Silica immobilized enzymes

Dezotti, M. Innocentini-Mei, L.H. Duran, N. Silica immobilized enzyme catalyzed removal of chlorolignins from eucalyptus Kraft effluent. J. Biotechnol. 1995, 43, 161-167. 

When a mixture containing AMP (0.5 nmol), ADP (0.2 nmol) and ADP-ribose (1 nmol) is applied to an alcohol dehydrogenase-silica column [see Section 4.2.3(iii)] under isocratic conditions, separation of the three species is achieved on the basis of the different strength of interaction between the silica-immobilized enzyme and the three nucleotides Figure 6) (21). 

The porosity of hybrid nanocomposites provides access of the substrates to immobilized enzyme and their proper functioning. It is attributable to the absence of volume shrinkage of synthesized materials after their preparation. Although the compacting does not occur as in the common two-stage processes (Figure 3.7), enzyme macromolecules are held inside the silica matrix and not easily washed out of it. 

Addition of third components to nanohybrids of proteins and mesoporous materials sometimes brings advantages in their functions. Kim, Hyeon, and coworkers immobilized enzyme molecules together with magnetite (Fe304) nanoparticles in hierarchically ordered, mesocellular, mesoporous silica (HMMS) (Figure 4.25)... 

Nanostructured silica and ordered mesoporous silicas have been envisaged as small enzyme immobilization supports [196]. The encapsulation approach is required either to further immobilize enzymes adsorbed in the channels by reducing the pore opening by further silylation or by encapsulating the enzyme directly [197]. 

Enzymes were immobilized onto silica gel by covalent crosslinking with glutaraldehyde in a procedure similar to that of Kondo et al. (3). Briefly, 1-4 g of silica gel was incubated in 1-13 wt% glutaraldehyde and in 100-1000 mL of enzyme solution for up to 48 h. The immobilized enzyme was recovered by filtration and washed to remove loosely bound enzyme. Samples of the soluble enzyme were collected before and after immobilization and assayed for activity, to provide an estimate of enzyme uptake onto the support. The a-amylases studied were Spezyme Fred (Genencor), Allyzme (Alltech), and Liquozyme (Novozymes). The cellulases studied were Spezyme CP and Spezyme CE (Genencor). 

Various lipase immobilization methods were tested with different silica matrices, and the immobilized enzyme samples were examined by morphologic, physicochemical, and biochemical characterization methods. The results allowed correlation of the activity-coupling yield of different immobilization methods in relation to the incorporation of lipase in the silica gels and showed that the most active biocatalyst resulted from the encapsulation of commercial CRL in the presence of PEG. 

Yao et al. reported a flow injection analytical system for the simultaneous determination of acetylcholine and choline that made use of immobilized enzyme reactors and enzyme electrodes [25]. Acetylcholineesterase-choline oxidase and choline oxidase were separately immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, and incorporated in parallel as the enzyme reactors in a flow injection system. The sample containing acetylcholine and choline in 0.1 M phosphate buffer (pH 8.3) carrier solution was injected into the system. The flow was split to pass through the two reactors, recombined, and mixed with 0.3 mM K4Fe(CN)6 reagent solution before reaching a peroxidase immobilized electrode. Because each channel had a different residence time, two peaks were obtained for choline and total acetylcholine and choline. Response was linear for 5 pM-0.5 mM choline, and for 5 pM 1 mM acetylcholine plus choline. The detection limits were 0.4 pM for choline and 2 pM for acetylcholine. 

Takahashi H, Li B, Sasaki T et al (2000) Catalytic activity in organic solvents and stability of immobilized enzymes depend on the pore size and surface characteristics of mesoporous silica. Chem Mater 12 3301-3305... 

Fig.. Repeated use of Immobilized lipase in non-aqueous system. Between each repetition the Immobilized enzyme adsorbed to a silica gel support matrix was washed with water. Fresh substrate (ethanol, butyric acid) was added In hexane. One cycle represents ca. 2 ) h.

By employing this integrated cofactor-regenerated approach, N-acetyl lacto-samine can be formed in multigram amounts with native [30] or immobilized enzymes on silica gel [32] or on a polyamide-based carrier [33]. 

By chemical modification of the silica surface it has become possible to design new highly-selective adsorbents and catalysts, active polymer fillers, efficient thickeners of dispersive media. Interest in the modified silicas, in particular, in the activated matrices based on functional organosilicas has quickened in the past few years as a result of the favorable prospects for their application for various kinds of chromatographic separation, preparation of grafted metal complex catalysts, immobilized enzymes and other biologically active compounds [1]. 

A distribution coefficient is used widely in various areas involving two-phase systems [43,44] to describe behaviour of immobilized enzymes, electrode systems, different kinds of chromatographic separation and, in particular, makes it possible to correlate analytically parameters describing equilibria on a surface with parameters of column and thin-layer chromatography, whose success is determined mostly by extensive use of pristine and modified silicas as adsorbents and supports. 

Galactosidase has been attached to glass in this way.56 The immobilized enzyme is useful in removing lactose from milk, for the benefit of those persons who are intolerant of lactose. A similar technique was used to attach glucose oxidase to magnetite (I i O-i) particles, starting with a trimethoxysilane.57 This permits easy separation with a magnet. The immobilized enzyme was 50% less active than the native enzyme, but kept 95% of its activity after 9 months at 4°C. Another way to attach an enzyme to a support is to treat the metal oxide support first with 2,4,6-trichloro 1,3,5 triazine (cyanuric chloride) (5.18), then with the enzyme.58 This was done on both alumina and on silica. 

There are some cases in which the immobilized enzyme has a higher activity than its free form. A lipase put on silica by the sol-gel method, using an alkyltrimethoxysilane, had twice the activity of the free enzyme.63 It retained 63% of its activity after 3 months (i.e., much more than that of the free enzyme). It was stable enough mechanically for use in Auidized beds. Inclusion of FesCL nanoparticles in the preparation made the enzyme easy to separate. Some enzymes trapped in silica by the sol-gel process are as active as the free enzymes and are much more stable thermally and to pH changes.64... 

Candida rugosa lipase Ionic liquid functionalized magnetic silica NPs Enzyme immobilization [179]... 

---