F. van Rantwijk

Glycosidase-catalysed synthesis of alkyl glycosides

Abstract Glycosidases catalyse the synthesis of anomerically pure alkyl glycosides in one step. In contrast, chemical synthesis of anomerically pure glycosides is circuitous and expensive. Two methodologies are used in enzymatic glycosylation: thermodynamically controlled reversed hydrolysis and kinetically controlled transglycosylation. The advantages and limitations of both approaches are delineated. Glycosidases exhibit broad specificity with regard to the aglycon: in addition to simple alcohols, hydroxy amino acids, nucleosides, ergot alkaloids and cardiac genins are glycosylated. Non-alcohol acceptors such as oximes and thiols also function as substrates whereas pyranoid glycals act as non-natural donors. Glycosidases exhibit absolute selectivity with regard to the stereochemistry at the anomeric centre and show a high degree of chemoselectivity for different hydroxyl groups, e.g., the order of reactivity is primary>secondary alcohols>phenols; tertiary alcohols are unreactive. Chiral primary alcohols are poorly discriminated, but the enantioselectivity towards a hydroxyl group that is directly attached to a (pro)chiral carbon atom is often high. The synthetic utility of glycosidases would be considerably improved if methods could be found for maintaining their catalytic activity in non-aqueous media.

Cross-linked enzyme aggregates with enhanced activity: application to lipases

Seven commercially available microbial lipases were immobilised as their cross-linked enzyme aggregates (CLEAs). Preparations with enhanced activity were obtained by a judicious choice of the precipitant [(NH4)2SO4, 1,2-dimethoxyethane or acetone] and by adding either a crown ether or surfactant, depending on the source of the enzyme. Thus, precipitation of the lipases from Thermomyces lanuginosus and Rhizomucor miehei with (NH4)2SO4 in the presence of SDS, followed by cross-linking with glutaraldehyde, afforded CLEAs with three and two times, respectively, the hydrolytic activity of the native enzymes. Preparations with up to ten times enhanced activity in organic medium were similarly prepared.

Cross-linked aggregates of penicillin acylase : robust catalysts for the synthesis of β-lactam antibiotics

A novel method for the immobilization of penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11) is reported. It involves the physical aggregation of the enzyme, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs). Compared with conventionally immobilized penicillin G acylases, these CLEAs possess a high specific activity as well as a high productivity and synthesis/hydrolysis (S/H) ratio in the synthesis of semi-synthetic antibiotics in aqueous media. Moreover, they are active in a broad range of polar and apolar organic solvents.

Characterisation of nitrilase and nitrile hydratase biocatalytic systems

Biocatalytic transformations converting aromatic and arylaliphatic nitriles into the analogous related amide or acid were investigated. These studies included synthesis of the β-substituted nitrile 3-hydroxy-3-phenylpropionitrile, subsequent enrichment and isolation on this substrate of nitrile-degrading microorganisms from the environment, and a comparative study of enzymatic reactions of nitriles by resting cell cultures and enzymes. Each biocatalyst exhibited a distinctive substrate selectivity profile, generally related to the length of the aliphatic chain of the arylaliphatic nitrile and the position of substituents on the aromatic ring or aliphatic chain. Cell-free nitrilases generally exhibited a narrower substrate range than resting whole cells of Rhodococcus strains. The Rhodococcus strains all exhibited nitrile hydratase activity and converted β-hydroxy nitriles (but did not demonstrate enantioselectivity on this substrate). The biocatalysts also mediated the synthesis of a range of α-hydroxy carboxylic acids or amides from aldehydes in the presence of cyanide. The use of an amidase inhibitor permits halting the nitrile hydratase/amidase reaction at the amide intermediate.

Improvement of the total turnover number and space-time yield for chloroperoxidase catalyzed oxidation

Chloroperoxidase from Caldariomyces fumago was applied for the oxidation of indole to oxindole using hydrogen peroxide as the oxidant in aqueous t-butyl alcohol medium. Different ways of adding the oxidant, various reactor types, and the use of a hydrogen peroxide-stat were compared, resulting in a 20-fold increase of the total turnover number (ttn) and space-time yield (sty). The highest ttn of >860,000 was obtained in a fed-batch reactor, whereas the highest sty of 120 g/(L . d) was reached in a continuously operated enzyme membrane reactor. The results were compared to other enzyme systems already established for the synthesis of amino acids and carbohydrates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 283-288, 1997.

Chloroperoxidase: Use of a Hydrogen Peroxide-Stat for Controlling Reactions and Improving Enzyme Performance

The use of a hydrogen peroxide-stat for controlling the hydrogen peroxide concentration has been demonstrated for the chloroperoxidase catalyzed oxidation of indole. It has been shown that in tert-butyl alcohol/water mixtures the oxidation can be effectively controlled by a hydrogen peroxide-stat. In this medium chloroperoxidase was stabilized towards oxidative destruction as long as indole was present in the reaction mixture but inactivation occurred in the absence of a reductant. In contrast with the results with to-r-butyl alcohol/water mixtures chloroperoxidase was inactivated in water by hydrogen peroxide even when indole was present in the reaction mixture and the oxidant concentration was as low as 30.

Chloroperoxidase catalyzed oxidations in t-butyl alcohol/water mixtures

Chloroperoxidase catalyzed oxidations of sulfides and indoles were performed in t-butyl alcohol/water mixtures at ambient temperature. t-Butyl alcohol/water (50 :50, v/v) proved to be a good solvent system for performing synthetic oxidations catalyzed by chloroperoxidase. The sulfoxidation of alkyl aryl sulfides and related compounds catalyzed by chloroperoxidase in t-butyl alcohol/water mixtures (50:50, v/v) was compared to the sulfoxidation in water. In both solvent systems, complete enantioselectivity to the R-sulfoxide (ee = 99%) was observed with hydrogen peroxide as oxidant when the size of the alkyl moiety was smaller than propyl. The uncatalyzed, racemic sulfoxidation did not proceed under these conditions. This is in contrast to literature data on sulfoxidation in water, where enantioselectivities were lower due to this uncatalyzed reaction. Reactions in water generally proceed faster than reactions in the cosolvent system except for substrates which dissolve poorly in water or for solid substrates for which diffusion becomes an important limiting factor in water. The lower activity in t-butyl alcohol/water for sulfoxidation and for indole oxidation is mainly due to an increase of the K m value (thermodynamically controlled). Also a decrease of k cat (catalytic turnover frequency) is observed, probably caused by a change in structure of the enzyme.

Chloroperoxidase-Catalyzed Oxidation of 5-Hydroxymethylfurfural

Abstract Chloroperoxidase (CPO) catalyzes the oxidation of 5-hydroxymethylfurfural (HMF) with hydrogen peroxide as the oxidant. The reaction proceeds with 60–74% selectivity to furan-2,5-dicarboxaldehyde (FDC). The main byproduct is 5-hydroxy-methyl-2-furancarboxylic acid (HFCA); a minor amount of 5-formylfuran-2-carboxylic acid (FFCA) was also detected. When H2 18O2 was used a virtually quantitative incorporation of 18O was observed in the HFCA product, whereas no 18O was incorporated from H2 18O. Hence, the CPO-catalyzed oxidation of aldehydes to acids proceeds with direct oxygen transfer from the iron-oxo complex of CPO. Controlling the H2O2-addition with a H2O2-stat facilitated the reaction procedure and a conversion of 87% of HMF was reached within 21 min.

Penicillin acylase-catalyzed resolution of amines in aqueous organic solvents

Abstract Penicillin acylase from Alcaligenes faecalis catalyzes the enantioselective acylation of amines with phenylacetamide in a kinetically controlled reaction in water at pH 11. Addition of cosolvent to the reaction mixture significantly improved the enantioselectivity in most cases. Penicillin acylase from E. coli also catalyzed the phenylacetylation of amines, but an order of magnitude less efficiently than with the enzyme of A. faecalis . Amine resolution via kinetically controlled acylation competes effectively with hydrolysis of N -acylated compounds and constitutes a synthetically useful alternative to existing lipase-based methods.

DYNAMIC KINETIC RESOLUTION OF PHENYLGLYCINE ESTERS VIA LIPASE-CATALYSED AMMONOLYSIS

Abstract Ammonolysis of d , l -phenylglycine methyl ester catalysed by Novozym 435 at 40°C in tert -butyl alcohol gave d -phenylglycine amide in 78% ee at 46% conversion, corresponding to an enantiomeric ratio ( E ) of 16. Lowering the temperature improved the enantioselectivity ( E =52 at −20°C). Combination of ammonolysis with pyridoxal-catalysed in situ racemisation of the unconverted ester (dynamic kinetic resolution), at −20°C, gave d -phenylglycine amide with 88% ee at 85% conversion. The amide racemised much slower than the ester at this low temperature.