Ole Kirk

One Biocatalyst–Many Applications: The Use of Candida Antarctica B-Lipase in Organic Synthesis

The application of the B-component lipase from the yeast Candida antarctica in organic synthesis is reviewed. This enzyme has been found to be a particularly efficient and robust lipase catalyzing a surprising diversity of reactions including many different regio- and enantio-selec-tive syntheses. Furthermore, the C. antarctica B-lipase is an example of an enzyme for which its specificity has been predicted based on the crystal structure and modeling of the active site region. This prediction is compared to experimental observations and a very close correlation is found.

The future impact of industrial lipases

Abstract Recognition of the advantages of the use of lipases relative to traditional chemical processes indicates that lipases may be expected to gain in importance in the enzyme market. Development of lipase technologies for the synthesis of novel compounds will probably also result in their expansion into new areas, and a major impact on a range of industries.

Lipase catalyzed synthesis of peroxycarboxylic acids and lipase mediated oxidations.

Abstract Lipase catalyzed synthesis of long chain peroxycarboxylic acids from hydrogen peroxide and free carboxylic acid was investigated. A 51% yield of peroxytetradecanoic acid was achieved when using a two phase system of toluene and water. The peroxy acids thus formed were applied for in situ oxidation of alkenes, in general leading to high yields of the corresponding epoxide. For example, a quantitative yield of cyclohexene oxide and a 94% yield of 1-hexadecene oxide was achieved in a solvent-free process.

Lipase-mediated formation of peroxycarboxylic acids used in catalytic epoxidation of alkenes

Epoxidation of alkenes was achieved under extremely mild conditions by employing peroxycarboxylic acids formed continuously in situ by lipase-catalysed perhydrolysis of the corresponding carboxylic acids.

A highly selective enzyme-catalysed esterification of simple glucosides

Regioselective 6-O-esterification of alkyl glucosides with long chain fatty acids, yielding more than 95% of 6-O-monoesters, can be achieved using lipases as catalysts in a solvent-free process.

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase...

EFFECT OF MUTATIONS IN CANDIDA ANTARCTICA B LIPASE

Three variants of the Candida antarctica B lipase have been constructed and characterized. The variant containing the T103G mutation, which introduces the consensus sequence G-X-S-X-G found in most other known lipases, shows an increased thermostability but retains only half the specific activity of the native enzyme. Also in ester synthesis the activity is lowered but the specificity and enantioselectivity remains unchanged. The W104H mutant, in which more space is introduced into the active site, has more dramatically changed properties. Both the thermostability and the specific activity are slightly reduced but the activity and specificity in ester synthesis is highly different from the native enzyme. In general, the activity is very low and the enantioselectivity is, furthermore, highly reduced. Finally, the mutation M72L was introduced to increase the oxidation stability of the enzyme. This variant did exhibit an increased resistance towards oxidation but the thermostability was, unfortunately, also reduced.

Enzyme Catalyzed Degradation and Formation of Peroxycarboxylic Acids

The ability of hydrolases to catalyze perhydrolysis, i.e. lysis of acyl substrates with hydrogen peroxide to form peroxycarboxylic acids, has been investigated. Lipases, esterases and cholinesterases were found to catalyze perhydrolysis but the preference of the enzymes for hydrogen peroxide relative to water as nucleophile was only 10–100 fold, even in the best cases. Hence, perhydrolysis proceeds with a very low efficiency in aqueous systems. Furthermore, all lipases, esterases and cholinesterases tested degrade peroxycarboxylic acids to the corresponding carboxylic acid and hydrogen peroxide. This reaction is most pronounced in the case of lipases while less so for cholinesterases. Consequently, cholinesterases are superior to the other hydrolases studied in catalyzing net formation of peracids in aqueous systems. In organic solvents, immobilized lipases efficiently catalyze formation of peracids from either triglycerides or the parent carboxylic acid. Proteases and phospholipase A-2 were found to neit...

Glucagon-like peptide-1(7–37) has a larger volume of distribution than glucagon-like peptide-1(7–36)amide in dogs and is degraded more quickly in vitro by dog plasma

SummaryThe pharmacokinetic properties of glucagon-like peptide-1(7–36)amide and GLP-1(7–37) were compared. Four beagle dogs received on 4 separate occasions s.c. bolus doses of 50 μg/kg, and 2 min i.v. infusions of 50μg/kg of each peptide. The plasma immunoreactivity of GLP-1 (P-GLP-1-IR) was measured by a sandwich enzyme-linked immunosorbent assay (ELISA). After i.v. infusion, the plasma half-life in the first-phase was 2.1±0.1 and 2.4±0.3 min, in the final-phase 68±6 and 81±3 min, the total plasma clearance 25±3 and 22±4 ml/kg. min, the volume of distribution at steady state 0.16±0.02 and 0.84±0.24 l/kg, and the mean residence time 6.2±0.3 and 36±5 min for GLP-1(7–36)amide and GLP-1(7–37), respectively. After s.c. administration, the maximum plasma concentration was reached after 15±5 and 19±4 min and the absolute bioavailability was 48±7 and 49±13% for GLP-1(7–36)amide and GLP-1(7–37), respectively. P-GLP-1-IR, measured by a radioimmunoassay (RIA), was considerably higher than when measured by ELISA. This discrepancy was due to cross-reactivity with metabolites of the parent peptide. The plasma degradation was studied in vitro in dog plasma at 37°C, and the half-lives were found to be 61±9 and 132±16 min for GLP-1(7–36)amide and GLP-1(7–37), respectively (n=6). Bacitracin inhibited the degradation of both peptides.

Determination of peroxycarboxylic acids by high-performance liquid chromatography with electrochemical detection

Abstract A sensitive high-performance liquid chromatographic method employing reversed-phase chromatography and amperometric detection of low levels of peroxycarboxylic acids is described. Detection limits of 0.1–0.6 μ M and linear dynamic ranges of at least 0.05–5 m M were obtained. As a consequence of the high sensitivity and selectivity provided by the electrochemical detector, the method is well suited for detection of various peroxycarboxylic acids even in the complex matrices represented by detergent solutions. As an illustration of the applicability of the system developed, the levels of various peroxycarboxylic acids were monitored in the course of a washing cycle performed with some commercially available detergents.