Biomedical subjects

R Hatti-Kaul

Publications and source records attributed to R Hatti-Kaul.

9 recordsLinked to original sources

PubMed2005

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-beta-hydroxybutyrate (PHB) by Halomonas boliviensis LC1. METHODS AND RESULTS: In this work, different amounts of the alpha-amylase (Amy 34) were utilized for starch hydrolysis, resulting in the production of mixtures of maltooligosaccharides (G1-G6) at varying ratios. The highest PHB accumulation (56 wt%) by H. boliviensis cultivated in shake flasks (with agitation at 160 rev min(-1)) was obtained when 6.4 U ml(-1) of the amylase was used for starch hydrolysis. When H. boliviensis cells were grown in a fermentor with no oxygen limitation the accumulation of PHB was decreased to 35 wt%. Although some improvements in PHB accumulation and cell mass concentration were reached by the addition of peptone and phosphate, respectively, major enhancements were attained when oxygen limitation was induced in the fermentor. CONCLUSIONS: Halomonas boliviensis uses preferentially maltose for PHB formation from starch hydrolysate. It is also able to hydrolyse higher sugars if no other simpler carbon source is available but with a significantly lower polymer yield. Furthermore, H. boliviensis is able to adjust its metabolism to oxygen limitation, most probably by directing the excess NAD(P)H to PHB accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: There have been no reports related to PHB production amongst the members of the genus Halomonas. The use of a maltooligosaccharide forming alpha-amylase, which is active at a temperature and pH close to that required for growth of H. boliviensis, and the versatility of this bacterium in the selection of the carbon source may provide an attractive alternative for the utilization of starch-derived raw materials.

3-Hydroxybutyric Acid↗

PubMed2002

Beta-galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis.

The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.

Acclimatization↗

PubMed2001

Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate.

Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated. The partition of the enzyme in a polyethylene glycol (PEG)-potassium phosphate system to the top phase was increased with lower molecular weight PEG. Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respectively. Addition of 0.5% (w/w) butyrate increased the partition coefficient from 17 to 135 and the purification factor from 10 to 23. The effect of butyrate was also confirmed by using the countercurrent mode of extraction. Recovery of cutinase from the top phase was achieved by a secondary extraction into a new salt phase at a lower pH or a lower temperature. A specific interaction of butyrate to the active site of the enzyme was demonstrated by fluorescence spectroscopy. Size exclusion chromatography showed the cutinase-butyrate complex to be over two times the size of the free enzyme.

Biochemistry↗

PubMed2001

Starch-hydrolyzing bacteria from Ethiopian soda lakes.

Alkaliphilic bacteria were isolated from soil and water samples obtained from Ethiopian soda lakes in the Rift Valley area--Lake Shala, Lake Abijata, and Lake Arenguadi. Starch-hydrolyzing isolates were selected on the basis of their activity on starch agar plate assay. Sixteen isolates were chosen, characterized, and subjected to 16S rRNA gene sequence analysis. All the isolates were gram positive and catalase- and beta-galactosidase positive. All isolates except one were motile endospore-forming rods and were found to be closely related to the Bacillus cluster, being grouped with Bacillus pseudofirmus, Bacillus cohnii, Bacillus vedderi, and Bacillus agaradhaerens. The one exception had nonmotile coccoid cells and was closely related to Nesterenkonia halobia. The majority of the isolates showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v) NaCl. Both extracellular and cell-bound amylase activity was detected among the isolates. The amylase activity of two isolates, related to B. vedderi and B. cohnii, was stimulated by ethylenediaminetetraacetic acid (EDTA) and inhibited in the presence of calcium ions. Pullulanase activity was expressed by isolates grouped with B. vedderi and also most of the isolates clustered with B. cohnii; cyclodextrin glycosyltransferase was expressed by most of the B. agaradhaerens-related strains. Minor levels of alpha-glucosidase activity were detected in all the strains.

Amylose↗

PubMed2001

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.

Biotechnology↗

PubMed2000

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.

Bacillus↗

PubMed2000

Stabilizing effect of chemical additives against oxidation of lactate dehydrogenase.

Oxidation is one of the major pathways for denaturation of proteins during storage, and also a potential problem in protein production, isolation and purification processes. In this study, a number of additives have been tested for their protective effects against oxidation in the presence of metal ions and hydrogen peroxide. Porcine muscle lactate dehydrogenase (LDH) was used as a model protein. Oxidation and denaturation of the enzyme were followed as activity loss, modification of certain amino acids, altered secondary structure and aggregation. Loss of activity during metal-catalysed oxidation was accompanied by structural damage of the enzyme, which was not the case during oxidation with peroxide. The best protectant during both modes of oxidation was found to be the polycation, poly(ethyleneimine) (PEI), followed by EDTA. Both chemicals also increased the enzyme's half-life during oxidation with a mixture of copper ions and hydrogen peroxide. Ammonium sulphate was an effective stabilizer during metal-catalysed oxidation, while sorbitol, sucrose and hydroxyectoine provided moderate stabilization. The ectoine was also stabilizing against oxidation with hydrogen peroxide, as was poly(ethylene glycol), whereas sorbitol enhanced the rate of enzyme inactivation. The stability of LDH towards denaturation by both oxidation and temperature was increased by addition of both PEI and sorbitol, as indicated by the melting-temperature profiles of the enzyme.

Animals↗

PubMed1999

Lactic acid production by immobilized Lactobacillus casei in recycle batch reactor: a step towards optimization.

Different nutritional and process parameters influencing lactic acid production by Lactobacillus casei, adsorbed to Poraver beads in a recycle batch reactor system, were studied in an attempt to set up a system having a long operational lifetime and permitting use of high substrate concentrations for maximal conversion to the product. The presence of lactose, even as a minor fraction of the total sugar amount, was necessary for complete utilization by the organism for growth and conversion to lactate. Hydrolysed whey protein constituted a richer source of nitrogen compared to yeast extract. Addition of lactate to the medium at the start of the process resulted in severe inhibition compared with the normal process. For a homofermentative process, pH 6.0 was found to be optimal. The overall productivity of the recycle system was higher under all conditions studied in comparison with the batch process using free cells. Enhancement in productivity in the recycle batch reactor was also accompanied by an increase in density of suspended cells. However, the contribution of the suspended cells to the overall reactor productivity was not noticeable. The bead size of the matrix was found to be important for operational stability of the reactor.

Hydrogen-Ion Concentration↗

PubMed1997

Towards a molecular level understanding of protein stabilization: the interaction between lysozyme and sorbitol.

The paper is investigating the mechanism of stabilization of proteins by polyols at the molecular level. It is addressing the interactions of sorbitol, a polyol commonly used as a protein stabilizing agent, with hen egg white lysozyme, a well studied protein. Differential scanning calorimetry shows an increase in denaturation temperature of lysozyme upon addition of sorbitol at a concentration of 250 mM and above. Increasing sorbitol concentration also caused an increase in signal intensity of the CD spectrum of lysozyme in the wavelength region of 280-300 nm. Two-dimensional nuclear magnetic resonance spectroscopy was used to examine interactions between lysozyme and sorbitol. Most significant changes are manifest in the anomalous relaxation properties of Ala and Thr methyl groups indicating modifications of local motions and possibly compression of the entire structure. This is further corroborated by new intra-protein nuclear Overhauser effects in the presence of sorbitol. There is also evidence that water is displaced from the enzyme surface close to Ile-88 upon addition of sorbitol. In combination these results reveal a complex interplay of different interactions. Comparison to NMR-spectra of lysozyme with a bound inhibitor (tri-N-acetyl-glucosamine) shows that the interaction with sorbitol affects spatially disparate regions of the protein.

Calorimetry, Differential Scanning↗