Effects of pore characters of mesoporous resorcinol–formaldehyde carbon gels on enzyme immobilization

This paper demonstrates, for the first time, the use of resorcinol–formaldehyde carbon gels (RFCs) as enzyme carriers. The immobilization behavior of Bacillus licheniformis serine protease in RFCs of different pore characters was investigated. RFCs derived with (RF1) and without (RF2) cationic surfactant (trimethylstearylammonium chloride; C18) resulted in predominantly microporous, and mesoporous characters, respectively. It was found that support pore size and volume were key parameters in determining immobilized enzyme loading, specific activity, and stability. RF2, with higher mesopore volume (V_mes: RF1 = 0.21 cm³/g; RF2 = 0.81 cm³/g) and mesopore size radius (RF1 = 1.7–3.8 nm; RF2 = 7.01 nm), accommodated approximately fourfold more enzyme than RF1. Serine protease loading in RF2 could reach as high as 21.05 unit/g support. In addition, RF2 was found to be a better support in terms of serine protease operation and storage stability. Suitable mesopore size likely helped preventing immobilized enzyme from structural denaturation due to external forces and heat. However, immobilized enzyme in RF1 gave 12.8-fold higher specific activity than in RF2, and 2.1-fold higher than soluble enzyme. Enzyme leaching was found to be problematic in both supports, nonetheless, higher desorption was observed in RF2. Enhancement of interaction between serine protease and RFCs as well as pore size adjustment will be necessary for repeated use of the enzyme and further process development.     

Effects of pH and pore characters of mesoporous silicas on horseradish peroxidase immobilization

Effects of immobilization pH and pore characters of mesoporous silicas (MPSs), MCM-41, SBA-15, and MCF, were simultaneously investigated for the immobilization of horseradish peroxidase (HRP; EC 1.11.1.7). MCM-41 and SBA-15 were rod-like with respective average pore diameters of 32, and 54 Å, while that of MCF with spherical cell and frame structure was 148 Å. Moreover, the MPSs synthesized were of identical surface functional groups and similar contents of free silanol groups. At immobilization pH 6 and 8 almost 100% HRP loadings were obtained and insignificant leaching were observed for all types of supports at pH 6. However, MCF was found to give both the highest enzyme loading and leaching at pH 10. Maximum and minimum HRP activities were obtained at respective immobilization pH 8, and 6. Activities of immobilized HRP increased with support pore diameters in the order: MCM-41 < SBA-15 < MCF. HRP immobilized at pH 8 gave the highest storage stability (both at 4 °C and room temperature), and in opposition to pH 6. In addition, HRP immobilized in MCF was found to be the most stable under storage. The finding should be useful for the creation of biocatalysts and biosensors.     

Chiral epoxide production using mycobacterium solubilized in a water-in-oil microemulsion

The application of many biotransformation processes is limited because the substrates/products are poorly water soluble, can be further metabolized, or are inhibitory. Hence non-aqueous media (e.g. two-phase systems, low water environments) are being examined to determine whether they can be used to overcome these problems. One novel approach is to encapsulate whole cells in water-in-oil (w/o) microemulsions (reverse micelles). In this study we have investigated the influence of key system parameters on system stability and epoxidation activity of Mycobacterium M156 cells in reverse micelles comprised of a mixture of Tween 85 and Span 80 (10-20 w%, with an hydrophilic/lipophilic balance [HLB] of 10 and a weight ratio of Tween 85 to Span 80 = 5.7) in n-hexadecane. It was found that the minimum allyl phenyl ether (APE) concentration required in the bulk hexadecane solvent phase for epoxidation to occur was 15 mM, whereas the minimum molar ratio of water to surfactant (W(0)) was 35. The optimum epoxidation rate achieved was 3.8 nmol/mg dwt-min with an APE concentration of 50 mM, and a W(0) of 50, with an enantiomeric excess (ee) of 86%. However, epoxidation was found to terminate approximately 3 h after initiation, and the causes for this were postulated to be either: the deleterious effect of the solvent on the Mycobacteria; inactivation of the energy generating system; an insufficient energy supply, or; the instability of the monooxygenase enzyme. It was concluded that on balance emulsion systems are not an economically viable system for producing phenyl glycidyl ether (PGE).