U.S. patent application number 12/740123 was filed with the patent office on 2010-10-21 for emulsion-derived particles.
This patent application is currently assigned to CSIR. Invention is credited to Dean Brady, Neil Stockenstrom Gardiner, Isak Bartholomeus Gerber, Justin Jordaan, Clinton Simpson.
Application Number | 20100267108 12/740123 |
Document ID | / |
Family ID | 40591572 |
Filed Date | 2010-10-21 |
United States Patent
Application |
20100267108 |
Kind Code |
A1 |
Jordaan; Justin ; et
al. |
October 21, 2010 |
EMULSION-DERIVED PARTICLES
Abstract
An emulsion-derived particle includes a lattice of polymeric
strands cross-linked by means of a cross-linking agent, and
interstitial openings adjacent and around the strands. Functional
groups are provided on the lattice and proteins and/or modified
proteins can react with these, thereby to be bonded to the lattice
and hence immobilized.
Inventors: |
Jordaan; Justin; (Edenvale,
ZA) ; Simpson; Clinton; (Manchester, GB) ;
Brady; Dean; (Kyalami Estates, ZA) ; Gardiner; Neil
Stockenstrom; (Pretoria East, ZA) ; Gerber; Isak
Bartholomeus; (Krugersdorp, ZA) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Assignee: |
CSIR
Pretoria
US
|
Family ID: |
40591572 |
Appl. No.: |
12/740123 |
Filed: |
October 29, 2008 |
PCT Filed: |
October 29, 2008 |
PCT NO: |
PCT/IB08/54458 |
371 Date: |
April 28, 2010 |
Current U.S.
Class: |
435/180 ;
525/417; 530/391.1 |
Current CPC
Class: |
C12N 11/08 20130101;
C08J 3/005 20130101; C08J 3/24 20130101; C08J 3/16 20130101; C12N
11/06 20130101; C08J 2379/02 20130101; C07K 17/08 20130101 |
Class at
Publication: |
435/180 ;
530/391.1; 525/417 |
International
Class: |
C12N 11/08 20060101
C12N011/08; C07K 17/08 20060101 C07K017/08; C08G 73/04 20060101
C08G073/04 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 29, 2007 |
ZA |
2007-09300 |
Claims
1. An emulsion-derived particle, which comprises a lattice of
polyethyleneimine strands cross-linked by means of a cross-linking
agent, interstitial openings adjacent and around the strands, and
functional groups on the lattice and with which proteins and/or
modified proteins can react, thereby to be bonded to the lattice
and hence immobilised.
2. A particle according to claim 1, wherein the functional groups
are present on the polyethyleneimine, and are selected so that
bonding of the proteins and/or modified proteins to the
polyethyleneimine can be effected through one or more of covalent
bonding, ionic bonding, hydrophobic bonding, and affinity bonding
which can be effected through modification of the lattice to
provide this functionality.
3. A particle according to claim 1, which includes at least one
protein and/or modified protein bonded to the lattice by means of
the functional groups, thereby being immobilised.
4. A particle according to claim 3, wherein a plurality of
different proteins and/or a plurality of different modified
proteins, are immobilised therein.
5. A particle according to claim 1, inclusive, which includes an
adjunct entrapped within the lattice.
6. A particle according to claim 5, wherein the adjunct is selected
from the group consisting of a co-factor, a modified co-factor, a
chemical mediator, magnetite and a magnetic substance.
7. A process for producing particles, which includes providing an
emulsion of droplets of a first liquid phase dispersed in a second
liquid phase, with the one liquid phase being an aqueous phase and
the other being an oil phase, and with the aqueous phase containing
a polymer dissolved therein as well as a cross-linking agent
dissolved therein; allowing the cross-linking agent to cross-link
strands of the polymer, thereby to form particles, each of which
includes a lattice of strands of the polymer, cross-linked by means
of the cross-linking agent, interstitial openings adjacent and
around the strands, and functional groups on the lattice and with
which proteins and/or modified proteins can react, thereby to be
bonded to the lattice and hence immobilised.
8. A process for producing particles, which includes providing a
first emulsion of droplets of a first liquid phase dispersed in a
second liquid phase, with the one liquid phase being an aqueous
phase and the other being an oil phase, and with the aqueous phase
containing a polymer dissolved therein; combining a second emulsion
of droplets of a first liquid phase dispersed in a second liquid
phase, with the one liquid phase being an aqueous phase and the
other being an oil phase, and with the aqueous phase containing a
cross-linking agent dissolved therein, with the first emulsion;
allowing the cross-linking agent to cross-link strands of the
polymer, thereby to form particles, each of which includes a
lattice of strands of the polymer, cross-linked by means of the
cross-linking agent, interstitial openings adjacent and around the
strands, and functional groups on the lattice and with which
proteins and/or modified proteins can react, thereby to be bonded
to the lattice and hence immobilised.
9. A process according to claim 7, wherein the polymer is
polyethyleneimine.
10. A process according to claim 7, which includes adding an
adjunct at least to one of the phases, so that the adjunct is
entrapped within the lattices of the particles.
11. A process according to claim 7, which includes recovering the
particles, and drying the recovered particles.
12. A process according to claim 11, which includes adding an
adjunct to the recovered particles before the drying of the
particles, so that the adjunct becomes entrapped within the
lattices of the particles.
13. A process according to claim 10, wherein the adjunct is
selected from the group consisting of a co-factor, a modified
co-factor, a chemical mediator, magnetite and/or a magnetic
substance.
14. A process according to claim 12, wherein the adjunct is
selected from the group consisting of a co-factor, a modified
co-factor, a chemical mediator, magnetite and a magnetic
substance.
15. A process according to claim 8, wherein the polymer is
polyethyleneimine.
16. A process according to claim 8, which includes adding an
adjunct at least to one of the phases, so that the adjunct is
entrapped within the lattices of the particles.
17. A process according to claim 8, which includes recovering the
particles, and drying the recovered particles.
18. A process according to claim 17, which includes adding an
adjunct to the recovered particles before the drying of the
particles, so that the adjunct becomes entrapped within the
lattices of the particles.
19. A process according to claim 16, wherein the adjunct is
selected from the group consisting of a co-factor, a modified
co-factor, a chemical mediator, magnetite and a magnetic
substance.
20. A process according to claim 18, wherein the adjunct is
selected from the group consisting of a co-factor, a modified
co-factor, a chemical mediator, magnetite and a magnetic substance.
Description
[0001] THIS INVENTION relates to emulsion-derived particles. It
relates also to a process for producing such particles.
[0002] Particles containing immobilised enzymes are typically used
for biocatalysis and for diagnostics, among other applications.
However, particles of which the Applicant is aware suffer from
drawbacks such as inadequate surface area for sufficient enzyme
immobilisation. It is thus an object of this invention to provide
particles whereby this drawback is at least alleviated, and a
process for producing such particles that have a high binding
capacity for proteins and can immobilise the proteins.
[0003] Thus, according to a first aspect of the invention, there is
provided an emulsion-derived particle, which comprises a lattice of
polymeric strands cross-linked by means of a cross-linking agent,
interstitial openings adjacent and around the strands, and
functional groups on the lattice and with which proteins and/or
modified proteins can react, thereby to be bonded to the lattice
and hence immobilised.
[0004] By `emulsion-derived` is meant that the particles have been
produced or formed using emulsion techniques such as, but not
limited to, the emulsion based processes of the second and third
aspects of the invention.
[0005] By "modified proteins" is meant proteins modified by
chemical means such as by the addition of di-aldehydes, or proteins
modified at a genetic level, such as by means of his-tags.
[0006] Thus, the particle includes functional groups on the
polymeric strands or fibres and/or on the cross-linking agent with
which proteins and/or modified proteins can react. More
specifically, the functional groups may be present on the polymer
of the strands or fibres, and may be selected so that bonding of
the proteins and/or the modified proteins to the polymer can be
effected through one or more of covalent bonding, ionic bonding,
hydrophobic bonding, and affinity bonding by modifying the
functional groups on the polymer.
[0007] The particle may thus include at least one protein and/or
modified protein bound or bonded to the polymer by means of the
functional groups, thereby being immobilised. The protein may be an
enzyme or a mixture of enzymes; an antibody or a mixture of
antibodies; or an antigen or a mixture of antigens or any other
protein which possesses functional or structural properties. A
plurality of different proteins and/or a plurality of different
modified proteins can thus, if desired, be immobilised within the
particle. When the protein is an enzyme, the particle provides a
means whereby the optimal pH of the enzyme can be shifted to the
acid or alkaline region, by immobilization of the enzyme in the
particle.
[0008] When the protein and/or modified protein is covalently
bonded to the polymer, this may be achieved, for example, by
epoxide or aldehyde interaction with amine groups of the protein
and/or the modified protein.
[0009] When the protein and/or modified protein is ionically bonded
to the polymer, this may be achieved by positively or negatively
charged functional groups on the polymer, ionically binding with
oppositely charged amino acid residues on the protein and/or
modified protein.
[0010] When the protein is hydrophobically bound to the polymer,
this may be achieved by aromatic or long chain alkane hydrophobic
groups on the polymer binding with hydrophobic amino acid on the
protein.
[0011] When the protein is affinity bonded to the polymer, this may
be achieved by affinity tags, such as divalent metals and/or
avidin, binding a histidine or biotinylated protein.
[0012] The particle may naturally, if desired, contain more than
one of the above types or categories of functional groups, for more
efficient binding of the protein.
[0013] The polymer of the strands or fibres may be a homopolymer,
and may be polyethyleneimine.
[0014] The cross-linking agent may be glutaraldehyde or another
aldehyde; an epoxide; or any other suitable compound having bi or
multi functional groups.
[0015] The particle may include an adjunct entrapped within the
interstitial openings or spaces of the lattice. The adjunct may be
selected from a co-factor, a modified co-factor, or a chemical
mediator, magnetite and/or a magnetic substance. By including, in
the particle, a suitable enzyme and/or a substrate as an adjunct,
continuous regeneration of co-factors used in a reaction can be
achieved, thereby permitting the reaction to reach equilibrium or
completion. By including magnetite or a magnetic substance as an
adjunct, recovery of the particles from the formation liquid medium
can readily be effected, using magnetic separation.
[0016] According to a second aspect of the invention, there is
provided a process for producing particles, which includes [0017]
providing an emulsion of droplets of a first liquid phase dispersed
in a second liquid phase, with the one liquid phase being an
aqueous phase and the other being an oil phase, and with the
aqueous phase containing a polymer dissolved therein as well as a
cross-linking agent dissolved therein; [0018] allowing the
cross-linking agent to cross-link strands of the polymer, thereby
to form particles, each of which includes a lattice of strands of
the polymer, cross-linked by means of the cross-linking agent,
interstitial openings adjacent and around the strands, and
functional groups on the lattice and with which proteins and/or
modified proteins can react, thereby to be bonded to the lattice
and hence immobilised.
[0019] The first liquid phase may be the aqueous phase, with the
second liquid phase thus being the oil phase, so that the emulsion
is a water(w)-in-oil(o) emulsion, ie a w/o emulsion. However, in
other embodiments of the invention, the emulsion may be an
oil-in-water (o/w) emulsion, a water-in-oil-in-water (w/o/w)
emulsion, or an oil-in-water-in-oil (o/w/o) emulsion.
[0020] The emulsion may be formed by admixing a first emulsion
comprising aqueous droplets, containing the polymer dissolved
therein, dispersed in an oil phase, with a second emulsion
comprising aqueous droplets, containing the cross-linking agent
dissolved therein, dispersed in an oil phase.
[0021] According to a third aspect of the invention, there is
provided a process for producing particles, which includes [0022]
providing a first emulsion of droplets of a first liquid phase
dispersed in a second liquid phase, with the one liquid phase being
an aqueous phase and the other being an oil phase, and with the
aqueous phase containing a polymer dissolved therein; [0023]
combining a second emulsion of droplets of a first liquid phase
dispersed in a second liquid phase, with the one liquid phase being
an aqueous phase and the other being an oil phase, and with the
aqueous phase containing a cross-linking agent dissolved therein,
with the first emulsion; [0024] allowing the cross-linking agent to
cross-link strands of the polymer, thereby to form particles, each
of which includes a lattice of strands of the polymer, cross-linked
by means of the cross-linking agent, interstitial openings adjacent
and around the strands, and functional groups on the lattice and
with which proteins and/or modified proteins can react, thereby to
be bonded to the lattice and hence immobilised.
[0025] At least one of the phases may include a detergent or
surfactant. The surfactant may be selected from a zwitterionic
surfactant, a neutral surfactant, a charged surfactant and/or a
polymeric surfactant. Anionic surfactants include an alkyl sulphate
such as sodium lauryl sulphate or sodium laureth sulphate, and an
alkyl ether sulphate. Cationic surfactants include centrimonium
chloride. Non-ionic surfactants include ethoxylated alkyl phenol
such as polyoxyethylene(10) iso-octylcyclohexyl ether (Triton X100)
or polyoxyethylene(9) nonylphenyl ether (Nonoxynol-9). Zwitterionic
or amphiphillic surfactants include decyl betaine. Polymeric
surfactants include sorbitol-(ethylene oxide) 80, ethylene
oxide-propylene oxide-ethylene oxide triblock copolymer, also known
as a poloxamer, such as that available under the trade name
Pluronic from BASF, and a propylene oxide-ethylene oxide-propylene
oxide triblock copolymer, also known as a meroxapol.
[0026] The oil of the oil phase(s) may, at least in principal, be
any suitable water immiscible organic solvent, a vegetable oil, a
mineral oil, a coal or crude oil derived oily component, or a
synthetic oil; however, it is preferably selected from a mineral
oil, paraffin, and a solvent such as iso-octane.
[0027] As hereinbefore described, the polymer may be
polyethyleneimine (PEI), while the cross-linking agent may be a
difunctional or multifunctional aldehyde such as glutaraldehyde,
succinaldehyde, dextran aldehyde, hexamethylene diisocyanate and
glyoxal. Other suitable cross-linking agents may be used for PEI or
derivitised PEI, or other polymers, such as isocyanates (including
hexamethylene diisocyanate or toluene diisocyanate, or
isothiocyanate); an epoxide (such as 2-chloromethyl oxirane); an
anhydride; epichlorohydrin, 1-ethyl-3,3-dimethylaminopropyl
carbodiimide; ethyl chloroacetate or the like. As unreacted
functional groups are used for the immobilisation of the protein
and/or the modified protein, the cross-linking agents may also be
considered to be derivitisation agents for polymer modification or
post-cross-linking modification. Other polymers or co-polymers may
be used, such as polyvinyl alcohol, nylon, alginate, other proteins
(such as albumin, collagen and such like) and such like, modified
or otherwise.
[0028] The process may include introducing a protein, such as an
enzyme, an antibody or an antigen and/or a modified protein, into
and onto the particles, so that the protein and/or the modified
protein react with functional groups on the polymeric strands or
fibres and/or on the cross-linking agent as hereinbefore described,
thereby to be bonded to the polymer of the strands or fibres and/or
to the cross-linking agents, and hence immobilised.
[0029] The process may include adding an adjunct to one of the
phases, so that the adjunct is entrapped within the lattices of the
particles. The adjunct may further be added before or after protein
linking to the lattice. As hereinbefore indicated, the adjunct may
be selected from a co-factor, a modified co-factor, a chemical
mediator, magnetite and/or a magnetic substance.
[0030] The process may include recovering the particles from the
oil phase. In particular, the recovery of the particles may be
effected by physical separation means, such as centrifugation or
filtration.
[0031] The process may include drying the recovered particles.
Drying of the particles may include acetone dehydration, air
drying, spray drying, or, preferably, lyophilisation or vacuum
drying.
[0032] The process may include adding an adjunct, as hereinbefore
described, to the recovered particles before the drying of the
particles, so that the adjunct becomes entrapped within the
lattices of the particles.
[0033] Drying of the recovered particles may be effected, either
before or after protein immobilisation, to achieve enhanced
stabilization of the protein and the particles, and/or for
entrapment of additives such as native or modified co-factors.
Drying may also result in improved cross-linking of the proteins or
modified proteins, by means of multipoint attachment. This in turn
may enhance stability of proteins such as enzymes.
[0034] It is envisaged that the particles of the invention can have
diverse uses or application, such as for biocatalysis, enzyme based
bioremediation, diagnostics, and for binding to a surface of a
solid support such as a membrane reactor or a protein
immobilisation matrix, to increase its surface area.
[0035] The invention will now be described in more detail with
reference to the following examples, and the accompanying
drawings.
[0036] In the drawings,
[0037] FIG. 1 is a microscopic photograph of the polymer particles
of glutaraldehyde cross-linked PEI, in accordance with Example 1,
showing the PEI support lattice of polymeric strands/fibres or
network backbone;
[0038] FIG. 2 is a graph which illustrates the results obtained for
laccase binding to fibrous lattices, in accordance with Example 2,
with binding efficiencies corrected for pH profile shifting;
[0039] FIG. 3 shows particle size distribution (average size)
analyses of particles manufactured using various oil phases, where
the particles had not been dried (wet), had been dried (using
lyophilisation), with in-line ultrasonication (US) or measured
after a pre-treatment with ultrasonication;
[0040] FIG. 4 shows particle size distribution analyses of
particles manufactured using various surfactants; determined by
light scattering as is, or analysis after ultrasonication (after
(US);
[0041] FIG. 5 shows particle size distribution for wet and dry
particles manufactured using a single emulsion; results show the
standard deviation of triplicate experiments; determined by light
scattering as is, with in-line ultrasonication (with US), or
analysis after sample pre-ultrasonication (after US);
[0042] FIG. 6 shows particle size distribution for wet and dry
particles manufactured at 20 ml mineral oil volume and 200 ml
mineral oil volume;
[0043] FIG. 7 shows activity maintenance of various enzymes bound
to particles, with and without substrates as potential
protectants;
[0044] FIG. 8 shows temperature optima for free laccase and wet and
dry laccase bound particles manufactured at PEI pH of 8 (FIG. 8A)
and 11 (FIG. 8B);
[0045] FIG. 9 shows pH stability (6 hours) for free laccase and wet
and dry laccase bound particles manufactured at PEI pH's of 8 and
11;
[0046] FIG. 10A shows laccase pH profiles of immobilised and free
enzymes on non-post-treated fibrous lattices or networks, in
accordance with Example 14, indicating the effect of varying
polyethyleneimine (`PEI`) concentration on pH profile shifting;
[0047] FIG. 10B shows laccase pH profiles of immobilised and free
enzymes on non-post-treated fibrous lattices or networks, in
accordance with Example 14, indicating the effect of varying or
glutaraldehyde concentration on pH profile shifting;
[0048] FIG. 11A shows laccase pH profiles of immobilised and free
enzymes on glutaraldehyde post-treated fibrous lattices or
networks, in accordance with Example 14, indicating the effect of
varying PEI concentration on pH profile shifting;
[0049] FIG. 11B shows laccase pH profiles of immobilised and free
enzymes on glutaraldehyde post-treated fibrous lattices or
networks, in accordance with Example 14, indicating the effect of
varying the glutaraldehyde concentration on pH profile
shifting;
[0050] FIG. 12 show the results obtained for peroxidase activity of
particles for experiments A to F.
EXAMPLE 1
Manufacturing of a Particle Consisting of a Network or Lattice of
Polymeric Strands/Fibres
[0051] This method involves the formation of a water-in-oil
emulsion in which an emulsion containing a polyamine polymer
(Polyethyleneimine) and another primary amine cross-linker
(Glutaraldehyde) are combined. The two reagents react to form
polymers in the form of microscopic particles or beads.
[0052] Chemicals
[0053] Glutaraldehyde (25% aqueous solution) was obtained from
Acros Organics (Geel West Zone 2, Janssen Pharmaceuticalaan 3a,
2440 Geel, Belgium). Polyethyleneimine (PEI) (50% aqueous solution,
Cat. No. P-3143, Mw 750,000 and Mn 60,000) was obtained from
Sigma-Aldrich (St Louis, Mo. 63178). Mineral oil (white oil
medicinal, 48031) was purchased from Castrol (8 Junction Avenue,
Parktown, 2193 Johannesburg, South Africa).
[0054] Method for Making Particles
[0055] Emulsion A Composition
[0056] 10 ml mineral oil (oil phase)
[0057] 0.05 ml nonoxyol-4 (surfactant)
[0058] 0.5 ml Polyethyleneimine (polyamine), (10% m/v aqueous
solution), pH 11
[0059] Stirred at 700 rpm, 30 min using a magnetic stirrer.
[0060] Emulsion B Composition
[0061] 10 ml mineral oil (oil phase)
[0062] 0.05 ml nonoxyol-4 (surfactant)
[0063] 0.5 ml glutaraldehyde, (25% m/v, grade II)
[0064] Stirred at 700 rpm, 30 min using a magnetic stirrer.
[0065] The two emulsions (A and B) were combined to permit the
polymer cross-linking reaction and stirred using a magnetic stirrer
bar at 700 rpm for 30 minutes to 1 hour to ensure the maintenance
of the emulsion. The emulsion was then centrifuged at 3000 rpm (10
minutes in a Beckman-Coulter J2-21ME fitted with a JA20.1 rotor) to
recover the particles formed. The pellet was re-suspended in
deionised water, diluted to 10 to 40 ml, and then centrifuged
again. This washing process was repeated twice more. The final
supernatant was clear. The final pellet was suspended in 10 ml of
Tris-Cl buffer (0.05 M, pH 8.0).
[0066] Results
[0067] Material from all emulsion preparations was recovered by
centrifugation and visualized by light microscopy. The result of
lattice formation is shown in FIG. 1, indicating that roughly
spherical particles were formed.
[0068] Influence of the PEI:Glutaraldehyde Concentration Ratio on
Particle Formation
[0069] The influence of the ratio of PEI to glutaraldehyde on
particle formation was investigated. Samples were prepared
according to Table 1.
[0070] Dry weight determination was performed by lyophilisation of
the fibrous backbone lattice or support and weighing. The results
of this experiment are tabulated in Table 1, and indicate that a
wide range of reactant combinations form particles.
TABLE-US-00001 TABLE 1 Quantities of PEI and Glutaraldehyde used
for fibrous polymeric backbone manufacture evaluation. PEI Backbone
(% of Glutaraldehyde Dry Weight Sample aqueous) (% of aqueous) (mg)
A 5 12.5 36 B 4.5 12.5 43.4 C 4 12.5 40.2 D 3.5 12.5 38.2 E 3 12.5
27.2 F 2.5 12.5 20.4 G 5 10 40.8 H 5 7.5 37.8 I 5 5 36.6 J 5 2.5
35.4
EXAMPLE 2
Binding of Laccase to PEI Support Lattice of Polymeric
Strands/Fibres or Network Backbone
[0071] Enzymes
[0072] DeniLite.TM., a laccase, was obtained from Novozymes
(Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark).
[0073] Enzyme Washing
[0074] Laccase was partially purified from DeniLite.TM. by
dissolving 5 g DeniLite II Base in 100 ml double distilled
H.sub.2O, while stirring at 200 rpm for 1 hour at 4.degree. C.
Suspended solids were removed by centrifugation at 10000 rpm for 1
hour at 4.degree. C. using a JA14 rotor in a Beckman-Coulter
J2-21ME centrifuge. The supernatant was removed and dialyzed
against 3 changes of 5 l of water at 4.degree. C. using
SnakeSkin.TM. (Pierce) dialysis tubing with a 10 kDa cut-off. The
first two changes lasted for 2 hours and the final dialysis for 12
hours. The enzyme was frozen in liquid nitrogen and lyophilized.
This laccase was then stored at 4.degree. C. until required.
[0075] Laccase Assays
[0076] Laccase assays were performed on centrifugal supernatants
after binding laccase to the support and the support immobilised
laccase to determine activity maintenance on binding. Laccase
reagent contained 1 mM guaiacol as the substrate in 100 mM
succinate-lactate buffer (pH 4.5) (Jordaan J, Pletschke B, Leukes
W. 2004 Purification and partial characterization of a thermostable
laccase from an unidentified basidiomycete. Enz. Microb. Technol.
34:635-641). Assays were performed in triplicate at 450 nm with an
extinction coefficient of 5 200 M.sup.-1.cm.sup.-1. Assays were
performed using a PowerWave HT Microtitre plate reader. One unit of
enzyme was defined as the quantity of enzyme required to oxidise 1
.mu.mol of substrate per minute.
[0077] Protein Determination
[0078] Protein loading was followed by determining the protein
concentration in solution by means of light absorbance at 280 nm
with laccase as the standard protein. Bound protein was defined as
total protein minus residual protein in solution.
[0079] Enzyme Immobilisation
[0080] Enzyme, laccase (1 ml of a 10 mg.ml.sup.-1 solution) was
bound to the support by mild agitation for 30 minutes at room
temperature. Enzyme was bound to the backbone dry weight indicated
in Table 2. Particles with bound laccase were recovered by
centrifugation at 700.times.g for 5 minutes. The immobilised enzyme
was washed 5 times with 50 ml water and recovered through the
aforementioned centrifugation.
[0081] Protein, in this case the enzyme laccase, was added to the
PEI-glutaraldehyde particles (derivatised or otherwise) in the form
of a buffer solution. Particles were prepared as described in
Example 1. The particles were allowed to react with the protein to
permit immobilisation of the protein to the polymer as mentioned
above. The particles had not been dried after recovery by
centrifugation and before use.
[0082] The results for protein binding to the various manufactured
protein supports are tabulated below (Table 2), while results for
laccase activity are indicated in FIG. 2.
[0083] Results
TABLE-US-00002 TABLE 2 Binding efficiency of laccase onto backbone
support Backbone Bound Protein PEI Glutaraldehyde Dry Weight
Protein Loading Sample (mg) (mg) (mg) (mg) (mg g.sup.-1) A 5 12.5
36 3.24 89.89 B 4.5 12.5 43.4 4.95 137.43 C 4 12.5 40.2 5.50 152.83
D 3.5 12.5 38.2 5.96 165.45 E 3 12.5 27.2 6.62 183.85 F 2.5 12.5
20.4 6.34 176.05 G 5 10 40.8 6.22 172.67 H 5 7.5 37.8 5.66 157.27 I
5 5 36.6 5.03 139.70 J 5 2.5 35.4 9.43 261.93
[0084] Another experimental set was prepared as in Table 1;
however, in this example the particles were post-treated with
glutaraldehyde and designated by the number two (i.e. A2-J2).
Laccase was subsequently bound to the particle according to the
method described above. Particles with bound laccase were recovered
by centrifugation at 700.times.g for 5 minutes.
[0085] The results for enzyme activity loaded onto the polymeric
support are indicated in FIG. 2.
[0086] This research indicates that the fibrous lattice or network
may be used as a protein immobilisation support with high protein
binding capacity while retaining functional activity of the
protein.
EXAMPLE 3
Manufacture of PEI Support Lattice of Polymeric Strands/Fibres or
Network Backbone Using Various Oils
[0087] The influence of variation in the oil phase of the emulsion
was investigated. The particles were manufactured by initially
preparing 2 separate emulsions A and B.
[0088] Emulsion A Composition
[0089] 10 ml mineral oil, paraffin oil, or isooctane (oil
phase)
[0090] 0.1 ml nonoxyol-4 (surfactant)
[0091] 0.5 ml Polyethyleneimine (polyamine), (10% m/v aqueous
solution), pH 11.
[0092] Stirred at 500 rpm, 25.degree. C., 30 min using a magnetic
stirrer.
[0093] Emulsion B Composition
[0094] 10 ml same oil phase as above.
[0095] 0.1 ml nonoxyol-4 (surfactant)
[0096] 0.5 ml glutaraldehyde, (25% m/v, grade II),
[0097] Stirred at 500 rpm, 25.degree. C., 30 min using a magnetic
stirrer.
[0098] Thereafter emulsion A was quickly added to emulsion B and
stirred for a further hour (700 rpm). The particles were recovered
from the emulsions by centrifugation (3000.times.g, Sorvall
benchtop centrifuge) for 10 minutes followed by washing 6 fold with
10 ml volumes of deionised water. After washing the final particles
were resuspended to 20 ml in deionised water, half of which was
dried by lyophilisation. Both the wet and dried particles were
analysed for particle size distribution (Malvern Mastersizer 2000).
The particle sizes were determined before, with and after in-line
sonication to investigate the presence of agglomeration. Mass
recovery after lyophilisation was also determined.
[0099] Results
[0100] Mass recovery of the particles manufactured with different
oil phases (20 ml total volume of each) was determined to be 111
mg, 90 mg and 79 mg for mineral oil, paraffin oil and isooctane
respectively. Comparison of non-sonicated wet and dry particles
manufactured in various oil phases revealed that after drying the
average particle size increased for mineral oil and paraffin oil
samples (FIG. 3). The particles manufactured in isooctane remained
relatively unchanged despite the drying step. In-line sonication
and pre-sonication showed large decreases in particle size
distribution of dried particles manufactured in mineral and
paraffin oils, indicating that after drying the particles were
agglomerating (FIG. 3), but could be separated by sonication.
Particle size analysis of the wet particles made in mineral and
paraffin oils also indicated decreased average particle size with
sonication treatment of the particles, although to lesser extent
than with dried particles (FIG. 3). Particles manufactured in
isooctane remained relatively unchanged irrespective of the drying
or sonication treatments.
[0101] In conclusion various oil phases can be used to manufacture
particles. The use of different oil phases (which presumably
influences emulsion droplet size) as well as various post treatment
techniques such as drying or sonication can be used to manipulate
their size.
EXAMPLE 4
Manufacture of PEI Support Lattice of Polymeric Strands/Fibres or
Network Backbone Using Various Surfactants
[0102] In the synthesis of the particles the surfactant type may
have influence. This was investigated.
[0103] The particles were prepared according to Example 3 with the
following exceptions--only mineral oil was used as the oil phase
and the surfactant type was varied.
[0104] Results
[0105] Mass recovery of the particles manufactured with various
surfactants in mineral oil (20 ml total volume) was determined to
be 111 mg, 72 mg and 92 mg for nonoxynol-4, CHAPS and Triton X-100
respectively. Drying of particles manufactured with different
surfactants revealed no real size difference when CHAPS and Triton
X-100 were used (FIG. 4). The drying of particles manufactured with
nonoxynol-4 as the surfactant increased particle size by
approximately 50% (FIG. 4). Furthermore size analysis after
sonication showed constant decreases in the particle sizes of the
particles manufactured with all the surfactants tested. Particle
size analyses of particles before and after drying after they had
been sonicated were similar.
[0106] In conclusion the use of various surfactants to manufacture
particles was possible. Moreover particle size could be manipulated
with various surfactants and post treatments.
EXAMPLE 5
Synthesis of Particles with Various Polyaldehydes as Polymer
Cross-Linkers
[0107] The cross-linker aldehyde used in the synthesis of the
particles can be varied. Hence glutaraldehyde, dextran aldehyde,
and hexamethylene diisocyanate were compared as cross-linkers.
[0108] The method for making particles was as in Example 3, with
the following exceptions: in one case the glutaraldehyde was
replaced with dextran aldehyde (1 ml of 15 mg/ml). In another it
was replaced with 0.5 ml hexamethylene diisocyanate (25% v/v).
[0109] To 5 ml of the particle suspension was added 6 ml of 5
mg.ml.sup.-1 purified Candida antarctica lipase B (CALB) in Tris-Cl
buffer (0.05 M, pH 8.0), which was stirred gently for 1 hour at
25.degree. C. The suspension was then centrifuged and washed twice
with 10 ml buffer at 4.degree. C. The suspension was then
centrifuged and the pellet resuspended in 10 ml Tris-Cl buffer. The
suspension (10 .mu.l) was assayed using p-nitrophenyl butyrate. For
comparison 10 .mu.l of 0.5 mg per ml purified Candida antarctica
lipase B in Tris-Cl buffer (0.05 M, pH 8.0) was also assayed, using
an assay based on hydrolysis of .rho.-nitrophenyl butyrate and its
subsequent analysis by spectrophotometry (Table 3).
[0110] Lipase Activity Assay
[0111] The activity of lipase involved the hydrolysis of a
p-nitrophenyl ester (p-nitrophenylbutyrate (PNPB) to p-nitrophenol
and butyric acid. The release of p-nitrophenol yields a yellow
colour which is measured with a UV/Vis spectrophotometer at 410 nm.
Activities were determined in triplicate. The solutions were
prepared as follows: solution A contained enzyme substrate
dissolved in 8 ml propan-2-ol; while solution B contained 267 mg
sodium deoxycholate dissolved in 50 mM Tris-buffer (pH 8.0)
followed dissolution of 66.7 mg gum arabic. Kinetic assays were
performed at 25.degree. C. using a PowerWave microtitre plate
reader (BioTek Instruments) with 240 .mu.l of a 1:10 (A:B) mixture
of the above mentioned solutions and 10 .mu.l of the immobilised
lipase suspension solutions or free enzyme.
[0112] Results
TABLE-US-00003 TABLE 3 comparison of particles made with various
aldehyde cross-linkers - activity of CALB. Hexa- methylene Free
enzyme Dextran di- Cross-linker (0.5 mg ml-1) Glutaraldehyde
aldehyde isocyanate Pellet colour Not applicable Orange Yellow
White Lipase U ml.sup.-1 U ml.sup.-1 U ml.sup.-1 U ml.sup.-1
activity Sample 1 8.74 9.45 2.31 6.13 Sample 2 5.72 9.10 2.10 6.13
Sample 3 5.99 7.50 2.52 7.56 Average 6.81 8.68 2.31 6.61
[0113] Although this process is not optimised, it demonstrates that
the particles can be generated using a range of poly-aldehyde
compounds.
[0114] This material could be recovered on a 0.45 .mu.m filter
(Sartorius) and reused, giving an average of 7.05 U.ml.sup.-1 for
the glutaraldehyde particle, or 81% of the original activity over 5
recycles.
[0115] A similar experiment was performed using the lipase from
Pseudomonas fluorescens (PFL). The assay for enzyme activity used
the p-nitrophenyl esters of butyric acid (PNPB) and palmitic acid
(PNPP) as substrates.
TABLE-US-00004 TABLE 4 comparison of particles made with various
aldehyde cross-linkers - activity of PFL. Total Activity (U)
Aldehyde PNPB PNPP Free enzyme 9.51 58.5 Hexa-methylene di- 1.04
0.7 isocyanate Glutaraldehyde 0.08 0 Dextran aldehyde 0.9 2
[0116] Hence various aldehydes could be used to provide both
effective cross-linking for particle formation and functional
groups to cross-link proteins to the particles. The selection of
cross-linker can influence the activity of the enzyme through
degree of enzyme denaturation, or degree of accessibility of the
particle to substrates and products. The optimum agent may be
selected based on the enzyme and the reaction substrate.
EXAMPLE 6
Manufacture of Particles Using a bi-Functional Epoxide
Cross-Linker
[0117] The cross-linker aldehyde used in the synthesis of the
particles can be replaced by other cross-linkers, such as
di-epoxides, for example 1,4-butanediol diglycidyl ether. This was
added (in lieu of glutaraldehyde) to the cross-linker emulsion (B)
as 0.5 ml of a neat, 50%, 25% or 12.5% v/v solution and reacted (as
per Example 3) at 40.degree. C. for 2 hours. The particles were
recovered from the emulsions by centrifugation (3000.times.g,
Sorvall RT7 benchtop centrifuge) for 10 minutes followed by washing
6 fold with 10 ml volumes of deionised water. After washing the
final particles were resuspended to 20 ml in deionised water. Those
prepared as above with the 12.5% or 25% v/v epoxide solution were
the most uniform in shape while those formed using neat of 50%
epoxide were large and diffuse.
[0118] The binding of protein was investigated using CALB (as per
previous example), which was dialysed overnight and reacted with
the epoxide based particles (prepared as above with the 12.5% v/v
epoxide solution) for one hour with gentle stirring. Enzyme
activity was analysed after recovery and washing of lipase bound
particles as described in Example 5. All analyses were performed
with PNPB as the substrate
[0119] Results
[0120] The use of butanediol diglycidyl ether as a cross-linker
resulted in the formation of white particles of approximately 0.1
mm in diameter. The particles were however somewhat irregular in
shape, indicating aggregation of particles with cross-linking or
subsequent cleaning procedures.
[0121] Particles produced with 12.5% v/v epoxide solution were
incubated overnight in the presence of the enzyme. These particles
yielded an activity of 0.28 U.ml.sup.-1. This demonstrates that
particles can be manufactured using an epoxide cross-linker
EXAMPLE 7
PEI Support Lattice of Polymeric Strands/Fibres or Network Backbone
Manufactured in a Single Emulsion
[0122] The method for formation of the particles can be adjusted
according to need. For example the particles may be formed through
application of a single emulsion.
[0123] The polymer and cross-linker can then be mixed
instantaneously in the aqueous phase by means of a dual injection
device into a mixing chamber. The simplest version of this device
could consist of two syringes that inject into a common line at the
same point. This can be injected directly into the oil phase. For
example, using 20 ml mineral oil in which 0.2 ml nonoxynol-4 was
dissolved for approximately 5 minutes and using magnetic stirring
at 500 rpm, such a set-up was evaluated. One syringe contained 0.5
ml PEI (10%) and the other contained 0.5 ml glutaraldehyde (20%,
grade II). The syringes were depressed simultaneously directly into
the mineral oil. The resultant emulsion was stirred for 1 hour and
then the particles were recovered as in example 3. The experiment
was performed in triplicate. The particles were divided into 2
equal fractions, one of which was freeze dried (Virtis) and
re-suspended to original volume before being analysed for particle
size distribution (Malvern Mastersizer 2000). The samples were
analysed for mass recovery after drying.
[0124] Results
[0125] The mass recovery for particles manufactured using a single
emulsion was calculated to be 152.+-.12 mg, which was approximately
1.4 fold higher than that obtained using the 2 emulsion strategy
(Refer to Example 3).
[0126] The formation of particles using a single emulsion was
possible and reproducible using the current manufacturing technique
(FIG. 5). The particles were found to be larger in particle size
distribution than those formed using a dual emulsion (FIG. 5). The
size distribution wet and dry obtained for single emulsion
particles were shown to be between 50 and 70% larger in size than
those obtained for the dual emulsion experiment (FIG. 3).
EXAMPLE 8
Scaled Manufacture of PEI Support Network or Lattice of Polymeric
Strands/Fibres
[0127] The objective was to linearly increase the scale of
particles manufacture 10 fold and evaluate the particle size. Two
separate batches of particles were prepared according to the
standard manufacturing method outlined in Example 3, except that
the second batch contained 10 fold more of each of the respective
constituents required. The batches were processed for particle
recovery as in Example 3 according to scale.
[0128] Results
[0129] The mass recovery for particles manufactured at 20 ml and
200 ml mineral oil volumes were calculated to be 0.111 g and 1.11 g
respectively which was exactly 10 fold difference. The manufacture
of particles at mineral oil volumes of 20 and 200 ml was possible
with the larger scale manufactured particles being consistently
only slightly smaller in particle size than those obtained at 20 ml
scale (FIG. 6). Interestingly the non-sonicated dried particles
manufactured at larger scale were around 50% smaller in size when
compared to the dried particles at the smaller scale (FIG. 6).
[0130] The manufacture of particles under standard conditions is
scalable by at least 10 fold based on mass recovery and particle
size analysis.
EXAMPLE 9
Application of a PEI Support Network or Lattice of Polymeric
Strands/Fibres to Immobilise a Range of Enzyme Classes
[0131] The objective was to bind different enzymes to the particles
backbone and calculate their binding percentages as well as the
enzymatic activity retained.
[0132] The particles manufactured according to example 3 were used.
The enzymes investigated were Laccase (Novozymes 51009,
Myceliopthora thermophilia), Glucose oxidase (Seravac Pty Ltd,
Aspergillus niger) and Lipase (CALB, Novozymes Candida antarctica).
For each experiment 5 mg (0.5 ml at 10 mg.ml.sup.-1) of the
respective enzyme was bound to 14 mg (0.5 ml at 28 mg.ml.sup.-1) of
particles with gentle shaking for 2 hours (25.degree. C.). Each
sample was centrifuged for 10 minutes using an Allegra X22R
centrifuge (2000.times.g). The particles were washed 6 times
consecutively, each time with 2 ml deionised water. The combined
supernatant fractions were analysed for total protein. Each of the
respective enzyme binding experiments was performed with and
without the inclusion of a particular substrate as a potential
protectant. For laccase the commercial mediator Denillite II Assist
(Novozymes) was the potential protectant (50 .mu.l of 100
mg.ml.sup.-1 pH adjusted to 6.8), for glucose oxidase the potential
protectant was glucose (50 .mu.l of 10% m/v glucose monohydrate)
and for CALB an 8 diasterioisomeric mix of
2-isopropyl-5-methylcyclohexanol (menthol) was used (50 .mu.l). The
particles were resuspended to 2 ml in deionised water. Particles
were assayed for their respective enzyme activities. All assays
were in triplicate.
[0133] Total Protein
[0134] Total protein assays were performed using the Bio-Rad Total
Protein Assay kit (Cat. No.: 500-0006) with each of the respective
enzymes used as standards.
[0135] Laccase activity was determined by using 1 mM
2,2_-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt (ABTS) as the substrate in 100 mM succinate-lactate buffer pH
4.5. The optical density of the solution was measured at 420 nm.
These assays were performed by adding 20 .mu.l of the samples to
180 .mu.l of ABTS reagent. Activity was followed
spectrophotometrically at 420 nm using a PowerWave HT (Biotek
Instruments) with incubation at 25.degree. C.
[0136] Glucose Oxidase
[0137] Glucose Oxidase (GOX) activity was measured using the
indirect oxidation of o-dianisidine by horseradish peroxidase
(HRP). The assays were performed according to Bergmeyer et al.,
1988. The following reagents were prepared: reagent A, 0.1 M
potassium phosphate buffer, pH 7, containing o-dianisidine.2HCl
(0.006%); reagent B, 10% aqueous solution of D-glucose (allowed to
mutarotate for 1 h before use); reagent C, 60 Uml.sup.-1 HRP
aqueous solution. Reagents A, B and C were mixed immediately prior
to assaying for glucose oxidase in the ratio 24:5:1, respectively.
The reaction contained 0.3 ml of the reaction reagent and was
initiated by the addition of 10 .mu.l of sample. The reaction was
measured kinetically at 436 nm (Powerwave HT microtiterplate
reader) at 25.degree. C. One unit of glucose oxidase activity is
defined as the amount of enzyme that catalyses the conversion of 1
.mu.mole .beta.-D-glucose to D-gluconolactone and H.sub.2O.sub.2
per minute at 25 .degree. C. and pH 7.
[0138] Lipase
[0139] The activity of lipase involved the hydrolysis of
p-nitrophenyl esters to .rho.-nitrophenol and an aliphatic
carboxylic acid. The release of p-nitrophenol yields a yellow
colour which is measured with a UV/Vis spectrophotometer at 410 nm.
Two p-nitrophenol esters were used, p-nitrophenylacetate (PNPA) and
p-nitrophenylbutyrate (PNPB) and activities were determined in
triplicate. The solutions were prepared as follows: solution A
contained enzyme substrate (11.6 mg PNPA or 24 mg PNPP) dissolved
in 8 ml propan-2-ol; while solution B contained 267 mg sodium
deoxycholate dissolved in 50 mM Tris-buffer (pH 8.0) followed by
66.7 mg gum arabic. Kinetic assays were performed at 25.degree. C.
using a PowerWave microtitre plate reader (BioTek) with 240 .mu.l
of a mixture of the above mentioned solutions and 10 .mu.l of the
spherezymes or free lipase solutions.
[0140] Results
[0141] The protein loading for all the enzymes tested for binding
to particles ranged between 30% to 36% (Table 5).
TABLE-US-00005 TABLE 5 Binding efficiency of various enzymes to
particles backbone support Protein Protein Particles Dry Bound
loading Loading Sample Weight (mg) Protein (mg) (mg g-1) (% m/m)
Laccase 14 4.77 341 34.06 Laccase - Substrate* 14 5 357 35.71
Glucose Oxidase 14 5 357 35.71 Glucose oxidase- 14 5 357 35.71
Substrate* CALB 14 4.27 305 30.5 CALB - Substrate* 14 4.6 329 32.89
*Substrate was added during enzyme immobilisation in order to
protect active site (refer to method above)
[0142] The inclusion of the menthol substrate for activity
maintenance of CALB towards PNPA was advantageous retaining
approximately 30% more activity (83%) (FIG. 7).
[0143] Laccase, glucose oxidase and CALB were all successfully
bound to particles with retention of activity, indicating that a
range of proteins can be effectively bound to the particles at high
protein loading. Furthermore, other enzymes, such as horseradish
peroxidise, protease and dehydrogenases, were also demonstrated to
bind to the particles (see examples below).
EXAMPLE 10
PEI Support Network or Lattice of Polymeric Strands/Fibres;
Particles with Multiple Enzymes Bound
[0144] The investigation was designed to demonstrate that more than
1 enzyme could be bound to the particles backbone with retention of
activity towards both enzymes. The glucose oxidase and horseradish
peroxidase system was chosen.
[0145] The particles were manufactured according to Example 3. The
enzymes investigated were glucose oxidase (Seravac Pty Ltd,
Aspergillus niger) and horseradish peroxidase (Serevac Pty Ltd).
For the example 5 mg (0.5 ml at 10 mg.ml.sup.-1) glucose oxidase
and 10 mg of horseradish peroxidase (1 ml at 10 mg.ml.sup.-1) was
bound to 28 mg (1 ml at 28 mg.ml.sup.-1) of particles backbone with
gentle shaking for 2 hours (25.degree. C.). Each sample was
centrifuged for 10 minutes using an Allegra X22 centrifuge
(200.times.g). The particles were washed 6 times consecutively each
with 2 ml deionised water. The combined supernatant fractions were
analysed for total protein. The activity of the particles was
determined according to the assay method for glucose oxidase in
Example 9, but without the inclusion of horseradish peroxidase in
the assay reagent.
[0146] Results
[0147] Glucose oxidase and horseradish peroxidase were successfully
bound to particles backbone and able to convert glucose at a rate
of 3 .mu.mole.min.sup.-1 (Table 6). As the assay detected activity,
this indicates that both glucose oxidase and horseradish peroxidase
were bound and active. Hence particles can be used to bind more
than one enzyme and where both enzymes retain activity.
TABLE-US-00006 TABLE 6 Binding efficiency and activity for the
glucose oxidase and horseradish peroxidase dual enzyme particles.
Particles Dry Bound Protein Protein Glucose Oxidase Weight Protein
Loading Loading Activity Sample (mg) (mg) (mg g.sup.-1) (% m/m)
(.mu.mole min.sup.-1) Glucose Oxidase and 28 6.7 240.7 24.1 3
horseradish peroxidase
EXAMPLE 11
PEI Support Network or Lattice of Polymeric Strands/Fibres
Manufactured Using Various Drying Methods
[0148] The aim was to manufacture particles and subsequently dry
them using different methods, such as lyophilisation, vacuum and
acetone drying.
[0149] The particles were manufactured under the standard
conditions as outlined in Example 1. Once washed, the particles
were dried by lyophilisation (Virtis Genesis freeze drier), vacuum
drying using a vacuum concentrator (Savant SpeedVac SC110, fitted
with a Savant RVT100 vapour trap), or by dehydrating with acetone
followed by air drying at 25.degree. C. for 12 hours. The acetone
dried particles could not be re-suspended in aqueous medium due to
agglomeration, and hence were not considered further.
[0150] However, vacuum drying and lyophilisation were both
successful techniques for drying the particles and could
subsequently be used for the attachment of a range of enzymes or
proteins (Table 7). To 5 ml of the particle suspension was added 6
ml of 5 mg per ml purified enzyme in Tris-Cl buffer (0.05 M, pH
8.0), which was stirred gently for 1 hour at 25.degree. C. The
suspension was then centrifuged and washed twice with 10 ml buffer
at 4.degree. C. The suspension was then centrifuged and the pellet
resuspended in 10 ml Tris-Cl buffer (0.05 M, pH 8.0). The particle
sizes were determined using a Malvern Mastersizer.
[0151] Results
TABLE-US-00007 TABLE 7 Average particle size after various drying
treatments. Particle size .mu.m (volume weight mean) Re-Suspended
Sample Drying Method Treatment Wet particles Dried Particles
Particles - No Lyophilisation None 22.432 17.662 immobilised
protein Sonication 13.141 14.556 Particles - CALB Lyophilisation
None 21.958 26.262 Sonication 14.199 13.734 Particles - BSA
Lyophilisation None 24.114 32.092 Sonication 20.071 21.129
Particles - Pseudomonas Lyophilisation None 18.705 21.648
fluorescens lipase Sonication 14.169 15.258 Particles - No Vacuum
drying None 17.662 66.613 immobilised protein Sonication 14.556
18.979 Particles - Pseudomonas Vacuum drying None 18.705 71.776
fluorescens lipase Sonication 14.169 18.84 Particles - Laccase
Lyophilisation None 38.081 34.491 Sonication 22.498 18.948
Particles - Alcalase Lyophilisation None 30.737 32.884 Protease
(Novozymes) Sonication 20.297 22.698
[0152] This example demonstrates that with drying, particularly
with lyophilisation, particles with bound enzyme do not agglomerate
to any great extent.
EXAMPLE 12
Characterisation of Laccase Binding to Support Network of or
Lattice of Polymeric Strands/Fibres Prepared from PEI at Various
pH's
[0153] The effect of drying on the properties of laccase after
binding bound particles on the enzymes characteristics was
determined.
[0154] Particles were manufactured according to the standard method
of manufacture outlined in Example 3 except that in addition to the
pH 11 preparation of the PEI (10%) a preparation adjusted to pH 8
was also evaluated. Thereafter laccase was immobilised to the
backbone as described in Example 9 with the following exceptions: 1
ml of laccase (Novozyme 51004, 50 mg.ml.sup.-1) was reacted with
6.25 ml particles (16 mg.ml.sup.-1), therefore 50 mg of laccase was
bound per 100 mg of particles. The laccase bound particles were
washed with 12.5 ml deionised water and resuspended to 20 ml using
deionised water. The particles were divided into 2 equal fractions
of 10 ml each, of which a sample was freeze dried for mass recovery
determination, and to investigate the characteristics of both wet
and dry laccase bound particles. The dried samples were resuspended
to their original volumes with deionised water. All assays were in
triplicate.
[0155] Total protein of the laccase supernatant samples was
determined using the Bio-Rad Total Protein Assay kit (Cat. No.:
500-0006) with laccase as the protein standard. Laccase activity
was determined according to Example 9.
[0156] Results
[0157] Drying of laccase bound particles by lyophilisation showed
that higher mass recovery was achieved when particles were
manufactured with PEI at pH 11 (Table 8), although more laccase
protein was bound to the particles using PEI at pH 8 for
manufacture. Optimum activity maintenance towards ABTS was achieved
with non-lyophilised particles manufactured using pH 8 PEI (16%),
but after lyophilisation the same sample only retained 3% of the
laccase activity. It was noted that particles manufactured using pH
8 PEI were difficult to re-suspend after drying, and therefore
probably agglomerate, thereby reducing surface to area ratio, and
hence reducing activity due to diffusional constraints on the
substrate and product. The same effect on activity was not observed
for particles generated using PEI at pH 11 (Table 8).
TABLE-US-00008 TABLE 8 Binding efficiency and activity maintenance
of laccase bound particles manufacture at various PEI pH values
with and without freeze drying. Particles Protein Laccase Laccase
particle Dry Loading Protein Activity preparation Weight Protein
(mg Loading Maintained conditions (mg) (mg) g.sup.-1) (%) (%)
particles wet pH 8 NA 25 383 38 16 particles dry pH 8 65 25 383 38
3 particles wet pH NA 25 295 30 8 11 particles dry pH 83 25 295 30
11 11
[0158] This experiment demonstrates that particles can be prepared
with PEI of varying pH's and that this changes the binding
properties of the particles for enzymes.
EXAMPLE 13
Stability of Laccase Bound to PEI Support Network or Lattice of
Polymeric Strands/Fibres Manufactured at pH 8 or 11 and
Lyophilised
[0159] The thermostability and pH stability of laccase bound to PEI
support network or lattice of polymeric strands/fibres manufactured
at pH 8 and 11 and lyophilised was determined (laccase bound
particles produced as described in Example 12).
[0160] Temperature Optima
[0161] The temperature optima profiles of free laccase and laccase
bound particles (at equivalent protein loading) was performed using
1 mM ABTS as the substrate in 100 mM succinate-lactate buffer (pH
4.5). Samples (100 .mu.l) were added to 1.9 ml of substrate
pre-equilibrated to the correct temperature in a water bath. Assays
were performed using a DU800 spectrophotometer (Beckman-Coulter,
420 nm) fitted with Peltier temperature controller. The
spectrophotometer was set to the temperature of interest and
cuvettes were allowed to equilibrate for 5 minutes prior to
addition of reagent equilibrated in a water bath.
[0162] pH Stability
[0163] The pH stability of free laccase and laccase bound particles
(at equivalent protein loading) was done with ABTS (1 mM) in 100 mM
succinate-lactate buffer (pH 4.5). The respective laccase samples
were incubated in the Britton-Robinson universal buffer at pH 2.5,
3 and 6. Samples (20 .mu.l) were periodically removed and assayed
(230 .mu.l assay reagent) at 420 nm using a PowerWave HT (Biotek
Instruments) with incubation at 25.degree. C. The 6 hour time
points for this experiment are illustrated in FIG. 9 below.
[0164] Results
[0165] The results are shown in FIGS. 8A, 8B and 9.
[0166] Both the laccase bound to particles and the free
(non-immobilised) laccase were optimally active at 70.degree. C.
(FIG. 8). However, laccase bound to particles indicated improved
thermostability at 90.degree. C. (55-65% activity) in comparison to
the free laccase (0% activity) in the time taken to prepare samples
for assay. There was also a minor improvement when the dried
particles were used, suggesting that with drying there is an
advantage. This may be due to more protein-particle links being
formed as water is removed. This in turn would increase the
multi-point covalent binding of the protein or enzyme, which is
known to provide greater stability, such as improved
thermostability [Improvement of enzyme activity, stability and
selectivity via immobilization techniques. Mateo, C., Palomo, J.
M., Fernandez-Lorente, G., Guisan, J. M., Fernandez-Lafuente, R.
2007 Enzyme and Microbial Technology 40 (6), pp. 1451-1463].
[0167] The pH stability of laccase bound particles and free enzyme
at pH 2.5, 3 and 6 was also determined. At pH 2.5 and pH 3 all the
laccase bound particle samples retained 80-110% activity while the
free enzyme had lost approximately 70 and 40% activity respectively
(FIG. 9). Hence the immobilisation provides improved pH stability.
All the samples including the free enzyme were stable at pH 6 after
6 hours.
[0168] Hence, the immobilisation of enzymes on particles of PEI
support network or lattice of polymeric strands/fibres can provide
additional stability in extremes of pH.
EXAMPLE 14
Enzyme pH Optimum Shift with Immobilisation to PEI Support Network
or Lattice of Polymeric Strands/Fibres
[0169] Enzymes have an optimum pH for activity. In some cases the
optimum pH of an enzyme does not coincide with the optimum pH for
other aspects of a reaction. For example the enzyme
substrate/reactant may be optimally soluble at another pH. Another
example is where more than one enzyme is used in a multi-step
one-pot reaction, and their pH optima may not coincide. Hence,
should an enzyme optimal pH change during immobilisation, this
could provide commercial and technical advantages. Hence the pH
optimum of a laccase was determined with and without immobilisation
on the particles.
[0170] Enzyme Assays
[0171] Laccase reagent contained 1 mM guaiacol in 50 mM
Britton-Robinson universal buffer (Davies T J, Banks C E, Nuthakki
B, Rusling J F, France R R, Wadhawana J D, Compton R G. 2002.
Surfactant-free emulsion electrosynthesis via power ultrasound:
electrocatalytic formation of carbon-carbon bonds. Green Chem.
4:570-577) adjusted to the pH values of interest. The universal
buffer was used to ensure that the same buffer system was present
and could effectively buffer across a wide pH range. Assays were
performed in triplicate at 450 nm with an extinction coefficient of
5 200 M.sup.-1.cm.sup.-1. pH Profiles of laccase immobilised to the
particles of support network or lattice as well as the free enzyme
were experimentally determined. Assays were performed using a
PowerWave HT Microtitre plate reader. One unit of enzyme was
defined as the quantity of enzyme required to oxidise 1 .mu.mol of
substrate per minute.
[0172] pH Profile Shifting
[0173] pH profiles were determined for laccase bound to the fibrous
lattice or network backbone support since pH profile shifting has
been known to occur during immobilisation. Particles were made as
per Example 3. The effect of varying glutaraldehyde and PEI
concentration on the pH optimum shift was also investigated as was
the effect of gluraldehyde post-treatment (Example 2, A2-J2).
[0174] Results
[0175] The results are shown in FIGS. 10A, 10B, 11A and 11B.
[0176] The general trend with respect to pH profile shifting is
towards a neutral to slightly alkaline pH. This example
demonstrates that the pH profile of enzymes may be shifted by
immobilisation to the particles.
EXAMPLE 15
Various Functionalities of PEI Network or Support Lattice of
Polymeric Strands/Fibres
[0177] The functionality of the particles backbone can be changed
to demonstrate hydrophobic, ionic and affinity based binding of
proteins.
[0178] The particles were manufactured by mixing an emulsion of 10
ml of mineral oil containing 0.1 ml nonoxynol-4 and 0.5 ml
polyethyleimine solution, pH 11, and an emulsion of 10 ml of
mineral oil containing 0.1 ml nonoxynol-4 and 0.5 ml glutaraldehyde
(Sigma grade II). Both emulsions had been agitated at 500 rpm for
30 min prior to mixing. The combined emulsion was agitated at 500
rpm for 30 min. This provided unmodified particles which were
recovered by centrifugation as described previously.
[0179] Ionic Binding
[0180] Ionic groups were generated on above unmodified particles by
functionalisation with glutaraldehyde (200 .mu.l of 25% aqueous
solution--washed with 3.times.20 ml of deionised water) followed by
treatment with ethylene diamine (1 ml of 0.33 M) to react with free
aldehyde residues (1 hour at room temperature with periodic
inversion). Particles were washed repeatedly with excess dionised
water and recovered by centrifugation at 2000.times.g for 10
minutes. Laccase (2.5 mg) was incubated with 5 mg of modified
particles for 30 minutes at 4.degree. C. with periodic inversion to
ensure adequate mixing. To determine ionic binding 1.0M NaCl (as a
counter-ion) was added to the laccase bound particles and mixed by
inversion for 5 minutes. Particles were recovered by centrifugation
(as mentioned above) and activity determined.
[0181] Hydrophobic Binding
[0182] Hydrophobic groups were added to the particles by incubating
the unmodified particles (5 mg) with epoxyoctane (0.1 ml) at
25.degree. C. for 4 hours. These were then repeatedly washed with
excess water and recovered by centrifugation (as mentioned above).
Hydrophobically bound protein (Pseudomonas fluorescens lipase, 5
mg) could be removed by addition of a surfactant (1% deoxycholate),
as determined by measuring protein absorbance at 280 nm.
[0183] Affinity Binding
[0184] Affinity binding of proteins is demonstrated in Example
17.
[0185] Results
[0186] Ionic Binding
[0187] 45% of the added laccase was bound to the modified particles
(as determined by the Bio-Rad protein assay dye reagent method,
500-0006, as per the manufacturers protocol). Of this 45%, 76.9% of
the laccase could be removed through addition of salts (1.0 M NaCl)
as a counter-ion. In comparison unmodified particles only bound 14%
of the laccase, and only 52.6% of this could be removed by the salt
solution.
[0188] Hydrophobic Binding
[0189] Protein was bound to the modified particles. Approximately
28% of the protein bound by the modified particles was removed by
the addition of deoxycholate, this being the hydrophobically bound
portion of the bound enzyme.
[0190] This example demonstrates that the functionality of the
particle matrix can be modified to effect alternative mechanisms of
protein binding to the particle matrix.
EXAMPLE 16
Co-Entrapment of Mediators and co-Factors
[0191] Inclusion of co-factor (or modified co-factor, or mediator)
permits co-entrapment of the co-factor with the enzyme after
cross-linking. Through selection of process conditions, the
porosity of the particle can be arranged to retain the co-factor
while permitting entry and exit of small molecules, such as
reactants, e.g. enzyme substrates, co-substrates, products and
co-products.
[0192] Amino acid dehydrogenase (AADH), formate dehydrogenase
(lyophilised) were purchased from Biocatalytics (USA). PEG
20000-NADH was obtained from Julich Fine Chemicals (Germany).
Mineral oil was obtained from Castrol (Germany). Nonoxynol-4 was
obtained from ICI (UK). Glutaraldehyde (Glut) (25% aqueous
solution) was obtained from Acros Organics (Belgium). Formic Acid
was obtained from Merck (Germany). Ethylenediamine (EDA),
Polyethyleneimine (PEI), 3-Methyl-2-oxobutyric acid (2-Ketovaline),
DL-valine, NADH and NAD.sup.+ were obtained from Sigma-Aldrich.
[0193] Polymeric particles were produced by cross-linking
polyethyleneimine (PEI) with glutaraldehyde. A water-in-oil
emulsion of the polyethyleneimine was prepared by emulsifying 800
.mu.l of 10% PEI in 40 ml of mineral oil containing 200 .mu.l of
pre-dissolved Nonoxynol-4 (20 minutes magnetic stirring in 100 ml
beaker with a 20 mm magnetic stirrer stirring at 500 rpm). A second
water-in-oil emulsion was prepared similarly using a 20%
glutaraldehyde solution. The two emulsions were mixed by adding the
glutaraldehyde emulsion to a rapidly stirring polyethyleneimine
emulsion (700 rpm). This was allowed to react for 30 minutes with
continuous stirring.
[0194] The polymeric particles were recovered by centrifugation at
2000.times.g for 10 minutes (Sorvall, RT7). The polymer particles
were washed 4 times with 45 ml of deionised water. Recovery during
washing was performed by centrifugation as in previous examples.
The product was re-suspended to a volume of 10 ml. This solution (1
ml) was aliquoted into eppendorf tubes and used in subsequent
experiments.
[0195] Protein solutions of each protein (formate dehydrogenase and
valine dehydrogenase) containing 10 mg.ml.sup.-1 were prepared.
These two solutions were subsequently mixed (200 .mu.l of each
solution) and incubated with the polymeric material. This solution
was mixed by inversion and allowed to react for 30 min with gentle
agitation. The particles containing immobilised enzyme were assayed
for activity determination using the methods described below.
[0196] The particles were washed with deionised water, and
recovered as mentioned above. The particles were mixed with 100
.mu.l PEG20000-NADH (obtained from Julich Fine Chemicals of Julich,
Germany) and incubated at room temperature with gentle agitation
for 2 hours. This solution was subsequently lyophilised. The
lyophilised product was washed twice with 2 ml of water and
recovered by centrifugation. The particles were re-suspended in 1
ml of 100 mM Tris-Cl buffer pH 8.0 containing 100 mg of lysine to
quench excess aldehyde functionality on the particles, and
incubated at room temperature for 1 hour. The particles were washed
5 times with 2 ml of Tris-Cl buffer (20 mM pH 8.0). This sample was
then tested for recycling ability using 1 ml of the recycling
reagent. The samples were washed 3 times with 2 ml volumes of water
between each cycle. Samples were analyzed for the production of
valine by amino acid TLC and HPLC as mentioned below.
[0197] These particles were reacted and recycled into fresh
reaction medium for subsequent reactions.
[0198] Recycling Reagent
[0199] Reagent for the recycling of PEG-20000-NADH for the
production of valine consisted of 50 mM formate (from 1 M stock of
sodium formate pH 8.0), 50 mM Tris-Cl buffer pH 8.0, 50 mM ammonium
tartrate, and 10 mM 2-ketovaline. The composition of this reagent
was formulated to ensure that valine was only produced if the PEG
NADH was recycled.
[0200] Analytical Methods
[0201] Amino acid TLC was performed on F254 silica gel plates
(Merck). The mobile phase used was 9:1 ethanol to glacial acetic
acid. The amino acid (valine) was stained using a solution of 2%
ninhydrin in acetone. The plates were heated at 120.degree. C.
until suitable resolution of valine (Rf 0.49) and ammonia (Rf 0.31)
was clearly visible.
[0202] HPLC was performed using the OPA derivitisation method
(o-phthalaldehyde reagent) for determination of amino acids.
Samples were derivatised in-line. Valine standards were
included.
[0203] Results
[0204] The reaction and recycle was a success according to TLC
data, with formation of valine spots for all of the consecutive
particle catalysed reactions. Positive TLC results were confirmed
and quantified using the HPLC OPA method, with the particles
converting 48, 35, 59, 43, 33, and 29% of 10 mM ketovaline to
valine respectively in six consecutive 16 hour reaction cycles
[0205] This example demonstrates the entrapment of co-factors in
the enzyme-particle matrix allowing enzymes that use co-factors to
maintain functionality and allowing re-cycling of entrapped
co-factors.
EXAMPLE 17
Binding of Antibodies by Particles of PEI Support Network or
Lattice of Polymeric Strands/Fibres
[0206] This example demonstrates the binding of antibody and/or
antigen on the particles. The binding of antibodies and antigens
demonstrates the suitability of the particles for affinity binding
of proteins via immobilised antibody or antigen. Further, the
plausibility of the particles for diagnostic applications, such as
ELISA, is demonstrated.
[0207] Polyethyleneimine (P3143; 50% aqueous solution),
glutaraldehyde grade 11 (G6257; 25% aqueous solution), mineral oil
(M8410), the antigen mouse interleukin 2 (I0523-2000; SL06092) and
the marker enzyme streptavidin-peroxidase from Streptomyces
avidinii (S5512-250UG; SL05181) were from Sigma-Aldrich. The
primary antibody, rat anti-mouse interleukin 2 (IL-2) MAB
(17663-27K1; L6080801) and the secondary antibody, rat anti-mouse
interleukin 2 (IL-2) Biotin MAB (17663-27M5; L6080803) were from
USBiologicals. Sodium chloride (S7653), sodium phosphate dibasic
(S0876), sodium phosphate monobasic (S0757), hydrogen peroxide
(21676-3), Tween 20 (P9416) and hydrochloric acid (H1758) were from
Sigma-Aldrich. 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonic
acid) (10102946001) was from Roche.
[0208] Particle Preparation
[0209] Crosslinked polyethyleneimine particles were prepared
according to Example 2, sample G. The only adjustment to a reagent
was that the polyethyleneimine solution was adjusted to pH 9 with
HCl before dilution to 10%. The experiment was directly scaled by a
factor of 4, thus requiring 20 ml mineral oil containing 200 .mu.l
of dissolved NP4 with 800 .mu.l of each of the emulsified
reactants. The resultant cross-linked polyethyleneimine particles
were washed with 6.times.50 ml of deionised water and recovered by
centrifugation at 5000.times.g for 5 min from the mineral oil and
between each wash step. The particle pellet was subsequently
resuspended to a volume of 10 ml with deionised water and 500 .mu.l
aliquots were used for experiments A to F.
[0210] Immobilisation of Proteins
[0211] The experiments were assigned letters according to Table 9
below. The addition of the various proteins was carried out in the
sequence indicated in the column below the experiment (Table 9).
The immobilisation of the first protein component for each
experiment was performed in deionised water (sequential addition
step 1, Table 9) at 4.degree. C. for 1 hour with inversion of the
samples every 10 minutes to ensure adequate mixing. The particles
were washed with 3.times.1 ml of deionised water and recovered as
mentioned above.
[0212] Subsequent protein binding, the protein treatments (Table 9,
rows 2 to 5), were performed in 10 mM phosphate buffer pH 6.8
containing 150 mM sodium chloride (binding buffer). This binding
was carried out at 37.degree. C. for 30 minutes with inversion
every 5 minutes to ensure adequate mixing. After each protein
binding the samples were washed with 2.times.1 ml of binding buffer
containing 0.05% Tween 20 to limit non-specific protein
interactions using mild agitation (IKA Vortex Genius, setting 2)
for 10 minutes. Recovery of the particles between each successive
step was achieved by centrifugation at 5000.times.g for 5 minutes.
The quantity of protein added for each of the protein treatment
steps (Table 9) were as follows: albumin--5 mg; rat anti-mouse
interleukin 2 MAB (A-IL2-MA)--50 pg; rat anti-mouse IL2 MAB Biotin
(B-A-IL2-MA)--25 pg; interleukin 2 (IL-2)--2 .mu.g;
streptavidin-peroxidase (strep-perox) 10 .mu.g. These quantities of
proteins were prepared in 1 ml of binding buffer.
TABLE-US-00009 TABLE 9 Sequence of protein binding for experiments
A to F. Sequential Addition Step A B C D E F 1 A-IL2-MA A-IL2-MA
IL-2 Albumin Albumin No treatment 2 Albumin Albumin Albumin -- IL2
-- 3 -- IL2 -- -- -- -- 4 B-A-IL2-MA B-A-IL2-MA B-A-IL2-MA
B-A-IL2-MA B-A-IL2-MA -- 5 Strep-Perox Strep-Perox Strep-Perox
Strep-Perox Strep-Perox --
[0213] Assay
[0214] The peroxidase assay reagent contained 2 mM ABTS and 2 mM
hydrogen peroxide in 10 mM phosphate buffer pH 6.8 with 150 mM
sodium chloride. Triplicate assays were measured at 420 nm and
30.degree. C. using a Powerwave HT microplate spectrophotometer
(Biotek Instruments) with 200 .mu.l of reagent and 50 .mu.l of
particle suspension per well.
[0215] Results
[0216] The binding of antibody to antigen was evaluated in
Experiment B (FIG. 12). This experiment indicates a positive
response which is interpreted as successful binding, of the primary
antibody (A-IL2-MA) to the surface of the particles, with
successive binding of the antigen (IL-2), secondary antibody
(B-A-IL2-MA) and streptavidin-peroxidase to the particles. This is
analogous to sandwich ELISA performed on a surface, such as the
well of a microtitre plate. Experiment C indicates that an antigen
may be bound to the surface of the particles and subsequently used
as a recognition element for antibody binding. These experiments,
when viewed in comparison with the various controls, indicates that
the particles can be used to bind antibody or antigen.
[0217] Experiment A is a control to indicate non-specific binding
of either the secondary antibody (B-A-IL2-MA) or
strepavidin-peroxidase to the particles with immobilised primary
antibody. The lower response when compared to B indicates that the
antigen (IL-2) enhances binding of the secondary antibody to the
particles. Experiment D and E are controls to indicate non-specific
binding of primary (A-IL2-MA) or secondary antibody (B-A-IL2-MA) to
the particle after albumin quenching. Experiment F contained
untreated cross-linked polyethyleneimine particles and was used as
an assay control.
[0218] This example indicates that the particles are a suitable
support immobilisation of antigens or antibodies. We further
demonstrate the immobilisation of antibody to antigen and
vice-versa through affinity interaction. This example thereby
indicates the feasibility of applications of the support for
affinity chromatography and diagnostics such as enzyme linked
immunosorbent assay.
EXAMPLE 18
Magnetite Incorporation into Particles of PEI Support Network or
Lattice of Polymeric Strands/Fibres
[0219] The inclusion of mediators and co-factors in the particles
has been demonstrated. Inclusion of magnetic particles in the
particles is demonstrated in this example.
[0220] Particle Preparation
[0221] Particles were prepared according to Example 17 above,
scaled linearly to 25 ml mineral oil per emulsion. Magnetite (250
mg) was incorporated into the 10% polyethyleneimine liquid solution
(pH 9.0) before emulsification. The cross-linked polyethyleneimine
particles were washed with 6 volumes 50 ml of deionised water and
recovered by centrifugation at 5000.times.g for 5 min from the
mineral oil and between each wash step. The particle pellet was
subsequently resuspended to a volume of 10 ml with deionised water
and functionalised for anion exchange by reaction with 500 .mu.l of
ethylenediamine for 30 minutes. The spheres were subsequently
washed with 3.times.50 ml aliquots of deionised water and recovered
by centrifugation as mentioned above. The final pellet was
resuspended to 10 ml in deionised water and used for albumin
binding experimentation below. The dry weight of the particles was
determined in triplicate by lyophilisation and weighing of 1 ml
aliquots of the 10 ml particle suspension.
[0222] Protein Binding and Quantification
[0223] Particles from the aliquots above were recovered by magnetic
separation on a magnetic stand (Magnetic Separation Stand--Promega;
Z5332) and the liquid removed. Particles were equilibrated with
2.times.2 ml washes of Tris-Cl buffer, pH 7.4 (50 mM). Bovine serum
albumin (BSA) was added to the particles to a final concentration
of 20 mg.ml.sup.-1. Ionic protein binding was allowed to take place
for 30 min at room temperature with end-over-end mixing. The
mixture was placed in a magnetic stand to retain the magnetised
resin on the side wall of the reaction tube and to allow removal of
the liquid from the sample. The resin was washed 5 times with 1 ml
50 mM Tris-Cl, pH 7.4 and recovered through the aforementioned
magnetic retainer. The ionically bound BSA was eluted from the
resin by the addition 2 volumes of 500 .mu.l of 1 M NaCl in 50 mM
Tris-Cl, pH 7.4.
[0224] Protein Quantification:
[0225] Protein quantification of the eluted fraction was performed
on a Qubit Fluorometer (Invitrogen) using the Quant-iT assay as per
the manufacturer's instructions (Table 10).
[0226] Results
TABLE-US-00010 TABLE 10 Binding of albumin to magnetite containing
particles (averages of triplicate data). Particle Dry Weight
Protein Binding Binding Efficiency (mg) (mg) (% m/m) 31.58 .+-.
0.90 1.07 .+-. 0.01 3.39
[0227] The results indicate that magnetite may be incorporated into
the particles and the magnetic properties of the particles may be
used for effective separation of these from a liquid suspension.
Further, these results indicate that the support may be used as an
efficient ion exchange resin. In the example provided here the
modification of the particle matrix with a positively charged
molecule such as an amine (ethylenediamine) allows the use of the
particles as an anion exchange resin. The use of negatively charged
molecules such as carboxyl containing molecules would allow use as
a cation exchange resin.
[0228] This example further provides an example of an alternative
recovery method by inclusion of magnetic particles into the
lattice, which would allow them to be attracted through application
of a magnetic field.
[0229] The particles of the invention thus include lattices of
polymeric strands or fibres cross-linked by means of a
cross-linking agent, and interstitial openings or spaces adjacent
and around the fibres. In other words, the invention provides a
fibrous interpenetrating network particle, preferably constructed
or made up from glutaraldehyde cross-linked polyethyleneimine. The
particle may be applied as an enzyme immobilisation matrix.
[0230] The particles are preferably produced using the emulsion
based technology or techniques of the second and third aspects of
the invention. The use of an emulsion based technology allows for
the benefits of size control such as the control of particle
surface area to volume ratio and defined size distribution, and
also advantageously permits a single step synthesis of the
particles. The particles offer a large surface area for
immobilisation and are applicable to, but not limited to,
biocatalysis of large and small substrates. This enzyme
immobilisation matrix exhibits a high immobilisation efficiency and
high enzyme activity maintenance after immobilisation.
[0231] The fibrous nature of the dendritic particles provides a
large internal surface area for enzyme binding. Also, the large
number of available attachment points per enzyme subunit provides
for the opportunity of significantly improved protein
stabilization, when compared to the backbone support material, eg
PEI, on its own. This, combined with the loose lattice or network,
allows for a high activity to weight ratio after biocatalyst
addition due to the large exposed surface area for immobilisation
and limited diffusional constraints for small and large substrates.
Furthermore, control of particle size allows for increased
reduction for diffusional constraints of the substrates should this
be a hindrance of this immovilisation matrix.
[0232] The process of preparation of the particles includes the
emulsification of the backbone support or lattice with or without
the cross-linking agent in the same phase. Preferably a bi-emulsion
system is used for the manufacture, as hereinbefore described. In
the case where the cross-linking agent is not included in the first
emulsion, it may be dissolved in the oil phase, or be incorporated
by mixing a second emulsion containing said cross-linking
agent.
[0233] The preferred process of manufacture is separate
emulsification of the backbone polymer (polyethyleneimine) in an
emulsion with an at least bifunctional cross-linking chemical
(glutaraldehyde) in a second emulsion. The spontaneous reaction
between the polymer and cross-linking agent results in a fibrous
lattice or network, containing in this case, excess aldehyde
functional groups, which are subsequently used to spontaneously
covalently link proteints to the lattice or support through
amine-aldehyde cross-reactivity. This aldehyde functionality can
further be extrapolated to link alternative compounds, or to impart
other properties such as hydrophobicity, thereby expanding its
application to binding a broader range of proteints, such as
hydrophobic proteints.
[0234] Protein immobilisation to matrices enhances the solvent,
thermal and pH stabilities of the enzymes. This stabilization may
further be enhanced by drying the support after protein
immobilisation to the dendritic support. It is believed that this
drying reduces the proximity of cross-reactive chemical functional
groups, thereby eliciting further spontaneous chemical coupling of
protein-backbone and backbone-backbone. Furthermore, additives may
be entrapped in the matrix during drying which could be of useful
for control of pore size or entrapment of functional molecules or
adjunct. High protein loadings are possible with the particles of
the invention i.e. relatively large quantities of protein can be
loaded in a small particle volume.
* * * * *