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M O Månsson

Publications and source records attributed to M O Månsson.

14 recordsLinked to original sources
PubMed1999

Use of molecularly imprinted polymers in a biotransformation process.

Molecularly imprinted polymers are highly stable and can be sterilised, making them ideal for use in biotransformation process. In this communication, we describe a novel application of molecularly imprinted polymers in an enzymatic reaction. The enzymatic condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to give Z-L-Asp-L-Phe-OMe (Z-aspartame) was chosen as a model system to evaluate the applicability of using molecularly imprinted polymers to facilitate product formation. When the product-imprinted polymer is present, a considerable increase (40%) in product yield is obtained. Besides their use to enhance product yields, as demonstrated here, we suggest that imprinted polymers may also find use in the continuous removal of toxic compounds during biochemical reactions.

Aspartame↗
PubMed1998

A new application of molecularly imprinted materials.

We have studied the possibility of shifting a thermodynamically unfavourable enzymatic equilibrium towards product formation via the addition of a highly specific adsorbent. The commercially interesting enzymatic condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to the sweetener aspartame was chosen as the model system. Extremely stable and specific adsorbents for the product Z-L-Asp-L-Phe-OMe (Z-aspartame) were prepared using the emerging technique of molecular imprinting. A considerable increase (40%) in the yield of product was obtained when such adsorbents were present during the enzymatic reaction. The message of this investigation is that the use of such specific, sterilizable adsorbents should be considered for enzymatic processes to increase the yield. Finally, the direct isolation of a product formed by the retrieval of the adsorbents carrying the product can be envisaged, especially if the adsorbents are magnetic.

Adsorption↗
PubMed1995

Horse liver alcohol dehydrogenase can accept NADP+ as coenzyme in high concentrations of acetonitrile.

The coenzyme specificity of horse liver alcohol dehydrogenase assayed in mixtures of acetonitrile and buffer, 0.01 M Tris/HCl pH 7.4, was investigated. The enzyme could accept NADP+ as coenzyme if it was first bio-imprinted with NADP+, i.e. precipitated from an aqueous buffer with 1-propanol in the presence of NADP+ dried, and then assayed in a system with less than 10% buffer. When assayed in a system with 25% buffer, no activity with NADP+ as coenzyme was observed. The activity was measured with a coenzyme regeneration assay with cinnamoyl alcohol and octylaldehyde as substrates. Other methods to prepare a binary complex of horse liver alcohol dehydrogenase and NADP+, i.e. if the enzyme was immobilized on silica and NADP+ added afterwards or if it was deposited on Celite together with NADP+, failed to show any bio-imprinting effect. The activity of horse liver alcohol dehydrogenase bioimprinted with NADP+ and assayed in acetonitrile/buffer mixtures with less than 10% buffer showed the same or higher activity than an enzyme preparation prepared by bio-imprinting with NAD+. The hypothesis is that the conformation of the active site of horse liver alcohol dehydrogenase is modified to one that is complementary to the ligand present during precipitation and drying. This is possible because of the restricted motility of the enzyme conformation in high concentrations of organic solvent. In the presence of more than 10% buffer the mobility increases in such a way that the imposed conformation with activity towards NADP+ disappears.

Acetonitriles↗
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↗
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↗
PubMed1983

Site-to-site directed immobilization of enzymes with bis-NAD analogues.

Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) have been crosslinked with glutaraldehyde on agarose beads. The crosslinking was performed while the two enzymes were spatially arranged with their active sites facing one another with the aid of a bis-NAD analogue. Subsequently the bis-NAD analogue was allowed to diffuse out. By using a third enzyme, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3), which was also coupled to the same beads and which competes with lactate dehydrogenase for the NADH produced by alcohol dehydrogenase, the effect of site-to-site directed immobilization was studied. It was found that much more NADH than was theoretically expected (50% instead of 19% of produced NADH) was oxidized by lactate dehydrogenase, which indicates that the NADH was preferentially channeled to lactate dehydrogenase due to the juxtapositioned active sites of the two enzymes.

Alcohol Oxidoreductases↗
PubMed1978

Covalent binding of an NAD analogue to liver alcohol dehydrogenase resulting in an enzyme-coenzyme complex not requiring exogenous coenzyme for activity.

1. The NAD analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-NAD, was covalently bound to horse liver alcohol dehydrogenase in a carbodiimide-mediated reaction and in such a way that it was active with the very same enzyme molecule to which it was coupled. 2. The degree of substitution, i.e. the number of NAD analogues per enzyme subunit, could be varied (0.3-1.6). In one preparation 1.6 coenzyme molecules were bound per subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess. 3. It was calculated that every fourth active site of this preparation was provided with a covalently bound functioning coenzyme analogue, and that this analogue had a cycling rate of about 40 000 cycles/h in a coupled substrate assay. 4. The presence of the covalently bound coenzyme made the active sites difficult to inhibit with a competitive inhibitor. For example, 10 mM AMP inhibited the activity of the preparation by 50% whereas a reference system containing native alcohol dehydrogenase was inhibited by 80% in spite of the fact that the reference system contained about 20 000 times as high a concentration of coenzyme.

Adenosine Monophosphate↗
PubMed1976

Continuous regeneration of NAD(P)+ by flavins covalently bound to sepharose.

Various flavins, FMN, FAD, and acriflavin, were immobilized to Sepharose using several different coupling methods. The only product stable enough to permit extended studies was acriflavin coupled to epoxy-substituted Sepharose. The nonenzymic oxidizing capacity towards NAD(P) H was investigated and a 25% specific activity, compared to that of free acriflavin, was observed. The reduced acriflavin was immediately auto-reoxidized in air and could thus be reused. It was shown that acriflavin-Sepharose preparations function as NAD(P)H oxidizing agents in a number of different dehydrogenase systems including lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), alanine dehydrogenase (alaDH), and glutamate dehydrogenase (GDH). The amount of expensive coenzyme necessary for high product formation of such systems was thereby markedly reduced.

Acriflavine↗
PubMed1975

Preparation of an alcohol-dehydrogenase--NAD(H)--sepharose complex showing no requirement of soluble coenzyme for its activity.

1. Horse liver alcohol dehydrogenase and an NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor. 2. The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40% of that obtained in excess of free coenzyme. 3. A molar ratio of 1:1 between the amount of bound enzyme was sufficient to obtain high activities in the absence of free coenzyme. 4. The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5. Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue. 6. The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.

Alcohol Oxidoreductases↗