Inorganic materials as supports for covalent enzyme immobilization: methods and mechanisms

Abstract

Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, the capability to form manageable particles with high surface area, reactivity towards derivatizing/functionalizing agents. Non-specific protein adsorption should be always considered when planning covalent immobilization on inorganic solids. A huge mass of experimental work has shown that silica, silicates, borosilicates and aluminosilicates, alumina, titania, and other oxides, are the materials of choice when attempting enzyme immobilizations on inorganic supports. More recently, some forms of elemental carbon, silicon, and certain metals have been also proposed for certain applications. With regard to the derivatization/functionalization techniques, the use of organosilanes through silanization is undoubtedly the most studied and the most applied, although inorganic bridge formation and acylation with selected acyl halides have been deeply studied. In the present article, the most common inorganic supports for covalent immobilization of the enzymes are reviewed, with particular focus on their advantages and disadvantages in terms of enzyme loadings, operational stability, undesired adsorption, and costs. Mechanisms and methods for covalent immobilization are also discussed, focusing on the most widespread activating approaches (such as glutaraldehyde, cyanogen bromide, divinylsulfone, carbodiimides, carbonyldiimidazole, sulfonyl chlorides, chlorocarbonates, N-hydroxysuccinimides).

Figure 1

Scheme of functionalization and activation of inorganic supports during covalent immobilization (functionalization with –NH₂ groups and activation with cyanogen bromide is reported as an example).

Figure 2

Several types of silanol functions can be found on the surfaces of silica-based materials: geminal silanols (a), vicinal silanols (b), isolated silanols (c). Silanetriols (d) have never been found on silica surfaces.

Scheme 1

Trialkoxyorganosilanes perform functionalization of silanols on the surface of inorganic supports.

Figure 3

The most widespread organosilanes for the functionalization of inorganic supports during protein immobilization.

Scheme 2

Alkylamine supports can be easily derivatized to carboxyl (using glutaric anhydride, left) or thiol (using N-acetyl-DL-homocysteine thiolactone, right) function.

Scheme 3

Possible mechanism for cyanogen bromide activation of silanol functions [35,173,174].

Scheme 4

Reaction pathway for cyanogen bromide activation of epoxy-functionalized supports.

Scheme 5

Reaction pathway for cyanogen bromide activation of amino-functionalized supports.

Figure 4

4-Nitrophenyl cyanate, N-cyanotriethylammonium bromide, and 1-cyano-4-dimethylaminopyridinium bromide have been described as effective cyanylating agents in alternative to BrCN [177].

Scheme 6

Activation of silanol functions with TCT.

Figure 5

The most common sulfonyl halides used in protein immobilization.

Scheme 7

Activation of primary alcoholic functions by sulfonyl halides.

Figure 6

The most used chlorocarbonates for the activation of hydroxyl-bearing supports. The molar extinction factor of the leaving group is reported.

Scheme 8

Mechanism of activation using chlorocarbonates.

Scheme 9

Mechanism of activation using thionyl chloride.

Scheme 10

The proposed mechanism for metal bridge activation of silanols [60].

Scheme 11

Glutaraldehyde can theoretically react with –NH₂ groups through two distinct mechanisms: Schiff base and Micheal-type addiction. However, the second is by far the most plausible [190,191].

Scheme 12

Carbodiimides activate carboxy-functionalized silicas, both in presence or absence of sulfo-N-hydroxysuccinimide [196].

Scheme 13

Divinylsulfone is able to activate mainly alcoholic –OH-modified silicas, allowing coupling with cysteine –SH functions [167].

Scheme 14

Mechanism of activation using p-benzoquinone.

Scheme 15

Mechanism of activation using disuccinimidyl suberate.

Figure 7

Several bifunctional N-hydroxysuccinimides esters containing cleavable cross-linking have been described.

Scheme 16

Mechanism of activation using succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

Scheme 17

Mechanism of activation using succinimidyl-3-(2-pyridyldithio)propionate (SPDP).

Scheme 18

Mechanism of activation using 2-2'-dipyridyldisulfide (DPDS).

Scheme 19

Mechanism of activation using 1,6-bismaleimidohexane (BMH).

Scheme 20

1-1'-Carbonyldiimidazole reacts with –OH from support forming an active ester, able to couple with protein lysines [216].

Scheme 21

Epoxy-functionalized support can be further functionalized with aromatic amine functions [219], that in turn undergo diazotization and coupling with protein tyrosine phenolic groups [35,218].

Scheme 22

Activation of aminated-silica with epichlorohydrin. Reaction with poly(ethylenglycol) allows the insertion of a molecular spacer long as required [221].