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DESCRIPTION OF THE INVENTION
This invention relates to an immobilized enzyme product suited for use in
an expanded or fluidized bed and to the process for production of such
enzyme product.
BACKGROUND OF THE INVENTION
Use of immobilized enzymes in an expanded or fluidized bed has certain
advantages over use of immobilized enzymes in a fixed bed. Reference is
made to U.S. Pat. Nos. 3,928,143 and 4,138,290 for detailed discussion of
how fluidized and expanded beds may be employed to carry out enzyme
catalyzed reactions. The most important advantages of operating in an
expanded or fluidized bed over the operating performance in a fixed bed is
that plugging is totally avoided in an expanded or fluidized bed and that
it is possible to use substrates with particulate materials therein in an
expanded or fluidized bed.
In spite of these advantages, almost all industrially enzyme catalyzed
continuous processes in which immobilized enzymes are used, to the date
hereof, are performed in fixed bed, inter alia because in an expanded or
fluidized bed the particles with the enzymatic activity are worn down
during operation as a result of collisions between the individual
particles.
Thus, a need exists for immobilized enzyme-containing particles suitable
for use in an expanded or fluidized bed which have a better abrasion
resistance than the previously known immobilized enzyme-containing
particles that were purported to be suitable for use in an expanded or
fluidized bed.
BRIEF DESCRIPTION OF THE INVENTION
The invention relates to a process for preparation of an immobilized enzyme
product, which process comprises first coating a particulate, dense
material with gelatine, treating the thus coated, dense material with
glutaraldehyde. Separately a pasty mixture of an enzyme product and an
aqueous polyethylene imine solution is prepared. The gelatine-coated and
glutaraldehyde-treated particulate material is admixed with the paste to
form a (still) pasty mixture. Then the thus formed pasty mixture is
treated with glutaraldehyde to generate a more solid mass; whereafter the
mass is granulated and dried.
Surprisingly coating of the particulate, dense material with gelatine and
the treatment of the thus coated, particulate, dense material with
glutaraldehyde in combination with the use of polyethylene imine on the
enzyme and the later treatment of the pasty mixture with glutaraldehyde,
provides an immobilized enzyme product wherein the enzymes adhere
extremely strongly to the particulate dense material and the ultimate
granules exhibit an exceptionally high abrasion resistance.
The invention also relates to the granular immobilized enzyme products of
the process, including notably so immobilized urease, inulinase and
lactase.
DETAILED DESCRIPTION OF THE INVENTION
In carrying out the process of this invention coating with gelatine is
usually commenced by soaking gelatine in water, then discarding excess
water, and thereafter melting the gelatine. The thus melted gelatine is
mixed with 100 parts by wt of the particulate, dense material, forming a
pasty mass of material. This pasty mass of material is treated with up to
about 1 part by wt of glutaraldehyde, whereby the gelatine is hardened and
the mass converts into a multiplicity of coated particles.
The paste of enzyme and polyethylene imine solution is formed by simply
mixing around 20 parts of the enzyme product and around 2 parts of a
polyethylene imine in solution. When this paste is then mixed with the
gelatine-coated, glutaraldehyde-hardened particulate, dense material,
another paste is formed, i.e., the mixture is still pasty, but after
treatment with about 2 parts of glutaraldehyde the paste falls apart into
granules. However, each granule contains therein two or more of the
particles of dense material. The individual particules of dense material
in each granule seem to be bonded together by the reaction product of the
polyethylene imine and glutaraldehyde.
The final immobilized enzyme (granular) product thus consists of a
multi-part granule in which the strength and abrasion resistance of the
granule derive from the dense particles which form the outer borderlines
of the granule, and in which the enzyme activity therein derive from the
cross-linked mass of enzyme-containing material between and around the
dense particles. The enzymatic activity is protected by the particulate
dense material acting as a shield for the (less abrasion resistant) mass
of enzyme-containing material.
As can be appreciated from the function of the dense material as a
weighting and abrasion resistant support substance, the chemistry of the
dense material forms no part of this invention, other than, of course, the
desirability of its being water insoluble and inert. Thus, the metallic
particles suggested in U.S. Pat. No. 3,928,143 may be employed. In
addition, mineral oxides are suitable for practice of this invention,
including notably, sand and titanium dioxide.
In a preferred embodiment of the invention the particulate dense material
is sand, a cheap relatively dense material.
In another preferred embodiment of the invention the particulate dense
material has a particle size of between 0.1 and 1 mm, a particle size
interval appropriate for most expanded bed or fluidized bed operations.
In another preferred embodiment of the invention the particulate, dense
material has a specific gravity exceeding 4 g/cm.sup.3. By using
particulate, dense material of more than 4 g/cm.sup.3 expanded or
fluidized bed enzymatic process can be controlled better, and liquids with
relatively high specific gravity can be processed without difficulty. The
particulate, dense material should be related to the specific gravity of
the solution or mixture treated. The higher the specific gravity of the
liquid to be processed in the expanded or fluidized bed, the higher should
be the specific gravity of the dense material. Titanium dioxide is dense,
highly inert and readily available; it is a preferred dense material for
practice of this invention.
In another preferred embodiment of the invention the weight of the gelatine
for the gelatine-coated particulate, dense material amounts to between
0.2% and 5% of the weight of the particulate, dense material, preferably
between 0.5% and 2% thereof. This proportion of gelatine is optimal in
relation to attainment of strong adhesion of the enzyme to the particles
of the dense material.
In another preferred embodiment of the invention the weight of the
glutaraldehyde with which the gelatine-coated particulate dense material
is treated amounts to between 2% and 25% of the weight of the gelatine,
preferably between 10% and 20% of the weight of the gelatine. This amount
of glutaraldehyde is optimal in relation to attainment of strong adhesion
of the enzyme to the particles of the particulate, dense material.
In another preferred embodiment of the invention the enzyme is urease.
Providing an immobilized urease product allows a urea-containing liquid
waste to be decomposed before being sent to the sewer, whereby
environmental pollution requirements can be met in a simple and cheap
manner.
Desirably the urease is prepared microbiologically from Bacillus pasteurii.
This urease is well suited for the urea decomposition purpose; it has a
good temperature stability and high activity at the pH levels of
urea-containing waste.
Another preferred enzyme is lactase. Immobilization of lactase according to
practice of this invention makes feasible a continuous conversion of the
lactose in whey to glucose and galactose by an expanded or fluidized bed
operation.
Another preferred enzyme is inulinase. Immobilization of inulinase
according to practice of this invention makes it possible to convert
inulin to fructose by an expanded or fluidized bed operation.
In another preferred embodiment of the invention the enzyme product
consists of dried enzyme containing whole bacterial cells. The cells
isolated from the enzyme fermentation broth in which the microorganism is
cultivated may be left with the concomitants and fillers which are normal
constituents of industrial enzyme products. Cultivation of the
microorganism and recovery of a crude cell product as the ultimate product
is a very cheap production method, as no treatment of the bacterial cells
is necessary.
In another preferred embodiment of the invention the enzyme product
consists of the crude whole bacterial cell product after subjecting the
cells to partial or total homogenization. The product still contains the
concomitants and fillers which are normal constituents of industrial
enzyme products. Such a product has the advantage that it may be employed
in substitution for a more expensive, more purified enzyme. Reference is
made to U.S. Pat. No. 3,980,521 for more detailed discussion of recovery
and homogenization methods applicable to intracellular enzymes such as
urease, inulinase, lactase as well as the glucose isomerase to which that
patent is directed.
Desirably the enzyme product, whether whole cells or homogenized cells, is
spray dried after recovery from the broth and homogenization. It is simple
and convenient to use a dry enzyme product for practice of this invention
and a spray dried product is well suited for the process according to the
invention.
In another preferred embodiment of the invention the weight of the
polyethylene imine amounts to between 2 and 50% of the enzyme substance,
i.e., cells and impurities, preferably between 5 and 15% of the enzyme
product weight. Less polyethylene imine than 2% of the enzyme substance
will not form an immobilized product with sufficient coherence, and more
polyethylene imine than 50% of the enzyme substance will not produce any
beneficial effect.
Desirably the polyethylene imine has a molecular weight between 300 and
100,000. These polyethylene imines are able to form an immobilized product
with the best coherence.
In another preferred embodiment of the invention the weight of the
glutaraldehyde, with which the pasty mixture of the imine treated enzyme
and coated dense particles is treated, is between 10% and 1000% of the
weight of the polyethylene imine, preferably between 50% and 200% of the
weight of the polyethylene imine. Less glutaraldehyde than 10% of the
weight of the polyethylene imine will not form a granule product with
sufficient abrasion resistance, and more glutaraldehyde than 1000% of the
weight of the polyethylene imine will not have any beneficial effect.
Drying of the granule product may be carried out as a fluid bed drying.
This is cheap, effective and fully satisfactory, particularly since the
abrasion resistance characteristic of the granules reduces drying losses.
The invention in its second aspect comprises a weighted immobilized enzyme
granular product suited for enzymatic treatment of a liquid substrate in
an expanded bed reactor. Such products are produced by the process of this
invention.
In a preferred embodiment of the invention the weighted immobilized enzyme
granular product is a weighted urease product suited for decomposition of
urea in aqueous phase in an expanded bed reactor. This urease product can
be produced as mentioned above and has utility for decomposition of urea
solutions before they are transported to the sewer. Other preferred
granular weighted enzyme products are lactase and inulinase.
For further understanding of this invention the following examples of
practice thereof are provided.
EXAMPLE 1
1.8 g of gelatine was soaked in demineralized water for 10-15 minutes, the
excess of water was discarded, and the gelatine was melted in a water
bath.
180 g of beach sand was heated to 60.degree. C. on a water bath.
Then the beach sand was poured into the gelatine with stirring in order to
form a homogenous paste. The gelatine-treated sand was divided in two
parts and treated with 0.2 and 0.1% glutaraldehyde, respectively (20% and
10% in relation to the amount of gelatine).
After the addition of glutaraldehyde, the mixture was agitated until the
mass gelled and a particulate material with particles of homogenous size
was formed, the time of gelation being around 1 minute.
2 g of spray dried urease with an activity of 6250 urease units/g and
produced from Bacillus pasteurii NCIB 8841 was mixed with (1) 2 ml 5% PEI
600 (polyethylene imine with molecular weight 40,000-60,000, purchased
from Dow Chemical Co.), (2) 2 ml 10% PEI 600 and (3) 3 ml 10% PEI 600 at
pH 7; when a paste was formed 10 g of the above described gelatine treated
beach sand was added, the mixing was then continued and thereafter
immobilization was completed by adding 1.25, 1.75 or 2.25 ml of 25%
glutaraldehyde with thorough mixing. The product was squeezed gently
whereby it fell apart into a granulate.
The urease activity unit is measured in the following way. The analysis is
performed in a pH-stat at pH 7.0 and 30.degree. C. with 3% (0.5 M) urea in
0.2 M phosphate as a substrate. The reaction can be represented by the
equation.
##STR1##
1 urease activity unit per definition splits 1 .mu.mol of urease per minute
under the above reaction conditions.
The table below indicates the conditions of immobilization and the activity
of the formed granular products.
TABLE
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Immobilizing
Sand Spray Yield of
Activity, batch pH 7
Used dried 25% glutar-
dry 300-700 .mu.
700-1000 .mu.
Amount
gelatine
glutaralde-
urease,
10% PEI
aldehyde
product, activity activity
g % hyde %
g 600, ml
ml g units/g
yield %
units/g
yield
__________________________________________________________________________
%
1 10 1 0.1 2 2 1.75 11.5 287 26 336 31
2 10 1 0.2 2 2 1.75 11.5 276 25 326 30
3 10 1 0.1 2 1 + 1m1H.sub.2 O
1.75 10.9 96 8.4 209 18
4 10 1 0.1 2 3 1.75 12.1 280 27 376 36
5 10 1 0.2 2 2 1.25 11.5 315 29 361 33
6 10 1 0.2 2 2 2.25 11.7 169 16 243 23
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The physical strength of all the immobilized enzyme granular products were
evaluated as good, no physical decomposition could be observed after 60
hours on a shaking table. A rougher manual treatment of the products
indicated that products Nos. 3 and 5 were somewhat weaker than the other
products. Product No. 6 is somewhat stronger than the others.
The products were tested as the active mass in a fluidized bed treatment of
an aqueous solution containing 1% urea as a substrate. The urea was
satisfactorily decomposed.
EXAMPLE 2
2 g lyophilized lactase with an activity of 973 lactase units/g and
produced from Bacillus sp: NRRL B-11, 229 was mixed with 3 ml 10% PEI 15 T
(polyethylene imine with molecular weight 700, purchased from Taihei
Sangyo Kaisha Ltd.) at pH 7.0. When a paste was formed 10 g of gelatine
treated sand, prepared as described in Example 1, was added. Mixing was
continued, and the immobilization was completed by adding 1.75 ml of 25%
glutaraldehyde. The product was squeezed gently whereby it fell apart as a
granulate. After drying the activity was 25.5 lactase units/g and the
activity yield was 16.3%.
Lactase activity is measured in the following way. The analysis is
performed in a shaking water bath at 60.degree. C. and pH 6.5 with 10%
lactose in milk buffer as a substrate. The reaction is terminated by
boiling for 10 minutes. The amount of glucose liberated in the supernatant
is determined by means of the glucose oxidase peroxidase method (Tech.
Bull. No. 510, Sigma Chemical Co.). 1 lactase activity unit per definition
liberates 1 .mu.mol of glucose per minute under the reaction conditions
mentioned above.
No disintegration of the particles of immobilized lactase could be
ascertained by visual inspection after continuous shaking in 20% lactose
solution for one week.
EXAMPLE 3
2 g of spray dried inulinase with an activity of 105 inulinase units/g
produced from Kluyveromyces fragilis (NRRL Y 1156), was mixed with 3 ml of
neutralized 10% polyethylene-imine solution (PEI 15 T, Taihei Sangyo
Kaisha Co., Ltd.). When a paste was formed 10 g of gelatine and
glutaraldehyde treated sand, prepared as in Example 1 was added. The
mixing process was continued, and the immobilization reaction completed by
adding 1.75 ml of 25% solution of glutaraldehyde and mixing thoroughly.
The product was squeezed gently, whereby it fell apart as a granulate, and
air dried.
The activity of the product was 13.5 inulinase units/g corresponding to an
activity yield of 77%.
The activity of the soluble enzyme was measured as .mu.mol reducing sugar
formed per minute at pH 4.7 and 50.degree. C. with a 2.5% solution of
inulin as a substrate.
The activity of the immobilized enzyme was measured as .mu.mol reducing
sugar formed per minute at pH 4.7 and 50.degree. C. from a 5% solution of
inulin as a substrate in a shaking bath.
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