple, facile and highly efficient method. The stepwise formation of enzyme-embedded hybrid nanoflowers and influence of experimental parameters viz. pH of solution mixture, enzyme and copper ion concentration on the activity of prepared hybrid nanoflowers were systematically investigated. The self-assembled hybrid glucoamylase nanoflowers were synthesized by mixing aqueous solution of copper sulphate (200mM) with PBS (pH 7.5, 5mM) containing glucoamylase (1mg/mL) in 24h at room temperature. These prepared nanoflowers were further characterized by FT-IR, SEM and XRD. The hybrid nanoflowers exhibited 204% enhanced activity recovery and two folds improvement in thermal stability in terms of half-life (in the range of 50-70°C) with respect to the free form. The hybrid glucoamylase nanoflowers retained 70% residual activity after eight successive cycles indicating their excellent durability. Additionally, the nanoflowers retained up to 91% residual activity upto 25 days of storage. Moreover, the conformational changes occurred in glucoamylase structure after preparing hybrid nanoflowers were evaluated by FT-IR spectroscopy data tools.

Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker

Pectinase was immobilized onto chitosan magnetic nanoparticles (CMNPs) by dextran polyaldehyde as a macromolecular cross-linking agent. The parameters like cross-linking concentration, time and CMNPs to enzyme ratio were optimized. Further, prepared magnetic pectinase nanobiocatalyst was characterized by FT-IR and XRD. The thermal kinetic studies for immobilized pectinase showed two folds improved thermal stability in the range of 55-75°C as compared to free form. The Vmax and Km values of immobilized pectinase were found to be nearly equal to native form which indicated that conformational flexibility of pectinase was retained even after immobilization. The residual activity of immobilized pectinase was 85% after seven successive cycles of reuse, while it retained upto 89% residual activity on storage of fifteen days which exhibited excellent stability and durability. The conformational changes in pectinase after immobilization were evaluated by FT-IR spectroscopy data analysis tools. Finally, magnetic pectinase nanobiocatalyst was employed for apple juice clarification which showed turbidity reduction upto 74% after 150min treatment.

Application of Response Surface Methodology for Optimization of Bromelain Extraction in Aqueous Two-Phase System

Extraction of bromelain from pineapple fruit in an aqueous two phase system (ATPS) composed of polyethylene glycol (PEG) 1500 and potassium phosphate has been studied using response surface methodology. The various process variables such as PEG, potassium phosphate and NaCl concentration, and pH were optimized using a central composite rotatable design (CCRD) of response surface methodology (RSM) based on the partition coefficient, % yield, and purification factor of an enzyme. An optimized ATPS composed of 14% (w/w) PEG 1500, 17.66% (w/w) potassium phosphate and 1 mM sodium chloride at pH 7.5 was used to purify bromelain from a pineapple fruit. With this system, a maximum enzyme partition coefficient of 12.62 and %yield of 90.33 in the top PEG-rich phase with a purification factor of 2.4 was predicted. The enzyme partition coefficient, % yield, and purification factor obtained from experimentation are 12.22, 89.65, and 2.8, respectively, in the top PEG phase. The response model is validated by the closeness between the predicted and experimental results.

Recent advances in enzyme extraction strategies: A comprehensive review

The increasing interest of industrial enzymes demands for development of new downstream strategies for maximizing enzyme recovery. The significant efforts have been focused on the development of newly adapted technologies to purify enzymes in catalytically active form. Recently, an aqueous two phase system (ATPS) is emerged as powerful tools for efficient extraction and purification of enzymes due to their versatility, lower cost, process integration capability and easy scale-up. The present review gives an overview of effect of parameters such as tie line length, pH, neutral salts, properties of polymer and salt involved in traditional polymer/polymer and polymer/salt ATPS for enzyme recovery. Further, advanced ATPS have been developed based on alcohols, surfactants, micellar compounds to avoid tedious recovery steps for getting desired enzyme. In order to improve the selectivity and efficiency of ATPS, recent approaches of conventional ATPS combined with different techniques like affinity ligands, ionic liquids, thermoseparating polymers and microfluidic device based ATPS have been reviewed. Moreover, three phase partitioning is also highlighted for enzymes enrichment as a blooming technology for efficiently integrated bioseparation techniques. At the end, it includes an overview of CLEAs technology and organic-inorganic nanoflowers preparation as novel strategies for simultaneous extraction, purification and immobilization of enzymes.

Facile synthesis of glucoamylase embedded metal-organic frameworks (glucoamylase-MOF) with enhanced stability

The self-assembled glucoamylase metal-organic framework (glucoamylase-MOF) was synthesized by facile one-step method within 20min by simply mixing aqueous solution of 2-methylimidazole (160mM), glucoamylase (5mg/mL) and zinc acetate (40mM) at room temperature (28±2°C). The prepared glucoamylase-MOF was characterized by using FT-IR, confocal scanning laser microscopy, XRD and SEM. The robustness and thermal stability of glucoamylase embedded MOF was evaluated in terms of half-life (in the range of 60-80°C) which showed 6 folds increment as against free form. Further, in Michaelis-Menten kinetics studies, glucoamylase entrapped MOF exhibited higher Km value and lower Vmax value as compared to native enzyme. Moreover, the immobilized glucoamylase exhibited up to 57% of residual activity after six consecutive cycles of reuse, whereas it retained 91% of residual activity till 25days of storage. Finally, the conformational changes occurred after the encapsulation of glucoamylase in the interior of MOF, which was analyzed by using FT-IR data analysis tools.

Separation of Bromelain by Aqueous Two Phase Flotation

Aqueous two phase flotation (ATPF) system of polyethylene glycol (PEG) and potassium phosphate is studied for the separation and partial purification of bromelain from the pineapple fruit (Annanus comosus L. Merryl). The effect of PEG molecular weight (1500–20000), concentration of phase forming components (PEG 12–18% w/w and potassium phosphate 14–20% w/w), system pH, nitrogen flow rate, and flotation time were studied and optimum conditions for ATPF were obtained. At optimum conditions of the system, i.e., 14% w/w PEG 1500, 18% w/w potassium phosphate, 80 mL/min of nitrogen flow rate and pH 7, maximum yield of 91.47% and purification fold of 4.26 were obtained. ATPF was found to be an effective technique for the purification of bromelain from pineapple fruit with higher extraction yield and purification fold as compared to aqueous two phase extraction (ATPE).

Purification and characterization of polyphenol oxidase from waste potato peel by aqueous two-phase extraction.

Potato peel from food industrial waste is a good source of polyphenol oxidase (PPO). This work illustrates the application of an aqueous two-phase system (ATPS) for the extraction and purification of PPO from potato peel. ATPS was composed of polyethylene glycol (PEG) and potassium phosphate buffer. Effect of different process parameters, namely, PEG, potassium phosphate buffer, NaCl concentration, and pH of the system, on partition coefficient, purification factor, and yield of PPO enzyme were evaluated. Response surface methodology (RSM) was utilized as a statistical tool for the optimization of ATPS. Optimized experimental conditions were found to be PEG1500 17.62% (w/w), potassium phosphate buffer 15.11% (w/w), and NaCl 2.08 mM at pH 7. At optimized condition, maximum partition coefficient, purification factor, and yield were found to be 3.7, 4.5, and 77.8%, respectively. After partial purification of PPO from ATPS, further purification was done by gel chromatography where its purity was increased up to 12.6-fold. The purified PPO enzyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Km value 3.3 mM, and Vmax value 3333 U/mL, and enzyme stable ranges for temperature and pH of PPO were determined. These results revealed that ATPS would be an attractive option for obtaining purified PPO from waste potato peel.

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis–Menten kinetic parameters (km and Vmax) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.