University of Groningen
Improved biocatalysts based on Candida antarctica lipase B immobilization
Miletic, Nemanja
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Publication date:
2009
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Miletic, N. (2009). Improved biocatalysts based on Candida antarctica lipase B immobilization Groningen:
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Summary
Energy consumption and environmental pollution have become important issues
in recent decades. At the moment, many researchers are focused on developing
novel processes that will deal with these problems. An important next step, that
will reduce both energy consumption and environmental pollution, is usage of
enzymes in the chemical industry. Replacing acids in the starch processing
industry, replacing alkalis or oxidizing agents in fabric desizing, reducing the use
of sulphide in tanneries, reducing waste, replacing chlorine and phosphates in
clothes cleaning, elimination of chemical treatments during production processes,
synthesizing of compounds that cannot be obtained via traditional chemistry are
just a few of the advantages that enzyme usage will provide.
On the other hand, application of enzymes has a number of drawbacks. The most
important one is the ease of enzyme denaturation due to the different operational
conditions in industry compared with nature. One of the solutions that may
improve enzyme performance is immobilization. This thesis deals with an
immobilization of the most widely used lipase, Candida antarctica lipase B (CalB), on different types of carriers. To this end, as starting carrier, a highly porous
epoxy-containing copolymer was synthesized: poly(glycidyl methacrylate-coethylene glycol dimethacrylate) [poly(GMA-co-EGDMA)]. This copolymer was
synthesized via suspension polymerization that allows control of the final
copolymer properties by altering with reaction conditions (composition of the inert
component, type and amount of the cross-linker, stirring speed, etc). The properties
of immobilized enzymes are highly influenced by the copolymer properties, in
particular, porosity characteristics (particle size, pore size, specific surface area and
specific volume). It was shown that Cal-B loading and activity can be increased by
decreasing the particle size, increasing the pore size and increasing the specific
surface area. We also proved that enzyme immobilization alters the porosity
parameters of the carrier that is also proof of successful immobilization.
The use of poly(GMA-co-EGDMA) was motivated by the presence of epoxy rings
in its structure that makes this copolymer highly reactive. Hence, various
modifications of epoxy-containing copolymer were carried out (with various
diamines, 2-fluoroethylamine, glutaraldehyde and cyanuric chloride). The reactive
130
Summary
epoxy group opens the possibility to obtain diverse carriers for Cal-B
immobilization: carriers that favor physical bonding between the enzyme and the
copolymer and carriers that favor chemical bonding between the enzyme and the
copolymer. The influence of the type of immobilization on enzyme loading and
enzyme activity is assessed. Cal-B demonstrated the highest activity, immobilized
on poly(GMA-co-EGDMA) modified with glutaraldehyde, where 61.1 % of Cal-B
was covalently attached. These results show that too high a degree of chemically
bound enzymes to the carrier, restricts the enzyme mobility such that they do not
have sufficient freedom to adopt the most suitable conformation for increased
activity. On the other hand, too weak interaction between the enzyme and the
carrier allows too much freedom to the enzymes, that does indeed give rise to
increased activity (compared with crude enzyme powder), but substantially less
than that of the enzyme immobilized on the glutaraldehyde-modified carrier.
After modifying the starting epoxy-containing copolymer, we turned our attention
to Cal-B itself and tried to develop carrierless immobilization methods. Since, it
was proved that Cal-B may successfully react with epoxy groups from the
poly(GMA-co-EGDMA), modification and cross-linking with epoxy-containing
compounds were chosen (various epoxides and various diepoxides). With these
treatments, in most of cases, activity and thermal stability of the Cal-B derivatives
were significantly improved. The length of the epoxide arm in the modification
process, as well as the length of the spacer arm in the cross-linking process
pronounced a large influence on the thermal stability of the novel species. Since the
epoxide arm is hydrophobic, a longer alkyl chain resulted in a large decrease in the
thermal stability behavior. On the other hand, a cross-linking process showed
enzyme mobility as the more important issue compared with hydrophobicity.
Namely, a spacer arm that is too short restricts enzyme mobility and thus
negatively influences enzyme activity.
Modifications of poly(GMA-co-EGDMA) with various diamines and 2fluoroethylamine (obtaining the carriers that favor the physical adsorptions with
enzymes) did not yield 100 % of conversion. Even with a high yield of
modifications, the resulting carriers are hydrophilic, although introduction of
fluorine into the copolymer structure increased the hydrophobicity of the carrier. At
this point, our attention turned to the carrier that is completely hydrophobic, with
no opportunity for chemical connection with enzymes. Hence, polystyrene
nanoparticles were synthesized via the nanoprecipitation process. Since the
hydrophobic interactions were the driving force of the immobilization process, it
turned out that the pH of adsorption has a significant influence on immobilized
enzyme activity. We attributed this pH-dependence to the favorable charge
distribution on the amino acid residues if the adsorption is carried out near the
isoelectric point.
Summary
131
Besides an assortment of carriers with different porosity properties, functional
groups, hydrophobicity, etc. it was important to move beyond general correlations
to a better understanding on a molecular level of how immobilization on surfaces
can stabilize and activate protein catalysts. The standard procedure to evaluate the
surface topography of immobilized enzyme and to characterize the architecture
down to the molecular level is enzyme immobilization on flat surfaces (e.g. silicon
surface). In order to immobilize Cal-B on silicon surfaces, pre-treatments with
aminopropyltriethoxysilane (APTES) and subsequently with glutaraldehyde (GLA)
were performed. The APTES-GLA modification approach is accompanied by a
number of drawbacks: it is difficult, but not impossible to control formation of the
APTES film, since APTES molecules have a tendency to enter into competing
reactions, including polymerization. Final enzyme properties are strictly
determined by the structure and architecture of the APTES film, and all subsequent
steps cannot significantly change the immobilization protocol. We have found the
reaction conditions (short reaction time, chloroform as a solvent, certain APTES
concentration, etc.) that favor creation of the perfect fully-covered APTES
monolayer (as evidenced by XPS, ellipsometry, AFM, IR and contact angle
measurements). However, subsequently immobilized Cal-B on a fully-covered
APTES monolayer does not exhibit the highest activity, due to the presence of
enzymes in the sublayers that cannot contribute in the catalytic activity.