Mats-Olle Månsson

High-performance liquid affinity chromatography of nucleosides, nucleotides and carbohydrates with boronic acid-substituted microparticulate silica

High-Performance Liquid Affinity Chromatography of Nucleosides, Nucleotides and Carbonhydrates with Boronic Acid-Substituted Microparticulate Silica

The synthesis of a D-amino acid ester in an organic media with α-chymotrypsin modified by a bio-imprinting procedure

SummaryIn this report the use of chymotrypsin in the synthesis of a D-amino acid ester is described. The enzyme was modified to accept the D-form of a tryptophane derivative, by precipitating the enzyme-inhibitor complex between chymotrypsin and N-acetyl-D-tryptophane in 1-propanol. The precipitate was then used to synthesize N-acetyl-D-tryptophane ethyl ester in cyclhexane. The rate of the D-esterification was 7.5 nmole/mg enzyme·h.

Recycling by a second enzyme of NAD covalently bound to alcohol dehydrogenase

The covalent coupling of an NAD-analogue to liver alcohol dehydrogenase (LADH) giving an enzymecoenzyme complex that does not require any exogenous NAD for activity has been described [I]. We report here on the interaction of such an enzymebound NAD with a second enzyme, lactate dehydrogenase (LDH) or malate dehydrogenase. In contrast to [2] where the enzyme and the coenzyme had been co-immobilized on a matrix, which prevents proper interaction with other enzymes, our preparation allows recycling of the covalently-bound NAD by a second enzyme. The cycling of the covalently-bound coenzyme has been studied mainly by fluorimetry and advantage has been taken of the fluorescent properties of the reduced form of the coenzyme [3] .

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.

Electrochemical Regeneration of NAD Covalently Bound to Liver Alcohol Dehydrogenase

Abstract An enzyme-coenzyme complex was immobilized on the surface of a glassy carbon electrode and investigated with cyclic voltammetry in ethanol-containing buffers. The complex consists of an Liver Alcohol Dehydrogenase molecule to which an NAD-analogue is covalently attached via its straight six-carbon, spacer. One cycle was observed but repeated recycling could not be carried out. presumably due to catalytic decomposition of the coenzyme at the electrode surface.

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. Copyright

Immobilized active coenzymes.

Publisher Summary This chapter describes general aspects related to immobilized coenzymes and also to provide a number of additional references. Coenzymes usually have to be modified to allow proper immobilization and regeneration. This modification of a coenzyme can be accomplished in basically two ways. One is the preassembly approach and the other has been called the solid-phase modular approach. In the preassembly approach, the coenzyme is first modified and then assembled with a spacer molecule to increase the steric availability for the coenzyme of the enzyme and subsequently coupled to a support. Immobilized active coenzymes, such as nicotinamide adenine dinucleotide (NAD), have found use in analytical systems as well as in enzyme reactors. The reactor of choice is the membrane reactor in which a semipermeable membrane retains the high molecular weight immobilized coenzyme together with the enzymes. However, an important issue both in the reactor systems and for analytical systems is the requirement for regeneration of the coenzyme because of its high cost and the fact that it is needed in stoichiometric amounts relative to the product formed. Regeneration can be accomplished chemically, electrochemically, or enzymatically. At present, enzymatic regeneration has the most advantages, especially because of its high-specificity in regeneration. However, electrochemical procedures are gaining in importance.

[77] Covalent enzyme—coenzyme complexes of liver alcohol dehydrogenase and NAD

Publisher Summary This chapter focuses on the synthesis of covalent enzyme-coenzyme complexes of liver alcohol dehydrogenase and nicotinamide adenine dinucleotide (NAD). The problems of retention and regeneration are solved by two methods used for the preparation of complexes between alcohol dehydrogenase and NAD. The first method consists of the immobilization of the reduced NAD analog, N6-[N-(6-aminohexyl)carbamoylmethyl]NADH, and the enzyme, liver alcohol dehydrogenase, to a support such activated agarose. The second method provides more alternatives for regeneration. The broad substrate specificity of liver alcohol dehydrogenase for redox reactions makes it a versatile enzyme for use in organic reactions. The complex of liver alcohol dehydrogenase-NADH and agarose is stable and easy to prepare and might be useful in the production of various ketones, aldehydes, and alcohols. The soluble complex of liver alcohol dehydrogenase and NAD also function in enzyme reactions with other NAD-dependent dehydrogenases where both enzymes share a common coenzyme, though it is covalently bound to liver alcohol dehydrogenase.

Immobilized and soluble site-to-site directed enzyme complexes composed of alcohol dehydrogenase and lactate dehydrogenase

Publisher Summary After cross-linking of the two enzymes with glutaraldehyde, the bis-NAD template is removed, leaving the active sites still positioned against one another. By such an active site arrangement of the two enzymes, it can be expected that the diffusion of the product of the first enzyme to the juxtaposed active site of the second enzyme would be facilitated when compared with “at random” immobilized enzyme systems. Such site-to-site enzyme systems might also serve as models for the enzyme complexes of consecutively operating enzymes, which are believed to be of importance in the channeling of labile intermediates and in the regulation of metabolism. The soluble site-to-site enzyme complex was also studied with the scavenger enzyme assay and in principle the same kind of results was obtained as for the immobilized system. The results obtained for both the immobilized system and the soluble system indicate that, given two alternative routes, the one to Lactate dehydrogenase (LDH) is preferred over the one to Lipoamide Dehydrogenase (LiDH) when LDH and ADH are site-to-site oriented.