activity is exhausted, the carrier may easily be reused by reloading it with fresh
biocatalyst.
Covalent Attachment
Covalent binding of an enzyme onto a macroscopic carrier leads to the irreversible
formation of stable chemical bonds, which prevents leakage completely. A disadvantage of this method is that rather harsh conditions are required since the protein must
undergo a chemical reaction. Consequently, some loss of activity is always observed
[472, 473]. As a rule of thumb, each bond attached to an enzyme decreases its native
activity by about one fifth. Consequently, residual activities generally do not exceed
60–80% of the activity of the native enzyme, and values of around 50% are normal.
The functional groups of the enzyme which are commonly involved in covalent
binding are nucleophilic, i.e., mainly N-terminal and ε-amino groups of lysine, but
also carboxy-, sulfhydryl-, hydroxyl-, and phenolic functions. In general, covalent
immobilization involves two steps, i.e., (i) activation of the carrier with a reactive
‘spacer’ group and (ii) enzyme attachment. Since viable cells usually do not survive
the drastic reaction conditions required for the formation of covalent bonds, this type
of immobilization is only recommended for isolated enzymes.
Porous glass is a popular inorganic carrier for covalent immobilization
[474]. Activation is achieved by silylation of the hydroxy groups using
aminoalkylethoxy- or aminoalkyl chlorosilanes as shown in Scheme 3.45. In a
subsequent step, the aminoalkyl groups attached to the glass surface are either
transformed into reactive isothiocyanates or into Schiff bases by treatment with
thiophosgene or glutardialdehyde, respectively. Both of the latter species are able to
covalently bind an enzyme through its amino groups.
Carriers based on natural polymers of the polysaccharide type (such as cellulose
[475], dextran [476], starch [477], chitin, or agarose [478]) can be useful alternatives to inorganic material due to their well-defined pore size. Activation is
achieved by reaction of adjacent hydroxyl groups with cyanogen bromide leading
to the formation of reactive imidocarbonates (Scheme 3.46). Again, coupling of the
enzyme involves its amino groups. To avoid the use of hazardous cyanogen
bromide, pre-activated polysaccharide-type carriers are commercially available.
During recent years, synthetic copolymers (e.g., based on polyvinyl acetate)
have become popular (Scheme 3.47) [479–481]. Partial hydrolysis of some of the
acetate groups liberates a hydroxyl functionality, which is activated by epichlorohydrin [482]. A number of such epoxy-preactivated resins are commercially available. Among them, Eupergit C™ is widely used. It is a macroporous copolymer of
N,N
0 -methylene-bi-(methacrylamide), glycidyl methacrylate, allyl glycidyl ether
and methacrylamide with an average particle size of 170 μm and a pore diameter
of 25 nm [483]. Enzyme attachment occurs with the formation of a stable C–N bond
via nucleophilic opening of the epoxide groups by amino groups of the enzyme
under mild conditions. In contrast to the majority of the above-mentioned covalent
immobilization reactions involving ε-amino groups of Lys, which remove a positive (ammonium) charge from the enzyme’s surface due to Schiff base formation or
N-acylation, this method preserves the charge distribution of the enzyme since it
constitutes an N-alkylation process.
3.3 Immobilization
371
biocatalyst.
Covalent Attachment
Covalent binding of an enzyme onto a macroscopic carrier leads to the irreversible
formation of stable chemical bonds, which prevents leakage completely. A disadvantage of this method is that rather harsh conditions are required since the protein must
undergo a chemical reaction. Consequently, some loss of activity is always observed
[472, 473]. As a rule of thumb, each bond attached to an enzyme decreases its native
activity by about one fifth. Consequently, residual activities generally do not exceed
60–80% of the activity of the native enzyme, and values of around 50% are normal.
The functional groups of the enzyme which are commonly involved in covalent
binding are nucleophilic, i.e., mainly N-terminal and ε-amino groups of lysine, but
also carboxy-, sulfhydryl-, hydroxyl-, and phenolic functions. In general, covalent
immobilization involves two steps, i.e., (i) activation of the carrier with a reactive
‘spacer’ group and (ii) enzyme attachment. Since viable cells usually do not survive
the drastic reaction conditions required for the formation of covalent bonds, this type
of immobilization is only recommended for isolated enzymes.
Porous glass is a popular inorganic carrier for covalent immobilization
[474]. Activation is achieved by silylation of the hydroxy groups using
aminoalkylethoxy- or aminoalkyl chlorosilanes as shown in Scheme 3.45. In a
subsequent step, the aminoalkyl groups attached to the glass surface are either
transformed into reactive isothiocyanates or into Schiff bases by treatment with
thiophosgene or glutardialdehyde, respectively. Both of the latter species are able to
covalently bind an enzyme through its amino groups.
Carriers based on natural polymers of the polysaccharide type (such as cellulose
[475], dextran [476], starch [477], chitin, or agarose [478]) can be useful alternatives to inorganic material due to their well-defined pore size. Activation is
achieved by reaction of adjacent hydroxyl groups with cyanogen bromide leading
to the formation of reactive imidocarbonates (Scheme 3.46). Again, coupling of the
enzyme involves its amino groups. To avoid the use of hazardous cyanogen
bromide, pre-activated polysaccharide-type carriers are commercially available.
During recent years, synthetic copolymers (e.g., based on polyvinyl acetate)
have become popular (Scheme 3.47) [479–481]. Partial hydrolysis of some of the
acetate groups liberates a hydroxyl functionality, which is activated by epichlorohydrin [482]. A number of such epoxy-preactivated resins are commercially available. Among them, Eupergit C™ is widely used. It is a macroporous copolymer of
N,N
0 -methylene-bi-(methacrylamide), glycidyl methacrylate, allyl glycidyl ether
and methacrylamide with an average particle size of 170 μm and a pore diameter
of 25 nm [483]. Enzyme attachment occurs with the formation of a stable C–N bond
via nucleophilic opening of the epoxide groups by amino groups of the enzyme
under mild conditions. In contrast to the majority of the above-mentioned covalent
immobilization reactions involving ε-amino groups of Lys, which remove a positive (ammonium) charge from the enzyme’s surface due to Schiff base formation or
N-acylation, this method preserves the charge distribution of the enzyme since it
constitutes an N-alkylation process.
3.3 Immobilization
371