Heiddy M. Alvarez

Stability and structural changes of horseradish peroxidase: microwave versus conventional heating treatment.

Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4-6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.

MICROWAVE ACTIVATION OF IMMOBILIZED LIPASE FOR TRANSESTERIFICATION OF VEGETABLE OILS

This work investigated the effect of microwave irradiation (MW) on the ethanolysis rate of soybean and sunflower oils catalyzed by supported Novozyme 435 (Candida antarctica). The effects of tert-butanol, water addition and oil:ethanol molar ratio on transesterification were evaluated under conventional heating (CH), and under optimum reaction conditions (with no added water in the system, 10% tert-butanol and 3:1 ethanol-to-oil molar ratio). The reactions were monitored up to 24 h to determine the conditions of initial reaction velocity. The investigated variables under MW (50 W) were: reaction time (5.0-180 min) and mode of reactor operation (fixed power, dynamic and cycles) in the absence and presence of tert-butanol (10% (w/w). The measured response was the reaction conversion in ethyl esters, which was linked to the enzyme catalytic activity. The results indicated that the use of microwave improved the activity at fixed power mode. A positive effect of the association of tert-butanol and MW irradiation on the catalytic activity was observed. The reaction rate improved in the order of approximately 1.5 fold compared to that under CH with soybean oil. Using soybean oil, the enzymatic transesterification under MW for conversion to FAEE (fatty acid ethyl esters) reached >99% in 3h, while with the use of CH the conversions were about 57% under similar conditions.

Biotechnological route for obtaining methyl esters from crambe oil (Crambe abyssinica)

The fatty acid esters synthesis by transesterification of oils to produce biodiesel commonly involves methanol or ethanol as acyl acceptor. The transesterification of vegetable oils catalyzed by lipases is an alternative process for obtaining biodiesel. These biocatalysts working under mild conditions of temperature, allow for easy recovery of glycerol, synthesis of specific alkyl esters and transesterification of triglycerides with high concentrations of free fatty acids. The seeds of crambe (Crambe abyssinica) have a high oil content and great potential for biodiesel production [1]. In this way, the present work aims at crambe oil transesterification with methanol catalyzed by immobilized lipase Novozyme 435 (Candida antarctica).

Horseradish peroxidase encapsulation in alginate microspheres in the presence of imidazolium ionic liquids.

Biocatalysis with free enzymes may not be economically viable due to the complexity of their recovery in the reaction medium. With the objective of enabling the total activity recovery yield of biocatalyst and to improve the operational characteristics, have been applied enzyme immobilization techniques. The main interest in the enzyme immobilization is to obtain a catalyst whose activity and stability is not affected during the process when compared to the free enzyme. The objective of this work was to immobilize horseradish peroxidase (HRP) by encapsulation method in alginate microspheres in the presence of imidazolium ionic liquid (ILs). The enzyme encapsulation was carried with the enzyme incorporation in an aqueous solution of sodium alginate to 0.05 % (w / v). The influence of the enzyme loading was studied in the range of 0.0406-0.65 mg HRP / g alginate. Alginate microspheres were obtained by dripping in calcium chloride solution (1M) according to literature [1]. The HRP immobilization in the presence of ILs was conducted using the enzyme loading more appropriate, incorporating in the immobilization medium 1% (w/v) of IL. The ILs studied as additives were: [C4mim]TF2N; [C4mim]BF4; [C4mim]HSO4; [C4mim]Ac e [C4mim]PF6. The activity of free and immobilized enzyme was verified by colorimetric method based on the change of absorbance at 470 nm due to the formation of product in guaiacol oxidation for three minutes. The results obtained for biocatalysts immobilized by encapsulation suggest that the use of IL as an additive in the immobilization process causes a significant increase in the total activity recovery yield (RA) HRP (increase from 5.2 to 23.5 %). The results for the HRP immobilization showed that RA was 23.5% and 20.40% for the LIs [C4mim] HSO4 and [C4mim]TF2N, respectively. [C4mim]Ac provided RA of 13.3 %, while the biocatalyst with [C4mim] PF6 showed RA of 17.3 %. The [C4mim] BF4 showed the lowest performance as an additive among the ILs studied. According to Diego et al.(2009) [2] there is not have a rule to predict the behavior of immobilized enzymes in the presence of ILs. The results obtained for biocatalysts immobilized by encapsulation in alginate microspheres show that the use of ionic liquids in this process had a positive effect on the activity of the immobilized biocatalyst. Additional studies in the structural characteristic of the immobilized biocatalyst are being conducted to evaluate the influence of ILs in the supporting structure.

Carboxylic acid chlorides as arylating agents in the palladium-catalyzed Heck reaction - The Blaser reaction

Heck reaction is a powerful tool in organic synthesis to perform C-C couplings by which functionalized alkenes can be obtained under mild conditions and great selectivity. Developing alternative methodologies where arylating agents different from the conventional aryl halides or triflates can be introduced with success is one of the areas of research nowadays. By these methodologies it is envisaged to provide better conditions for the industrial application of the palladium-catalyzed Heck C-C coupling. One of the aims of this research is to introduce non expensive arylating agents as well as to minimize waste. In this regard we describe in this paper some studies present in the literature where acid chlorides and other arylcarboxylic acid derivatives are employed as arylating agents in the Heck reactions.