Morten Würtz Christensen

Enzymatic acylation of di- and trisaccharides with fatty acids: choosing the appropriate enzyme, support and solvent

Enzymatic synthesis of fatty acid esters of di- and trisaccharides is limited by the fact that most biological catalysts are inactivated by the polar solvents (e.g. dimethylsulfoxide, dimethylformamide) where these carbohydrates are soluble. This article reviews the methodologies developed to overcome this limitation, namely those involving control over the reaction medium, the enzyme and the support. We have proposed the use of mixtures of miscible solvents (e.g. dimethylsulfoxide and 2-methyl-2-butanol) as a general strategy to acylate enzymatically hydrophilic substrates. We observed that decreasing the hydrophobicity of the medium (i.e. lowering the percentage of DMSO) the molar ratio sucrose diesters versus sucrose monoesters can be substantially enhanced. The different regioselectivity exhibited by several lipases and proteases makes feasible to synthesise different positional isomers, whose properties may vary considerably. In particular, the lipase from Thermomyces lanuginosus displays a notable selectivity for only one hydroxyl group in the acylation of sucrose, maltose, leucrose and maltotriose, compared with lipase from Candida antarctica. We have examined three immobilisation methods (adsorption on polypropylene, covalent coupling to Eupergit C, and silica-granulation) for sucrose acylation catalysed by T. lanuginosus lipase. The morphology of the support affected significantly the reaction rate and/or the selectivity of the process.

ENZYME CATALYSED SYNTHESIS IN AMBIENT TEMPERATURE IONIC LIQUIDS

The activity of three different lipases, a glycosidase and a protease in ionic liquids has been studied. Ambient temperature ionic liquids are a new class of solvents that are nonvolatile and nonflammable and thus an interesting alternative to classical organic solvents. Monitoring the synthesis of a simple ester, all lipases were found to exhibit both excellent activity and stability in the non-polar ionic liquid 1-butyl-3-methylimidazohum hexaflurophosphate ([bmin][PF6], 1). Furthermore, β-galactosidase from E. coli and the Subtilisin protease SavinaseTM were both found to exhibit a hydrolytic activity in a 50% aqueous solution of the water-miscible ionic liquid 1-butyl-3-methyhmidazoUum tetra-fluoroborate ([bmin][BF4], 2) comparable to the activity observed in 50% aqueous solutions of ethanol and acetonitrile.

Effect of the immobilization method of lipase from Thermomyces lanuginosus on sucrose acylation

Lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa ) was immobilized using granulation by incubating low-particle-size silica with the lipase. Granules with a particle diameter in the range 0.3-1 u mm were obtained. The immobilized lipase was tested in the acylation of sucrose with vinyl laurate in mixtures of tert -amyl alcohol: dimethyl sulfoxide. Results were compared with immobilization of enzyme by adsorption on polypropylene (Accurel EP100), deposition on Celite by precipitation, and covalent attachment to Eupergit C. Granulated lipase converted >95% of sucrose into 6- O -lauroylsucrose in 6 u h. Accurel-lipase was also very active, converting 70% of sucrose into monoester in 2 u h. The residual activity of granules after five reaction cycles under the best reaction conditions was 72%; this value was considerably higher than the one observed for the same lipase adsorbed on Accurel (15% residual activity after five cycles).