Rajeshwari Sinha

Interaction and nanotoxic effect of ZnO and Ag nanoparticles on mesophilic and halophilic bacterial cells

The toxicity of two commonly used nanoparticles, silver and zinc oxide on mesophilic and halophilic bacterial cells has been investigated. Enterobacter sp., Marinobacter sp., Bacillus subtilis, halophilic bacterium sp. EMB4, were taken as model systems. The nanotoxicity was more pronounced on Gram negative bacteria. ZnO nanoparticles reduced the growth of Enterobacter sp. by 50%, while 80% reduction was observed in halophilic Marinobacter sp. In case of halophiles, this may be attributed to higher content of negatively charged cardiolipins on their cell surface. Interestingly, bulk ZnO exerted minimal reduction in growth. Ag nanoparticles were similarly cytotoxic. Nanotoxicity towards Gram positive cells was significantly less, possibly due to presence of thicker peptidoglycan layer. The bacterium nanoparticle interactions were probed by electron microscopy and energy dispersive X-ray analysis. The results indicated electrostatic interactions between nanoparticles and cell surface as the primary step towards nanotoxicity, followed by cell morphological changes, increase in membrane permeability and their accumulation in the cytoplasm.

Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: Differential role of metal ions in stability and activity

A moderately halophilic protease producer, Bacillus sp. strain isolated from sea water is described. The protease is purified to homogeneity by ammonium sulphate precipitation and CM cellulose chromatography. The serine protease has a molecular mass of 29 kDa. Enzymatic characterization of protease revealed K(m) 2.22 mg mL(-1), Vmax 1111.11 U mL(-1), pH optimum 9.0, t1/2 190 min at 60°C and salt optima 1% (w/v) NaCl. The protease is remarkably stable in hydrophilic and hydrophobic solvents at high concentrations. The purified preparation is unstable at room temperature. Ca(2+) ions are required for preventing this loss of activity. Interestingly, the activity and stability are modulated differentially. Whereas, divalent cation Ca(2+) are involved in maintaining stability in solution at room temperature by preventing unfolding, monovalent Na(+) and K(+) ions participate in regulating the activity and assist in refolding of the enzyme. Application of the protease is shown in efficient removal of blood stain.

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants.

EFFICIENT PROTEOLYSIS AND APPLICATION OF AN ALKALINE PROTEASE FROM HALOPHILIC Bacillus sp. EMB9

A salt-stable alkaline protease from moderately halophilic Bacillus sp. EMB9, isolated from the western coast of India, is described. This protease was capable of efficiently removing silver from used/waste X-Ray films, as well as hydrolyzing defatted soy flour with 31% degree of hydrolysis (DH). Production of the protease was optimized by using response surface methodology. Ca2+ and NaCl were the most critical factors in enhancing the yield. Under optimized culture conditions, a maximum of 369 U protease/mL was obtained, which is quite comparable to the yields of commercial proteases. The elevated production level coupled with ability to efficiently hydrolyze protein-laden soy flour and complete recovery of silver from used X-Ray films makes it a prospective industrial enzyme.

Differential interactions of halophilic and non-halophilic proteases with nanoparticles

BackgroundIncrease in the industrial use of nanomaterials and nanoparticles (NPs) make their release into the environment inevitable. This may lead to environmental contamination and exposure of the biological/ microbial diversity. Nanoparticles have been reported to impregnate the cells and interact with cellular biomolecules especially proteins and DNA, leading to nanotoxicity in many cases. The present work targets to study nanoparticle-protein interactions in-vitro, especially to assess their effects on extracellularly secreted enzymes. The primary extracellular enzymes viz. hydrolases and proteases could be the first to come in contact with environmentally released nanoparticles.ResultsTwo halophilic proteases from Geomicrobium sp. EMB2 and Bacillus sp. EMB9 and one non-halophilic protease, subtilisin from Bacillus licheniformis have been investigated for their interaction with silver and zinc oxide nanoparticles as model systems. The activities of Geomicrobium sp. and Bacillus sp. protease were unaffected while that of non-halophilic subtilisin was lost by 70% and 30% in presence of Ag and ZnO nanoparticles respectively. The secondary and tertiary structure of halophilic proteases was unchanged on exposure to ZnO and Ag nanoparticles. Non-halophilic protease showed significant loss in α-helical structure with changes in the microenvironment of the protein as observed by CD and fluorescence spectroscopy. The greater stability and structural integrity may be attributed to higher negative charges on the surfaces of halophilic proteins.ConclusionsHalophilic extracellular proteases were more stable and did not lose proteolytic activity. Their secondary structure remained unaffected by interaction with ZnO and Ag nanoparticles. Alterations in structure and loss of activity in non-halophilic protease have been quite prevalent on exposure to nanoparticles. The extracellular halophilic nanostable enzymes thus offer a promising robust system to counter nanotoxicity. A precise understanding of nanoparticle interaction with extracellular enzymes will pave the way for designing of novel enzymes and creating appropriate system to protect microbial diversity against nanoparticle disposal.

Heavy Metal Bioremediation and Nanoparticle Synthesis by Metallophiles

Metallophiles are a group of extremophiles which are resistant to high concentration of metals. These microbes inherently possess properties that can influence the biomineralization processes by changing the physicochemical behaviour of the surrounding, metal speciation, mobility and toxicity. Metallophiles possess an endogenous ability to exquisitely regulate their physiology to overcome the toxic effect of the external metal environment and hence perceived to be excellent bioresource for different biotechnological applications. Their biotechnological applications in different bioprocesses have been quite promising. Recent studies have shown that in combination with bioremediation and biomineralization, these metallophiles can be harnessed to convert environmentally problematic metals into ‘high-end’ important and functional metallic materials. The current chapter describes some of the novel metallophilic microbes and their potential usages in bioremediation of problematic heavy metals from the environment and nanoparticle synthesis. The current level of understanding about metal–microbe interaction and plausible mechanism of metal bioremediation and nanoparticle biosynthesis have also been comprehended in this chapter.