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Biomedical subjects

Rajni Hatti-Kaul

Publications and source records attributed to Rajni Hatti-Kaul.

At least 19 recordsLinked to original sources

Solvent-free enzymatic synthesis of fatty alkanolamides.

An environmentally benign and volume efficient process for enzymatic production of alkanolamides is described. Immobilized Candida antarctica lipase B, Novozym435, was used to catalyze the condensation of lauric acid with monoethanolamine. The reaction temperature of 90 degrees C was required to keep the reactants in a liquid state. Stepwise addition of the amine minimized problems caused by the formation of a highly viscous amine/fatty acid ion-pair. The enzyme was both very active and stable under the reaction conditions, with about half of the activity remaining after 2 weeks. The maximum amide yield obtained when using equimolar amounts of the reactants was 75%, which could be increased to 95% upon water removal. Special precautions to avoid co-distillation of the amine were required. Two different strategies to avoid the amine loss are presented.

Amides↗

Optimizing refolding and recovery of active recombinant Bacillus halodurans xylanase in polymer-salt aqueous two-phase system using surface response analysis.

An experimental design was used to determine optimal conditions for refolding of a recombinant thermostable and alkaline active xylanase from Bacillus halodurans in PEG-phosphate two-phase system. The influence of different experimental variables on the enzyme recovery has been evaluated. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology with a face-centered central composite design (CCF) was defined based on the conditions found by preliminary tests that resulted in the highest refolding yield. The adequacy of the calculated model for the response was confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, polyethylene glycol (PEG) molecular weight and concentration, and salt concentration for different enzyme loads revealed different effects of these five factors on the studied parameters. Recovery of more than 92% active xylanase was predicted for a system with 18.3% (w/w) PEG 1000, 14.4% (w/w) phosphate at pH 8.5, and enzyme load corresponding to a protein concentration of about 0.05 mg/g system. The yield of the refolded enzyme was found to be optimal at 22 degrees C. The validity of the response model was verified by a good agreement between predicted and experimental results.

Analysis of Variance↗

Optimizing conditions for poly(beta-hydroxybutyrate) production by Halomonas boliviensis LC1 in batch culture with sucrose as carbon source.

Halomonas boliviensis LC1 is able to accumulate poly(beta-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l(-1) CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l(-1) and 14 g l(-1), respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l(-1) in 17 h but reduced the PHB content in the cells to 52 wt.%.

Culture Techniques↗

Purification of plasmid DNA with polymer-salt aqueous two-phase system: optimization using response surface methodology.

An experimental design was used to optimize plasmid purification from an alkaline lysate of Escherichia coli cells using PEG-sodium citrate aqueous two-phase systems (ATPS), and to evaluate the influence of pH, PEG molecular weight, tie line length, phase volume ratio, and lysate load. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology (RMS) with an orthogonal rotatable central composite design was defined based on the conditions found for the highest purification by preliminary tests. The adequacy of the calculated models for the plasmid recovery and remaining RNA were confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, PEG molecular weight, tie line length, and cell lysate load for three different phase volume ratios revealed different effects of these five factors on the studied parameters. Plasmid recovery of 99% was predicted for a system with PEG 400, pH 6.9, tie line length of 38.7%, phase volume ratio of 1.5, and lysate load of 10% (v/v). Under these conditions the predicted RNA removal was 68%.

Citrates↗

Fusion of carbohydrate binding modules from Thermotoga neapolitana with a family 10 xylanase from Bacillus halodurans S7.

Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)(6) tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.

Amino Acid Sequence↗

Nesterenkonia aethiopica sp. nov., an alkaliphilic, moderate halophile isolated from an Ethiopian soda lake.

Strain DSM 17733(T), isolated from the shore of Lake Abjata in Ethiopia, is a heterotrophic, alkaliphilic, moderately halophilic, Gram-positive, strictly aerobic, non-motile,non-endospore-forming bacterium. The organism grows optimally at 30-37 degrees C, pH 9 and 3 % (w/v) NaCl. Analysis of the cell wall showed the presence of murein of the type L-lys-gly-L-Glu, variation A4alpha. The G + C content of the genomic DNA was 69.0 mol%. Sequence analysis of 16S rRNA gene sequence of strain DSM 17733(T) placed the isolate in the genus Nesterenkonia. DNA-DNA hybridization of DSM 17733(T) with those organisms with the closest phylogenetic affiliation, i.e. Nesterenkonia halobia, Nesterenkonia lacusekhoensis and Nesterenkonia xinjiangensis, gave relatedness values of 48.5 %, 63.7 % (repetition, 57.2 %) and 35.7 % (repetition, 29.3 %), respectively. On the basis of both phenotypic and phylogenetic criteria and the low levels of DNA-DNA relatedness with the phylogenetically closest species N. xinjiangensis and N. halobia, it is proposed that the isolate be classified in a novel species, Nesterenkonia aethiopica sp. nov. The type strain is DSM 17733(T) (=CCUG 48939(T)).

Ethiopia↗

Cloning, sequence analysis, and expression of a gene encoding an endoxylanase from Bacillus halodurans S7.

The gene encoding an alkaline active xylanase of Bacillus halodurans S7, containing an open reading frame of 1188 nucleotides encoding 396 amino acids, was cloned and expressed in Escherchia coli. On the basis of sequence similarity, possible -10 and -35, ribosome binding, and transcription terminator regions were identified. Analysis of the deduced amino acid sequence revealed that the protein was a single domain enzyme belonging to family 10 and designated as xyn10A. The calculated molecular mass and isoelectric point (pI) of the mature peptide were 42.6 and 4.5 kDa, respectively. Xylanase activity expressed by the recombinant organism was detected in the cytoplasm, periplasm and the extracellular medium. In an 18-h old culture, about 39% of the xylanase was detected in the medium. The stability and activity profile of the recombinant xylanase was similar to the properties of the enzyme produced by the wild-type organism.

Amino Acid Sequence↗

Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris.

A gene encoding a lipolytic enzyme amplified from the alkaliphilic bacterium Bacillus halodurans LBB2 was cloned into the pPICZalphaB vector and integrated into the genome of the protease deficient yeast strain Pichia pastoris SMD1168H. This previously undescribed enzyme was produced in active form, and cloning in frame with the Saccharomyces cerevisiae secretion signal (alpha-factor) enabled extracellular accumulation of correctly processed enzyme, with an apparent molecular mass of 30 kDa. In shake-flask cultivations, very low production levels were obtained, but these were significantly improved by use of a "batch-induced" cultivation technique which allowed a maximum enzyme activity of 14,000 U/l using p-nitrophenyl butyrate (C-4) as a substrate and a final extracellular lipolytic enzyme concentration of approximately 0.2 g/l. Partial characterization of the produced enzyme (at pH 9) revealed a preference for the short-chain ester (C-4) and significant but lower activity towards medium (C5-C6) and long (C16 and C18) fatty acid chain-length esters. In addition, the enzyme exhibited true lipase activity (7,300 U/l) using olive oil as substrate and significant levels of phospholipase activity (6,400 U/l) by use of a phosphatidylcholine substrate, but no lysophospholipase activity was detected using a lysophosphatidylcholine substrate.

Amino Acid Sequence↗

Analysis of fatty acid epoxidation by high performance liquid chromatography coupled with evaporative light scattering detection and mass spectrometry.

Conventionally, epoxidation of unsaturated fatty acids has been studied either with titrimetric methods or in a lengthy procedure involving derivatization followed by gas chromatography (GC). We have developed a more rapid and descriptive analysis procedure for the substances using high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). Chemo-enzymatic epoxidation of unsaturated fatty acids (oleic, linoleic and linolenic acid, respectively) has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym 435). The fatty acids and their epoxidation products were separated by HPLC on a C-18 reversed-phase column using methanol-water containing 0.05% acetic acid as mobile phase. The method facilitated the simultaneous determination of fatty acids and epoxides differing from each other in the number of epoxide rings, the degree of unsaturation and the position of the epoxide rings and double bonds. An important aspect of the method development was the use of electrospray ionization and tandem mass spectrometry to confirm the structure of the epoxide products. It is suggested that the HPLC method, providing more information about the kind and concentration of fatty acids and their epoxides, represents a powerful complement to the existing methods for monitoring epoxidation processes on fatty acids.

Chromatography, High Pressure Liquid↗

High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format.

Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples.

Cell Culture Techniques↗

Bacillus bogoriensis sp. nov., a novel alkaliphilic, halotolerant bacterium isolated from a Kenyan soda lake.

Strain LBB3(T) isolated from Bogoria soda lake in Kenya is an alkaliphilic, Gram-positive, strictly aerobic, non-motile, spore-forming bacterium. It was identified as a member of the genus Bacillus on the basis of phenotypic and phylogenetic analyses. The organism grows optimally at 37 degrees C and pH 10. The G+C content of the genomic DNA is 37.5 mol%. 16S rRNA gene sequence analysis showed 95 and 96 % sequence similarity with Bacillus pseudofirmus (DSM 8715(T)) and Bacillus alcalophilus (DSM 485(T)), respectively. Furthermore, DNA-DNA hybridization against these two Bacillus species showed 39.0 and 55.5 % similarity, respectively. Based on our observations, strain LBB3(T) is proposed to represent a novel species of the genus Bacillus, for which the name Bacillus bogoriensis sp. nov. is proposed. The type strain of B. bogoriensis is LBB3(T) (=ATCC BAA-922(T)=LMG 22234(T)).

Bacillus↗

Agarose-coated anion exchanger prevents cell-adsorbent interactions.

A common problem during recovery of bioproducts by adsorption from particulate broths is fouling of the adsorbent material as a result of the interaction of cells and cell debris, which present negative charges, with the positively charged anion exchangers commonly used in bioprocesses. The effect of shielding an adsorbent with a layer of agarose on reducing the binding of cells while still allowing the low-molecular-mass bioproducts to be adsorbed was studied. Coating the anion-exchange resin Amberlite IRA-400 with agarose followed by cross-linking the agarose layer effectively prevented the binding of Escherichia coli, Saccharomyces cerevisiae, and Lactobacillus casei cells but allowed binding of lactic acid to the adsorbent. The cross-linked agarose layer was stable during recycling of the adsorbent.

Adsorption↗

Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts.

Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for beta-galactosidase activity, were used for the synthesis of 1-hexyl-beta-d-galactoside and 1-octyl-beta-d-galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6-36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50mM lactose for at least a week.

Alcohols↗

Lactate production in an integrated process configuration: reducing cell adsorption by shielding of adsorbent.

The problem of binding of microbial cells to an adsorbent matrix during in situ recovery of bioproducts from a fermentation broth has been addressed by shielding the adsorbent with a thin layer of a non-ionic polymer. Extractive bioconversion of lactic acid by integrating ion-exchange adsorption with the fermentation stage was studied. The effect of coating of the ion-exchanger with agarose on product recovery and cell adsorption was evaluated. Extractive fermentation with both uncoated and coated resin resulted in an increase in reactor productivity as compared to the normal fermentation. The free cell density in the system with agarose-coated beads was similar to that in control fermentation, but was significantly lower in the system with the uncoated ion-exchanger. Electron microscopic scanning of the bead surface after passage of the fermentation broth showed cells attached to the native adsorbent but not to the coated one.

Bacterial Adhesion↗

Lipase-producing microorganisms from a Kenyan alkaline soda lake.

Lipolytic enzyme production of 150 isolated strains from samples of Lake Bogoria (Kenya) was examined. Among these, fifteen isolates were selected on the basis of their lipolytic activities and subjected to morphological and 16S rRNA gene sequencing analyses for their identification. All the microorganisms have been selected under culture conditions with pH ranges between 7-10 and temperatures of 37-55 degrees C. Most of them showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v). Ten of the isolates were Gram-negative, nine of which were closely related to the Pseudomonas cluster and one to the Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining Gram-positive isolates were closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis. Four members of the Bacillus cluster and the Halomonas sp. produced lipolytic activity under alkaline conditions, while others did so at neutral pH values.

Bacillus↗

Starch hydrolysing Bacillus halodurans isolates from a Kenyan soda lake.

Fourteen obligate alkaliphilic and halotolerant bacterial isolates, exhibiting extracellular amylase activity at 55 degrees C and pH 10, were isolated from hot springs around Lake Bogoria, Kenya. From 16S rDNA sequence analysis, nine isolates shared 100% identity with Bacillus halodurans strain DSM 497T, while the rest shared 99% identity with alkaliphilic Bacillus species A-59. PCR of the intergenic spacer region between 16S and 23S rRNA genes (ISR-PCR) divided the isolates into two groups, while tDNA-PCR divided them into three groups. Bacillus halodurans DSM 497T had a different ISR pattern from the isolates, while it had a tDNA-PCR profile similar to the group that shared 99% identity with alkaliphilic Bacillus species A-59. All isolates hydrolysed soluble starch as well as amylose, amylopectin and pullulan. The amylase activity (1.2-1.8 U ml(-1)) in the culture broths had an optimum temperature of 55-65 degrees C, was stimulated by 1 mm Ca2+, and was either partially (16-30%) or completely inhibited by 1 mM EDTA. Activity staining of the cell-free culture supernatant from the isolates revealed five alkaline active amylase bands.

Amylases↗

Halomonas boliviensis sp. nov., an alkalitolerant, moderate halophile isolated from soil around a Bolivian hypersaline lake.

Halomonas boliviensis sp. nov. is proposed for two moderately halophilic, psychrophilic, alkalitolerant bacteria, LC1(T) (=DSM 15516(T)=ATCC BAA-759(T)) and LC2 (=DSM 15517=ATCC BAA-760), both of which were isolated from a soil sample around the lake Laguna Colorada, located at 4300 m above sea level in the south-west region of Bolivia. The bacteria are aerobic, motile, Gram-negative rods that produce colonies with a cream pigment. Moreover, they are heterotrophs that are able to utilize various carbohydrates as carbon sources. The organisms reduce nitrate and show tryptophan deaminase activity. The genomic DNA G+C contents were 51.4 mol% for isolate LC1(T) and 52.6 mol% for isolate LC2. Based on 16S rDNA sequence analysis, isolates LC1(T) and LC2 were identified as members of the genus Halomonas and clustered closely with Halomonas variabilis DSM 3051(T) and Halomonas meridiana DSM 5425(T). However, DNA-DNA relatedness between the new isolates and the closest related Halomonas species was low.

Base Composition↗

Chromohalobacter sarecensis sp. nov., a psychrotolerant moderate halophile isolated from the saline Andean region of Bolivia.

A moderately halophilic, aerobic, motile, Gram-negative, rod-shaped bacterium (strain LV4(T)) was isolated from saline soil around the lake Laguna Verde in the Bolivian Andes. The organism is a heterotroph, able to utilize various carbohydrates as a carbon source. It showed tryptophan deaminase, oxidase and catalase activity, but was unable to produce indole or H(2)S; nitrate was not reduced. The G+C content of the genomic DNA was 56.1 mol%. The pH range for growth was 5-10, temperature range was 0-45 degrees C and the range of NaCl concentrations was 0-25 % (w/v). On the basis of 16S rRNA gene sequence analysis, strain LV4(T) was found to be closely related to Chromohalobacter canadensis DSM 6769(T) and Pseudomonas beijerinckii DSM 7218(T); however, its DNA-DNA relatedness with these type strains was low. Strain LV4(T) resembled other Chromohalobacter species with respect to various physiological, biochemical and nutritional characteristics but also exhibited differences. Thus, a novel species, Chromohalobacter sarecensis sp. nov., is proposed, with LV4(T) (=CCUG 47987(T)=ATCC BAA-761(T)) as the type strain.

Amino Acid Oxidoreductases↗