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Biomedical subjects

K Mosbach

Publications and source records attributed to K Mosbach.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of a recombinant bifunctional enzyme, galactose dehydrogenase/bacterial luciferase, displaying an improved bioluminescence in a three-enzyme system.

The two structural genes encoding galactose dehydrogenase (Pseudomonas fluorescens) and the beta subunit of luciferase (Vibrio harveyi) were fused in-frame in order to prepare and subsequently characterize an artificial bifunctional enzyme complex. This hybrid enzyme exhibited both galactose dehydrogenase activity and bioluminescence when expressed in Escherichia coli together with the alpha subunit of luciferase. The purified conjugate was used to study possible proximity effects in a sequential three-enzyme reaction with the bifunctional enzyme catalyzing the first and the last reaction. The intermediate enzyme, diaphorase, was added separately. The engineered enzyme system, comprising the galactose dehydrogenase/luciferase conjugate, could display a twofold higher bioluminescence in the overall enzyme reaction compared to a corresponding reference system with separate native enzymes. The increased bioluminescence obtained for the engineered enzyme system is proposed to be due to an improved organization of the enzyme in solution.

Dihydrolipoamide Dehydrogenase↗
PubMed1992

Peptide screening.

Since late 1990, there have been several advances in preparing and screening large numbers of various peptides. Developments have continued in methods of peptide screening based on peptides exposition on coat proteins, produced via fusion coliphage constructs. Further developments have been made in increasing the multitude of peptides produced by the chemical synthetic strategy, including light-directed, spatially addressable chemical synthesis, single-bead, single-peptide synthesis, as well as iterative peptide selection and synthesis.

Amino Acid Sequence↗
PubMed1991

The oligopeptide (Gly-Pro)2-Ala-(Gly-Pro)2 increases the internal proline level and improves NaCl tolerance when produced in Escherichia coli.

We have produced various proline-containing peptides as translational fusions with beta-glucuronidase (beta Glu) in Escherichia coli. When these linkers were introduced in the 5'-end of the beta Glu-encoding gene, the production of the enzyme was increased substantially in vivo. The peptides carrying repetitive Gly-Pro sequences could also stimulate the growth of the transformants in media with inhibitory concentrations of NaCl. Furthermore, the freezing tolerance could be improved.

Escherichia coli↗
PubMed1991

Metal affinity precipitation of proteins carrying genetically attached polyhistidine affinity tails.

In this study, galactose dehydrogenase (EC 1.1.1.48) was chosen as a prototype target protein to investigate the capability of metal affinity precipitation to facilitate the purification of genetically engineered proteins. A DNA fragment encoding five histidine residues was fused to the 3'-terminal end of the galactose dehydrogenase gene from Pseudomonas fluorescens and thereafter expressed in Escherichia coli. The additional five histidines functioned as an affinity tail and the modified enzyme could be purified using metal affinity precipitation when the metal-chelate complex with ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetra-acetic acid, EGTA(Zn)2, was added to the protein solution. The affinity tail could also be applied for the purification of the fusion protein utilising immobilised metal affinity chromatography. After purification, the pentahistidine affinity tail could be removed enzymatically by carboxypeptidase A. Furthermore, growth rate experiments demonstrated that the expression of the metal-binding affinity tail in E. coli cells enhanced the tolerance to zinc ions when added to the growth medium.

Amino Acid Sequence↗
PubMed1991

Preparation of a genetically fused protein A/luciferase conjugate for use in bioluminescent immunoassays.

The genes encoding staphylococcal protein A and bacterial luciferase (Vibrio harveyi) were fused in-frame in order to obtain a general marker enzyme for bioluminescent immunoassays. Two constructs were made where protein A was ligated to the first and the 12th amino acid residue, respectively, of the N terminus of the beta subunit of luciferase. Only the first fusion protein encoding the entire beta subunit was able to form an enzymatically active luciferase complex when expressed together with the alpha subunit. The fusion of protein A to luciferase did not notably alter the emitted wavelength spectrum or its stability to urea treatment. The fusion protein was found to retain at least 50% of the specific bioluminescent activity compared to native luciferase. In preliminary tests, this hybrid protein was shown to be useful in bioluminescent immunoassays.

Animals↗
PubMed1991

Multienzyme systems obtained by gene fusion.

The preparation of artificial bi- and polyfunctional enzymes by gene fusion appears to have great potential in enzyme technology. Their purification is facilitated compared with that of their naturally occurring counterparts and they exhibit favourable enzyme kinetics. Applications can be found in biochemical analysis, enzyme process technology and metabolic engineering.

Cloning, Molecular↗
PubMed1991

Continuous regeneration of NAD(H) covalently bound to a cysteine genetically engineered into glucose dehydrogenase.

We introduced a cysteine residue on the surface of glucose dehydrogenase from Bacillus subtilis using site-directed mutagenesis. To this mutant, an NAD-analogue was covalently attached by a disulphide bridge so that it was active intramolecularly. The glucose dehydrogenase-cys44-NAD complex, which contained one reactive NAD molecule per subunit of glucose dehydrogenase, was operated together with lactate dehydrogenase in a coupled enzymatic regeneration of NAD(H) in a hollow fiber reactor. L-lactate and gluconic acid were continuously produced from pyruvate and D-glucose, respectively, with a turnover number of 45 cycles per minute for each NAD molecule. The total turnover per coenzyme was 135,000 for the first 2.5 days.

Bacillus subtilis↗
PubMed1990

Construction and characterization of a recombinant tripartite enzyme, galactose dehydrogenase/beta-galactosidase/galactokinase.

The in-frame gene fusion between 3 enzymes, galactose dehydrogenase, beta-galactosidase and galactokinase, is described. The purified artificial tripartite enzyme displayed all three enzymic activities. Two major forms of the hybrid protein were found, consisting of 4 and 8 subunits respectively, but other forms could also be identified. Each subunit was made up of one monomer each of galactose dehydrogenase, beta-galactosidase and galactokinase. Proximity effects exhibited by the hybrid enzyme could be demonstrated using [14C]galactose as a reporter molecule.

Base Sequence↗
PubMed1990

Enantiomeric resolution on molecularly imprinted polymers prepared with only non-covalent and non-ionic interactions.

Molecular imprints were prepared utilizing only weak bonds between the print molecule and functional monomers; the bonding forces used in the imprinting process were only those weaker than covalent and ionic bonds. Methacrylate-based molecular imprints were prepared using a number of chiral compounds, including N-protected amino acid derivatives, as print molecules. Methacrylic acid was used as the functional monomer because the acid function of the monomer forms hydrogen bonds with a variety of polar functionalities, such as carboxylic acids, carbamates, heteroatoms and carboxylic esters, of the print molecule. Bulk polymers were prepared, ground and sieved to particles of size less than 25 microns, packed into high-performance liquid chromatographic (HPLC) columns and used for enantiomeric separations in the HPLC mode. The polymers were shown to effect efficient enantiomeric resolution of a racemate of the print molecule in addition to substrate selectivity for the print molecule in a mixture of substrates with very similar structures. For example, the enantiomers of Cbz-aspartic acid and Cbz-glutamic acid (Cbz = carbobenzoxy) were resolved with separation factors of 1.9 and 2.5, respectively, on polymers with molecular imprints of the L-form of the respective compounds. In addition, these polymers, prepared against Cbz-L-aspartic acid and Cbz-L-glutamic acid, respectively, had the ability to bind selectively the print molecule from a mixture of both racemates, although the two compounds differ only by one methylene group. The results presented represent a substantial widening of the scope of molecular imprinting in that it may now be possible to prepare molecular imprints against a very large number of compounds.

Chromatography, High Pressure Liquid↗
PubMed1990

Enantiomeric resolution of amino acid derivatives on molecularly imprinted polymers as monitored by potentiometric measurements.

Potentiometric measurements have been applied to the detection of enantiomeric separations on molecularly imprinted polymers. A flow-through column electrode, based on the use of polymers imprinted against L-phenylalanine anilide, is described. The electrode consisted of a glass column in which the polymer was packed and where the end frits constituted the electrodes. The flow stream potential across the column can be continuously recorded as solvent is pumped through the system. The column resolved the enantiomers of phenylalanine anilide as detected by both UV absorption and potentiometric measurements and the recorded signals could be correlated with the concentration of phenylalanine anilide. The calibration graphs obtained for the UV absorption of phenylalanine anilide were linear over the concentration range investigated, whereas the potentiometric signal was shown to be exponentially linear with concentration. The application of molecular imprints to the preparation of supports suitable for chromatographic separations of enantiomers and for the preparation of specific electrodes is discussed.

Amino Acids↗
PubMed1990

Purification and site-specific immobilization of genetically engineered glucose dehydrogenase on thiopropyl-Sepharose.

The gene encoding glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was inserted in a plasmid 1.0 kb downstream from a lac promoter, resulting in a 70-fold higher production of the enzyme when expressed in Escherichia coli. A glucose dehydrogenase mutant containing a cysteine residue at position 44 could also be expressed at the same high level. This single cysteine residue was used as an 'affinity tag' to simplify the purification procedure as well as for site-specific immobilization of glucose dehydrogenase on Thiopropyl-Sepharose. This enzyme was purified to homogeneity with a final recovery of 65% and a specific activity of 240 U/mg. The oriented immobilization resulted in increased thermal stability.

Affinity Labels↗
PubMed1990

Polyacrylic polyhydrazides as novel reagents for detection of antibodies in immunoblotting assays.

By the glycoprotein specific staining method introduced recently (Heimgartner et al., 1989, Anal. Biochem. 181, 182-189) it is also possible to detect an antibody bound to its antigen on a membrane. The antibody is oxidized by periodate prior to incubation. Next, a polyacrylic polyhydrazide is coupled to the aldehyde groups generated in the sugar part of the antibody molecule. A periodate oxidized glycoenzyme, such as horseradish peroxidase, is then coupled to the remaining hydrazide groups of the polymer and incubation with a suitable enzyme substrate visualizes the glycoenzyme-polyhydrazide-antibody-antigen complex. The sensitivity of the detection is most critically dependent on the antibody class and the polyhydrazide reagent. Oxidation conditions are less important and the antibody does not need to be purified prior to periodate oxidation. Under standard conditions, the sensitivity obtained with IgG type antibodies was about ten times lower than with peroxidase-labeled secondary antibodies. However, a similar if not higher sensitivity can be expected with more glycosylated antibodies, such as IgM or IgE, or with chicken antibodies. This approach is advantageous because antibodies of all classes and from all species can be detected with the same reagent, the polyhydrazide, no foreign molecule is introduced in the antibody before its binding to the antigen and no conjugate needs to be prepared in advance.

Acrylates↗
PubMed1990

Reversible and irreversible cross-linking of immunoglobulin heavy chains through their carbohydrate residues.

After periodate oxidation and incubation with a dihydrazide, cross-linking of the two heavy chains of immunoglobulins G from several species proceeds specifically through their oligosaccharides. We have used malonic acid dihydrazide, adipic acid dihydrazide and dithiodipropionic acid dihydrazide. The last compound is introduced in this work as a cleavable-carbohydrate-specific cross-linker. It was found that in rabbit and human immunoglobulins the degree of cross-linking was strongly dependent on the oxidation conditions but only very weakly dependent on the concentration and size of the dihydrazides. Papain cleavage of the cross-linked rabbit IgG indicated that the cross-linking occurred predominantly, if not exclusively, in the Fc region, probably through the two glycans linked to Asn-297 in the CH2 domain of each of the two heavy chains. The immunoglobulins from sheep, pig, goat and guinea pig show a comparable cross-linking pattern, indicating that the sugar chains from these immunoglobulins have a spatial structure closely related to that of rabbit and human IgG. When dithiodipropionic acid dihydrazide was used as the cross-linker, the cross-link could be cleaved by mercaptoethanol.

Animals↗
PubMed1990

Enzymatic synthesis of dipeptide units of the D-D-configuration in aqueous media.

The enzymatic synthesis of dipeptide units of the D-D-configuration in aqueous media, catalysed by muramoyl-pentapeptide carboxypeptidase (E.C.3.4.17.8), is described. Ac-L-Lys(Ac)-D-Ala-D-Lac-OH and Ac-D-Ala-OMe were used as acyl-components. Neutral, basic, and hydrophobic amino acids acting as nucleophiles were incorporated. The enzyme is stereospecific in that only the D-enantiomers of amino acids or amino acid derivatives were incorporated. As nucleophiles, the unmodified amino acids resulted in higher product yields compared with using the corresponding amino acid derivatives. Product yields ranged from 40 to 87%.

Amino Acid Sequence↗
PubMed1990

Characterization of a D-amino acid oxidase with high activity against cephalosporin C from the yeast Trigonopsis variabilis.

We describe an improved method to purify D-amino acid oxidase with activity towards cephalosporin C. The protein has a carbohydrate content of 1.3% and two molecules of non-covalently bound flavin cofactor per protein molecule. HPLC profiles and enzymatic analysis have indicated that the cofactor is FAD, even though fluorescence spectroscopy shows a slightly altered spectral profile in the 400-500 nm range compared to authentic FAD. N-terminal sequencing of the protein revealed a high level of similarity (56% identity in 25 amino acids) between the fungal and mammalian oxidase, and probably represents a "Rossman fold" with a beta-alpha-beta structure for the binding of the adenosyl moiety of the cofactor.

Amino Acid Sequence↗
PubMed1989

Construction of an artificial bifunctional enzyme, beta-galactosidase/galactose dehydrogenase, exhibiting efficient galactose channeling.

The in-frame fusion between two oligomeric enzymes, beta-galactosidase and galactose dehydrogenase, is described. The lacZ gene was fused to the 3' end of the galdh gene with a linker encoding only three amino acids. The purified artificial bifunctional enzyme displayed the enzymic activity of both gene products. The hybrid protein was found in two major forms, consisting of four and six subunits, but other forms could also be identified. The molecular weight of each subunit was determined to be 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bifunctional enzyme shows kinetic advantages over the identical native system in conversion of lactose to galactonolactone. A higher steady-state rate and a reduction of the transient time are observed. This phenomenon is especially pronounced at low initial substrate concentrations and when the pH is adjusted to a level at which the galactose dehydrogenase activity is much higher than that of the beta-galactosidase.

Carbohydrate Dehydrogenases↗