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Cross-linked crystals of hydroxynitrile lyase as catalyst for the synthesis of optically active cyanohydrins

Purified hydroxynitrile lyase (HNL) from Manihot esculenta was crystallized by the sitting-drop vapour-diffusion method. The bipyramidal crystals formed (10-20 μm) were cross-linked with different amounts of glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic

Improved oligosaccharide synthesis by protein engineering of β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type β-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type β-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out

Modeling the performance of immobilized α-chymotrypsin catalyzed peptide synthesis in acetonitrile medium

A model was developed which describes simultaneous reaction and internal diffusion for kinetically controlled, immobilized α-chymotrypsin-catalyzed, oligopeptide synthesis in acetonitrile medium. The model combines the equations that describe the intrinsic kinetics of four different reactions and the physical characteristics of three different support materials, as determined experimentally, to pr

Enhanced transglucosylation/hydrolysis ratio of mutants of Pyrococcus furiosus β-glucosidase : Effects of donor concentration, water content, and temperature on activity and selectivity in hexanol

The transglucosylation reaction catalyzed by wild-type β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus and active site mutants (M424K, F426Y, M424K/F426Y) was studied. The conversion of pentyl-β-glucoside to hexyl-β-glucoside in hexanol was used as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction. The activity (rates of hydrolysis and transglucosylation)

Optimization of galactooligosaccharide production from lactose using β-glycosidases from hyperthermophiles

The maximal production of galactooligosaccharides (GOS) from lactose by (β-glycosidases from the hyperthermophilic archaea, Sulfolobus solfataricus (LacS) (derived from lacS gene) and Pyrococcus furiosus (CelB) (derived from celB gene) was optimized. The performance of these enzymes under extreme reaction conditions, temperatures up to 95°C and lactose concentrations up to 90% (w/v), were studied.

Lipase-catalyzed reactions in organic and supercritical solvents : Application to fat-soluble vitamin determination in milk powder and infant formula

The aim of this study was to thoroughly investigate the possibility of using enzyme catalyzed hydrolysis and alcoholysis of ester bonds in vitamin A and E esters to facilitate their determination in different food formulas. Two vitamin esters, retinyl palmitate and α-tocopheryl acetate were used as model compounds and two food formulas, milk powder and infant formula, were used as model matrices.

Stability and stabilization of hydroxynitrile lyase in organic solvents

The stability of hydroxynitrile lyase from Hevea brasiliensis has been studied in organic solvents. In dry solvents, the enzyme had half-lives in the range 1400-2500 hours. The enzyme half-life was one order of magnitude lower if the medium was water saturated. The substrates, aldehyde and hydrogen cyanide, were found to promote enzyme deactivation. The deactivation increased with substrate concen

Effect of mass-transfer limitations on the selectivity of immobilized α-chymotrypsin biocatalysts prepared for use in organic medium

The selectivity of preparations of α-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass- transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donor

Stabilisation of chloroperoxidase towards peroxide dependent inactivation

The addition of polyethyleneimine with a molecular weight of 2000 to chloroperoxidase from Caldariomyces fumago dramatically improved the stability of the enzyme towards peroxide dependent inactivation. The rate constant for the H 2 O 2 -dependent inactivation of chloroperoxidase decreased from 0.0016s -1 to 1.1 * 10 -5 s -1 in the presence of 1% polyethyleneimine. The stabilising effect towards

Hydrolytic and transphosphatidylation activities of phospholipase D from Savoy cabbage towards lysophosphatidylcholine

The hydrolysis and transphosphatidylation of lysophosphatidylcholine (LPC), with a partially purified preparation of phospholipase D (PL D) from Savoy cabbage, was investigated. These reactions were about 20 times slower than the hydrolysis of phosphatidylcholine (PC) in a micellar system. For the transfer reaction, 2 M glycerol was included in the media, which suppressed the hydrolytic reaction.

Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa

Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lip

Lipase-catalyzed fatty acid exchange in digalactosyldiacylglycerol. Improvement of yield due to the addition of phenylboronic acid

Rhizopus arrhizus lipase immobilized on porous polypropylene was used in an acidolysis reaction in toluene at low water activity (0.11) to exchange the fatty acid in the sn-1-position of digalactosyldiacylglycerol (DGDG). Without extra precautions, an yield of only about 20% was obtained, mainly due to side reactions; incorporation of an extra acyl group on the primary hydroxyl of the digalactosyl

Candida antarctica lipase B-catalysed synthesis of dihydroxyacetone fatty acid esters

The enzymatic syntheses of 1-lauroyl-dihydroxyacetone and 1,3-dilauroyl-dihydroxyacetone were investigated. Lipase B from Candida antarctica (SP435) was used to catalyse the acylation of dihydroxyacetone (DHA) with lauric acid in organic solvent media at controlled water activity. High conversions of dihydroxyacetone (> 90%) are achieved when the water activity is 0.11 or below in solvents of vari

Lysophosphatidylcholine synthesis with Candida antarctica lipase B (Novozym 435)

Immobilized lipase from Candida antarctica lipase B (Novozym 435) was effective in the synthesis of lysophosphatidylcholine (LPC). The transesterification of L-α-glycerophosphorylcholine (GPC) and vinyl laurate was carried out in a solvent free system or in the presence of 50% (v/v) t- butanol. High conversions (>95%) were easily achieved. The lipase was selective for the sn-1 position of the glyc

Enzymatic fatty acid exchange in digalactosyldiacylglycerol

Six different lipases were screened for their ability of acidolysis between digalactosyldiacylglycerol (DGDG) and heptadecanoic acid in toluene. Lipases from Geotrichum candidum, Alcaligenes sp. and Penicillium camembertii did not catalyse the acidolysis reaction. Rhizopus arrhizus and Rhizomucor miehei (Lipozyme) catalysed the acidolysis but produced a mixture of DGMG, DGDG, acyl-DGMG and acyl-DG

Two-enzyme system for the synthesis of 1-lauroyl-rac-glycerophosphate (lysophosphatidic acid) and 1-lauroyl-dihydroxyacetonephosphate

A combination of two enzymes, phospholipase D (PL D) and C (PL C), was investigated for the production of two lysophospholipids, 1-lauroyl-rac- glycerophosphate (1-LGP) and 1-lauroyl-dihydroxyacetonephosphate (1-LDHAP). The high transphosphatidylation ability of phospholipase D from Streptomyces sp. allowed the formation of 1-lauroyl-phosphatidylglycerol (1-LPG) and 1- lauroyl-phosphatidyldihydrox