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Novel extracellular phytase was produced by Aspergillus niger NCIM 563 under submerged fermentation conditions at 30°C in medium containing dextrin and glucose as carbon sources along with sodium nitrate as nitrogen source. Maximum phytase activity (41.47 IU/mL at pH 2.5 and 10.71 IU/mL at pH 4.0) was obtained when dextrin was used as carbon source along with glucose and sodium nitrate as nitrogen source. Nearly 13 times increase in phytase activity was observed when phosphate in the form of KH 2 PO 4 (0.004 g/100 mL) was added in the fermentation medium. Physic-chemical properties of partially purified enzyme indicate the possibility of two distinct forms of phytases, Phy I and Phy II. Optimum pH and temperature for Phy I was 2.5 and 60°C while Phy II was 4.0 and 60°C, respectively. Phy I was stable in the pH range 1.5-3.5 while Phy II was stable in the wider pH range, 2.0-7.0. Molecular weight of Phy I and Phy II on Sephacryl S-200 was approximately 304 kDa and 183 kDa, respectively. Phy I activity was moderately stimulated in the presence of 1 mM Mg 2+ , Mn 2+ , Ca 2+ and Fe 3+ ions and inhibited by Zn 2+ and Cd 2+ ions while Phy II activity was moderately stimulated by Fe 3+ ions and was inhibited by Hg 2+ , Mn 2+ and Zn 2+ ions at 1 mM concentration in reaction mixture. The Km for Phy I and II was 3.18 and 0.514 mM while Vmax was 331.16 and 59.47 lmols/min/ mg protein, respectively.

Aspergillus niger NCIM 563 produced two different extracellular phytases (Phy I and Phy II) under submerged fermentation conditions at 30°C in medium containing dextrin-glucose-sodium nitrate-salts. Both the enzymes were purified to homogeneity using Rotavapor concentration, Phenyl-Sepharose column chromatography and Sephacryl S-200 gel filtration. The molecular mass of Phy I and II as determined by SDS–PAGE and gel filtration were 66, 264, 150 and 148 kDa respectively, indicating that Phy I consists of four identical subunits and Phy II is a monomer. The pI values of Phy I and II were 3.55 and 3.91, respectively. Phy I was highly acidic with optimum pH of 2.5 and was stable over a broad pH range (1.5–9.0) while Phy II showed a pH optimum of 5.0 with stability in the range of pH 3.5–9.0. Phy I exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. Similarly Phy II was strongly inhibited by Ag+, Hg2+ (1 mM) metal ions and Phy I was partially inhibited. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF also indicated that both the proteins were totally different. The Km for Phy I and II for sodium phytate was 2.01 and 0.145 mM while Vmax was 5,018 and 1,671 μmol min−1 mg−1, respectively. The N-terminal amino acid sequences of Phy I and Phy II were FSYGAAIPQQ and GVDERFPYTG, respectively. Phy II showed no homology with Phy I and any other known phytases from the literature suggesting its unique nature. This, according to us, is the first report of two distinct novel phytases from Aspergillus niger.

Most of the self-assembly studies have hitherto explored the aqueous media as fluid phase for self-assembly of amphiphilic biomacromolecules, wherein architectural modification of biomolecules is generally a prerequisite for self-assembly of modified biomolecules. We demonstrate for the first time that ionic liquids can act as nonaqueous designer solvents to self-assemble amphiphilic biomacromolecules without requiring their prior modification. To this end, we show that enzyme (phytase) molecules self-assembled in the presence of an appropriate ionic liquid, resulting in the formation of enzyme capsules. Phytase capsules synthesized using this approach were further used as templating nanoreactors for the synthesis of enzyme-containing hollow silica nanocontainers. In situ immobilized phytase enzyme in the silica nanocontainers, when subjected to enzyme-reusability application, establishes them as excellent reusable biocatalysts.

The intricate, hierarchical, highly reproducible, and exquisite biosilica structures formed by diatoms have generated great interest to understand biosilicification processes in nature. This curiosity is driven by the quest of researchers to understand nature's complexity, which might enable reproducing these elegant natural diatomaceous structures in our laboratories via biomimetics, which is currently beyond the capabilities of material scientists. To this end, significant understanding of the biomolecules involved in biosilicification has been gained, wherein cationic peptides and proteins are found to play a key role in the formation of these exquisite structures. Although biochemical factors responsible for silica formation in diatoms have been studied for decades, the challenge to mimic biosilica structures similar to those synthesized by diatoms in their natural habitats has not hitherto been successful. This has led to an increasingly interesting debate that physico-chemical environment surrounding diatoms might play an additional critical role towards the control of diatom morphologies. The current study demonstrates this proof of concept by using cationic amino acids as catalyst/template/scaffold towards attaining diatom-like silica morphologies under biomimetic conditions in ionic liquids.

The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

We present an extensive overview of the evolution and progress made in the field of microstructures and nanostructures preparation using microfluidic techniques in recent times. A microfluidic system creates particles that are within a narrow range of shape and size distribution. It enables controlling the shape, size and composition of nanomaterials (NMs) for various applications. A brief evaluation of the advantages of both droplet-based and continuous flow synthesis of nanoparticles (NPs) is discussed in detail and compared with the traditional wet chemical batch synthesis approach. Due to increasing applications of biosensing, nanobiotechnology, nanomedicine and diagnostics devices, special attention should be paid to metal NPs developed through microfluidic routes.

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