PubMed Health⌕ Search

Biomedical subjects

M R Kula

Publications and source records attributed to M R Kula.

At least 37 records · Page 2Linked to original sources
PubMed1999

Pilot scale production and isolation of recombinant NAD+- and NADP+-specific formate dehydrogenases.

The expression of the recombinant wild-type NAD+- and mutant NADP+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from the methanol-utilizing bacterium Pseudomonas sp. 101 in Escherichia coli cells has been improved to produce active and soluble enzyme up to the level of 50% of total soluble proteins. The cultivation process for E. coli/pFDH8a and E. coli/pFDH8aNP cells was optimized and scaled up to a volume of 100 L. A downstream purification process has been developed to produce technical grade NAD+- and NADP+-specific formate dehydrogenases in pilot scale, utilizing extraction in aqueous two-phase systems.

Biomass↗
PubMed1999

Cell/adsorbent interactions in expanded bed adsorption of proteins.

Expanded bed adsorption (EBA) is an integrated technology for the primary recovery of proteins from unclarified feedstock. A method is presented which allows a qualitative and quantitative understanding of the main mechanisms governing the interaction of biomass with fluidized resins. A pulse response technique was used to determine the adsorption of various cell types (yeast, Gram positive and Gram negative bacteria, mammalian cells and yeast homogenate) to a range of commercially available matrices for EBA. Cells and cell debris were found to interact with the ligands of agarose based resins mainly by electrostatic forces. From the adsorbents investigated the anion exchange matrix showed the most severe interactions, while cation exchange and affinity adsorbents appeared to be less affected. Within the range of biologic systems under study E. coli cells had the lowest tendency of binding to all matrices while hybridoma cells attached to all the adsorbents except the protein A affinity matrix. The method presented may be employed for screening of suitable biomass/adsorbent combinations, which yield a robust and reliable initial capture step by expanded bed adsorption from unclarified feedstock.

Adsorption↗
PubMed1998

Studies of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Effect of inhibitors and low temperature.

The effect of nicotine and diphenylamine on astaxanthin biosynthesis in Xanthophyllomyces dendrorhous was studied. The effects were analysed under standard and low temperature conditions. It was found that 10 mM-nicotine inhibits the cyclization of lycopene and de novo protein synthesis was not needed to reverse the inhibition. The oxidation of beta-carotene was irreversibly inhibited by 10 microM-diphenylamine while the dehydrogenation of phytoene was reversibly inhibited by 60 microM-diphenylamine. The simultaneous exposure to low temperature (4 degrees C) overcomes the inhibition of beta-carotene oxidation at low diphenylamine concentration.

Antioxidants↗
PubMed1998

Combined preparative enzymatic synthesis of dTDP-6-deoxy-4-keto-D-glucose from dTDP and sucrose.

dTDP-6-deoxy-4-keto-D-glucose (1), the common intermediate in the biosyntheses of the manifold deoxysugars, was synthesized on a gram-scale by the combination of sucrose synthase and dTDP-D-glucose 4,6-dehydratase in a fed batch, starting the reaction with dTDP. This process allowed a dTDP conversion with a 100% rate. An easy and efficient three-step purification with anion-exchange chromatography and gel filtration gave 1.1 g of 1 in an overall yield of 73%. This work realizes a first step for an economic access to activated deoxysugars.

Biotechnology↗
PubMed1998

Analysis of hybridoma cell culture processes by SDS/gel capillary electrophoresis and matrix-assisted laser desorption ionization-time-of-flight MS.

SDS/gel capillary electrophoresis (SDS/gel CE) and matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF-MS) were employed to analyse changes in the culture broth during batch and continuous cultivation of hybridoma cells. The stability of IgG was analysed by SDS/gel CE and capillary zone electrophoresis (CZE). The results obtained by the new analytical procedures reflect the changes in the cultivation conditions very well, indicating that these tools can be used to follow animal cell culture processes. A new technique to collect fractions of very small volumes during the CZE separation is described, and the successful off-line coupling of CZE and MALDI-TOF-MS for the analysis of biotechnological processes is demonstrated.

Animals↗
PubMed1997

Rapid SDS-Gel capillary electrophoresis for the analysis of recombinant NADP(+)-dependent formate dehydrogenase during expression in Escherichia coli cells and its purification.

The level of expression in Escherichia coli cells and different steps of purification of the recombinant NADP(+)-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from bacterium Pseudomonas sp.101 was analyzed by rapid SDS-Gel capillary electrophoresis (SDS-Gel CE) and compared with SDS polyacrylamide gel electrophoresis (SDS PAGE). First standard proteins were separated in the short capillary and the calibration curve generated, then fractions taken during the fermentation and purification process were analysed. The main advantages of SDS-Gel CE are short analysis time, high sensitivity, the possibility to quantify proteins at different ultraviolet wavelength, and small injection volumes. The data for each step of the fermentation process and during the purification were controlled by spectrophotometric analysis of enzyme activity and protein concentration as well as standard SDS PAGE. The molecular mass of the purified FDH was determined as 44,078 Da by matrix-assisted laser desorption/ ionisation time of flight mass spectrometry.

Calibration↗
PubMed1997

Cloning, sequencing and overexpression of the leucine dehydrogenase gene from Bacillus cereus.

The L-leucine dehydrogenase gene from Bacillus cereus (DSM 626) was cloned from a partial genomic library and sequenced. The open reading frame has 1101 bp and codes for a protein of 39.9 kDa. The deduced amino acid sequence of the LeuDH from B. cereus shares 70-80% identity with LeuDH's from the thermophilic strains B. stearothermophilus and Thermoactinomyces intermedius. The active protein was overexpressed in Escherichia coli to yield approximately 30% of the total soluble protein.

Amino Acid Oxidoreductases↗
PubMed1997

Sodium dodecyl sulfate-polymer capillary electrophoresis for the analysis of cell culture proteins.

The application of sodium dodecyl sulfate (SDS)-polymer capillary electrophoresis (CE) to the analysis of proteins of hybridoma cell culture supernatant is demonstrated. All steps of the developed analysis are shown and discussed. Following optimization of sample preparation and concentration, as well as of the mode of injection all main protein components were separated with high resolution and selectivity. Proteolytic degradation of IgG, due to microbial contamination, was observed on SDS-gel CE and capillary zone electrophoresis (CZE). The technique was applied to monitor the cell cultivation process and concentrations of main components are demonstrated. IgG was found to increase from initially 5 mg/L to 20 mg/L after 4 days and to > 130 mg/L after 14 days of cultivation. The techniques may be applied to other biotechnological processes for the production or purification of proteins.

Calibration↗
PubMed1996

Isolation of monoclonal antibodies from cell containing hybridoma broth using a protein A coated adsorbent in expanded beds.

A novel expanded bed adsorbent carrying a recombinant protein A ligand was used for the isolation of monoclonal antibodies from cell containing hybridoma fermentation broth. The untreated effluent from a continuous hybridoma cultivation was applied to the stable expanded adsorbent (Streamline rProteinA), which proved to have a high capacity for the MAb studied (14 mg MAb per ml of adsorbent). A clarified and highly concentrated (up to 50 fold) eluate of high purity was obtained. A scale up of the MAb purification is demonstrated from lab scale (250 mg MAb per purification cycle) to a small pilot scale (2 g MAb per cycle). Low product concentration in the broth in combination with the high capacity of the adsorbent caused long sample application cycles (10-11 h). Experimental problems arising from these long cycle times are discussed with regard to a large scale application of the method.

Antibodies, Monoclonal↗
PubMed1996

Investigations on the specificity of thiophilic interaction for monoclonal antibodies of different subclasses.

A comparative study was carried out to investigate the influence of different mouse antibody subclasses on the chromatographic behaviour on thiophilic supports. Cell-free supernatants from different mouse-mouse hybridoma cultures in a standard medium were purified on thiophilic agarose and Fractogel EMD TA. The adsorption capacities and purification factors were monitored under optimised adsorption conditions. The different isotypes did not differ significantly regarding capacity of the thiophilic matrix, but the purity of the eluted antibody fractions was significantly lower for the IgG2a subclass compared to all other murine antibodies. A significant copurification of proteins from cell culture supernatant with antibodies of the IgG2a subclass indicated a restriction in the universal nature of thiophilic interaction.

Animals↗
PubMed1995

The replacement of Trp392 by alanine influences the decarboxylase/carboligase activity and stability of pyruvate decarboxylase from Zymomonas mobilis.

The bulky tryptophan residue 392 located in the deep cleft leading to the active center of pyruvate decarboxylase (PDC) from Zymomonas mobilis was changed to alanine which is found in the equivalent position of PDC from yeast. The mutation reduced the decarboxylase activity towards pyruvate by a factor of two (60-70 U/mg), whereas the Km (1.1 mM in Mes/KOH buffer) remains unchanged compared with the wild-type enzyme. The apparent Km for thiamine diphosphate (thiamin-P2) in the presence of 5 mM MgSO4 was increased by a factor of 10 (84 microM in Mes/KOH buffer) and the tetrameric mutant protein was less stable, as indicated by urea denaturation experiments. The mutation enhanced the carboligase activity of the enzyme towards benzaldehyde by a factor of four. The resulting alpha-hydroxyketone was identified as (R)-phenylacetylcarbinol.

Acetone↗
PubMed1995

Application of thiophilic membranes for the purification of monoclonal antibodies from cell culture media.

The application of thiophilic membranes for the purification of monoclonal antibodies from hybridoma culture media was studied. Affinity filtrations were performed with membrane stacks and also in a cross flow module with a spiral filtration channel. Purification factors up to five and concentration factors of about eight could be achieved. The flux behaviour was analysed and interpreted according to existing models of filtration. The results were confirmed by scanning electron microscopy. The binding capacity of the membranes differed considerably with the mode of operation. The main component responsible for membrane fouling was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid sequence analysis as bovine serum albumin or its fragments.

Antibodies, Monoclonal↗
PubMed1995

Purification and characterization of a newly screened microbial peptide amidase.

A microbial peptide amidase was found in a limited screening and purified about 500-fold from Stenotrophomonas maltophilia. The native enzyme has a molecular mass of 38 kDa (gel filtration). The sequence of the first 16 amino acids was determined by Edman degradation. The isoelectric point was found to be around 5.8. The peptide amidase exhibited a pH optimum of 6.0 and a temperature optimum of about 39-45 degrees C. The enzyme is stable in 50 mM TRIS/HCl, pH 7.5, at 30 degrees C, and the residual activity was found to be above 90% after 1 week of incubation. The biocatalyst is not inhibited by potential inhibitors like Hg2+, EDTA, D-cycloserine or dithiothreitol and only weakly influenced by inhibitors of serine proteases. The peptide amidase deamidates selectively C-terminal amide groups in peptide amides without hydrolysing internal peptide bonds or amide functions in the side-chain of glutamine or asparagine. Unprotected amino acid amides are not hydrolysed. The enzyme is stereoselective with regard to L-enantiomers in the C-terminal position.

Amidohydrolases↗
PubMed1995

Performance of a Graesser contactor in the continuous extraction of whey proteins: mixing, mass transfer and efficiency.

The performance of a Graesser Raining Bucket Contactor in an extraction using aqueous two-phase systems was characterized by axial mixing coefficients and counter current mass transfer for whey protein purification. The influence of rotor speed, phase ratio and phase velocity on the salt phase axial mixing coefficients was determined applying a dispersion model to the residence time distribution experiments. When dissolving whey powder in the salt-rich phase of the system, alpha-lactalbumin was extracted to the polyethylene-glycol-rich phase, whereas beta-lactoglobulin predominantly remained in the bottom phase, making the process suitable for removal of the major allergen beta-lactoglobulin. The mass transfer in the phase system was determined from the steady state data of alpha-lactalbumin extraction. On the basis of a diffusion model, the efficiency of the counter current extraction under different process conditions was calculated from results of the mass transfer experiments.

Cheese↗
PubMed1994

Reversible dissociation and unfolding of pyruvate decarboxylase from Zymomonas mobilis.

The denaturation and renaturation process of pyruvate decarboxylase (PDC) from Zymomonas mobilis (ATCC 29191) has been investigated using guanidine hydrochloride and urea as denaturing agents. The quarternary structure of the homotetramer is strongly stabilized by the cofactors Mg2+ and thiamine diphosphate (TDP). The structural transitions were monitored by activity measurements, fluorescence spectroscopy, circular dichroism and gel-filtration chromatography. A three-step denaturation process, described as follows, is indicated by non-coincidental denaturation curves: (a) inactivation of the tetramer upon dissociation of cofactors (> 0.4 M guanidine hydrochloride, > 1 M urea); (b) dissociation of the tetramer into monomers (> 1 M guanidine hydrochloride, > 3 M urea); (c) complete unfolding of these (> 2.5 M guanidine hydrochloride, > 5 M urea). The refolding process initiated by rapid dilution of fully denatured protein in renaturation buffer involves the rapid reassociation of an inactive intermediate followed by the reconstitution of the active site.

Amino Acid Sequence↗
PubMed1994

Purification of UDP-glucose pyrophosphorylase from germinated barley (malt).

UDP-glucose pyrophosphorylase was purified from germinated barley (malt) using anion exchange and hydrophobic interaction chromatography followed by preparative gel filtration. Gel filtration and SDS-PAGE revealed a molecular mass of 51 to 54 kDa for the monomeric protein. Microsequencing of the blotted protein by Edman degradation gave 20 N-terminal amino acids. UDP-glucose pyrophosphorylase from malt could be markedly stabilized by the addition of bovine serum albumin. The enzyme preparation is free of contaminating nucleoside triphosphatases (UTPases) and can be utilized for the enzymatic synthesis of activated sugars.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of a novel carbonyl reductase isolated from Rhodococcus erythropolis.

During growth on n-tetradecane a novel NADH-dependent carbonyl reductase is induced in the Gram-positive bacterium Rhodococcus erythropolis (Peters, P., Zelinski, T. and Kula, M.R. (1992) Appl. Microbiol. Biotechnol. 38, 334-340). The enzyme has been purified to homogeneity using fractional pH precipitation, anion exchange chromatography and affinity chromatography. The isoelectric point of the oxidoreductase is 4.4. The apparent molecular mass of the native enzyme is 161 kDa, that of the subunits 40 kDa as determined by SDS gel electrophoresis. A tetrameric structure of the carbonyl reductase is consistent with these results. Important biochemical data concerning the application of the reductase are: a broad pH-optimum, temperature optimum at 40 degrees C and stability at room temperature for more than 5 days. The oxidoreductase accepted as substrate aliphatic and aromatic ketones, keto esters (esters of keto carboxylic acids) and halogenated carbonyl compounds and reduced them to the corresponding hydroxyl compounds with (S)-configuration with more than 98% enantiomeric excess. The NAD(+)-dependent oxidation of primary alcohols was not catalyzed by the carbonyl reductase, whereas secondary alcohols and hydroxy acid esters were oxidized to the corresponding carbonyl compounds at about 10-fold slower reaction rates compared to the reduction.

Alcohol Oxidoreductases↗