Meryam Sardar

Enzyme Immobilization: An Overview on Nanoparticles as ImmobilizationMatrix

Immobilization process is to optimize the operational performance of an enzyme for industrial applications. So far different matrices have been described in the literature to improve the performance of the immobilized enzymes. With the advent of nanotechnology, the nanomaterials because of their unique physico-chemical properties constitute novel and interesting matrices for enzyme immobilization. The nanomaterials possess ideal characteristics to equilibrate principal factors which determine biocatalysts efficiency, including specific surface area, mass transfer resistance and effective enzyme loading. This review presents the current scenario and techniques in enzyme immobilization. An overview of the main methods used to combine proteins/enzymes with nanoparticles is given in the study. The advantages and disadvantages of nanoparticles as immobilization matrix are also discussed.

Alpha amylase assisted synthesis of TiO2 nanoparticles: Structural characterization and application as antibacterial agents

The enzyme alpha amylase was used as the sole reducing and capping agent for the synthesis of TiO2 nanoparticles. The biosynthesized nanoparticles were characterized by X-ray diffraction (XRD) and transmission electron microscopic (TEM) methods. The XRD data confirms the monophasic crystalline nature of the nanoparticles formed. TEM data shows that the morphology of nanoparticles depends upon the enzyme concentration used at the time of synthesis. The presence of alpha amylase on TiO2 nanoparticles was confirmed by FTIR. The nanoparticles were investigated for their antibacterial effect on Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentration value of the TiO2 nanoparticles was found to be 62.50 μg/ml for both the bacterial strains. The inhibition was further confirmed using disc diffusion assay. It is evident from the zone of inhibition that TiO2 nanoparticles possess potent bactericidal activity. Further, growth curve study shows effect of inhibitory concentration of TiO2 nanoparticles against S. aureus and E. coli. Confocal microscopy and TEM investigation confirm that nanoparticles were disrupting the bacterial cell wall.

Modulation of antioxidant machinery in α-tocopherol-enriched transgenic Brassica juncea plants tolerant to abiotic stress conditions

The antioxidant machinery in plants consists of several components with unique or overlapping functions that combat the deleterious production of reactive oxygen species (ROS) induced by stress conditions. Tocopherols are a group of powerful antioxidants having additional roles in signaling and gene expression, with α-tocopherol being the most potent form. In the present study, we used wild-type (WT) and α-tocopherol-enriched transgenic (TR) Brassica juncea plants grown under salt, heavy metal, and osmotic stress to compare their relative tolerance to these stresses and to assess the effects of increased α-tocopherol content on the other antioxidative enzymes and molecules. The oxidative damage caused by induced stress was lower in TR plants compared to WT plants as assessed by their higher relative water content and lower electrolyte leakage, malondialdehyde content as well as H2O2 accumulation. Lesser superoxide and H2O2 accumulation was also observed by histochemical staining in TR seedlings exposed to stress. Though no significant differences were evident under normal growth conditions, TR plants showed higher activities and transcript levels of antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase than WT plants under similar stress conditions. A decrease in ascorbate and glutathione content with marginally higher reductive ratios of these compounds was also observed in TR plants under the stress conditions. Our findings implicate the role of higher α-tocopherol levels in conferring better tolerance against salt, heavy metal, and osmotic stresses and also establish the existence of interplay between this lipid-soluble antioxidant and other water-soluble components of plant antioxidant defense.

Exploiting unusual affinity of usual polysaccharides for separation of enzymes on fluidized beds

Two polysaccharides, alginate and chitosan, showed unusual affinity and bound alpha-amylase (from various sources) and Aspergillus niger cellulase, respectively. The beads prepared from these polymers were successfully used for the purification of the respective enzymes by fluidized bed affinity chromatography. alpha-amylase from wheat germ could be purified by 58-fold with about 90% recovery of activity. Aspergillus niger cellulase, on the other hand, was purified by 30-fold with 80% recovery of enzyme activity. Both purified preparations show single band on SDS-PAGE.

Evaluation of antiplasmodial activity of green synthesized silver nanoparticles.

In the present study silver nanoparticles (silver(np)) were synthesized from AgNO3 through simple green routes using either purified Alpha Amylase or aqueous leaf extracts of Ashoka and Neem respectively. The use of plant extract/enzyme for synthesis of nanoparticles is a single-step, cost effective and eco-friendly process. The silver(np) obtained by these three different ways were characterized using UV-visible spectroscopy, DLS, TEM, XRD and FTIR. These nanoparticles were found to be antiplasmodial with IC50 (μg/ml) 3.75 (Amylase(np)), 8 (Ashoka(np)) and 30 (Neem(np)) whereas plant extracts or amylase alone did not show any activity up to 40 μg/ml. Although AgNO3 was also found to have intrinsic antiplasmodial activity (IC50 0.5 μg/ml), the hemolytic tendencies appeared to be higher for AgNO3 (MHC10: 10 μg/ml) against the nanoparticulate preparations (MHC10: >40 μg/ml).

Alginate beads as an affinity material for alpha amylases

Calcium-alginate beads were found to bind a variety of enzymes in a nonspecific fashion. However, alpha amylases from porcine pancreas, Bacillus subtilis (BAN 240L) and wheat germ bound at a significant level and B. subtilis and wheat germ amylases could be eluted with 1M maltose. The wheat germ alpha amylase could be purified 45 fold with 70% recovery. The SDS - PAGE pattern showed significant purification by this single step strategy.

A smart bioconjugate of alginate and pectinase with unusual biological activity toward chitosan

The commercial preparation of pectinase (Pectinex Ultra SP‐L) was conjugated to alginate by noncovalent interactions by employing 1% alginate during the conjugation protocol. The optimum “immobilization efficiency” was 0.76. The pH optimum and the thermal stability of the enzyme remained unchanged upon conjugation with alginate. The soluble bioconjugate showed a 3‐fold increase in Vmax/Km as compared to the free enzyme when the smart biocatalyst was used for chitosan hydrolysis. Time course hydrolysis of chitosan thus showed higher conversion of chitosan into reducing oligosaccharides/sugars. The smart bioconjugate could be reused five times without any detectable loss of chitosanase activity.

Cellulase assisted synthesis of nano-silver and gold: Application as immobilization matrix for biocatalysis

In the present study, we report in vitro synthesis of silver and gold nanoparticles (NPs) using cellulase enzyme in a single step reaction. Synthesized nanoparticles were characterized by UV-VIS spectroscopy, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD), Circular Dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR). UV-visible studies shows absorption band at 415nm and 520nm for silver and gold NPs respectively due to surface plasmon resonance. Sizes of NPs as shown by TEM are 5-25nm for silver and 5-20nm for gold. XRD peaks confirmed about phase purity and crystallinity of silver and gold NPs. FTIR data shows presence of amide I peak on both the NPs. The cellulase assisted synthesized NPs were further exploited as immobilization matrix for cellulase enzyme. Thermal stability analysis reveals that the immobilized cellulase on synthesized NPs retained 77-80% activity as compared to free enzyme. While reusability data suggests immobilized cellulase can be efficiently used up to sixth cycles with minimum loss of enzyme activity. The secondary structural analysis of cellulase enzyme during the synthesis of NPs and also after immobilization of cellulase on these NPs was carried out by CD spectroscopy.

A reusable multipurpose magnetic nanobiocatalyst for industrial applications

A multipurpose magnetic nanobiocatalyst is developed by conjugating Pectinex 3XL (a commercial enzyme containing pectinase, xylanase and cellulase activities) on 3-aminopropyl triethoxysilane activated magnetic nanoparticles. The nanobiocatalyst retained 87% of pectinase, 69% of xylanase and 58% of cellulase activity after conjugation on modified nanoparticles as compared to their soluble counterparts. Thermal stability data at 70°C showed increase in enzyme stability after conjugation to nanoparticles and the kinetic parameters (Km and Vmax) remain unaltered after immobilization. The immobilized enzyme system can be successfully used upto 5th cycle after that slight decrease in enzyme activities was observed. The nanobiocatalyst retained high pectinase activities in organic solvents and chemical reagents as compared to free enzymes. DLS data shows that the nanoparticles size increases from 63nm to 86nm after immobilization. Atomic Force Microscopy data confirms the deposition of enzymes on the nanoparticles. The nanobiocatalyst was used for the clarification of pine apple and orange juice and was also used for the production of bioethanol. Hydrolysis of pretreated wheat straw produced 1.39g/l and 1.59g/l after treatment with free Pectinex 3xL and nanobiocatalyst respectively. The concentration of bioethanol also increases by 1.4 fold as compared to the free enzyme.

Reusable magnetic nanobiocatalyst for synthesis of silver and gold nanoparticles.

In the present work, we describe a simple procedure for the biosynthesis of nanosilver and gold by the reduction of silver nitrate and auric chloride respectively using a nanobiocatalyst. The nanobiocatalyst was prepared by covalent coupling of alpha amylase on (3-aminopropyl)triethoxysilane (APTES) modified iron oxide magnetic nanoparticles. The nanobiocatalyst retains 77% of its activity as compared to free alpha amylase. The nanobiocatalyst can be used up to three consecutive cycles for the synthesis of nano silver and gold. The biosynthesized nanoparticles after each cycle were characterized by UV-vis spectrophotometer, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscope (TEM), X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Silver and gold nanoparticles of same morphology and dimensions were formed in each cycle. The procedure for synthesis of nanoparticles using an immobilized enzyme is eco-friendly and can be used repeatedly.