Changes on enantioselectivity of a genetically modified thermophilic lipase by site-directed oriented immobilization

Biocatalysis at Extreme Temperatures: Enantioselective Synthesis of both Enantiomers of Mandelic Acid by Transterification Catalyzed by a Thermophilic Lipase in Ionic Liquids at 120 °C

“…A similar behavior was reported by Fernandez-Lorente et al [64] with BTL2 immobilized on octylagarose; these same authors described the use of immobilized chemicallyaminated BTL2 derivatives covalently linked to CNBr-agarose or adsorbed on glyoxylagarose, and in this case (S)-2 (with only one exception) was the major enantiomer [65]. On the other hand, Godoy et al [72], using wild-type BTL2 as well as some mutants produced by introducing a unique cysteine into different positions of the protein surface, reported a low enantioselectivity (24% ee of the corresponding (R)-2 acid for the soluble wild-type enzyme and ranging from 19-38% for the different mutants) in the hydrolysis of (R, S)-1 at pH=7 and 25°C. Remakably, the direct immobilization of BTL2 and its mutants on disulfide supports slightly increased the enantioselectivity (ee fluctuating from 20 to 50%), while the use of aldehyde-disulfide supports dramatically increased the enantiomeric discrimination up to ee values higher than 99% [72].…”

“…Moreover, derivatives of (R)-mandelic acid have been also used as chiral synthons in the preparation of some drugs with different therapeutical activities: platelet/antithrombotic agents (clopidogrel [103]), vasodilator (cyclandelate [104]), anti-tumor (complex of cis-[Pt{2-(α-hydroxy)benzylbenzimidazole))2Cl2] [105], antiobesity [106,107] or CNS-stimulant dopaminergic agents ((R)-pemoline [108]). Conversely, (S)-mandelic acid is used for the production of non-steroidal anti-inflammatory drugs such as deracoxib and celecoxib [109] Selection of (R, S)-1 as model substrate was based in many previous studies in which BTL2 (mainly immobilized) had been tested for catalyzing the stereoselective hydrolysis (Figure 1(b)) [61,65,67,72,74]. Thus, Palomo et al [61] reported the hydrolysis of (R, S)-1 using several preparations of BTL2 immobilized on different supports (octadecyl agarose, glyoxyl agarose or polyethyleniminemodified (PEI) sepabeds [63]), at different pH values (5, 7 and 9) and different temperature (4, 25 and 37°C).…”

“…On the other hand, Godoy et al [72], using wild-type BTL2 as well as some mutants produced by introducing a unique cysteine into different positions of the protein surface, reported a low enantioselectivity (24% ee of the corresponding (R)-2 acid for the soluble wild-type enzyme and ranging from 19-38% for the different mutants) in the hydrolysis of (R, S)-1 at pH=7 and 25°C. Remakably, the direct immobilization of BTL2 and its mutants on disulfide supports slightly increased the enantioselectivity (ee fluctuating from 20 to 50%), while the use of aldehyde-disulfide supports dramatically increased the enantiomeric discrimination up to ee values higher than 99% [72].…”

New Strategy for the Immobilization of Lipases on Glyoxyl–Agarose Supports: Production of Robust Biocatalysts for Natural Oil Transformation

“…The activity values (1.01–1.88 μmol min −1 ·g catalyst −1 ) were close to those of previously obtained BTL2 derivatives using other immobilization strategies [ 22 ]. As reported for other lipases in hydrolytic reactions, immobilization conditions influence derivative activity and selectivity [ 50 , 51 ]: the aminated enzyme immobilized at pH 8.0 with DTT and basified (at pH 10.1) results in the best biocatalyst in terms of selectivity, with values close to those of the aminated BTL2 mutant immobilized by site-directed multipoint covalent attachment at the surface comprising native Gln40 residue (eicosapentaenoic acid (EPA) to docosahexaenoic acid (DHA) ratios, EPA/DHA of 2.32 and 2.4, respectively) [ 22 ]. These properties, along with results concerning stability ( Section 2.2 ), suggest that both derivatives may share a similar enzyme–support orientation and interaction strength, however, each derivative was obtained through different strategies which are much less simple than that presented here.…”

Asymmetric hydrolysis of dimethyl-3-phenylglutarate in sequential batch reactor operation catalyzed by immobilized Geobacillus thermocatenulatus lipase

“…Lipase from Geobacillus thermocatenulatus (BTL2) is a non-commercial intracellular enzyme presenting two "lids" covering the active site, a feature also reported for lipases from Pseudomonas stutzeri [2], Pseudomonas aeruginosa [3], Staphylococcus simulans [4], Serratia marcenses [5], Pseudomonas sp MIS38 [6] and T1 from Geobacillus zalihae [7]. The interest in this lipase is based on its stability at high pH and temperature and in the presence of organic solvents, and the ability to carry out selective reactions [8,9].…”