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M R Kula

Publications and source records attributed to M R Kula.

At least 73 records · Page 4Linked to original sources
PubMed1991

Analysis of polymer molecular weight distributions in aqueous two-phase systems.

The partitioning of proteins and other biomaterials between two aqueous phases containing polyethyleneglycol and dextran is a strong function of the molecular weight of the two polymers. Although both polymers are polydispersed (especially Dx) most theoretical treatments refer only to the average molecular weight (number or mass) and assume that the molecular weight distribution of each polymer is the same in both phases. In this work the molecular weight distribution of each polymer is the same in both phases. In this work the molecular weight distributions of four stock solutions of PEG (4000, 6000, 10,000 and 20,000) and four stock solutions of Dx (10,000, 40,000, 110,000 and 500,000) were measured using High Performance Gel Chromatography. The measurements were repeated on the phases formed by the polymer solutions after they were mixed and allowed to equilibrate. The molecular weight distribution of the Dx differed in the top and bottom phase; both differed from that of the stock solution. Although we believe that the molecular weight distribution for PEG also differs in the top and bottom phases, we were unable to determine this within the resolution of our instruments.

Dextrans↗
PubMed1991

Temperature dependence of the partition coefficient of proteins in aqueous two-phase systems.

We report the partition coefficients of lysozyme, chymotrypsinogen-A, albumin and catalase in sixty four Polyethyleneglycol/Dextran/Water systems at 4, 25 and 40 degrees C. We found that the partition coefficients of the four proteins generally increase with increasing temperature. The influence of temperature on the partition coefficient seems to be highly dependent on the kind of protein which is partitioned and on the total polymer concentration, but does not, in general, depend on the molecular weight of the polymers. The partition coefficients of small and hydrophilic proteins like lysozyme and chymotrypsinogen-A are only slightly affected by changes in temperature, while the partition coefficients of bigger and more hydrophobic proteins like albumin and catalase are strongly affected by changes in temperature. The results suggest the incorporation of attractive forces (possible electrostatic) into a model previously reported by us.

Albumins↗
PubMed1991

The partitioning of cholesterol oxidase in Triton X-114-based aqueous two-phase systems.

Cholesterol oxidase from various bacterial sources (membrane-bound and extracellular) was studied in Triton X-114R solutions above the cloud point. The influence of temperature, salt, enzyme concentration and source, and pH on phase equilibrium and enzyme partitioning was investigated in this detergent-based aqueous two-phase system. The method combines remarkable recovery (over 70% and 90% in the detergent-rich phase for the extracellular and membrane-bound forms, respectively) and 10 to 20-fold concentration of the enzyme in just one purification step. The results from cholesterol oxidase are compared with other proteins, both hydrophobic and hydrophilic. The system shows considerable promise for selectively partitioning proteins based on their surface hydrophobicity.

Biotechnology↗
PubMed1991

Investigation of the UDP-glucose dehydrogenase reaction for a coupled assay of UDP-glucose pyrophosphorylase activities.

An optimized coupled enzyme assay for UDP-glucose pyrophosphorylase (EC 2.7.7.9) using UDP-glucose dehydrogenase (EC 1.1.1.22) is presented. This optimized assay was developed by a detailed investigation of the kinetics of the UDP-glucose dehydrogenase reaction. In addition the data provide a basis for the enzymatic synthesis of UDP-glucuronic acid. The results demonstrate that the two binding sites of the dehydrogenase differ since a different modulation of the enzyme activity and stability is observed after preincubation with UDP-glucose or NAD+ at various pH values. This is of general interest for the preparation of assay mixtures where UDP-glucose dehydrogenase is used as an auxiliary enzyme.

Enzyme Stability↗
PubMed1991

Influence of water miscible organic solvents on orange flavedo peptide amidase.

Due to the broad substrate spectrum and stability in some water-miscible organic solvents, the peptide amidase from orange flavedo was found to be a suitable enzyme for experiments in water-miscible organic solvents. Immobilization and the kind of organic solvent had no significant influence on the kinetic data while the polarity of the substrate caused a great difference in the apparent Km values. The results are explained by inadequate partition of the substrate between the microenvironment of the enzyme and the reaction solution.

Amidohydrolases↗
PubMed1991

Immunoaffinity partitioning: synthesis and use of polyethylene glycol-oxirane for coupling to bovine serum albumin and monoclonal antibodies.

Polyethylene glycol (PEG)-oxirane was synthesized by reacting aminated monomethoxy-PEG 5000 (NH2-MPEG 5000) with butanediol diglycidyl ether and used to derivatize bovine serum albumin (BSA) and monoclonal antibodies (mAb) against horseradish peroxidase (HRP) and porcine lactate dehydrogenase isoenzyme 5, respectively. Determination of oxirane end groups revealed a very high number, which arise from the chain breaks of the polymer. Covalent coupling of PEG-oxirane to BSA resulted in 30-50 times higher partition coefficients under optimized conditions. The mAb investigated could be modified with PEG-oxirane while retaining its binding properties and could be used as an affinity ligand for selective extraction of Ag in immunoaffinity partitioning. However, a high degree of modification results in a lower binding constant of mAb anti-HRP and higher [mAb]/[Ag] concentration ratios in immunoaffinity partition experiments.

Antibodies, Monoclonal↗
PubMed1990

Investigation of affinity partition chromatography using formate dehydrogenase as a model.

The enzyme formate dehydrogenase (FDH) was purified from the crude extract of Candida boidinii by affinity partition chromatography. The partition coefficient, K, of the enzyme was selectively increased by adding polyethylene glycol-Procion Red HE3b as an affinity ligand to the mobile phase in the chromatographic column. The increased K value led to early elution of the enzyme-ligand complex and separated the target protein from the main peak of the contaminants.

Candida↗
PubMed1990

Application of carboxypeptidase C for peptide synthesis.

Carboxypeptidase C partially purified from the flavedo of citrus fruit by a new, simple procedure was studied as a catalyst for peptide-bond formation. Dipeptides were obtained in high yields (80-95%) with Bz--Tyr--OEt as carboxyl-compound, and amino acid amides and amino acid alkylesters as nucleophiles. To characterize the synthesis reaction, a number of parameters such as pH, excess of the nucleophile, and the molarity of the buffer were evaluated. The yield of dipeptides depends on the side chain of the amino acid alkylester used as the carboxyl component as well as on the N-terminal protecting group. Esterase activity was minimal in the absence of a nucleophile, suggesting a modified mechanism for the synthesis reaction compared to other serine proteases. No secondary hydrolysis of the peptides formed was observed.

Carboxypeptidases↗
PubMed1990

Monitoring of enzymes during chromatographic separations.

An on-line enzyme assay is presented based on flow injection techniques combined with fluorimetric detection. It allows to monitor NAD-dependent oxidoreductases during the purification of microbial crude extracts or partially purified enzymes by fast protein liquid chromatography (FPLC) in a near real-time mode. The arrangement is simple and can be easily integrated in the chromatographic system avoiding dead volumes. A high measuring frequency (up to 180 samples h-1) and a short response time (10-30 s) are achieved. The method has a low limit of detection (approximately 0.01 U ml-1), and a good reproducibility (1-4%), the injected sample volume is only 2 microliters.

Alanine Dehydrogenase↗
PubMed1990

Pilot- and process-scale techniques for cell disruption.

Microorganisms are a source of protein with catalytic and/or biological activity, which are of increasing commercial interest for applications in industry or therapy. For the isolation of intracellular products cell disruption is necessary. In principle, chemical, biological, or physical means may be employed to release proteins from cells. These different approaches are reviewed with special emphasis on scale-up and possible industrial operation. Mechanical devices have been improved considerably in recent years and appear most universally suited to cell disintegration. Chemical extraction or enzymatic lysis offers improved selectivity but requires individual procedures for each product. For a final process design, product yield and cost of the unit operation must be considered as well as the possible implications for the subsequent steps in product recovery, especially on solid/liquid separation.

Cell Membrane Permeability↗
PubMed1990

Optimization of enzyme-mediated peptide bond formation.

Enzyme-catalyzed peptide bond formation requires thorough examination and optimization of each coupling step. In order to identify factors influencing the selectivity between aminolysis and hydrolysis, a systematic study was carried out for the kinetically controlled peptide synthesis. The reaction temperature, the type of C-terminal protecting group, and different organic cosolvents showed little influence on the selectivity. The enzyme, excess nucleophile, pH, N-terminal protecting group, and ionic strength of the solution were identified as major factors controlling the selectivity and, therefore, the yield of the dipeptide synthesis. Under optimized conditions, the selectivity of the chymotrypsin-catalyzed synthesis of PheSer could be increased from 35 to 100%.

Carboxypeptidases↗
PubMed1988

Enzymatic determination of L-phenylalanine and phenylpyruvate with L-phenylalanine dehydrogenase.

An enzymatic method is described for the determination of L-phenylalanine or phenylpyruvate using L-phenylalanine dehydrogenase. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine or the reductive amination of the 2-oxoacid, respectively. The stoichiometric coupling of the coenzyme allows a direct spectrophotometric assay of the substrate concentration. The equilibrium of the reaction favors L-phenylalanine formation; however, by measuring initial reaction velocities, the enzyme can be used for L-phenylalanine determination, too. Standard solutions of L-phenylalanine in the range of 10-300 microM and of phenylpyruvate (5-100 microM) show a linearity between the value for dENADH/min and the substrate concentration. Besides phenylalanine, the enzyme can convert tyrosine and methionine, and their oxoacids, respectively. The Km values of these substrates are higher. The influence of tyrosine on the determination of phenylalanine was studied and appeared tolerable for certain applications.

Amino Acid Oxidoreductases↗
PubMed1987

Studies on the enzymatic hydrolysis of amino acid carbamates.

Several commercially available enzymes were tested for their ability to hydrolyze amino acid carbamates. No activity was found with pig liver esterase, the hydantoinase from Pseudomonas fluorescens DSM 84, or the urease from jack beans. A stereoselective cleavage of the carbamyl group yielding L-amino acids was observed by acylase and acetylcholinesterases from bovine and human erythrocytes. Racemic mixtures of N-(methoxycarbonyl)-DL-alanine, N-(ethoxycarbonyl)-DL-alanine, and the corresponding valine carbamates are hydrolyzed to L-alanine and L-valine, respectively, by acylases leaving the D-amino acid carbamates unchanged. The lysine carbamates were not hydrolyzed by acylases. In contrast only the methoxycarbonyl amino acids were split by acetylcholinesterases, which, however, also cleave alpha, epsilon-(N-methoxycarbonyl)-DL-lysine stereoselectively at the alpha position, yielding epsilon-N-methoxycarbonyl-L-lysine. The optimum pH for enzymatic activity of hog kidney acylase was 7.5 and a Km value of 8.2 mM for N-(methoxycarbonyl)-DL-alanine was determined. For the acetylcholinesterases the reaction rate reaches an optimum between pH 7.5 and 8. The Km value was 68 mM for N-(methoxycarbonyl)-DL-alanine.

Acetylcholinesterase↗