Manuel Bernabé

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.

Quantitative enzymatic production of 6-O -acylglucose esters

Selective production of emulsifiers from glucose and fatty acids has been achieved using an immobilized Candida antarctica lipase. Optimization of process selectivity considers the solubilities of the sugar and its monoesters in acetone at different temperatures, the percentage of this organic solvent in the reaction mixture, and the reaction temperature. The solvent (acetone) is both easily eliminated and accepted by the European Community for use in the manufacture of foods and/or food additives. Different fatty acids with a longer length chain than that of caprylic acid may be employed. For saturated fatty acids longer than lauric acid, continuous precipitation of the monoester as it is formed at 40 degrees C permits nearly complete conversion (98%) of glucose to the monoester within 2-3 days. The procedure does not require total dissolution of the sugar, and precipitation of the monoester permits selective conversion of charges of glucose higher than 100 mg/mL solvent. A scaleup of the process under the optimum conditions gives high yields of 6-O-lauroyl glucose, which may be readily prepared on a gram scale. Copyright 1998 John Wiley & Sons, Inc.

A Simple Procedure for the Regioselective Synthesis of Fatty Acid Esters of Maltose, Leucrose, Maltotriose and n-Dodecyl Maltosides

Abstract The enzymatic acylation of several di- and trisaccharides with acyl donors ranging from 8 to 18 carbon atoms was carried out by transesterification with the corresponding vinyl esters. The reaction was performed in 2-methyl-2-butanol/dimethylsulfoxide mixtures using the lipase from Humicola lanuginosa (immobilized on Celite). Maltose, maltotriose and n-dodecyl maltosides were specifically acylated in the primary hydroxyl of the non-reducing-end glucose moiety; the acylation of leucrose occurred preferentially in the primary hydroxyl of the glucose ring.

Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides

Abstract.The polysaccharides obtained from the alkali-extractable, water-soluble fraction (F1SS) from the cell wall of Myrothecium verrucaria and Myrothecium atroviride were shown to be composed of β-(1→6)-galactofuranose fully substituted at O-2 by terminal residues of α-glucopyranose and α-glucuronic acid. Glucuronic acid was substituted at O-4 by glucopyranose in the Myrothecium species M. inundatum, M. setiramosum, M. prestonii, M. tongaense and M. roridum. The acidic polysaccharides from Phaeostilbella atra (=Myrothecium atrum) and Myrothecium gramineum lacked the backbone of 2,6 di-O-substituted galactofuranose and contained a high amount of O-5-substituted β-galactofuranose. The structures of the polysaccharides isolated from Myrothecium cinctum and Myrothecium penicilloides were unrelated to each other and to the polysaccharides from the other species analysed. The usefulness of these polysaccharides as a characteristic for delimitation of the genus Myrothecium is discussed.

Quantitative enzymatic production of 1,6-diacyl fructofuranoses

Three different 1,6-diacyl fructofuranoses have been prepared enzymatically. At low temperature (5°C), the synthesis produces quantitative yields of the diester by simple addition of the original sugar to a solution of the fatty acid in a solvent (acetone) which is accepted by the EEC for use in the manufacture of food additives. A strategy to reduce the reaction times is also reported. The method is not limited by the low solubility of the sugar in the medium. In contrast with alternative enzymatic methods, the indicated method minimizes the solvent/sugar ratio. The stability of the biocatalyst (Novozym 435) is high relative to the required reaction time.

Regioselective deacylation of 1,6-anhydro-β-D-galactopyranose derivatives catalyzed by soluble and immobilized lipases

Abstract The regioselectivity of the hydrolysis of secondary acyl esters of carbohydrates by lipases has been investigated. 1,6-Anhydro-2,3,4-tri- O -butanoyl-β- D -galactopyranose ( 1 ) and 2,3,4-tri- O -acetyl-1,6-anhydro-β- D -galactopyranose ( 8 ), used as conformationally rigid model compounds, were treated with soluble lipases from pig pancreas and with lipases from C. cylindracea and Mucor miehei immobilized on agarose and ion exchange resin, respectively. Analysis of the reaction mixtures by 1 H-n.m.r. spectroscopy indicated that the axially oriented acyl esters at C-2 react faster than the equatorially oriented acyl esters at C-4, and these faster than the axially oriented acyl esters at C-3. The corresponding 3,4-di-, and 3-mono- O -acyl esters could be isolated in yields from moderate to excellent depending on the starting material and the reaction conditions.

STRUCTURE OF COMPLEX CELL WALL POLYSACCHARIDES ISOLATED FROM TRICHODERMA AND HYPOCREA SPECIES

The structure of fungal polysaccharides isolated from the cell wall of Trichoderma reesei, T. koningii, and Hypocrea psychrophila, have been investigated by means of chemical analyses and 1D and 2D NMR spectroscopy. The polysaccharides have an irregular structure, idealized as follows: [formula: see text] The proportions of the different side chains vary from a species to another, being n above some three times larger in H. psychrophila than in T. reesei or T. koningii.

Synthesis of asymmetric 1-amino-2-phenylcyclopropanecarboxylic acids by diastereoselective cyclopropanation of highly functionalized homochiral olefines

Abstract Homochiral benzylidenediketopiperazine and α-benzoylaminocinnamic esters react with diazomethane giving high to good diastereomeric ratios of spiropyrazoline derivatives which, on photolysis and acid hydrolysis of the resulting spirocyclopropyl compounds gave, respectively, (+)- and (−)-1-amino-2-phenylcyclopropanecarboxylic acids.

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus.

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit:[→ 3)-β-D-Galf-(1 → 5)-β-D-Galf-(1 →]n → mannan core.The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae:[→ 5)-β-D-Galf-(1 →]n → mannan core.The mannan cores have also been investigated, and are constituted by a (1 → 6)-α-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 → 2) linked α-mannopyranoses. Published in 2004.

Structural studies of fungal cell-wall polysaccharides from two strains of Talaromyces flavus

The water-soluble cell-wall polysaccharides isolated from strains CBS 352.72 and 310.38 of Talaromyces flavus have been investigated by chemical analyses and NMR studies. Two different skeletons coexist, having the structures: [formula:see text]. The small differences between the polysaccharides isolated from both strains are probably due to slight diminution of branching in strain 352.72, as compared with strain 310.38.