Chemistry Reference
In-Depth Information
formation [
22
]. This bioinspired method provides a green and one step process for
lipase immobilization [
23
].
Among all of the lipases that have been reported in the literature for organic syn-
thesis, CALB has proved to be the most efficient and highly stable in organic media
during esterification and transesterification reactions [
24
,
25
]. CALB is a globular
protein with approximate molecular dimensions of 30 Å × 40 Å × 50 Å, and relative
mass of 33 kDa [
26
]. CALB has a Ser-His-Asp catalytic triad in its active site when
in an 'open' conformation with a restricted entrance. The active site is responsible
for the substrate specificity and high degree of stereo-specificity of CALB, which
is an
α/β
type protein that has many features in common with other lipase structures
and related enzymes. However, the primary sequence has no significant homol-
ogy to any other known lipase and it deviates from the sequence around the active
site serine that is found in other lipases. CALB was physically immobilized within
a macroporous resin (Lewatit VP OC 1600) poly(methyl methacrylate-
co
-divinyl-
benzene) and sold under the brand name Novozym-435
®
. The polymeric resin has
a reported average particle size of 315-1000
μ
m, a surface area of 130 m
2
g
−1
, and a
pore diameter of ~ 150 Å, respectively [
15
]. However, Novozym-435 has been re-
ported to exhibit poor mechanical stability and the enzyme was found to be leaching
out during the reactions carried out in organic media [
24
,
25
].
In this chapter we report the immobilization of CALB using two approaches.
Firstly, CALB was immobilised onto silica gel particles that had been surface modi-
fied porous by 3-APS, followed by chemically crosslinking with glutaraldehyde. The
second approach adopted a bioinspired green method. The catalytic activity and the
thermal stability of these particles were studied using an esterification model reac-
tion between 1-octanol and lauric acid in isooctane. The results were then assessed
and compared to those for free CALB and to the commercial Novozym-435
®
system.
5.2 Experimental
5.2.1 Materials
All of the chemicals were analytical grade and were used as received. Immobilized
lipase B from
Candida antarctica
, Novozym-435
®
(Lot#047K1672 with an activ-
ity of 11,200 PLU (propyl laurate units)/g), 3-aminopropyl)triethoxysilane (3-APS
or
γ
-APS), silica gel (Davisil
®
, Grade 646, pore size 150 Å, 35-60 mesh, particle
size 250-500 µm, surface area 300 m
2
/g, pore volume 1.15 cm
3
/g, specifications
provided by Aldrich Co.), glutaraldehyde and Brilliant Blue G-250 were purchased
from Aldrich Co. HPLC/Spectro grade toluene and tetrahydrofuran (THF) were
purchased from Tedia Co. Inc. Polyallylamine hydrochloride (PAH) and polyeth-
ylenimine (PEI), which were used for bioinspired silica synthesis, were obtained
from Sigma-Aldrich. The free CALB was kindly provided to us by our collaborator
Professor Richard A. Gross, Polytechnic Institute of NYU, Brooklyn, NY.
