Note: Descriptions are shown in the official language in which they were submitted.
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SILICEOUS SUPPORT MEDIA FOR IMMOBILIZATION OF ENZYMES
Backaround of the Invention
This invention relates to a method for the
immobilization of enzymes onto siliceous support
materials and the use of such immobilized enzymes in
continuous enzyme-based processes such as the production
of pharmaceuticals. Immobilized enzymes prepared
according to the invention have potential applications,
however, in a wide range of synthetic and materials
treatment processes such as the production of specialty
commodity chemicals, waste water treatment and pulp and
paper processing.
The industrial use of enzymes is often limited by
their high cost and rapid inactivation. In particular,
the use of soluble enzymes necessitates regular -
replenishment of the enzymes, lost with the product at
the conclusion of a process. To improve their economic
feasibility in industrial processes, enzymes are
generally immobilized onto a matrix. Immobilization
facilitates re-use of the enzymes, and may affect the
selectivity and stability of the enzyme. Immobilization
research has focused upon means to enhance the transfer
of enzymes onto the support, and upon means to ensure
that the transferred enzymes remain active.
A number of different organic and inorganic support
matrices and enzyme immobilization techniques have been
tried with a view to achieving a high level of enzyme
uptake with a minimum of enzyme degradation or
inactivation. One such approach is the immobilization of
an enzyme by its physical entrapment within a gel,
microcapsule or similar polymeric structure. An example
is afforded by U.S. Patent No. 3,850,751 (Messing) which
teaches the adsorption of an enzyme to the inner surface
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of a porous, essentially non-siliceous ceramic body
having an average pore diameter at least as large as the
largest dimension of the enzyme.
While entrapment is a simple process and generally
affords a high uptake of enzyme without appreciable
inactivation during the immobilization process, the
enzyme once bound is surrounded by a matrix imposing a
mass transfer barrier. In the result, the observed
activity may be much lower than the intrinsic activity of
the enzyme. On the other hand, direct physical
adsorption of the enzyme to a substrate, without any
entrapment, is generally characterized by relatively weak
binding between enzyme and support, leading to
significant enzyme desorption.
Another approach is the direct covalent bonding of
an enzyme to a suitably chemically modified support
medium. While this leads to strong bonding between the
enzyme and the support, a labour-intensive and expensive
multi-step procedure is usually involved (including the
step of "activating" the support). Too, low enzyme
yields are not uncommon, owing to inactivation of the
enzyme by the harsh conditions employed in the
immobilization process.
A further, widely used approach to enzyme
immobilization might be referred to as the "covalent
cross-linking" process and is exemplified by U.S. Patents
Nos. 4,071,409 (Messing et al.); 4,258,133 (Mirabel et
al.); and 4,888,285 (Nishimura et al.). According to the
teachings of these patents a support medium is modified
or coated to present functionalities which can then be
linked by way of a cross-linking agent to free functional
groups on the enzyme. Thus, Nishimura et al. modifies a
silica gel or porous glass support surface by reaction
with an aminosilane derivative in an organic solvent.
The resulting aminated support is then linked to the
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enzyme in the presence of a polyfunctional cross-linking
agent (glutaraldehyde), a phenoxycarboxylic acid (tannic
acid) and, optionally, a basic polysaccharide (e. g.
chitosan). Nishimura et al. asserts that the tannic acid
and chitosan stabilize the enzyme, so as to reduce
inactivation by the cross-linking agent during the
immobilization process.
According to the aforementioned Mirabel patent,
which affords a second example of the covalent cross-
linking technique, an inorganic support having surface
hydroxyl groups (e. g. brick, alumina, aluminosilicates)
is modified with compounds containing an alcohol or
phenol group (e.g. monoethanolamine, amino-1 pentanol, p-
aminophenol) to generate an ester linkage on a "grafted"
support. The resulting grafted support is then coupled
to the enzyme, usually in the presence of a bifunctional
reagent.
Known enzyme immobilization proceedings employing
covalent cross-linking involve in many cases, time
consuming modifications to the substrate surface and/or
the use of expensive or hazardous reagents (either
solvents or the grafting agents themselves).
It is an object of the present invention to provide
a simple and efficient method for the immobilization of
enzymes on siliceous supports, requiring no prior
modification of the support material to avoid the
disadvantages attendant on such modification. Our
approach is based on the discovery that polyaldehyde
cross-linking agents may be used to immobilize enzymes
onto previously unmodified siliceous support materials,
to produce water-insoluble immobilized enzyme complexes
exhibiting high yields of enzyme activity and stability.
It is a further object of the invention to provide
immobilized enzymes which may usefully be applied to
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continuous enzymatic reactions with a variety of
industrial applications, including waste water treatment,
production of pharmaceuticals and other commodity
chemicals, and pulp and paper processing.
Summary of the Invention
With a view to achieving these objects and
overcoming the disadvantage of known enzyme
immobilization techniques, there is provided a method for
preparing a stable immobilized enzyme having a high yield
of enzyme activity, which comprises the steps of (i)
incubating a siliceous support material having surface
hydroxyl groups with a first aqueous solution containing
a polyaldehyde cross-linking agent, under conditions
suitable to prepare a support material modified by having
at least a portion of the cross-linking agent bound
thereto; (ii) removing the modified support material from
the solution containing cross-linking agents; (iii)
allowing the modified support material to come into
contact with an aqueous solution of the enzyme sought to
be immobilized, thereby to bind enzyme to free aldehyde
functions on the cross-linking agent bound to the support
material; and (iv) removing the immobilized enzyme from
the enzyme solution, for use in the desired enzymatic
reactions.
In a particular useful embodiment of the invention,
glutaraldehyde is used to cross-link tyrosinase to an
unmodified siliceous support material such as zeolites,
sodium aluminosilicate or silica gel, and the immobilized
tyrosinase is used in the production of L-DOPA.
General Description of the Invention
The present invention is not limited by any
particular theory as to the reasons for enhancement of
stability of enzymes immobilized on previously unmodified
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siliceous supports. However, this increased stability is
believed to be due to the fact that silicates, including
aluminosilicates, are excellent scavengers of superoxide
anion, a species believed to be responsible for the
inactivation of many enzymes [S. D. Kinrade et al., "The
Peroxysilicate Question: 29SI-NMR Evidence for the Role of
Silicates in Alkaline. Peroxide Brightening of Mechanical
Pulp", J. Wood Chem. Technol. 15(2), 203-222 (1995)]. The
protective mechanism is as follows:
Hydroxyl radicals (~OH) can react with the enzyme (E) to
yield an inactive oxidized enzyme, Murray R. Gray,
"Substrate Inactivation of Enzymes In Vitro and In Vivo",
Biotech. Adv. vol. 7, pp. 527-575, (1989),:
~OH + E -~ E-0 + H+ (1)
Hydroxyl radicals required for reaction (1) can be
generated from the reaction of hydrogen peroxide with
metals (usually Cu or Fe) within the enzyme (Gray, 1989; J.
Gierer et al., "Formation of Hydroxyl Radicals from
Hydrogen Peroxide and Their Effect on Bleaching of
Mechanical Pulp", J. Wood Chem. Technol. 13, 561 (1993):
Cu+ + H202 -a Cu2+ + OH + ~ OH ( 2
Hydrogen peroxide may be native to the reaction (as in the
production of gluconic acid from glucose mediated by
glucose oxidase), or it may be produced indirectly from
superoxide anion (OZ-~), via the following two-step process
(Gray, 1989)
02 ~ ~ 02 + O2 ( 3 )
02 + 2 H20 -a H202 + 20H ( 4 )
The superoxide anion required for reaction (3) is generated
by oxidation of metal, which may be part of the enzyme, or
an impurity in the reaction solution:
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Cu+ + OZ ~ Cu2+ + 02 ~ ( 5 )
Reaction (5) is inevitable. However, by trapping the
superoxide anion generated by reaction (5), the sequence
of steps leading to an inactive enzyme (i.e., reaction 3,
then 4, then 2, then 1) can be blocked, and enzyme
activity can be maintained. The ability of silicates to
selectively trap superoxide anion thus leads to
dramatically enhanced stability of enzymes when bound to
siliceous supports.
The supports covered in this invention include all
forms of siliceous materials, including, but not limited
to, silica gel, amorphous aluminosilicate, natural or
synthetic zeolites (including substituted forms), and
natural or synthetic sodium aluminosilicates.
The immobilization method involves the use of a
multifunctional cross-linking agent such as
glutaraldehyde or other polyaldehydes to couple the
enzyme to the support.
Any enzyme may be used in this invention, as long as
it is stable during the coupling process between the
enzyme and the support. Said enzyme includes, for
example, glucose oxidase, polyphenol oxidase, xylanase,
catalase, peroxidase, and cellulase.
The immobilized enzyme is prepared via a three-step
process. Initially, the support is incubated in an
aqueous-based solution containing the cross-linking agent
for several hours, at near neutral pH. The concentration
of the cross-linking agent is normally up to 3.0% (w/v),
but ideally, is approximately 0.5 to 1.5% (w/v). The
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resulting modified support is then isolated by
filtration, and dried.
The second step involves coupling the enzyme to the
modified support via the remaining unbound functional
groups on the polyfunctional cross-linking agent. In
this step, the modified support is incubated in an
enzyme-containing aqueous solution (hereafter referred to
as the immobilization solution) for several hours at a
temperature between 5°C and 50°C, depending upon the
thermal stability of the enzyme. The pH of the
immobilization solution is in the range where the enzyme
is not inactivated (normally between pH 5 and pH 9). The
incubation time is between 1 and 24 hours. For every
millilitre of the immobilization solution used, 2 to 5
milligrams of modified support is required.
In the final step, the resulting immobilized enzyme
is recovered from the immobilization solution by
filtration, then rinsed with water, dried, and stored.
This final step removes any loosely bound enzyme from the
support. The hydroxyl groups of the support are linked
to the amino groups of the protein via the polyfunctional
groups of the cross-linking agent, such that the
resulting bond between the enzyme and the support is very
strong.
According to this invention, as described above, an
enzyme can be efficiently and strongly immobilized on a
siliceous support. The immobilization procedure provides
a high yield of enzyme transferred to the support, and
the resulting immobilized enzyme is stable both during
storage and during operation. Thus, the immobilized
enzyme obtained via this process is well-suited to long-
term continuous enzymatic reactions, enhancing
productivity while reducing the quantity of enzyme
required to achieve a particular degree of conversion.
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A detailed description of the invention follows
below, with reference to examples for methods for the
immobilization of xylanase and tyrosinase onto
crystalline sodium aluminosilicate (zeolite A) and onto
calcium aluminosilicate.
Detailed Description of Preferred Embodiments
Example 1: Immobilization of xylanase on crystalline
sodium aluminosilicate (zeolite A)
A glutaraldehyde/buffer solution was prepared by
mixing sufficient glutaraldehyde in 0.050M phosphate
buffer to produce a pH 7 buffer solution containing 1%
(w/v) glutaraldehyde.
20mg of zeolite A was mixed with 50 mL of
glutaraldehyde/buffer solution, and incubated at 20°C for
four hours. The mixture was then washed with HPLC grade
water, and the remaining (modified) zeolite was recovered
by vacuum filtration. The recovered zeolite was dried
overnight at room temperature, and weighed.
An enzyme solution was prepared by dissolving
0.0032g (8000 units) of xylanase in 100mL of 0.050M
citrate buffer (pH 6.5). The resulting immobilization
solution had an activity of eight standard units per
millilitre of solution.
To immobilize the enzyme, the recovered modified
zeolite was incubated in 5mL of the enzyme solution at
20°C for 8 hours, under gentle stirring. The mixture was
then washed with 0.050M citrate buffer (pH 6.5), and the
resulting immobilized enzyme was recovered by vacuum
filtration. The recovered enzyme-zeolite powder was left
to dry overnight at room temperature.
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The fraction of the available xylanase transferred
onto the zeolite support was determined by comparing the
initial xylanase activity in the immobilization solution
with the activity after the immobilization process was
complete.
Example 2: Immobilization of tyrosinase on
crystalline sodium aluminosilicate or
calcium aluminosilicate
A glutaraldehyde/buffer solution was prepared by
mixing sufficient glutaraldehyde in 0.050M phosphate
buffer to produce a pH 7 buffer solution containing 1%
(w/v) glutaraldehyde.
mg of the zeolite support was mixed with 50 mL of
glutaraldehyde/buffer solution, and incubated at 20°C for
15 four hours. The mixture was then washed with HPLC grade
water, and the remaining (modified) zeolite was recovered
by vacuum filtration. The recovered zeolite was dried
overnight at room temperature, and weighed.
An enzyme solution was prepared by dissolving 0.16
20 mg of tyrosinase (545 Units) in 5.0 mL of 0.050M
citrate/HC1 buffer solution (pH 6.5). The resulting
solution had an activity of 108 standard units per
millilitre of solution.
To immobilize the enzyme, the recovered modified
zeolite was incubated in 5mL of the enzyme solution at
20°C for 24 hours, under gentle stirring. The mixture was
then washed with 0.050M citrate buffer (pH 6.5), and the
resulting immobilized enzyme was recovered by vacuum
filtration. The recovered enzyme-zeolite powder was left
to dry overnight at room temperature.
The fraction of the available tyrosinase transferred
onto the zeolite support was determined by comparing the
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initial tyrosinase activity in the immobilization
solution with the activity after the immobilization
process was complete.
Comparative Example 2: Immobilization of tyrosinase on
nylon 6,6
Membranes of nylon 6,6 were modified by soaking in
water for 24h, then in 3. OM HC1 for 10 minutes, and again
in water. The membrane was then incubated for 24h in a
50 mL solution of N,N'dicyclohexylcarbodiimide (1% w/v)
and 3,3',5,5'-tetramethylbenzidine (1% w/v) in methylene
chloride. The membrane was then washed with, in order,
methylene chloride, acetone, and water. The modified
nylon membrane was then immersed in a 50 mL solution of
glutaraldehyde (3% w/v) in O.lOM phosphate buffer (pH 8),
and soaked for 2.5h at 4°C, with gentle stirring. The
membrane was the washed with phosphate buffer, and
incubated for 24h at 20°C in a 30mL solution of O.lOM
phosphate buffer (pH 7) containing 12,300 units of
tyrosinase. The membrane with the immobilized tyrosinase
was washed in phosphate buffer, then stored in a saline
solution (9g/L) at 4°C until needed for use.
The fraction of the available tyrosinase transferred
onto the modified nylon support was determined by
comparing the initial tyrosinase activity in the
immobilization solution with the activity after the
immobilization process was complete.
Example 3: L-DOPA production using tyrosinase
immobilized on crystalline sodium
aluminosilicate (zeolite A)
The immobilized enzyme was circulated throughout a
batch reactor containing 2.5mM L-tyrosine and 2.5mM L-
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ascorbate in phosphate buffer (pH 6.5) at 22°C. The rate
of L-DOPA production was monitored over 7 hours.
Comparative Example 3: L-DOPA production using
tyrosinase immobilized on nylon
6, 6
The nylon membrane (containing the enzyme) was
immersed in a batch reactor containing 2.5mM L-tyrosine
and 2.5mM L-ascorbate in phosphate buffer (pH 6.5) at
22°C. The rate of L-DOPA production was monitored over 7
hours.
Example 4: Stability of tyrosinase immobilized on
crystalline sodium aluminosilicate and
calcium aluminosilicate (zeolites)
The stability of the enzyme under standard operating
conditions was determined by comparing the activity of
the enzyme at various stages during repeated-batch
production of L-DOPA from L-tyrosine. The half life is
defined as the time required for the initial activity to
be reduced by 505.
Results:
(A) Up to 40% of the available xylanase was
transferred to the zeolite support. Studies with the
immobilized enzyme demonstrated that the enzyme was
active and stable.
(B) In Table 1, the fraction of available
tyrosinase transferred to zeolite supports is compared
with the immobilization yield observed using other
supports and immobilization methods.
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Table 1:
Immobilization Yield of
T rosinase on Various
Su orts
Support; Immobilization MethodImmobilization
Enz me Units Re uired' Yield
Nylon 6 gels; chemically Covalent cross-linking33-76%
modified with benzidine, to chemically modified
isonitrile, nylon gels using
either
dimethylpropanediamine, glutaraldehyde,
or
diaminodiphenylmethane acetaldehyde, or
(88,400 units) nitrite
Collagen membranes; covalent cross-linking75%
chemically modified with to chemically modified
dimethyladipimidate or membranes using
ethyl
acetimidate (1496000 units)lutaraldeh de
Enzacryl AA'; activated covalent cross-linking79%
by
diazotation with nitric to arylamine groups
acid (5 on
units of proenzyme, the support via
subse uentl activated) t rosine residues
Magnetite; activated withcovalent cross-linking70-80%
3
aminopropyltriethoxysilaneusing glutaraldehyde
(3400 units)
Nylon 6,6 membranes; covalent cross-linking63-73%
chemically modified with to modified support
benzidine and carbodiimideusing glutaraldehyde
(12320 units)
Zeolites covalent cross-linking82-89%
(545 units) to unmodified support
usin of aldeh des
1. Enzyme Units Required (in parentheses) indicates the quantity of
enzyme in the solution at the beginning of the immobilization
3 0 process.
The rate of L-DOPA production using tyrosinase
immobilized on a variety of supports is illustrated in
Table 2. Clearly, the tyrosinase immobilized on zeolites
trademark for polyacrylic support material
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provides superior productivity, while reducing the enzyme
requirement dramatically.
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Table 2:
Production
of L-DOPA
Using Tyrosinase
Immob.
on Various
Supports
Support Enzyme Average Duration Maximum
Requirement.Production Production
Units' Rate, m L'' Rate, m L''
h'' h''
Nylon 6,6 12,300 33 7h 88
membranes
Enzacr I 5' 27.6 8h 70
AA
Zeolite 545 34 7h 92
A i
Zeolite 545 54 40 215 i
A,
repeated I
batch
' Enzyme
Requirement
refers
to the
amount
of enzyme
required
for immobilization;
the immobilized
enzyme,
when used
to produce
L-DOPA,
leads to
the specified
production
rates.
Unless
s ecified,
Mushroom
t rosinase
was used.
z Proenzyme
from frog
epiderra;is,
which is
subsequently
activated
using
trypsin
and sepharose*
C) L-DOPA Stability
As shown in Table 3, the observed stability of
tyrosinase immobilized on zeolites is much superior to
the stability of tyrosinase immobilized on other
supports, when used for the same biochemical
transformation.
*Trademark
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Table
3:
Half Life of Tyrosinase
on Various Supports
Support Half Life
Nylon 6 gels 3.5-1 1 h
Collagen membranes 1.5h
Enzacryl AA 6.5h
Nylon 6,6 membranes 46h
Zeolites No loss of activity
over 40h
