Chemistry Reference
In-Depth Information
immobilised in such a way has been shown to retain high levels of activity, and
demonstrate improved stability [
37
,
42
].
Lipase has been adsorbed onto highly hydrophobic octyl-agarose supports with
success. Very high levels of lipase activity have been observed (with claims of 'hy-
peractivation' such that the immobilised lipase displays greater activity than free
enzyme), and immobilisation occurred rapidly, making the method favourable. The
strength of lipase adsorption was also strong enough to allow efficient use under
mild conditions in a range of media (including organic solvents), and an improve-
ment in thermal stability compared to free enzyme was observed [
16
].
The physical adsorption of lipase onto mesoporous silica nanoparticles has
also been demonstrated. Lipase was physically adsorbed to SBA-15 silica spheres
through hydrophobic interactions, by placing the spheres in a lipase solution. The
silica nanoparticles used were lengthy to produce and entailed harsh conditions
(temperatures of 550 ºC). The activity maintained depended on pore size, with larg-
er pores (24 nm) resulting in higher activity than smaller pores (5 nm) [
17
,
18
,
60
];
the activity of the lipase immobilized in larger pores was very similar to that of the
free enzyme, whereas it was just 20-30 % in smaller pores. This was thought to be
due to mass transfer limitations associated with smaller pore sizes [
17
]. Further
studies suggest that for similar routes, the stability of the lipase can be improved
by chemically pre-treating the enzyme with ethylene glycol bis (succinimidyl suc-
cinate) or glutaraldehyde; the half-life of glutaraldehyde treated lipase was 8 times
that of non-treated lipase [
27
].
Lipase has also been adsorbed onto fumed silica supports. Fumed silica has a
large surface area and high absorption capacity for proteins, therefore making it a
suitable support for enzymes, and adsorption is thought to occur through electro-
static interactions. Adsorption was found to depend on factors such as pH, ionic
strength and silica availability. The activity of the material compared well with the
commercial Novozym-435, however, the thermal stability was poorer, and the ma-
terial had less storage potential (in organics) [
19
].
4.3.5 Cross-Linking
With cross-linking, additional reagents are added to bring about inter-molecular
cross-linking, and if significant enough, the enzyme becomes insoluble. First of
all, a precipitating agent is added to precipitate the enzyme out of solution to cre-
ate physical enzyme aggregates. These are, however, unstable and would readily
dissolve back into solution. A cross-linking agent is therefore added to cross-link
these physical enzyme aggregates, and it works by reacting with the reactive groups
on the enzyme, therefore creating cross-linked enzyme aggregates (CLEA) [
48
].
Suitable chemicals to bring about physical aggregation include salts and organic
solvents, such as acetone and ethanol [
41
]. Glutaraldehyde is the most common
cross-linking agent, and is suitable for immobilising a range of enzymes including
amylase, lipase, invertase, nitrilases and hydrolase [
2
,
61
]. Alternative cross-linking
agents include dimethyl suberimidate, toluene diisocyanate and hexamethylene di-
