Immobilization of Lipase CALB in Organically Modified Silica

In order to improve the activity of enzymes immobilized in silica, additives such as polyethylene glycol (PEG) can be introduced in the sol-gel process. This addition aims to protect the enzymes from denaturing effects by forming protection between the protein and the reaction medium. Thus, the aim of the work was to evaluate the effect of the use of the PEG additive in the process of immobilization of the commercial lipase of Candida antarctica B (CALB) in xerogel and aerogel obtained by the sol-gel technique. The mathematical model for the process was validated, and the optimum points determined were 0.09 g/ml of enzyme and 0.15 g/ml of polyethylene glycol additive for the xerogel and 0.12 g/ml of enzyme and 0.20 g/mL of polyethylene glycol additive for the aerogel. The maximum esterification activity and yield values were 544 U/g, 585%, and 266 U/g, 140% for xerogel and aerogel, respectively. Polyethylene glycol showed better performance in the esterification activity and stability as an additive when used in the xerogel, that is, when the process of obtaining the support uses the removal of the solvent only by evaporation. Regarding aerogel, a reduction in enzyme activity was observed, which may be due to the interaction of PEG with CO2 in the drying process.

Effect of Reaction Parameters on the Lipase-Catalyzed Kinetic Resolution of (RS )-Metoprolol

Racemic metoprolol is a selective ß1-blocker, which is used in cardiovascular disease treatment. It has been found that (S)-metoprolol has a higher affinity to bind the ß-adrenergic receptor compared to (R)-metoprolol. Moreover, the regulatory authorities’ high market demand and guidelines have increased the preference for single enantiomer drugs. In this work, the lipase-catalyzed kinetic resolution of racemic metoprolol was performed to obtain the desired enantiomer. The type of lipase, acyl donor, and solvent were screened out. This was achieved by Candida antarctica B lipase-catalyzed transesterification of racemic metoprolol in hexane and vinyl acetate as the solvent and an acyl donor, which gave maximum conversion of (S)-metoprolol (XS) of 52%, enantiomeric excess of substrate, (ees) of 92% and product (eeP) of 90% with enantiomeric ratio (E) of 62. This method can be considered as green chemistry, which can be applied to produce other enantiopure beta-blockers.

Immobilization of Candida antarctica B (CALB) in Silica Aerogel: Morphological Characteristics and Stability

Enzymes have been extensively used due to their catalytic properties, and immobilization is a promising technique to enhance their catalytic activity and stability. Lipases are enzymes naturally efficient, can be employed for the production of many different molecules, and have a wide range of industrial applications thanks to their broad selectivity. The objective of the present study was to characterize the Candida antarctica B CALB immobilized obtained using the aerogel technique regarding the morphological characteristics of the aerogel silica and its stability. For this purpose, analyzes of XRD, adsorption-desorption isotherms, TGA, SEM, and stability (storage, operational, and thermal) were performed. The supports obtained have an amorphous structure and isotherm type IV. Regarding TGA, two distinct regions were obtained and studied. Aerogels showed an increase in thermal, storage, and operational stability in relation to the free enzyme and demonstrated between 8 and 12 cycles of reuse. The contribution of this work was to present the stability advantages of the immobilized CALB enzyme through the sol-gel technique.

Processo enzimático para produção de acetato de isoamila livre de solvente orgânico

Nos últimos anos, a crescente demanda por produtos naturais na indústria de alimentos incentivou esforços notáveis no desenvolvimento de processos biotecnológicos para a produção de compostos aromatizantes. O presente estudo relata a maximização da produção de acetato de isoamila por esterificação de álcool isoamílico e ácido acético em um sistema isento de solventes usando duas lipases diferentes como catalisador. A lipase Novozyme NZL-102-LYO-HQ (Candida antarctica B imobilizada em poliuretano – PU) foi comparada com a comercializada Novozym 435 e foi avaliada em termos de desempenho, rendimento e estabilidade operacional. Os efeitos nas taxas de reação mostraram valores maximizados diferentes, empregando esses dois catalisadores, no entanto as condições reacionais no que se refere a razão molar do substrato (ácido acético: álcool isoamílico), massa de catalisador e tempo de reação foram distintos. Embora Novozym 435 exibisse maior conversão inicial com relação a NZL-102-LYO-HQ, ambas formas lineares e apresentaram uma conversão superior a 90%. Para o catalisador Novozym 435 (razão molar 1:3 de ácido acético:álcool isoamílico e massa de catalisador 17% em peso) em 240 minutos e a Novozyme NZL-102-LYO-HQ (Candida antarctica B imobilizada em poliuretano – PU) (razão molar 1:5 de ácido acético:álcool isoamílico e massa de catalisador 7% em peso) em 360 minutos de reação com 10 ciclos de reutilização. Sendo assim esses resultados são de grande valia para a produção industrial, sobretudo na síntese de ésteres de cadeia média, como acetato de isoamila em diferentes preparações enzimáticas, tornando uma fonte promissora comparado com estudo apresentados na literatura aberta.

Biodegradable Oligoesters of ε-Caprolactone and 5-Hydroxymethyl-2-Furancarboxylic Acid Synthesized by Immobilized Lipases

Following the latest developments, bio-based polyesters, obtained from renewable raw materials, mainly carbohydrates, can be competitive for the fossil-based equivalents in various industries. In particular, the furan containing monomers are valuable alternatives for the synthesis of various new biomaterials, applicable in food additive, pharmaceutical and medical field. The utilization of lipases as biocatalysts for the synthesis of such polymeric compounds can overcome the disadvantages of high temperatures and metal catalysts, used by the chemical route. In this work, the enzymatic synthesis of new copolymers of ε-caprolactone and 5-hydroxymethyl-2-furancarboxylic acid has been investigated, using commercially available immobilized lipases from Candida antarctica B. The reactions were carried out in solvent-less systems, at temperatures up to 80 °C. The structural analysis by MALDI TOF-MS, NMR, and FT-IR spectroscopy confirmed the formation of cyclic and linear oligoesters, with maximal polymerization degree of 24 and narrow molecular weight distribution (dispersity about 1.1). The operational stability of the biocatalyst was explored during several reuses, while thermal analysis (TG and DSC) indicated a lower thermal stability and higher melting point of the new products, compared to the poly(ε-caprolactone) homopolymer. The presence of the heterocyclic structure in the polymeric chain has promoted both the lipase-catalyzed degradation and the microbial degradation. Although, poly(ε-caprolactone) is a valuable biocompatible polymer with important therapeutic applications, some drawbacks such as low hydrophilicity, low melting point, and relatively slow biodegradability impeded its extensive utilization. In this regard the newly synthesized furan-based oligoesters could represent a “green” improvement route.

Synthesis, characterization and enzymatic degradation of copolymers of ε-caprolactone and hydroxy-fatty acids

New copolymers of ε-caprolactone with three hydroxy-fatty acids, 12-hydroxy stearic acid, 16-hydroxyhexadecanoic acid and ricinoleic acid, were synthesized by catalytic polyesterification. The reactions were carried out in solvent-free systems and in organic solvents as well, using tin(II) 2-ethylhexanoate as catalyst, at different temperatures and molar ratios of the comonomers. Cyclic and linear polymeric products with medium molar weight of about 2000 Da have been synthesized and their chemical structures were confirmed by FT-IR, NMR and MALDI-TOF MS analysis. The synthesis parameters were optimized and the ε-caprolactone/hydroxy acid molar ratio was set as 5:1, according to mass spectrometry results. The biodegradability of the newly synthesized polymers was studied in the presence of Candida antarctica B lipase in phosphate buffer solutions (pH=7.4), at 37°C. The weight-loss profile emphasized the degradation of the 16-hydroxyhexadecanoic acid based polymer samples at more than 50% of their initial weight in 18 days of incubation in the presence of the lipase. The composition of the degradation products was assessed using the GC-MS technique and displayed residues of the comonomers moieties.