Elsevier

Carbohydrate Research

Volume 337, Issue 13, 16 July 2002, Pages 1179-1184
Carbohydrate Research

Effect of fatty acid chain length on initial reaction rates and regioselectivity of lipase-catalysed esterification of disaccharides

https://doi.org/10.1016/S0008-6215(02)00112-XGet rights and content

Abstract

In a reaction medium mixture of 9:11 t-BuOH and pyridine (v/v) the effect of fatty acid chain length (C-4–C-12) on C. antarctica lipase B (Novozym 435, EC 3.1.1.3) catalysed esterification was studied. α and β maltose 6′-O-acyl esters in an anomeric molar ratio of 1.0:1.1 were synthesised independently of the chain length, but the initial specific reaction rate increased with decreasing chain length of the acyl donor. The product yield followed the same trend with a lauryl ester yield of 1.1% (mol/mol) and a butyl ester yield of 27.6% (mol/mol) after 24 h of reaction. With sucrose as the acyl acceptor the 6′-O-acyl and 6-O-acyl monoesters were formed with fatty acids of chain length C-4 and C-10 while the 6′,6-O-acyl diester was formed only with butanoic acid (C-4:0) as acyl donor. The 6′-O-acyl and 6-O-acyl monoesters and the 6′,6-O-acyl diester of butanoic acid were produced in a molar ratio of 1.0:0.5:0.2 and with decanoic acid (C-10:0) the 6′-O-acyl and 6-O-acyl monoesters were formed in the ratio of 1.0:0.3. The highest initial reaction rate and yield were obtained with the shortest chain length of the acyl donor. Initial reaction rates and ester yields were affected by the solubility of the disaccharide, with higher reaction rates and yields with maltose than with sucrose, while no formation of esters were observed with either cellobiose or lactose as acyl acceptors.

Fatty acid esters of sucrose and maltose were synthesised in a lipase-catalysed esterification process. R=(CH2)nCH3n=3 and 9.

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Introduction

Carbohydrate esters are an interesting group of compounds with surfactant and emulsifying properties. They are biodegradable, non-toxic and can be produced from renewable resources (fatty acids and native carbohydrates). The specific properties of the ester, including the ẖydrophilic:ḻipophilic ḇalance (HLB value) is controlled by the type of acyl group, the degree of substitution and the degree of polymerisation of the carbohydrate. In fact, a broad range of functional properties can be obtained with different types of esters, with the hydrophilic, carbohydrate part being important to the HLB value. In contrast to conventional available food emulsifiers it is possible to produce carbohydrate fatty acid esters that covers a wide range of HLB values (from 2 to 18).1

Synthesis of carbohydrate esters can be catalysed by hydrolytic enzymes (EC 3) in a reversed hydrolysis reaction provided that the water activity is controlled and kept sufficiently low. In particular, lipases and proteases are being applied for the regioselective acylation of mono and disaccharides. Enzyme-catalysed acylation has been performed in solvent based systems by transesterification using vinyl laurate as acyl donor2 and by reversed hydrolysis with native carbohydrates and fatty acids as substrates3 which also has been reported for solvent free systems.4 Compared to conventional chemical catalysis the enzyme catalysis can be performed at milder process conditions and with higher specificity of the reaction.

The activity and specificity of hydrolases in organic solvents is highly influenced by the nature of the solvent.5 Previous research has tried to identify a general relationship between the ability of enzymes to catalyse reactions in organic solvents and the physico-chemical properties of the solvents. The parameters investigated have included the dielectric constant, the dipole moment, the water solubility and miscibility and hydrophobicity (log P). Consistent correlations have been obtained with the solvent hydrophobicity and it has been shown that enzymes for example lipases generally have higher activities at high log P values.6., 7., 8. A hydrophobic solvent is however not necessarily a good choice for achieving optimal rates and degrees of conversion, depending on the solubility of the hydrophilic substrate.9 Therefore optimal reaction conditions are a compromise between enzyme activity and substrate solubility, for which investigation of the effect of solvent mixtures and medium engineering is required. In the present study a mixed reaction medium, favouring the solubility of carbohydrate, was used to study the effect of fatty acid chain length on a lipase-catalysed esterification of native disaccharides using an immobilised preparation of C. antartica lipase B. Reactions were characterised with regard to the products obtained, the yields and reaction rates.

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Results and discussion

For all fatty acids investigated (C-4–C-12) the immobilised preparation of C. antarctica lipase B (Novozym 435) catalysed the formation of the corresponding 6′-O-acyl monoester with maltose as acyl acceptor (Fig. 1). Independently of the acyl donor chain length α and β maltose 6′-O-acyl esters were synthesised in the anomeric molar ratio of 1.0:1.1. The identification of the presence of mono acylated maltose ester by mass spectroscopy was confirmed by 13C NMR spectra of the purified compound as

Synthesis of carbohydrate fatty acid esters

The enzyme reactions were carried out in 25 mL glass-stoppered Erlenmeyer flasks in 5 mL solutions of 9:11 t-BuOH:pyridine (v/v) which contained 30 mM fatty acid and 100 mg of either maltose, lactose, sucrose or cellobiose. 0.5 g molecular sieves (3 Å) were added to remove the water from the reaction mixture, which was equilibrated at 45 °C on a rotary shaker table (250 rpm) for 24 h. The reaction was then initiated by adding 50 mg of immobilised Novozym 435. The reaction mixture was incubated

Acknowledgements

This work was a part of a Ph.D. program supported by Aalborg University. Drs. Oene Robert Veltman and Marilyn Wiebe are acknowledged for their critical reading of the manuscript.

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