The specificity of the A-lipase from Candida antarctica (CALA) has been characterized to further clarify the scope of the biocatalyst. The lipase was found to exhibit an almost uniform activity towards various straight-chained primary alcohols and carboxylic acids, only exhibiting a low activity towards very short-chained acids. More interestingly, the enzyme was found to exhibit a high activity towards a surprising diversity of sterically hindered alcohols, including both secondary and tertiary alcohols. These results indicate that CALA can have a unique applicability for the conversion of highly branched substrates where most other lipases fail to display any activity. A new, potentially highly cost-effective, immobilization technology using silica-based granulation has been applied in the immobilization of the B-lipase from the same yeast (CALB). Highly stable particles were obtained with an activity comparable to that of the commercially available immobilized preparations of this enzyme.

The lipases from Thermomyces lanuginosus (immobilized by granulation with silica) and Candida antarctica B (adsorbed on Lewatit, "Novozym 435") were comparatively assayed for the synthesis of sugar esters by transesterification of sugars with fatty acid vinyl esters in 2-methyl-2-butanol:dimethylsulfoxide mixtures. We found that lipase from C. antarctica B is particularly useful for the preparation of 6,6'-diacylsucrose, whereas T. lanuginosus lipase catalyzes selectively the synthesis of 6-Oacylsucrose. The granulated T. lanuginosus lipase retained more than 80% of its initial activity after 20 cyles of 6 hours. Both lipases were similarly effective for the regioselective synthesis of 6'-O-palmitoylmaltose and 6-O-lauroylglucose. The effect of the synthesized sugar esters on the growth in liquid medium of various microorganisms (Gram-positive, Gram-negative and yeasts) was evaluated. 6-Olauroylsucrose and 6'-O-lauroylmaltose inhibited the growth of Bacillus sp. at a concentration of 0.8 mg/ml, and of Lactobacillus plantarum at 4 mg/ml. Sucrose dilaurates and 6-O-lauroylglucose did not show antimicrobial activity, probably due to their low aqueous solubility. As regards the inhibition of yeasts, none of the tested carbohydrate esters inhibited significantly the growth of Zygosaccharomyces rouxii and Pichia jadinii.

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 vs. sucrose monoesters can be substantially enhanced. The different regioselectivity exhibited by several lipases and proteases makes feasible to synthesize 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 catalyzed by T. lanuginosus lipase. The morphology of the support affected significantly the reaction rate and/or the selectivity of the process.

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