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M R Kula

Publications and source records attributed to M R Kula.

At least 55 records · Page 3Linked to original sources
PubMed1994

Purification and characterisation of a microbial L-carnitine amidase.

A novel enzyme, L-carnitine amidase, was purified about 140-fold from a newly screened microorganism (DSM 6320) to yield a homogeneous protein. The native enzyme has a molecular mass of 125 kDa (gel filtration) and consists of two identical subunits as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Edman degradation. The pH optimum was found around pH 8.5. Out of 60 chemicals tested as substrates (amides of various aliphatic and aromatic acids, nitriles, amino acid amides and dipeptide amides) the amidase hydrolysed only L-carnitine amide. The Michaelis constant (Km) was found to be 11.6 mM, and the pure protein had a specific activity of 328 units/mg. Complex kinetics were observed with the racemic mixture of D,L-carnitine amide as starting material during enzymatic hydrolysis.

Amidohydrolases↗
PubMed1994

A kinetic study and application of a novel carbonyl reductase isolated from Rhodococcus erythropolis.

The newly described carbonyl reductase from Rhodococcus erythropolis (RECR) accepts a broad range of substrates. Based on the kinetic constants of a variety of methyl and ethyl ketones a hypothetical model of the substrate-binding site is proposed. Whether a substrate of interest may be reduced by the RECR can be predicted from this model together with the kinetic data. A study of initial velocities and product inhibition is presented, which shows that the kinetics of the RECR follow a Theorell-Chance mechanism. The pro-R hydride of NADH is transferred by the enzyme to the re face of the carbonyl compounds yielding (S)-alcohols. The reduction of methyl 3-oxobutanoate and ethyl 4-chloro-3-oxobutanoate catalyzed by the oxidoreductase lead to the corresponding hydroxy compounds with high enantiomeric purity [enantiomeric excess (e.e.) > or = 99%]. The synthesis of ethyl (2R,3S)-3-hydroxy-2-methylbutanoate was accomplished with high diastereoselectivity (diastereomeric excess = 95%) and enantioselectivity (e.e. > or = 95%).

Alcohol Oxidoreductases↗
PubMed1994

Multichannel flow-injection-analysis biosensor system for on-line monitoring of glucose, lactate, glutamine, glutamate and ammonia in animal cell culture.

The application of a chemiluminometric method for the on-line monitoring of a hybridoma cell culture is described. Enzyme sensors for glucose, lactate, glutamine, glutamate and ammonia, based on oxidase-catalysed reactions, were developed and connected to a flow-injection-analysis (f.i.a.) biosensor. H2O2 produced by the oxidase-catalysed enzyme reaction was detected by luminol chemiluminescence with a fibre-optic H2O2 biosensor. The system has been used to monitor animal cell cultures. A continuous hybridoma cell cultivation for the production of monoclonal antibodies is presented as an example. It was possible to monitor the bioprocess over a period of 15 days. A complete analysis of all five components could be performed within 42 min. The enzyme sensors were stable during the whole cultivation time without significant loss of activity. The computer-controlled biosensor f.i.a. works with good reliability. The precision for all five components ranged between 2.2 and 4.5%. It was possible to determine glutamine in one step using an anti-interference enzyme reactor. Endogenous glutamate was completely removed up to a level of 0.5 mM.

Ammonia↗
PubMed1993

Microbial synthesis of 3-deoxy-D-erythro-hex-2-ulosonic acid 6-phosphate.

A microbial route was explored for the synthesis of 3-deoxy-D-erythro-hex-2-ulosonic acid 6-phosphate (2-keto-3-deoxy-6-phosphogluconate, KDPG). Two strains of bacteria, Alcaligenes eutrophus H16 F34 (DSM 529) and Escherichia coli DF 71 (CGSC 4880), lacking in KDPG-aldolase activity were tested for excretion of KDPG. Using pyruvate and gluconate as carbon sources, Alcaligenes eutrophus H16 F34 accumulated and excreted 3-deoxy-D-erythro-hexulosonic acid 6-phosphate into the culture broth, while the E. coli strain, using pyruvate and glucuronate, failed. KDPG was isolated from the culture supernatant of Alcaligenes eutrophus H16 F34 in 78% yield and 5 g scale with respect to the consumed gluconate.

Alcaligenes↗
PubMed1993

Studies on the substrate specificity of a peptide amidase partially purified from orange flavedo.

We recently reported the isolation and some properties of an unusual enzyme called peptide amidase (Steinke, D. and Kula, M. R. Angew. Chem. Int. Ed. Engl. 1990, 29, 1139-1140). Here we describe the partial purification of the enzyme from the flavedo of orange fruits and discuss results of a detailed study of the substrate range of the peptide amidase, which is extremely wide and useful for a C-terminal enzymatic deprotection in peptide synthesis under very mild conditions. The substrate spectrum includes protected or unprotected peptide amides and N-protected amino acid amides. The chain length of the substrate peptide amide, as well as the amino acid composition, including the C-terminal amino acid side chain, are of minor importance. The peptide amidase is stereoselective with regard to the C-terminal position, since only L-amino acid amides are accepted as substrates, with the exception of proline. Notably, side chain amides are not deamidated. The peptide amidase is free of any proteolytic activity, which would hydrolyze internal peptide bonds of substrate peptides. In the penultimate position D-amino acids are tolerated; peptide modifications toward the N-terminal region do not abolish the enzymatic deamidation at the C-terminus.

Amidohydrolases↗
PubMed1993

A novel NADH-dependent carbonyl reductase with an extremely broad substrate range from Candida parapsilosis: purification and characterization.

A novel oxidoreductase catalyzing the NADH-dependent reduction of a variety of carbonyl compounds, especially keto esters, was found in Candida parapsilosis DSM 70125. The enzyme was purified by fractional poly(ethylene glycol) precipitation, anion exchange, and affinity chromatography. The enzyme was enriched about 3100-fold and appeared to be homogeneous as judged by native and sodium dodecyl sulfate gel electrophoresis. The carbonyl reductase from C. parapsilosis is a dimeric enzyme with an apparent molecular mass of about 135 kDa. Important properties concerning the application of the enzyme are the relatively broad pH optimum between pH 6.5 and 9.0, temperature optimum between 36 and 42 degrees C, and good stability. Besides keto esters, the new enzyme reduces other aliphatic, aromatic, and cyclic ketones, as well as aldehydes and ketoacetals with high reaction rates. 4-Halo-3-hydroxybutanoates, which are promising chiral intermediates for the chemical synthesis of L-carnitine, alkaloids and pharmaceuticals, are now accessible by enzymatic reduction, as well as several phenyl-ethanol derivatives, which are important for the synthesis of pharmaceuticals and agrochemicals. The preparative applicability of the enzyme was demonstrated in a coupled enzyme system with regeneration of coenzyme. Methyl 3-oxobutanoate was converted into methyl (S)-(+)-3-hydroxybutanoate (98.5% ee), a versatile chiral building block for the synthesis of pheromones and different antibiotics.

Acetoacetates↗
PubMed1993

Protein partitioning in detergent-based aqueous two-phase systems.

Aqueous solutions of nonionic polyoxyethylene detergents form two liquid phases upon temperature increase above the cloud point. One of these phases is detergent-enriched and called the coacervate phase, whereas the other is detergent-depleted. Protein partitioning in such detergent-based aqueous two-phase systems was studied systematically and quantitatively, employing a series of similar polyoxyethylene detergents and proteins of varying hydrophobicity. Increasing the detergent alkyl chain length, temperature or salt concentration leads to an increase of detergent separating into the coacervate phase and a concomitant increase of protein. The positive correlation between protein hydrophobicity and partitioning into the coacervate phase confirms that protein-detergent interactions in such systems are primarily hydrophobic. These detergent-based systems can be applied to membrane proteins as well as water-soluble proteins which possess hydrophobic domains.

Animals↗
PubMed1993

Investigation of sucrose synthase from rice for the synthesis of various nucleotide sugars and saccharides.

The unique character of the plant glucosyltransferase sucrose synthase, to catalyse in vitro the synthesis and cleavage of sucrose under appropriate conditions, can be exploited for the enzymatic synthesis of carbohydrates. The present paper describes the potential utilization of sucrose synthase from rice for the enzymatic synthesis of activated sugars and saccharides. In the cleavage reaction of sucrose, the nucleoside diphosphates can be used in the order UDP > TDP > ADP > CDP > GDP to obtain the corresponding activated glucoses. In batch reactions, > 90% conversion of UDP and TDP could be achieved. Substituting different di- and trisaccharides for sucrose in the cleavage reaction with UDP 2-deoxysucrose was the most promising substrate. Sucrose synthase was combined with UDP-galactose 4'-epimerase and beta 1-4 galactosyltransferase to synthesize N-acetyllactosamine with in situ regeneration of UDP-glucose. In the synthesis reaction of sucrose synthase, different donor (UDP-sugars) and acceptor substrates were investigated. UDP-N-acetylglucosamine and UDP-xylose could be used in combination with fructose as acceptor. D-Xylulose, D-tagatose, D-lyxose, D-psicose, L-sorbose, D-mannose, L-arabinose, 1,6 anhydroglucose, lactulose, raffinose and isomaltulose can serve as acceptors for UDP-glucose.

Disaccharides↗
PubMed1993

Improved purification of an (R)-oxynitrilase from Linum usitatissimum (flax) and investigation of the substrate range.

The purification of (R)-oxynitrilase (EC 4.1.2.10) from Linum usitatissimum (flax) has been improved considerably. The enzyme is obtained from seedlings in 60% yield by fractional salt precipitation followed by ion-exchange and hydrophobic-interaction chromatography. Final gel-permeation chromatography yields a protein with a specific activity of 53 units/mg at pH 4.1. The N-terminal sequence is reported and microheterogeneity demonstrated. The substrate range was investigated using (R)-oxynitrilase immobilized on Eupergit and t-butyl methyl ether as solvent. The addition of HCN to various aliphatic ketones and aldehydes is catalysed by the enzyme, while aromatic ketones are not converted. (R)-Butan-2-one cyanohydrin was synthesized on a preparative scale and the product characterized.

Aldehyde-Lyases↗
PubMed1992

Purification of S-oxynitrilase from Sorghum bicolor by immobilized metal ion affinity chromatography on different carrier materials.

The purification of the hydroxynitrile lyase (EC 4.1.2.11, S-oxynitrilase) from Sorghum bicolor is compared using different strategies. A new procedure is presented, which exploits the affinity of S-oxynitrilase towards metal ions as a key step in purification. The metal ions are immobilized by chelators on different carrier materials, e.g. Sepharose beads, microporous membranes or poly(ethylene glycol). A systematic examination demonstrates the excellent potential of immobilized metal affinity chromatography as a preparative separation method.

Aldehyde-Lyases↗
PubMed1992

Studies on the enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester.

The enzymatic reduction of N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester, the key intermediate in the stereoselective synthesis of a statinanalogue, was studied with Hansenula anomala and Hansenula silvicola. Using whole cells of H. anomala gives complete conversion and a diastereomeric excess of 88% of the desired 3S, 4S statinanalogue. The strain contains two NADPH-dependent oxidoreductases, that can be separated by ion exchange chromatography or gelfiltration, yielding the 3S, 4S or 3R, 4S stereoisomers, respectively, with > 99% diastereomeric excess (DE). In the crude extract the 3S, 4S oxidoreductase is very unstable and could be purified with << 1% yield only. In contrast, H. silvicola, which gave poor conversions using whole cells, exhibited about 80-fold higher specific activity in the crude extract than H. anomala. The NADPH-dependent oxidoreductase was purified 317-fold in 12% yield. A single enzyme of 54 kDa reduces the substrate with 97.4% DE. Besides the statinanalogue a wide range of other compounds could be reduced, most notably diones and chinones such as isatin or campherchinone. It was demonstrated that the enzymes often discussed for the reduction of beta-ketoesters with yeast e.g. L-3-hydroxyacyl CoA dehydrogenase (EC 1.1.1.35), the beta-ketoreductase of the fatty acid synthase complex and also the 3-hydroxy-3-methyl glutaryl-CoA dehydrogenase (EC 1.1.1.34) are separated during the purification steps from the oxidoreductase acting on N-Boc-4S-amino-3-oxo-5-phenylpentanoic acid methylester. The physiological role of the new enzyme is still unknown.

Amino Acids↗
PubMed1992

The use of detergent-based aqueous two-phase systems for the isolation of extracellular proteins: purification of a lipase from Pseudomonas cepacia.

The partitioning of a variety of extracellular lipases, both pro- and eucaryotic, in detergent-based aqueous two-phase systems was examined. The results revealed that all procaryotic lipases showed a clear preference for the detergent-rich coacervate phase. In contrast, all eucaryotic lipases were significantly excluded from this phase, most probably caused by their glycosylation. The potential of such detergent-based systems for the isolation of extracellular lipases directly from cell-free culture broth was analyzed using the bacterium Pseudomonas cepacia (DSM 50181). This strain was identified after a limited screening for lipase activity. About 76% of the lipase could be extracted into the coacervate phase in just one purification step, leading to a four-fold concentration of lipase and a purification factor of 24.

Animals↗
PubMed1992

Sequential membrane-based purification of proteins, applying the concept of multidimensional liquid chromatography (MDLC).

A purification method for formate dehydrogenase from Candida boidinii has been designed by using a sequence of membrane-based adsorbents. The membranes carrying ion-exchange and dye ligands (Sartobind) are general-purpose adsorbents for proteins. The membranes were used in an on/off mode. A sequence of cation exchange, dye-ligand and anion exchange allows to purify the enzyme as judged by SDS-PAGE and assay of specific activity. The sequence of ligands and the buffer conditions were chosen in such a way that the fraction eluted from one membrane could be directly loaded on the next membrane. Thereby an integrated version of MDLC was realized with membrane-based adsorbents. The excellent scaleability from micro- to laboratory scale (factor 40) indicates that the method could afford a general approach to develop purification trains since the micro-scale allows for a rapid screening of adsorbent types and sequences.

Candida↗
PubMed1992

Purification and primary structure of pyruvate decarboxylase from Zymomonas mobilis.

Pyruvate decarboxylase (E.C. 4.1.1.1), the key enzyme in the glycolytic pathway to ethanol, was isolated in gram amounts from Zymomonas mobilis for structural studies. The primary structure was determined by automated Edman degradation and compared with that deduced from the DNA sequence of the structural gene, previously published by two groups (A. D. Neale, R. K. Scopes, R. E. H. Wettenhall, and N. J. Hoogenraad, 1987, Nucleic Acids Res. 15, 1753-1761; M. Reynen, and H. Sahm, 1988, J. Bacteriol. 170, 3310-3313). The peptide data differ from the published DNA sequences, which also deviate from each other. Crystals diffracting to about 0.3 nm resolution have been obtained by the hanging drop vapor diffusion method. The space group was identified as P4(1)22 or its enantiomorphs containing presumably one tetramer per asymmetric unit.

Amino Acid Sequence↗
PubMed1991

Recycling of salts in partition protein extraction processes.

Aqueous two-phase extraction systems were developed to separate intracellular proteins from cell debris. To economize on the use of chemicals as well as to minimize environmental pollution, a closed mode of operation was sought. Various approaches to achieve recycling of salt from the primary lower phase containing 30-60% (w/w) of the added salt together with cell debris, proteins and nucleic acids were studied. Techniques assessed included microfiltration, heat treatment and the extraction with phase systems formed by aliphatic alcohols. With 20% (w/w) primary lower phase it was found to be possible to separate 95% of the salt in 3-4 theoretical steps using counter-current extraction.

Ethanol↗
PubMed1991

Partition of free and monoclonal-antibody-bound horseradish peroxidase in a two-phase aqueous polymer system--novel procedure for the determination of the apparent binding constant of monoclonal antibody to horseradish peroxidase.

The principle that the antigen and the antibody prefer different phases in an aqueous two-phase system is the analytical basis of the work presented here. The antigen horseradish peroxidase, which is bound to a monoclonal antibody (mAb), is separated from free Ag in an aqueous phase system (polyethylene glycol (PEG)/dextran) as a function of the concentration of mAb. The plot of the partition coefficient kappa of horseradish peroxidase versus the concentration of mAb yields a sigmoidal curve similar to the curve obtained by enzyme-linked immunosorbent assay (ELISA). Comparing the plots normally used for ELISA in order to determine the apparent binding constant of mAb and the number of epitopes on the Ag we derived a relationship between the difference in partitioning of the free Ag and the bound Ag (delta kappa) and the concentration of mAb. The new linear plot of reciprocal delta kappa versus reciprocal concentration of mAb gives the apparent binding constant of mAb, which is evaluated from the slope. From the intercept at the ordinate the maximum difference of the partition coefficient of the free and bound antigen is derived and the apparent partition coefficient of the free monoclonal antibody can be calculated.

Animals↗
PubMed1991

Formation of peptide bonds by carboxypeptidase c from orange leaves.

Carboxypeptidase c partially purified from orange leaves was studied as a catalyst for enzymatic peptide synthesis. Various N-protected ester- and nucleophile compounds were evaluated in order to determine the substrate specificity. For further characterization of the synthetic reaction, optimum pH and the influence of the N-terminal protecting group were studied. Kinetic investigations revealed considerable differences in Km and Vmax for the nucleophile when the N-terminal protecting group of the substrate was varied.

Amino Acid Sequence↗