Chemistry Reference
In-Depth Information
the support, which react well with the amine groups on the enzyme. This therefore
leads to the enzyme becoming covalently bound to the support [
7
,
43
,
44
]. Glu-
taraldehyde and aminopropyltriethoxysilane can also be used [
45
]. There are pre-
activated supports commercially available, such as Sepabeads, which are composed
of epoxy resins. These are frequently encountered in industry, and can be used to
support a wide range on enzymes, including lipase, however, they are expensive
[
7
]. Furthermore, naturally occurring supports such as the polysaccharides cellu-
lose, chitosan, agarose and dextran are gaining popularity due to their low cost and
availability [
42
,
46
].
Compared to other methods, covalent binding has the benefit of strong covalent
bonds (200-1000 kJ/mol) [
47
] between the support and the enzyme. This there-
fore results in minimal levels of leaks/leaching of enzyme from the support; this is
beneficial since enzyme losses are minimal and product purity is maintained [
48
].
These strong bonds also regularly result in increased stability of the enzyme over a
wider range of temperatures (and often pH), since the strong bonds help resist en-
zyme deformation. Covalent binding also results in high levels of contact between
the enzyme and substrate since the enzyme is firmly localised on the surface of the
support [
41
].
Covalent binding does, however, have its drawbacks. The strong bonds may re-
sult in conformational deformation of the enzyme, as well as hindering enzyme
movement. Attachment of the enzyme to the support can also block active sites.
These factors can result in reduced enzyme activity. The chemicals used for activa-
tion also mean that the route is usually not considered green [
2
]. Furthermore, the
immobilisation process can be often time-consuming as illustrated with an example
of mesoporous silica as a support in Fig.
4.4
.
Glyoxyl agarose, as an example of covalent binding, has been reported to be an
effective support for immobilising lipase. Agarose is a suitable support due to its
high surface area and adequate porosity, and can be activated with epoxide reagents
to create glyoxyl groups on the surface [
26
]. Reports suggest that 38-86 % of the
enzyme's activity can be retained during immobilisation, and significant improve-
ments in enzyme stability compared to the free enzyme have been seen, in terms
of pH and temperature [
7
,
26
,
50
]. Stabilisation factors of up to 150 have been
reported [
7
].
Commercial Eupergit resin support beads are also successful for lipase immobil-
isation, and they typically bind to the enzyme through epoxy, carboxyl or sulfhydryl
groups [
7
,
51
]. Retention of up to 80 % of enzyme activity has been reported [
52
].
This, however, depends on immobilization conditions, such as ionic strength and
the presence of metal chelates.
Recently, green coconut fibres have been demonstrated as suitable support for
covalent binding of lipase. For this, the fibres were first activated through silanisa-
tion using 3-glycidoxypropyltrimethoxysilane (GPTMS). Improved enzyme stabil-
ity was seen (up to 360 times that of the free enzyme), and it also showed very good
storage potential, with only small drops in activity [
25
].
The use of genetic engineering has also been demonstrated as means for im-
proving immobilisation of lipase. Lipase has been genetically modified to display a
free cysteine at a defined position, which was then attached to a glass surface [
53
].
