Rajni Hatti-Kaul

Aqueous two-phase systems. A general overview.

Biphasic systems formed by mixing of two polymers or a polymer and a salt in water can be used for separation of cells, membranes, viruses, proteins, nucleic acids, and other biomolecules. The partitioning between the two phases is dependent on the surface properties and conformation of the materials, and also on the composition of the two-phase system. The mechanism of partitioning is, however, complex and not easily predicted. Aqueous two-phase systems (ATPS) have proven to be a useful tool for analysis of biomolecular and cellular surfaces and their interactions, fractionation of cell populations, product recovery in biotechnology, and so forth. Potential for environmental remediation has also been suggested. Because ATPS are easily scalable and are also able to hold high biomass load in comparison with other separation techniques, the application that has attracted most interest so far has been the large-scale recovery of proteins from crude feedstocks. As chemicals constitute the major cost factor for large-scale systems, use of easily recyclable phase components and the phase systems generated by a single-phase chemical in water are being studied.

Purification and characterization of cellulases produced by two Bacillus strains.

Cellulases produced by two Bacillus strains, CH43 and HR68, isolated from hot springs in Zimbabwe, were purified to homogeneity from culture supernatants. Both enzymes had molecular mass of 40 kDa and isoelectric point of 5.4. The enzymes also resembled each other in N-terminal amino acid sequence which was Ala-Gly-Thr-Lys-Thr-Pro-Val-Ala-Lys-Asn-Gly-Gln, showing 100% homology with that of endoglucanases from Bacillus subtilis belonging to glycoside hydrolase family five. The cellulases were optimally active in the pH range of 5-6.5. The optimum temperature was 65 and 70 degrees C for the endoglucanase of CH43 and HR68, respectively. The CH43 enzyme was stable at 50 degrees C in a pH range of 6-10, and HR68 at pH 6-8. Both the enzymes retained complete activity for at least 24 h at 50 degrees C. The enzymes showed highest activity with beta-glucan as substrate followed by carboxymethylcellulose. Significant activity was also observed with crystalline forms of cellulose such as filter paper and Avicel, particularly for HR68 cellulase. For carboxymethycellulose, the CH43 and HR68 cellulases had a Km of 1.5 and 1.7 mg ml(-1), respectively, and Vmax of 0.93 and 1.70 mmol glucose min(-1) mg protein(-1) respectively. The activity of the enzymes was not influenced by most metal ions at 1 mM concentration, but was increased by about 38% by Co2+. The inhibition by Hg2+ and Mn2+ was higher for CH43 than for HR68 enzyme. Ag+ inhibited the CH43 activity but stimulated the HR68 activity. The CH43 cellulase was inhibited by N-bromosuccinimide and iodoacetamide while HR68 was unaffected.

Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather

Two alkaline protease producing alkaliphilic bacterial strains, designated as AL-20 and AL-89, were isolated from a naturally occurring alkaline habitat. The two strains were identified as Nesternkonia sp. and Bacillus pseudofirmus, respectively. Both strains grew and produced alkaline protease using feather as the sole source of carbon and nitrogen. Addition of 0.5% glucose to the feather medium increased protease production by B. pseudofirmus AL-89 and suppressed enzyme production by Nesternkonia sp. AL-20. The enzymes from both organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight, determined using SDS–PAGE, was 23 kDa for protease AL-20 and 24 kDa for protease AL-89. Protease AL-20 was active in a broad pH range displaying over 90% of its maximum activity between pH 7.5 and 11.5 with a peak at pH 10. The enzyme is unique in that unlike all other microbial serine proteases known so far, it did not require Ca2+ for activity and thermal stability. Its optimum temperature for activity was at 70 °C and was stable after 1 h incubation at 65 °C both in the presence and absence of Ca2+. These properties make protease AL-20 an ideal candidate for detergent application. Protease AL-89 on the other hand require Ca2+ for activity and stability at temperature values above 50 °C. Its optimum activity was at 60 and 70 °C in the absence and presence of Ca2+, respectively. It displayed a pH optimum of 11 and retained about 70% or more of its original activity between pH 6.5 and 11. B. pseudofirmus AL-89, and the protease it produce offers an interesting potential for the enzymatic and/or microbiological hydrolysis of feather to be used as animal feed supplement.

Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects

Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.

Protein stabilising effect of polyethyleneimine

Abstract The effect of polyethyleneimine (PEI), a cationic polymer, on protein stability was investigated. Shelf stability of lactate dehydrogenase, stored as an aqueous solution, at 36°C was distinctly improved in the presence of 0.01–1% (w/v) polymer (except at pH 5.0). PEI also prevented aggregation of the protein during long-term storage. Both high and low molecular weight (ca. 600 000 and 2000, respectively) PEI were equally effective. Stabilising effect was also observed for other proteins including alcohol dehydrogenase, β-galactosidase and trypsin. Circular dichroism scan in the far UV range confirmed the protection offered by PEI against complete loss of protein secondary structure during storage. Studies with LDH revealed that the presence of polymer did not increase its denaturation temperature, but on the other hand, hindered the oxidation of free sulfhydryl groups catalysed by metal ions and resultant inactivation of the enzyme. Protection by the polymer was also seen during drying of LDH but not during freeze-thawing.

Purification and characterization of a thermostable xylanase from Bacillus amyloliquefaciens

Abstract A Bacillus amyloliquefaciens strain isolated from soil produced xylanolytic enzymes in the extracellular medium when grown on xylan and nitrate. No cellulase activity was detected. Xylanase was purified from the culture supernatant in three steps which comprised anion-exchange adsorption, ammonium sulfate precipitation, and hydrophobic interaction chromatography. The pure enzyme appeared as two close protein bands on SDS-PAGE with molecular weights of 18.4 and 19.6 kD, respectively. The molecular weight obtained by sedimentation equilibrium under native conditions was about 18.5 kD. The isoelectric point was 10.1. The enzyme contains a relatively high content of aspartate, glycine, and threonine. Tryptophan seems to be essential for xylanase activity. The presence of carbohydrate was noted in the pure enzyme. Circular dichroism studies indicated that the protein contains almost equal proportions of α-helix, β-sheet, and β-turns. The optimum pH of activity was 6.8–7.0. The enzyme exhibited good stability even at pH 9.0. Excellent stability was observed at 50°C even though optimal activity was at 80°C. The activity was completely inhibited by Hg 2+ ions and was reduced drastically in the presence of Cu 2+ and Fe 3+ ions. Mn 2+ ions, EDTA, β-mercaptoethanol, and dithiothreitol up to 5 m m stimulated the enzyme activity.

Synergistic relationships in algal-bacterial microcosms for the treatment of aromatic pollutants.

The potential of algal-bacterial microcosms was studied for the biodegradation of salicylate, phenol and phenanthrene. The isolation and characterization of aerobic bacterial strains capable of mineralizing each pollutant were first conducted. Ralstonia basilensis was isolated for salicylate degradation, Acinetobacter haemolyticus for phenol and Pseudomonas migulae and Sphingomonas yanoikuyae for phenanthrene. The green alga Chlorella sorokiniana was then cultivated in the presence of the pollutants at different concentrations, showing increasing inhibitory effects in the following order: salicylate < phenol < phenanthrene. The synergistic relationships in the algal-bacterial microcosms were clearly demonstrated, since for the three contaminants tested, a substantial removal (>85%) was recorded only in the systems inoculated with both algae and bacteria and incubated under continuous lighting. This study presents, to our knowledge, the first reported case of photosynthesis-enhanced biodegradation of toxic aromatic pollutants by algal-bacterial microcosms in a one-stage treatment.

Mushroom immunomodulators: unique molecules with unlimited applications

For centuries, mushrooms have been used as food and medicine in different cultures. More recently, many bioactive compounds have been isolated from different types of mushrooms. Among these, immunomodulators have gained much interest based on the increasing growth of the immunotherapy sector. Mushroom immunomodulators are classified under four categories based on their chemical nature as: lectins, terpenoids, proteins, and polysaccharides. These compounds are produced naturally in mushrooms cultivated in greenhouses. For effective industrial production, cultivation is carried out in submerged culture to increase the bioactive compound yield, decrease the production time, and reduce the cost of downstream processing. This review provides a comprehensive overview on mushroom immunomodulators in terms of chemistry, industrial production, and applications in medical and nonmedical sectors.

Poly(beta-hydroxybutyrate) production by a moderate halophile, Halomonas boliviensis LC1 using starch hydrolysate as substrate

Aim:  The objective of the present work was to enable the use of starch hydrolysate, generated by the action of a recombinant maltooligosaccharide forming amylase from Bacillus halodurans LBK 34, as the carbon source for the production of poly‐β‐hydroxybutyrate (PHB) by Halomonas boliviensis LC1.

Cell Chromatography: Separation of Different Microbial Cells Using IMAC Supermacroporous Monolithic Columns

Supermacroporous monolithic columns with Cu2+‐IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross‐linked with methylenebis(acrylamide) and has a network of large (10–100 μm) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild‐type Escherichia coli(w.t. E. coli) and recombinant E. coli cells displaying poly‐His peptides (His‐tagged E. coli) and of w.t. E. coli and Bacillus haloduranscells. Wild‐type E. coli and His‐tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8‐fold enriched with His‐tagged E. colicells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. colicells. Haloalkalophilic B. haloduranscells had low affinity to the Cu2+‐IDA cryogel column and could be efficiently separated from a mixture with w.t. E. colicells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.