José V. Sinisterra

Thermal stabilization of immobilized lipase B from Candida antarctica on different supports: Effect of water activity on enzymatic activity in organic media

Abstract Covalent immobilization of C. antarctica lipase B (CALB) on sepharose, alumina, and silica was undertaken. The thermal stability of these covalently immobilized catalysts were studied and compared to adsorbed derivatives from Novo Nordisk at 50°C under wet conditions. Native enzyme and Novozym 435 follow a deactivation model E → E 1 whereas covalently immobilized derivatives and SP435A follow the model E → E 1 → E 2 . This different behavior is related to the nature of the support and the immobilization methodology. Water absorption isotherms of dry solid biocatalysts in air or isooctane were used to predict the optimum preequilibrium a w value to obtain the highest rate in the esterification of ( r,s )-ibuprofen.

Applied Biotransformations in Green Solvents

The definite interest in implementing sustainable industrial technologies has impelled the use of biocatalysts (enzymes or cells), leading to high chemo-, regio- and stereoselectivities under mild conditions. As usual substrates are not soluble in water, the employ of organic solvents is mandatory. We will focus on different attempts to combine the valuable properties of green solvents with the advantages of using biocatalysts for developing cleaner synthetic processes.

Novel microbial lipases: catalytic activity in reactions in organic media.

2,000 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Culture broths were initially tested with 1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufinyl ester, a chromogenic substrate specific for lipases. Fourteen lipase-producing microorganisms were selected and their taxonomic identification was carried out. Hydrolysis of tributyrin or olive oil and the esterification of oleic acid with heptanol were selected to preliminary evaluate the catalytic activity of these lipases. All the selected lipases catalysed this esterification reaction with good yields. Resolution of (R,S)-2-(4-isobutylphenyl) propionic acid, (R,S)-1-phenylethanol, (R,S) 1-phenylethylamine and of (R) or (S) glycidol were performed to evaluate the stereoselectivity of these novel enzymes as biocatalysts in reactions in organic media. Lipases from the fungi Fusarium oxysporum and Ovadendron sulphureo-ochraceum gave the best yields and enantioselectivities in the resolution of racemic ibuprofen and 1-phenylethanol. Several lipases displayed a high stereoselectivity in the resolution of chiral amines by an alcoxycarbonylation reaction.

2-Methyltetrahydrofuran as a suitable green solvent for phthalimide functionalization promoted by supported KF

An efficient chemoselective nitrogen functionalization of phthalimides by using KF-Alumina in 2-methyltetrahydrofuran, a solvent obtained from renewal sources, is described.

Regioselective enzymatic acylation of pharmacologically interesting nucleosides in 2-methyltetrahydrofuran, a greener substitute for THF

1-β-Arabinofuranosyl uracil, 9-β-arabinofuranosyl adenosine, 2′-O-(2-methoxyethyl)-5-methyl uridine, adenosine and uridine were enzymatically acylated with hexanoic anhydride and vinyl esters by CALB lipase (lipase B from Candida antarctica) with excellent regioselectivity in many cases and analytical reaction yields above 90%. The influence of the stereochemistry of the hydroxyl group on C-2′ was studied. Some of these esterifications were carried out in 2-methyltetrahydrofuran (MeTHF), which is described as an excellent substitute for THF in biocatalysed processes in organic media. This application for this green solvent is a proof-of-concept opening the use of MeTHF in biotransformations.

A controlled fed-batch cultivation for the production of new crude lipases from Candida rugosa with improved properties in fine chemistry

A controlled constant feeding rate fed-batch strategy using oleic acid as inducer produced a crude lipase preparation from Candida rugosa (CRL-UAB) with higher protein content, carbohydrate content and lipase activity than commercial Sigma type VII CRL. CRL-UAB was partially characterised and tested in selective biotransformations of chiral compounds in aqueous (2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE)) and organic media (2-phenyl propionic acid and ketoprofen). CRL-UAB showed higher substrate specificity and enantioselectivity in aqueous media compared to Sigma CRL. Also, higher specific initial rates with 2-phenyl propionic acid and ketoprofen were observed in organic media. The influence of water on the esterification of ketoprofen was not relevant with CRL-UAB under the conditions tested, whereas a dramatic influence was observed in Sigma CRL. Different CRL-UAB batches obtained under the same cultivation controlled conditions were identical from the point of view of chromatographic behaviour, immobilisation rates and catalytic properties, indicating that a reproducible C. rugosa lipase extract had been obtained.

Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

ABSTRACT A novel type II nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% α-helix, 10% β-strand, 16% β-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (Tm) of 64°C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40°C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2′-fluorodeoxyribonucleosides was carried out.

Water adsorption isotherm as a tool to predict the preequilibrium water amount in preparative esterification

SummaryThe cross point of the water adsorption isotherms of the lyophilised enzyme in air and of the lyophilised enzyme in the organic solvent gives a criterium to predict the equilibrium aw value to obtain the maximum yield in an esterification reaction in preparative conditions. The esterification of several (R,S) 2-arylpropionic acids is tested.

Immobilisation/stabilisation on different hydroxilic supports of lipase from Candida rugosa

Lipase from Candida rugosa has been covalently immobilized on tosyl activated matrix (agarose and corn cob) and optimum immobilisation conditions determined. The immobilized derivatives exhibited greater residual activity than the ones reported previously. Studies on the activity and stability of the different insolubilized derivatives prepared showed that the enzymatic derivative immobilized on small corn cob is resistant to inactivation by temperature, at 50°C. This derivative was 100 times more stable than its soluble counterpart. The insolubilized derivatives are more active and stable at higher temperatures (&>; 35°C) than soluble enzyme. Lipase activity (using olive oil emulsion) and esterase activity (using p-nitrophenyl butyrate) have been determined. Kinetic studies have been carried out with soluble and immobilized derivatives. The influence of Na (I) and Ca (II) on the lipase and esterase activities is discussed.

Heptyl oleate synthesis as useful tool to discriminate between lipases, proteases and other hydrolases in crude preparations

Heptyl oleate synthesis is a good test to compare crude lipases. Being a reaction selective for lipases, esterases and proteases are not active in this esterification. The heptyl oleate synthesis has been carried out with crude lipases and other hydrolases (such as proteases and esterases) from different suppliers and origins, in order to explore the methodology scope. While the initial rate of esterification seems to be mainly dependent on the concentration of lipases in the sample tested, the final synthetic yield depends on the total amount of water in the reactor. The standard protocol to evaluate crude lipases is described.