S.K. Khare

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.

Enzymes from solvent-tolerant microbes: Useful biocatalysts for non-aqueous enzymology

Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent’s toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.

Citric acid production from Okara (soy-residue) by solid-state fermentation

Abstract Okara (soy-residue), a cellulosic byproduct of the soymilk and tofu (soypaneer) industry, was used for the production of citric acid by solid-state fermentation using a cellulolytic Aspergillus terreus and citric-acid-producing Aspergillus niger . Okara supplemented with ammonium sulphate (0·10% N) when fermented by A. niger with simultaneous saccharification using A. terreus at pH 8·3 and incubation temperature of 30°C resulted in production of 5·10 g citric acid/100 grams dry solids (gds) by the eleventh day.

Degradation of phorbol esters by Pseudomonas aeruginosa PseA during solid-state fermentation of deoiled Jatropha curcas seed cake.

Large amount of seed cake is generated as by-product during biodiesel production from Jatropha seeds. Presence of toxic phorbol esters restricts its utilization as livestock feed. Safe disposal or meaningful utilization of this major by-product necessitates the degradation of these phorbol esters. The present study describes the complete degradation of phorbol esters by Pseudomonas aeruginosa PseA strain during solid state fermentation (SSF) of deoiled Jatropha curcas seed cake. Phorbol esters were completely degraded in nine days under the optimized SSF conditions viz. deoiled cake 5.0 g; moistened with 5.0 ml distilled water; inoculum 1.5 ml of overnight grown P. aeruginosa; incubation at temperature 30 °C, pH 7.0 and RH 65%. SSF of deoiled cake seems a potentially viable approach towards the complete degradation of the toxic phorbol esters.

Immobilization of Rhizopus japonicus lipase on celite and its application for enrichment of docosahexaenoic acid in soybean oil

Rhizopus japonicus lipase was immobilized on Celite, the immobilized enzyme acquired 6.34 units of transesterification activity over the native enzyme which does not show any significant transesterification activity in n-hexane. However, there was no improvement in the thermal stability of Celite-immobilized lipase. The preparation is shown to be promising for its end application in producing structured lipid. An incorporation of 25% docosahexaenoic acid (DHA) into soybean oil was observed in n-hexane media in 24 h with the immobilized lipase.

Screening and isolation of halophilic bacteria producing industrially important enzymes

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Purification and characterization of maltooligosaccharide-forming α-amylase from moderately halophilic Marinobacter sp. EMB8.

Maltooligosaccharides especially maltotriose and maltotetraose producing amylases are highly desirable for application in bread making and other food industries. A maltotriose and maltotetraose producing amylase from moderately halophilic Marinobacter sp. EMB8 is described. Under optimized culture conditions, 48.0 IU/mL amylase was obtained. The enzyme was purified to homogeneity by ultrafiltration, DEAE cellulose and Sephadex G-75 column chromatography with 52% yield and 76-fold purification. It was a monomeric protein of 72 kDa. The amylase had many novel features viz. stability up to 20% NaCl, 80 °C temperature, pH 6.0-11.0 and in wide range of organic solvents at high concentrations. The enzyme efficiently hydrolyzed starch into maltooligosaccharides rich in maltotriose and maltotetraose. These novel properties make the Marinobacter sp. amylase a potentially useful enzyme.

Mercury bioaccumulation and simultaneous nanoparticle synthesis by Enterobacter sp. cells

A mercury resistant strain of Enterobacter sp. is reported. The strain exhibited a novel property of mercury bioaccumulation with simultaneous synthesis of mercury nanoparticles. The culture conditions viz. pH 8.0 and lower concentration of mercury promotes synthesis of uniform sized 2-5 nm, spherical and monodispersed intracellular mercury nanoparticles. The remediated mercury trapped in the form of nanoparticles is unable to vaporize back into the environment thus, overcoming the major drawback of mercury remediation process. The mercury nanoparticles were recoverable. The nanoparticles have been characterized by high resolution transmission electron microscopy, energy dispersive X-ray analysis, powder X-ray diffraction and atomic force microscopy. The strain can be exploited for metal bioaccumulation from environmental effluent and developing a green process for nanoparticles biosynthesis.

Hydrolysis of rice hull by crosslinked Aspergillus niger cellulase

A. niger cellulase was crosslinked by glutaraldehyde to obtain a heat-stable enzyme preparation for rice hull cellulose hydrolysis. Under optimized crosslinking conditions of 0.12 M glutaraldehyde, pH 7.0, temperature 40 degrees C and at 45 min of crosslinking, a preparation having 15% more activity than free enzyme was obtained which also had considerable improvement in heat stability at 65 degrees C and 70 degrees C. Whereas the free enzyme lost 80% of its activity in 4 h at 65 degrees C, the crosslinked preparation lost only 30% activity. The crosslinked preparation hydrolyzed cellulosic biomass more effectively giving 2.2 mg/ml glucose and 52% corresponding saccharification in 4 h at 65 degrees C as compared to 14% saccharification by free enzyme under similar conditions.

An efficient purification process for sweet potato beta-amylase by affinity precipitation with alginate

beta-amylases are used in production of maltose syrup. It is shown that sweet potato beta-amylase can be purified by affinity precipitation with alginate with 80% activity yield and 44 fold purification. SDS-PAGE of the purified protein showed a single band and a subunit weight of 50 kDa. Preliminary data with soybean and barley enzymes indicate that this may be a general method for purification of beta-amylases.