Bo Mattiasson

Facile synthesis of fatty acid esters in high yields

The facile synthesis of esters by lipase-catalysed esterification of fatty acids and ethanol is demonstrated. Evaporation of the water generated in the reaction allowed the rapid production of esters of >99% yield in refluxing pentane or hexane. Water was trapped by condensing the refluxing vapor phase and passing it over activated molecular sieves in a reflux trap. High yields were rapidly obtained in synthesis of ethyl esters of oleic, linoleic, α-linolenic, and arachidonic acids without peroxidation of double bonds. The wax ester oleyl oleate was also synthesized rapidly with little peroxidation. A molar excess of 1.25 to 1.5 of ethanol allowed the most rapid ester synthesis. Synthesis of ethyl stearate was carried out on a preparative scale (50 g). Greater than 99% conversion was obtained in 50 min.

Lipase-catalyzed transesterification of phosphatidylcholine at controlled water activity

The incorporation of a free fatty acid into thesn-1 position of phosphatidylcholine by lipase-catalyzed transesterification was investigated. The thermodynamic water activity of both the enzyme preparation and the substrate solution was adjusted to the same value prior to the reaction. The reaction rate increased with increasing water activity but the yield of modified phosphatidylcholine decreased due to hydrolysis. By using a large excess of the free fatty acid (heptadecanoic acid), the hydrolysis reaction was slowed down, so a higher yield was obtained at a given degree of incorporation. The best results were obtained withRhizopus arrhizus lipase immobilized by adsorption on a polypropylene support. With this preparation, a yield of 60% and nearly 50% incorporation of heptadecanoic acid (100% incorporation in thesn-1 position) was obtained at a water activity of 0.064. The enzyme preparation had good operational stability and position specificity. Little incorporation (<1%) was observed in thesn-2 position, when almost all the fatty acid in thesn-1 position was exchanged.

Triglyceride interesterification by lipases. 3. Alcoholysis of pure triglycerides

Lipase-catalyzed alcoholysis of triolein dissolved in ethanol or isopropanol for the formation of ethyl and isopropyl esters was investigated. Of 16 lipases screened, Amano lipase from P. fluorescens was selected for investigation of the effects of basic reaction conditions on alcoholysis yields. Ethanolysis yields were only slightly affected by water additions to immobilized lipase preparations. Isopropyl ester yields decreased with water addition. Good operational stability was observed over 17 days. Changes in initial triolein concentration in the range 5–50 mM had very little effect on ester yields. The ionic strength of the phosphate buffer used in lipase immobilization affected ethanolysis and isopropanolysis yields in opposite ways. The highest ethanolysis yields were obtained with lipases immobilized from 250 mM buffer, while isopropyl ester yields were highest with lipases immobilized from water. In addition, the quantities and isomers of monoglyceride intermediates in ethanolysis were affected by the immobilization buffer strength. Larger quantities of 2-monoglycerides were formed in ethanolysis reactions with lipase preparations immobilized from water.

Process monitoring by flow-injection immunoassay: Evaluation of a sequential competitive binding assay

A new variation on the theme of flow-injection binding assays is presented, namely the sequential competitive binding assay, in which the sample containing the native antigen is first introduced into the flow system followed by a pulse of labelled antigen. The flow-injection binding assay was used in monitoring the effluent from column chromatographic separations of proteins. By applying a computer-based evaluation system, concentrations are automatically read and compensation is made for denaturation in the affinity sorbent.

Modification of the microenvironment of enzymes in organic solvents. Substitution of water by polar solvents

Enzyme catalysis in water-immiscible organic solvents is strongly influenced by the amount of water present in the reaction mixture. Effects of substitution of part of the water by other polar solvents were studied. In an alcoholysis reaction catalyzed by chymotrypsin deposited on celite, it was possible to exchange half of the water by formamide, ethylene glycol or dimethyl sulfoxide with often increased initial reaction rate. Furthermore, these substitutions caused the suppression of the competing hydrolysis reaction. However, formamide caused enzyme inactivation, and ethylene glycol participated as a reactant in the alcoholysis to some extent, hence dimethyl sulfoxide was considered the best water substitute among the solvents tested. These effects were noted for chymotrypsin catalyzed alcoholysis in several water immiscible solvents and also for interesterification reactions catalyzed by Candida cylindracea lipase on celite. In the latter case a change in the stereoselectivity was observed. At a low water content a high stereoselectivity was observed; when the amount of polar solvent was increased, either by doubling the water content or adding an equal amount of DMSO, the stereoselectivity decreased.

Complex Formation Between Chymotrypsin and Polymers as a Means to Improve Exposure of the Enzyme to Organic Solvents

Abstract When mixing chymotrypsin and a polymer, e.g. ethyl cellulose and drying them together, complexes are formed that are soluble in organic solvents. The enzyme maintains its activity and is even stabilized as compared to a preparation in which the enzyme is added as a dry powder to the solvent. In both cases, minute amounts of water are added afterwards. The system containing a polymer shows a markedly reduced sensitivity to additions of water to the system. The complex formation is reversible and the enzyme can conveniently be recovered from the organic solvent by adding enough water to form a separate phase.

Stabilization of Adsorbed Enzymes Used as Biocatalysts in Organic Solvents

Abstract Enzymes immobilized by deposition onto a solid support at low enzyme loadings were shown to be stabilized by additives, such as proteins and PEG. The additive protects the enzyme from deactivation during immobilization. The amount of additive required to obtain full stabilization was dependent on the type of support material used and corresponded approximately to a monolayer coverage of the additive on the surface of the support. The stabilizing effect of the additive was less pronounced at high enzyme loadings.

Cryo-Bioorganic Synthesis - Enzyme Catalysis at low Temperature and in Low Water Content Environments

Abstract A combination of low water content (2.5–5 %, v/v) and subzero temperatures has been utilized to drastically suppress the hydrolytic side-reaction in a peptide synthesis reaction. Almost no hydrolytic side-reaction occured at -22 ° or at -35 ° starting with equimolar concentrations of amino acid ester (donor) and amino acid amide (acceptor). In this way, nearly complete conversion to peptide was obtained.