Alini Tinoco Fricks

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.

The novel mesoporous silica aerogel modified with protic ionic liquid for lipase immobilization

Mesoporous silica supports (aerogels) were used to immobilize Burkholderia cepacia lipase (BC) by encapsulation (EN or ENIL), physical adsorption (ADS or ADSIL) and covalent binding (CB or CBIL) into or onto the aerogel modified with protic ionic liquid (PIL). Yield immobilization (Ya) and operational stability were determined by the hydrolytic reaction of olive oil. Ya (37% to 83% by physical adsorption) and operational stability (2 to 23 batches by encapsulation) increased when the support was modified with PIL. For immobilized derivates observed by the BET method, in this case ADS and CB for ADSIL and CBIL, increased pores size was observed, possibly due to the higher amount of BC immobilized conferring Ya and operational stability. This effect was probably attributed to the entry of the enzyme into the pores of the silica aerogel structure. SEM images showed a change in the structure and properties of immobilized lipase derived with PIL. A characteristic FTIR band was obtained for the silanol groups and amides I, IV and V, demonstrating the efficiency of immobilization of BC. The most efficient biocatalysts were ADSIL with regard to yield immobilization and ENIL for operational stability.

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.

Screening of immobilization method in aerogel matrix in the presence of protic ionic liquid

Aerogel and xerogel are formed via hydrolysis and polycondensation reactions of silica precursors, such as tetraethylorthosilicate (TEOS), always careful not to cause collapse, reduction in surface area and pore size. Several studies shown the use sol-gel entrapment possesses a number of desirable attributes, as the enzyme is physically entrapped in a rigid glass framework that permitted stabilization of the enzymes, terciary structure owing to the tight gel network. In this study, have focused on the screening of immobilization method in aerogel matrix in the presence of protic ionic liquid (PIL) with evaluation catalytic efficiency and operational stability. The novel mesoporous silica supports (aerogels) obtained by the sol-gel technique was used to immobilize commercial Burkholderia cepacia (BC) lipase by physical adsorption, covalent binding and encapsulation in the absence and presence de protic ionic liquid (N-methylmonoethanolamine pentanoate - C5). Catalytic efficiency was determined in the analyses of hydrolytic activities were carried out on the lipase loading solution and immobilized enzyme to determine the total activity recovery yield, Ya (%). For physical adsorption the recovery of activity with PIL was 82.95% (ADSLI) and in the absence 70.31% (ADS). Operational stability of the enzyme has also been examined and obtained similar values a half-life of 0.73 h to ADSLI and 0.88 h for ADS occurs with 2 batches. And other immobilized technique, covalent binding the results showed that in aerogels supports in the presence of C5 obtained 69.13% (CBLI) of total activity recovery yield and 39.11% (CB). Under such conditions, the operational stability tests indicated that a small enzyme deactivation occurs after 15 batches, revealing a biocatalyst half-life of 7.5 h for CBLI. For encapsulation, the recovery of activity with PIL (ENLI) was 45% and 37% in the absence PIL (EN). ENLI exhibited a half life of more than 23 batches with 70% of its activity remaining, with around 11.5 h. And EN the half-life was 6.5 hours, totaling 13 batches. Therefore, ADSLI presented good efficiency with total activity recovery yield but for operational stability ENLI showed the best between immobilization methods studied. Hence, it is rather appropriate for application in hydrolytic processes.