Characterization of sol-gel bioencapsulates for ester hydrolysis and synthesis.

Candida rugosa lipase was entrapped in silica sol-gel particles prepared by hydrolysis of methyltrimethoxysilane and assayed by p-nitrophenyl palmitate hydrolysis, as a function of pH and temperature, giving pH optima of 7.8 (free enzyme) and 5.0-8.0 (immobilized enzyme). The optimum temperature for the immobilized enzyme (50-55 degrees C) was 19 degrees C higher than for the free enzyme. Thermal, operational, and storage stability were determined with n-butanol and butyric acid, giving at 45 degrees C a half-life 2.7 times greater for the immobilized enzyme; storage time was 21 d at room temperature. For ester synthesis, the optimum temperature was 47 degrees C, and high esterification conversions were obtained under repeated batch cycles (half-life of 138 h).

Studies on immobilized lipase in hydrophobic sol-gel.

The hydrolysis of tetraethoxysilane using the sol-gel process was used to produce silica matrices, and these were tested for the immobilization of lipase from Candida rugosa by three methods: physical adsorption, covalent binding, and gel entrapment in the presence and absence of polyethylene glycol (PEG-1450). The silica matrices and their derivatives were characterized regarding particle size distribution, specific surface area, pore size distribution (Brunauer, Emmett, and Teller [B.E.T.] method), yield of grafting (thermogravimetric analyzer [TGA]), and chemical composition (Fourier transform infrared). Immobilization yields based on recovered lipase activity varied from 3.0 to 32.0%, and the highest efficiency was attained when lipase was encapsulated in the presence of PEG.

Evaluation of supports and methods for immobilization of enzyme cyclodextringlycosyltransferase.

An experimental design with factorial planning was used for the immobilization of the enzyme cyclodextringlycosyltransferase (CGTase) from Bacillus firmus (strain no. 37) to select the best combination of support, method of immobilization, and conditions that gives primarily higher average values for the specific immobilized enzyme activity, and secondarily, higher average values for the percentage of protein fixation. The experimental design factors were as follows: supports-controlled-pore silica, chitosan, and alumina; immobilization methods-adsorption, and two covalent bonding methods, either with gamma-aminopropyltriethoxysilane or hexamethylenediamine (HEMDA); conditions-7 degrees C without agitation and 26 degrees C with stirring. The best combination of factors that lead to higher average values of the response variables was obtained with immobilization of CGTase in silica with HEMDA at 7 degrees C. However, immobilization in chitosan at 7 degrees C gave the highest immobilized CGTase specific activity, 0.25 micromole of beta-CD/ (min mg protein). Physical adsorption gave low specific enzyme activities, and, in general, a high load of enzyme leads to lower specific enzyme activity.

Influence of substrate and product concentrations on the production of cyclodextrins by CGTase of Bacillus firmus, strain no. 37.

The influence of substrate or product level on the initial velocity of cyclodextrin (CD) production by cyclodextringlycosyltransferase from a Brazilian isolate of Bacillus firmus was studied. Our results indicate that the product gamma-CD is a stronger inhibitor to the reaction than beta-CD. Small saccharides could also inhibit CD production, although to a lesser extent than the products, and maltose was the strongest inhibitor among small saccharides. Increasing substrate concentration resulted in greater reduction on enzyme activity for the formation of beta-CD than for gamma-CD. We modeled the kinetics of CD production with a set of four reversible reactions including the cyclization/coupling reaction that forms/opens CDs, and three disproportionation reactions. Our model on the initial velocity data explained well the substrate inhibition phenomenon. Kinetic parameters were determined by fitting the initial velocity data into our model.

Esterification activity and stability of Candida rugosa lipase immobilized into chitosan.

Microbial lipase from Candida rugosa immobilized into porous chitosan beads was tested for esterification selectivity with butanol and different organic acids (C2-C12), and butyric acid and different aliphatic alcohols (C2-C10). After 24 h, the acids tested achieved conversions of about 40-45%. Acetic acid was the only exception, and in this case butanol was not consumed. Different alcohols led to butyric acid conversions >40%, except for ethanol, in which case butyric acid was converted only 26%. The system's butanol and butyric acid were selected for a detailed study by employing an experimental design. The influence of temperature, initial catalyst concentration, and acid:alcohol molar ratio on the formation of butyl butyrate was simultaneously investigated, employing a 2(3) full factorial design. The range studied was 37-50 degrees C for temperature (X1), 1.25-2.5% (w/v) for the catalyst concentration (X2), and 1 and 2 for the acid:alcohol molar ratio (X3). Catalyst concentration (X2) was found to be the most significant factor and its influence was positive. Maximum ester yield (83%) could be obtained when working at the lowest level for temperature (37 degrees C), highest level for lipase concentration (2.5% [w/v]), and center level of acid:alcohol molar ratio (1.5). The immobilized lipase was also used repeatedly in batch esterification reactions of butanol with butyric acid, revealing a half-life of 86 h.