U.S. patent number RE33,441 [Application Number 07/512,823] was granted by the patent office on 1990-11-13 for immobilization of biologically active material with glutaraldehyde and polyazaetidine.
This patent grant is currently assigned to Novo Industri A/S. Invention is credited to Henrik Mollgaard, Mogens Wumpelmann.
United States Patent |
RE33,441 |
Wumpelmann , et al. |
November 13, 1990 |
Immobilization of biologically active material with glutaraldehyde
and polyazaetidine
Abstract
Immobilized biologically active material in particle form is
prepared by cross-linking with glutaraldehyde and polyazetidine. An
aqueous dispersion or solution of biologically active material is
partially cross-linked with glutaraldehyde, a wet pasty mass is
recovered by dewatering and the mass is sub-divided into discrete
particles. A polyazetidine prepolymer is added before, at the
beginning or subsequent to partially cross-linking but prior to
subdividing the pasty mass into particles, and the prepolymer is
allowed to cross-link.
Inventors: |
Wumpelmann; Mogens (Herlev,
DK), Mollgaard; Henrik (Lyngby, DK) |
Assignee: |
Novo Industri A/S (Bagsvaerd,
DK)
|
Family
ID: |
26066670 |
Appl.
No.: |
07/512,823 |
Filed: |
April 23, 1990 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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874141 |
Jun 13, 1986 |
|
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Reissue of: |
213773 |
Jun 30, 1988 |
04892825 |
Jan 9, 1990 |
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Foreign Application Priority Data
|
|
|
|
|
Jun 14, 1985 [DK] |
|
|
2692/85 |
Jul 1, 1987 [DK] |
|
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3369/87 |
|
Current U.S.
Class: |
435/180; 435/177;
435/182 |
Current CPC
Class: |
C12N
9/88 (20130101); C12N 11/08 (20130101); C12P
13/227 (20130101); C12P 19/24 (20130101); G01N
33/543 (20130101); G01N 33/54353 (20130101) |
Current International
Class: |
C12N
9/88 (20060101); C12P 19/24 (20060101); C12P
19/00 (20060101); C12P 13/00 (20060101); C12P
13/22 (20060101); C12N 11/08 (20060101); C12N
11/00 (20060101); G01N 33/543 (20060101); C12N
011/08 (); C12N 011/02 (); C12N 011/04 () |
Field of
Search: |
;435/94,174,177,180,182 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Naff; David M.
Attorney, Agent or Firm: Fidelman; Morris Wolffe; Franklin
D.
Parent Case Text
This application is a continuation in part of copending application
Ser. No. 874,141 filed June 13, 1986, now abandoned.
Claims
We claim:
1. A process for forming polyazetidine cross-linked immobilized
biologically active materials in particle form which consists
essentially of:
partially cross-linking an aqueous dispersion or solution of a
biologically active material with glutaraldehyde, to produce a two
phase system of flocculated partially cross-linked solids
containing said biologically active material and water, dewatering
said two phase system and recovering the solids phase as a wet
pasty mass,
sub-dividing said pasty mass into discrete particles each of which
is essentially homogeneous adding a polyazetidine prepolymer
before, or at the beginning of partially cross-linking or
subsequent thereto but prior to subdividing said pasty mass into
particles, and
thereafter curing said particles whereby said polyazetidine
prepolymer undergoes cross-linking.
2. The process of claim 1 wherein the glutaraldehyde content in
said dispersion or solution comprises 5-40% by weight of the
biological material.
3. The process of claim 1 wherein said polyazetidine prepolymer is
added to said aqueous dispersion prior to instituting said partial
cross-linking.
4. The process of claim 3 wherein the polyazetidine is 0.5-5% w/w
of total dry matter.
5. The process of claim 1 wherein said polyazetidine prepolymer is
added after said partial cross-linking but prior to dewatering.
6. The process of claim 5 wherein the polyazetidine is 0.5-5% w/w
of total dry matter.
7. The process of claim 1 wherein said pasty mass is mixed with
aqueous prepolymer.
8. The process of claim 7 wherein the polyazetidine is 10-20% w/w
dry matter basis of the biologically active material.
9. The process of claim 1 wherein water content of said particles
is reduced to below about 25% by weight during the curing
thereof.
10. The process of claim 1 wherein said biologically active
material is an enzyme.
11. The process of claim 10 wherein said enzyme comprises
enzymatically active whole, fragmented or homogenized microorganism
cells.
12. The process of claim 11 wherein the microorganism is a strain
from a species selected from the genus group consisting of
Streptomyces, Bacillus, Actinoplanes, Fusarium, Rhodococcus,
Pseudomonas and Brevibacterium.
13. The process of claim 11 wherein said enzyme comprises cell
bound glucose isomerase.
14. The process of claim 10 wherein said enzyme is selected from
the group consisting of glucose isomerase, penicillin acylase and
nitrilase.
15. The process of claim 10 wherein said enzyme is in solution when
partial cross-linking begins.
16. The process of claim 1 wherein a flocculating agent is added to
said dispersion or solution before, during or after beginning said
partial cross-linking, but prior to said dewatering.
17. The process of claim 1 wherein partially cross-linking said
biologically active material is carried out in the presence of one
or more auxiliary cross-linking agents selected from the group
consisting of polyethylene imine, gelatine, albumin and
carboxymethyl cellulose.
18. An immobilized enzyme product made according to the process of
claim 10.
Description
This invention relates to a method for immobilizing biological
materials by cross-linking with polyazetidine and, in a preferred
mode, to a method for converting cell bound enzymes into cell mass
enzyme particles.
BACKGROUND OF THE INVENTION
Immobilized enzyme products, especially immobilized enzyme products
intended for use in a column has been a rapidly growing field as of
the date hereof. Research efforts have been directed towards
producing immobilized enzyme products of ever lower price, better
physical strength, higher unit activity and of particle size and
shapes giving rise to a minimum pressure drop during column
operation as well as a high particle strength against abrasion. As
of the date hereof, workers in the art have made available a
substantial number of reasonably satisfactory methods to immobilize
enzymes.
This invention is directed, in a preferred mode thereof, to the
conversion of cell bound microbial enzymes into particle form
immobilized enzymes made from the cell mass of the microorganism.
The discussion of enzyme immobilization hereinafter provided is
largely within a context of this type of immobilized enzyme
product.
On the whole, as the art has advanced, product and method
deficiencies, such as non-optimum particle size distribution, lack
of control over particle shape and the cost factor of relatively
low product yield, long considered to be unimportant defects in the
immobilization process become major defects, which must be
obviated. For example, glutaraldehyde has been employed in
commercial practice for cross-linking cell bound enzymes according
to the teachings of Amotz U.S. Pat. No. 3,980,521. Yet,
glutaraldehyde is not an ideal cross-linking agent, for cell bound
enzymes at least, reacting only with --NH.sub.2 and --SH groups.
The cells of many microorganism species react poorly with
glutaraldehyde.
A polyazetidine prepolymer may be employed advantageously for
cross-linking purposes, such being suggested by Wood et al. U.S.
Pat. No. 4,436,813 and by "A Novel Method of Immobilization and Its
Use in Aspartic Acid Production," Wood et al., Bio/Technology,
December 1984, pp. 1081-1084. This Patent and Paper ar incorporated
by reference herein. The polyazetidine prepolymer cross-linking
system is more widely applicable to immobilization of cell bound
enzymes than is glutaraldehyde because cross-linking reactions take
place between the polyazetidine prepolymer and --COOH and --OH
groups as well as --NH.sub.2 and --SH groups.
The instances to which practice of this invention is directed in
particular are those when the desired enzyme form constitutes
particles made from the microorganism cells, and cellular
substances, and cross-linking reagent(s), and optionally, auxiliary
cross-linking agents, e.g., proteins and/or agglomerating agents,
e.g., polyelectrolytes, and/or finely-divided filler materials. The
particles are essentially homogeneous. Such enzyme product form are
variously termed herein as cell mass particles and/or cell mass
particulate form. It is noted parenthetically, that the process of
above-referenced Wood et al. Patent and Paper is directed
principally to immobilizing the enzymatically active microorganism
cells and cellular substances on carrier particles, and that the
inventors hereof strongly prefer the cell mass particle form over a
carrier base particle form of immobilized enzyme product the latter
not being essentially homogeneous particles.
Efforts by the inventors hereof to employ polyazetidine prepolymer
cross-linking to generate cell mass particulate form enzyme
products evidenced existence of material deficiencies to the
process taught by the prior art. The reaction mixture constitutes
an aqueous dispersion of the polyazetidine prepolymer in solution
and individual microorganism cells along with any cellular
substances present, or other biological material necessitating
conduct of the curing reaction en mass. By crushing the reaction
product and sieving, a desired particle size fraction may be
recovered, but overall the yield of the wanted particle size
fraction is usually low, and also, the shape of the individual
particles is not controlled. Thus, cross-linking a non-particulate
cell mass composition gives rise to immobilized enzyme product
wherein particle shape and size is not controlled.
Although the foregoing discussion of the background of the
invention and the description of the invention which now follows is
couched in terms of cell bound enzymes and a cell mass particulate
product, such is done to facilitate understanding of the invention
and to describe preferred practice of the invention in fulsome
fashion. It is emphasized that practice of the invention is
applicable to biological materials more generally, including
notably, homogenized cell sludge, enzymes in solution (e.g., extra
cellular enzymes), co-enzymes and anti-bodies.
OBJECT OF THE INVENTION
The object of the invention is the provision of a method adapted to
produce a particulate form of biological material in high yield, of
particles with high physical strength, whereby also the shape and
size of the particles can be controlled.
Enzymes, soluble and cell bound alike, are preferred biological
materials and cell mass immobilized enzyme products are
particularly preferred products of the invention.
Enzymes of particular interest to the inventors hereof are glucose
isomerase, penicillin acylase and nitrilase.
STATEMENT OF THE INVENTION
In brief, the method of this invention comprises partially
cross-linking enzymatically active microorganism cells or some
other biological material in aqueous solution or suspension through
reaction with glutaraldehyde, followed by dewatering of the
resulting flocculated solids, resulting in a pasty consistency mass
of a (consistency) and coherency suitable for particle shaping.
Then, (wet) particles of desired shape and size are generated from
the mixture followed by drying. The polyazetidine prepolymer
solution is incorporated before or after dewatering, more
preferably the former. Curing of the polyazetidine prepolymer which
occurs during the drying step, converts the pasty mass particles
into cured particles of high physical strength.
Immobilized enzyme products prepared according to the invention
employed in packed bed exhibit a very small pressure drop (e.g., a
pressure drop which is only around 50% of a comparable prior art
product) and a high physical strength and resistance against
abrasion, and moreover, the immobilized enzyme products are
relatively inexpensive to make due to the high yield of usable
particles. Also, it has been found that preferred embodiment
immobilized enzyme product prepared according to the invention
exhibit a high volumetric activity.
DETAILED DESCRIPTION OF THE INVENTION
The cross-linking reactions between enzymatically active cellular
substances and a polyazetidine prepolymer to crosslink the enzyme
into a composition capable of repeated use is taught by the
above-referenced U.S. Pat. No. 4,436,813 and Paper of Wood et al.,
(see also G. J. Carlton et al, Biotechnology, vol. 4, pp. 317-320
(1986)) and, therefore, need not be discussed in detail here.
The polyazetidine polymer used for the practice of this invention
may be any water-soluble polymer with a substantial content of
reactive azetidine rings, such as those prepared by reacting a
polyamide with epichlorohydrin according to German patent
publication No. DT-AS-1,177,8254. Examples of commercially
available products are Polycup 2002, Kymene 557H and Reten 304
(these are all trade marks of Hercules Inc., U.S.A.).
A representative polyazetidine prepolymer in aqueous solution, such
as is depicted below, e.g., Polycup 172 (Hercules, Inc.). ##STR1##
is cross-linked by heat, H.sub.2 O removal or pH adjustment to an
alkaline pH value. Some of the curing reactions are reaction of the
prepolymer with available --NH.sub.2, --OH, --SH, --COOH groups of
cellular substances on or from the enzymatically active
microorganism cell.
Unless the reaction mass is sub-divided into individual particles
prior to curing a cross-linked cell mass product will be a single
block of material, i.e., a coherent mass that must then be
sub-divided into the desired particle from enzyme product. As has
already been pointed out, such a conversion results in relatively
low yield of the desired size range particles and, in addition, the
shape of the individual particles is not controlled at all.
Forming the cell mass into discrete particles prior to
cross-linking of the polyazetidine prepolymer, would, of course, be
desirable. Unfortunately, the fluid aqueous mixture of
polyazetidine prepolymer and cell mass, e.g., cell sludge, is not
well adapted to being formed into coherent particles.
As has been pointed out, the improvement of this invention is
directed to conversion of a biological material such as
microorganism cells into a wet past mass followed by subsequent
subdivision of the mass into discrete particles. The present
process can be considered to be a pretreatment procedure that is
carried out prior to curing the polyazetidine. Subsequently, as the
particles dry, the polyazetidine cross-linking reactions take place
and the particles assume their ultimate coherency, hardness etc.
Conversion, e.g., of the microorganism cells, into a relatively
coherent pasty mass is accomplished by carrying out partial
cross-linking of the biological material in aqueous dispersion by
reaction with glutaraldehyde, followed by dewatering.
Biological materials that can be immobilized by the method of this
invention include enzymes soluble or cell bound alike,
microorganism cell (intact or disrupted cells, viable or
non-viable), antibodies and coenzymes. The biological material to
be immobilized should be in an aqueous solution or dispersion and
may have been purified as desired by conventional techniques. The
degree of purification is not critical to the practice of the
invention.
It has been found that the quantity of water present in the
(pretreatment) partial cross-linking reaction mixture of this
invention is not critical. Excess water will be removed from the
flocculated partially cross-linked solid phase substance in the
reaction product mixture during dewatering without any serious loss
of active material. Thus, water may be added to the solution or
dispersion of the biological material to obtain a convenient
consistency for the partial cross-linking reaction, then excess
water is removed by dewatering. The term dewatering is employed
herein within a context of physical removal of water, such as, for
example, decanting, filtration, centrifugation and the like.
A convenient preferred starting material for practice of this
invention is the enzymatically active cell sludge recovered from a
fermenter through filtering or centrifuging the culture broth. The
cell sludge may be used as such or first be homogenized. Since the
fermenter may not be in close proximity to the immobilization
facility, it is noted that the optionally homogenized cell sludge
may be stored in frozen state. Indeed, freezing, then thawing of
the cell sludge maybe advantageous rather than be an activity
losing detriment in the overall process sequence.
The detailed chemistry of the reactions involved in partial
cross-linking through reaction of the microorganism cells and
cellular substances with glutaraldehyde are not known to the
inventors hereof. Indeed, insofar as the inventors hereof are
aware, the chemistry of cross-linking with glutaraldehyde is not
fully elucidated. Reference is made to Douglas J. Ford, 2. Reaction
of Glutaraldehyde with Proteins, University of Cincinnati; and
Hardy, The Nature of the Cross-linking of Proteins by
Glutaraldehyde, Part I, Journal of the Chemical Society, Perkin
Transactions, Vol. 1, Pg. 958, 1976. However, the art is familiar
with practical results from reacting glutaraldehyde with
microorganism cells.
Glutaraldehyde has been suggested to the enzyme art for
cross-linking to generate cell mass particle form enzymes, as
witness the teachings in the aforementioned U.S. Pat. No.
3,980,521. Glutaraldehyde has also been suggested for stabilizing
cell bound enzymes on the microorganism cells as witness the
teachings in U.S. Pat. No. 3,779,869. The usage of glutaraldehyde
in practice of this invention is related to the teachings of both
above-referenced patents, yet is quite different therefrom.
Although occurrence of cross-linking reactions is desired, in large
measure the exact extent to which reaction with glutaraldehyde
prevents loss of enzyme from individual microorganism cells (see
U.S. Pat. No. 3,779,869), or can convert cells and cell fragments
into a coherent covalently linked matrix (see U.S. Pat. No.
3,980,521), is not material for practice of this invention.
The purposes of treatment with glutaraldehyde in practice of this
invention is generation of a reaction product mixture that contain
flocculated solids which then can be dewatered and so doing
generates a pasty mass adapted for subdivision into discrete
particles. The pasty mass is capable of admixture with an aqueous
polyazetidine prepolymer and then be a mixture of a coherent
consistency from which particles may be formed. Generation of the
particle forming capability is the objective sought. It should be
appreciated that the term "pasty mass" as employed herein is both
descriptive of the partially cross-linked (still wet) dewatered
product and connotes existence of cohesiveness and a particle
forming capability.
Incident to treatment of a cell sludge or other biologic material
with glutaraldehyde, auxiliary cross-linking agents containing
--NH.sub.2 groups may be added to the reaction mixture e.g.,
polyethylene imine, chitosan, albumine, gelatine. Also,
flocculating agents may be added. Further, it may be advantageous
to treat the cell sludge with a metal ion complexing agent such as
EDTA. The exemplary details hereinafter provided about preferred
embodiments of this invention are not likely to be applicable in
all their detail to other cell bound enzymes. Cut and try tests
within the preferred glutaraldehyde ranges are suggested to
ascertain whether inclusion of auxiliary cross-linking agents
and/or inclusion of polymeric flocculating agents is advisable, or,
perhaps, is necessary to achieve a workable consistency and the
proper water content in the dewatering partially cross-linked cell
mass.
It may be noted also that finely divided filler materials and/or
enzyme stabilizers (e.g., metal ions) when presence of such is
desired in the ultimate cell mass immobilized enzyme product may
best be incorporated into the cell mass incident to the partial
cross-linking with glutaraldehyde.
It has been found that the quantity of water in the cell sludge and
that added with the glutaraldehyde and with any optional agent in
the partial cross-linking reaction mixture such as flocculating
agent, auxiliary cross-linking agent, enzyme stabilizer ions, etc.
is not a critical factor. All water in excess will be filtered or
centrifuged off from the partially cross-linked pasty mass.
However, the relative proportions of cell sludge dry matter and
glutaraldehyde are important.
According to one preferred mode of the invention, the polyazetidine
prepolymer solution is incorporated into the pasty mass.
In such preferred embodiment of the method according to the
invention, the amount of glutaraldehyde is between 5% and 40% w/w
in regard to cell sludge dry matter, preferably between 10% and 20%
w/w. If the amount of glutaraldehyde is below 5% w/w, the
filterability of the partially cross-linked cell mass may be
inferior, and if the amount of glutaraldehyde is above 40% w/w, the
enzyme yield recovery in the immobilized enzyme product maybe
unsatisfactory.
The dewatered partially cross-linked cell mass has a water content
of 70-90% w/w, preferably 80-85% w/w. If the water content is less
than 70% w/w, the pressure drop characteristics of the particulate
immobilized enzyme product may not be satisfactory, and if the
water content is above 90% w/w, performance of the particle shaping
step may be unsatisfactory. Thus, the relatively narrow 70%-90%
water content in the dewatered cell mass is relatively critical in
practice of the invention.
The practitioner of this invention will soon recognize the most
workable consistency area. The particle forming capability is
somewhat poor at both ends of the 70-90% water content range. It is
noted that water content for the most workable consistency will
vary enzyme to enzyme.
The water content range provided above for the dewatered partially
cross-linked cell mass takes into account that the polyazetidine
prepolymer added will be as an aqueous solution of 10-15% solids.
The preferred range of 80-85% water virtually assumes about a 12%
polyazetidine prepolymer solution and the more preferred
polyazetidine prepolymer content in regard to the dry weight of the
cells will be employed.
The polyazetidine prepolymer is added in an amount of between 5%
and 30% w/w (dry matter basis), more preferably 10-20% w/w (dry
matter basis) in regard to the cell sludge. If the amount is below
55% w/w, the pressure drop reduction improvement obtained in the
particulate product is unsatisfactorily low, and if the amount is
above 30% w/w the enzyme yield in the immobilized enzyme
particulate product is unsatisfactorily low. Thus, should a
particular cell-bound enzyme require less than 10% or more than 20%
of the polyazetedine prepolymer for yield or product stability
reasons, arbitrary adoption of the above given most preferred
80-85% water content range is not advised.
It is to be understood that the foregoing discussion of proportions
is within a context of incorporating the polyazetidine prepolymer
solution into a partially cross-linked pasty mass. Such has not
been found to be necessary, and a more preferred mode is to add the
polyazetidine prior to dewatering, at all of which times the
aqueous solution or dispersion of the biological material is still
in fluid state.
Although, according to this mode of the invention, the
polyazetidine prepolymer solution is added prior to dewatering, it
has been found that very little of the polyazetidine is lost during
dewatering. Apparently polyazetidine binds to the biological
material, e.g., to enzymatically active microorganism cells, by
acting as a cationic flocculent, or the polyazetidine can be made
to bind to the material through selection of appropriate
flocculent(s). In any event, the need for a flocculent as well as a
suitable type and quantity of flocculent is readily determined by
those skilled in the art for whatever particular enzyme or other
biological material is being cross-linked. Thus, optionally but
desirably, a flocculent is also added while the fluid state
exists.
Aside from a requirement for a lesser amount of polyazetidine for
optimum product properties when polyazetedine prepolymer is added
before the dewatering step, the process remains the same. The
processing conditions for partial cross-linking with glutaraldehyde
already described apply, as for example temperature
0.degree.-60.degree. C., pH 5-9, buffer as needed, cross-linking
for 5-60 minutes, auxiliary crosslinking agents when advisable or
when desired (to dilute the enzyme for instance). The auxiliary
cross-linking agents may be present in quantities of up to 100% of
the biological material by weight dry matter, preferably much less,
depending on the auxiliary agent, notably 20% or less for
polyethylene imine, 50% or less for albumin or gelatine and 10% or
less for carboxymethyl cellulose. Inclusion of flocculant as
appropriate.
The great advantage of incorporating the polyazetidine prepolymer
into the pre-dewatered and still fluid mixture is reduction in the
proportion of polyazatidine required and for better control over
the properties of the filter cake and of immobilized enzyme
product.
Thus, a relatively high proportion of polyazetidine in the product
generally yields physically strong particles, whereas a lower
proportion yields particles with lower diffusion restriction and
therefore with higher activity. The most suitable amount of
polyazetidine for this mode of the invention will usually be in the
range about 0.1-about 10% by weight of total dry matter in the
solution or dispersion, typically about 0.5-5%, e.g., preferably
about 0.5 to 3%.
In both modes of the invention discussed above, the partially
cross-linked dewatered mixture is a pasty mass that exhibits a
coherency and a consistency suited to particle shaping. A preferred
particle shaping technique is to extrude the dewatered mixture,
then partially dry (drying cures the polyazetidine prepolymer),
thereafter spheronize, followed by supplementary drying, the last
being optional. The marumerizing practice of Great Britain No.
1,362,265 may be followed.
Partially cross-linking the suspended biological material particles
changes their physical character, makes them sticky so that upon
dewatering of the two phase aqueous mixture the solids fuse into a
relatively coherent pasty mass. Comparably. partially cross-linking
a dissolved biological material, such as for example enzyme in
solution, causes precipitation of the biological material. Upon
dewatering of the two phase aqueous mixture, the precipitate
converts into a relatively coherent pasty mass. Thus, in each
event, dewatering, e.g., by filtration, removes much of the water
phase generating the desired pasty mass. Prior to dewatering the
precipitated solids or the partially cross-linked particles which
even collect in bunches, i.e., become flocculated. Inclusion of
flocculant is to improve flocculation of the partially cross-linked
solid phase. Flocculants, when present and polyazetedine
prepolymer, when present, are mostly in the pasty mass.
To repeat the pasty mass made according to practice of this
invention exhibits a significant level of coherency, allowing the
mass to be subdivided, e.g., extruded, and the extrudate ribbons do
not fuse together before curing of the polyazetidine prepolymer has
been effected.
Additional water is removed during the curing step, e.g., by
evaporation as the cross-linking reaction proceeds, so that a
physically strong relatively dry product is obtained. Preferred
techniques are air drying or fluidized-bed drying, generally at
15.degree.-80.degree. C. In case of very sensitive biological
materials, low temperature drying or freeze-drying may be
needed.
The pasty mass is a homogeneous mixture. Any particulate matter
therein, e.g., finely, divided filler particles, is uniformly
dispersed. The composition of any one portion of the pasty mass
will be no different from any other portion. Such uniformity
carries through to the product particles. Each will have the same
composition. Also, the center of each particle is of the same
composition as the particle periphery. In this sense, the product
particles are essentially homogeneous.
In all preferred embodiments of the method according to the
invention, the particle form immobilized enzyme is dried to a water
content of about 15-25% w/w. With an ultimate water content of
above around 25% the microbial stability of the product is
unsatisfactory. Furthermore, as previously indicated, with water
content above around 25% the cross-linking with the polyazetidine
prepolymer may not have taken place or may not be complete; the
particles may tend to aggregate over time in storage. Drying to a
water content of below about 15% may cause loss in enzyme
activity.
UTILITY
The process of this invention is widely applicable to
immobilization of biological materials, such as enzymes,
particularly, glucose isomerase, penicillin acylase and nitrilase,
cell mass, coenzymes and antibodies (monoclonal or polyclonal).
Enzymes which may be immobilized can be in the form of
enzymatically active cells, or partly or fully homogenized cell
paste, or as a largely cell-free enzyme solution. Some instances
where the method of the invention is particularly advantageous
are:
glucose isomerases from Streotomcyes sp.. e.g. from the following
species:
______________________________________ S. murinus (EP 0 194 760) S.
flavovirens S. achromogenus S. echinatus S. wedmorensis S. albus
(U.S. Pat. No. 3,616,221) S. olivochromogenes S. venexueloe (U.S.
Pat. No. 3,622,463) S. griseoflavus (U.S. Pat No. 4,137,126) S.
galbus S. gracilis S. margensis S. niveus S. plantensis (Hungarian
patent 12,415) S. violaceoniger (German patent 2,417,642) S.
acidodurans (U.S. Pat. No. 4,399,222) S. phaeochromogenes S.
fradiae S. roseochromogenes S. olivoceus S. californicus S.
vanoceus S. virginioe (Japanese patent publication 69-28,473) S.
olivaceus (U.S. Pat. No. 3,625,828)
______________________________________
glucose isomerase from Bacillus coagulans. (see U.S. Pat. No.
3,979,261).
glucose isomerase from Actinoplanes sp., especially A.
missouriensis.
glucoamylase from Asperoillus sp., especially from black Aspergilli
and more especially from A. niger (see U.S. Pat. No. 3,677,902), or
from Rhizoous sp.. especially Rh. delemar or Rh. niveus.
penicillin-V acylse from Fusarium sp., especially F. uioides, F.
aroiilaceum, F. avenaceum, F. bulbioenum, F. coeruleum, F.
eouiseti, F. lateritium, F. minimum, F. monoliforme, F. oxysporum,
F. sambucinum, F. semisectum, F. solani, and F. sulphureum (see GB
No. 891,173).
penicillin acylase from Eschericia coli, Proteus rettoeri, Kluyvera
citrophila, Bacillus sohaericus. Bovista plumbea or Bacillus
megaterium.
lactase from Kluyveromyces sp., especially from K. fragilis or K.
lactis.
cyanide hydratase according to Danish DK No. 87/1283
nitrilase, nitrile hydratase or amidase, especially from
Rhodococcus sp., pseudomonas sp. or Brevibacterium sp.
See U.S. Pat. No. 4,001,081, EP Nos. 0 093 782 and 0 188 316.
The cell mass immobilized into cell mass particle form by the
process of the invention may be viable. or non-viable intact cells
as well as in the form of homogenized cell paste. The cells are
preferably of microbial or plant orgin. Some preferred examples
follow:
viable cells for use in bioconversion, e.g., yeast for ethanol
fermentation.
cells with enzymatic activity, e.g., fungal mycelium containing
cyanide hydratase, see EP Nos. 0 061 249 and 0 116 423.
cell mass preparations for use in adsorptive removal, e.g., of
heavy metals, see EP No. 0 181 497, U.S. Pat. Nos. 4,320,093,
4,298,334, 4,293,333 and JP-A No. 49-104,454.
EXEMPLARY APPLICATIONS OF THE INVENTION
As has already been pointed out, a preferred mode of this invention
is directed to instances when the art desires to convert cell bound
enzymes into a cell mass particle form. Within the above-described
parameters for practice of this invention is sufficient variability
to make practice of the invention applicable to any microorganism
source cell bound enzyme. For example, glucose isomerase a well
known cell bound enzyme, has been produced on a large scale
cultivation of Bacillus coagulans. An excellent glucose isomerase
is elaborated by glucose isomerase producing strain belonging to
the genus Streotomcyes, e.g., a Streotomcyes murinus cluster
strain. The cell bound enzyme from Bacillus coaoulans can be
immobilized readily by reaction with glutaraldehyde; see the
aforementioned U.S. Pat. No. 3,980,521. However, the cell bound
enzyme produced by strains of the genus Streptomyces, including
notably the Streptomyces murinus cluster is characterized by an
insufficient cross-linking capability with glutaraldehyde to
produce a satisfactory cell mass particle form immobilized enzyme.
However, either of these cell bound enzymes may be immobilized in
cell mass particle form through practice of this invention. It
follows, of course, that practice of this invention is particularly
suited to the Streptomyces murinus enzyme. Glucose isomerase is a
commercially important enzyme, which is to say, that immobilization
of the Streptomyces murinus glucose isomerase is one preferred mode
practice of this invention.
The widespread applicability of this invention is also exemplified
hereinafter by a disparate commercially important cell bound
enzyme, i.e., trytophan synthetase derived from a strain of E.
coli. Preparation of this enzyme, too, is a preferred mode practice
of the invention. None of the prior art immobilization methods
investigated by the inventors hereof resulted in an immobilized
tryptophan synthetase product of satisfactory physical strength. It
is noted that tryptophan synthetase requires a cofactor. Such is
present in the cytoplasm. Since disruption of the cell would cause
loss of the cofactor, immobilization of whole cells is employed in
the instance of the tryptophan synthetase enzyme.
Enzyme granules made according to practice of this invention,
particularly according to preferred practices of the invention,
exhibit superior physical properties. In packed bed they exhibited
a pressure drop for the liquid flowing therethrough which is only
around 50% of a comparable prior art product. (In this test study,
the comparable prior art product was a glutaraldehyde cross-linked
Bacillus coaoulans glucose isomerase made according to the
teachings of U.S. Pat. No. 3,980,521, a product that is in
widespread commercial usage.) In addition, high physical strength
and resistance against abrasion were found.
For further understanding of the practice of this mode of the
invention, the following specific Examples are presented.
EXAMPLE 1
Glucose isomerase containing cells of Streptomyces murinus, DSM
3252 were cultivated in a conventional medium comprising glucose, a
complex nitrogen source, minerals and trace elements.
After pH adjustment of 7.0-7.5 the cells were recovered from the
fermentation broth by centrifugation and homogenized after addition
of MgSO.sub.4, 7H.sub.2 O in an amount of 0.5% w/v by means of a
Manton-Goulin homogenizer. The homogenized cell sludge was kept in
-18.degree. C. and thawed immediately before use in a
immobilization experiment.
300 g of homogenized cell sludge with a dry matter content of 6.7%
was diluted to 750 ml with 1.5% MgSO.sub.4, 7H.sub.2 O, and pH was
adjusted to 7.5. 30 g Corcat p-18 polyethylene imine flocculent
(Cordova Chem. Co.) was added. Then 9.24 g of 50% glutaraldehyde
was added; pH was maintained at 7.4-7.6 for one hour. The flocs
were collected by filtration.
The filter cake containing approximately 15.8% DM was divided into
two equal parts in terms of dry matter content. The two parts were
mixed with 0 and 15% w/w (dry matter basis) respectively of
Polycup.RTM. 1884 polyazetidine prepolymer solution, pH 7.5 (from
Hercules, Inc., Delaware) calculated on filter cake dry matter.
Both parts were extruded through 0.8 mm orifices. The extruded
material was allowed to dry at room temperature to a dry matter
content of 83-85% w/w. The 300-700 u fraction was obtained by sieve
fractionation.
The glucose isomerase activity (measured according to NOVO Document
F 850399) recovered in the two immobilized enzyme preparations was
approximately the same. Pressure drop (in g/cm2) measured according
to NOVO, AF 166 is given in the following Table 1.
TABLE 1 ______________________________________ Pressure drop versus
percentage of polyazetidine, calculated as dry matter on filter
cake dry matter. ______________________________________ %
polyazetidine 0 15 Pressure Drop (25 h/50 h) 14/17 6/7
______________________________________
EXAMPLE 2
A tryptophan synthetase producing strain of E. coli, ATCC 15491,
was grown on an agar slant at 37.degree. C. and from there
transferred to a preculture in shake flasks at 37.degree. C. The
preculture was inoculated on a medium prepared as follows. The
composition of the medium was:
______________________________________ (NH.sub.4).sub.2 SO.sub.4 8
g/l KH.sub.7 PO.sub.4 1.6 g/l Na.sub.2 HPO.sub.4.2H.sub.2 O 5.6 g/l
Trisodiumcitrate, 2H.sub.2 O 0.5 g/l NaCl 3 g/l MgSO.sub.4.7H.sub.2
O 0.5 g/l CaCl.sub.2.2H.sub.2 O 00.2 g/l FeCl.sub.3 2H.sub.2 O 90
mg/l ZnSO.sub.4.7H.sub.2 O 20 mg/l MgSO.sub.4.4H.sub.2 O 24 mg/l
MnSO.sub.4.4H.sub.2 O 22 mg/l CuSO.sub.4.5H.sub.2 O 4 mg/l KI 4
mg/l NaMoO.sub.4.2H.sub.2 O 4 mg/l H.sub.3 BO.sub.3.6H.sub.2 O 1.2
mg/l CoCl.sub.2.6H.sub.2 O 6 mg/l NiCl.sub.2.6H.sub.2 O 6 mg/l
*Biotin 4 .mu.g/l *Calcium pantothenate 800 .mu.g/l *Folic acid 4
.mu.g/l *Inositol 4000 .mu.g/l *Niacin 800 .mu.g/l *p-aminobenzoic
acid 400 .mu.g/l *Pyridoxine HCl 800 .mu.g/l *Riboflavin 400
.mu.g/l *Thiamine HCl 800 .mu.g/l
______________________________________
The medium was sterilized at 121.degree. C. for 25 minutes except
for the vitamins (*), which were added by sterile filtration after
cooling together with dextrose (10 g/l) and indole in 48% ethanol
(125 mg/l). The submerged fermentation was conducted under aseptic
conditions at 37.degree. C. with aeration at 1
volume/volume/minute, agitation at 500 rpm and pH controlled by
acid/base addition at pH 7.0 for 40 hours. 24 hours after
inoculation a further addition of dextrose (40 g/l) and indole in
48% ethanol (875 mg/l) was carried out. Cells were harvested after
40 hours by centrifugation and immediately used for the
immobilization experiments described as Example 3 hereinafter.
EXAMPLE 3
60 g of wet cells recovered as described in Example 2, having a dry
matter content of 17% w/w were resuspended in 1200 ml of 0.2M EDTA
(adjusted to pH 7.5) and left for 30 min at room temperature and
then centrifuged. The aqueous EDTA washing procedure was repeated
once.
The wet cells were mixed with 300 of 85 mM KH.sub.2 P.sub.4, pH
7.5; 8.4 g of polystyrene from Kodak (200-400 mesh, cross-linked
with 2% w/w divinylbenzoic acid), 96 mg of pyridoxal phosphate and
9.6 g of a 12.5 w/v glutaraldehyde solution. The pH value was
maintained at 7.5 by addition of base throughout the immobilization
procedure. Then 60 g of Corcat P-150 polyethylene imine solution
from Cordova Chemical Company of Michigan, adjusted to pH 7.5 with
10N NaOH, was added, followed by the same amount of glutaraldehyde
solution as before. Dilution was carried out with 900 ml of 50 mM
KH.sub.2 PO.sub.4, pH 7.5 after 1 hour from the first addition of
glutaraldehyde. Then flocculation was performed by addition of 250
ml of 1% w/v Superloc A 130 from American Cyanamide. Partially
cross-linked and flocculated cells were recovered by filtration.
The filter cake containing approximately 19% w/w of dry matter was
divided into two equal parts. One part was mixed with 7.2 g of
Polycup.RTM. 172 (Hercules, Inc., Delaware) pH 7.5, corresponding
to 16.5% w/w of dry polyazetidine prepolymer in regard to the dry
matter in the wet cells recovered by centrifugation from the
fermentation.
Both parts were extruded through 0.8 mm holes.
The extruded material (both parts) was allowed to dry at room
temperature to a dry matter content of 90% w/w. The 300-700 um
fraction was obtained by sieve fractionation.
The tryptophan synthetase activity recovered in the two immobilized
enzyme product was approximately the same. The pressure drop (in
g/cm2) measured according to NOVO AF 166 of the product with
polyazetidine wa 14(25 h)/15 (50 h) and of the product without
polyazetidine 23 (25 h)/24 (50 h).
The examples which now follow are directed to the more preferred
mode of the invention wherein the polyazetidine prepolymer is added
prior to dewatering, including being present during the partial
cross-linking treatment. All other (dry substance) ingredients also
are present in the pretreatment reaction mixture.
It may now be appreciated better that the underlying rationale to
this more preferred mode of practice of this invention is to
achieve conversion of an enzyme (or other biologic material) in
aqueous solution or uniform dispersion together with any other
dissolved or dispersed ingredients into a two phase mixture wherein
desirably the (partially cross-linked) solid phase will contain all
of the (dry basis) substances desired in the final product and the
aqueous phase nothing but water and undesired ions, etc. Dewatering
converts the solid phase substances into a pasty mass for forming
into coherent particles. Thus, flocculants, auxiliary cross-linking
agent and other optional ingredients are added to the aqueous
solution or dispersion when their presence facilitates generation
of a more suitable two phase dewaterable inhomogeneous mixture.
The examples which follow exemplify most preferred practice of the
invention and in addition illustrate the effect of varying the
ingredients in the (partial crossinking) reaction mixture so as to
be suggestions to those skilled in the art how best to approach
immobilizing some biological material not exemplified herein.
EXAMPLE 4
Glucose isomerase containing cells were produced by fermentation of
Streptomyces murinus, strain DSM 3253, according to Example 1.
The cells were harvested by centrifugation of the culture broth.
The cell sludge had a dry substance content of 7.0%.
The general immobilization procedure was as follows; To 300 g cell
sludge was added 300 g deionized water containing 1.5% MgSO.sub.4,
7H.sub.2 O. pH was adjusted to 7.5. The indicated amount of
polyethyleneimine (Sedipur, product of BASF, West Germany) was
added, and after thorough mixing the mixture was cross-linked by
addition of 15% active glutaraldehyde based on cell sludge dry
substance plus polyethyleneimine dry substance. After 1 hour the
polyazetidine prepolymer (Polycup .RTM.2002) was added and
thoroughly mixed with the cross-linked cell suspension.
The mixture was then flocculated by addition of a cationic
flocculent, Superfloc C521 (Cyanamid Int.). The cross-linked enzyme
was recovered by filtration, formed into particles by extrusion
through a 0.8 mm screen and dried at room temperature.
The glucose isomerase activity was measured by NOVO analysis method
F-855310 (available on request from Novo Industri A/S, Denmark) and
the physical stability determined as pressure drop over a
column.
The pressure drop was measured over a column with a diameter of 24
mm and an enzyme bed height of 4 cm (5 g enzyme). The solution, 45%
glucose in demineralized water with 1 g MgSO.sub.4 S/1, was pumped
through the column at a rate of 40 g/min at 60.degree. C. The
pressure drop (in mm of liquid) describes the physical stability of
the enzyme particle, i.e., a low pressure drop corresponds to a
good physical stability. The results are shown in the table
below.
______________________________________ Glucose isomerase Pressure
drop activity (.mu.mol/min/g) (mm)
______________________________________ 5% 0% poly- 614 400 poly-
azetidine ethylene- 2.5% poly- 667 99 imine azetidine 5% poly 586
57 azetidine 10% 0% poly- 692 105 poly- azetidine ethylene- 2.5%
poly- 578 11 imine azetidine 5% poly 506 10 azetidine
______________________________________
Preparations with pressure drops exceeding about 20 mm liquid are
not considered to be well suited to industrial glucose isomerase
columns. Activity decreases slightly with increasing polyazetidine
concentration.,
This example clearly shows the improvement of physical stability of
the immobilized preparations obtained with polyazetidine.
EXAMPLE 5
A similar experiment to that described in Example 4 was performed
using a higher yielding descendant of DSM 3253.
The dry substance content of the cell sludge was 5.3%, and the
cells were partially disrupted by homogenization. Otherwise the
immobilization was performed as described in Example 4.
The results are given in the Table below.
______________________________________ Glucose isomerase Pressure
drop activity (.mu.mol/min/g) (mm)
______________________________________ 5% 0% poly- 556 16 poly-
azetidine ethylene- 1% poly- 855 13 imine azetidine 2.5% poly- 839
13 azetidine 5% poly- 707 8 azetidine 10% 0% poly- 651 12 poly-
azetidine ethylene- 1% poly- 975 10 imine azetidine 2.5% poly- 911
5 azetidine 5% poly- 853 3 azetidine
______________________________________
From these experiments, it can be concluded that the polyazetidine
improves the physical stability of even very physically stable
formation.
EXAMPLE 6
Bacillus coagulans containing glucose isomerase was immobilized
with and without polyazetidine.
Homogenized cell paste of B. coaoulans prepared according to U.S.
Pat. No. 3,979,261 was suspended in 0.1% MgSO.sub.4 to a final dry
substance concentration of 3%. Glutaraldehyde was added to a final
concentration of 0.5%. After 60 min with mixing at room temperature
polyazetidine prepolymer (Polycup.RTM. 2002) wa added followed by
floccillation with Superfloc C521. The cross-linked enzyme was
recovered by filtration, formed into particles by extrusion and
dried at room temperature.
Activity and pressure drop were measured as described in Example 1.
Resistance to grinding was measured as turbidity (optical density)
at 600 nm after 1 hour of vigorous stirring of 0.5 g enzyme in 20
ml 50 mM phosphate, pH 7 with a propeller. Before the assay the
enzyme product had been swelled and washed in 6% NaCl in 50 mM
phosphate, pH 7.0.
______________________________________ Amount of 0 1 3
polyazetidine (% dry substance) activity (.mu.mole/min/g) 540 506
493 pressure drop (mm liquid) 5 3 2 25 grinding 0.111 0.068 0.062
______________________________________
Both resistance to grinding and physical stability were improved by
adding polyazetidine, while only a small activity loss was
observed.
EXAMPLE 7
To exemplify immobilization of a soluble enzyme the
amyloglucosidase from Aspergillus niger has been chosen. This
enzyme is extracellular.
A commercial preparation AMG 400 L HP (Novo Industri A/S, Denmark)
was dialyzed against 50 mM phosphate pH 7.0. The preparation was
diluted to a dry substance concentration of 2% w/v, and an equal
ammount of egg albumen was added (i.e., 2% w/v). Glutaraldehyde was
added to a concentration of 0.6% w/v. After one hour of stirring,
polyazetidine prepolymer (Polycup.sup.R 2002) was added to a final
concentration of 0.08% w/v, and the mixture was flocculated with
Filtrafloc (Servo B.V., Netherlands). Enzyme was recovered as
described in previous examples.
Activity was measured as described in Novo Analysis Method AF159/2.
Pressure drop was measured as described in Example 1, but at
35.degree. and with 11% w/w glucose in 50 mM phosphate, pH 7.5.
______________________________________ polyazetidine, % w/v 0 0.08
activity (.mu.mol/min/g)* 196 160 20 pressure drop (mm liquid) 9 6
______________________________________ *Particle fraction: 425-710
.mu.m
EXAMPLE 8
Cell paste of Fusarium sp. containing penicillin acylase activity
(prepared according to British patent specification No. GB
891,173), which had been washed thoroughly with 0.9% NaCl, was
suspended in 50 mM phosphate pH 7.0 to give a final dry substance
content of 3%. Polyethyleneimine (Sedipur) was added to give a
final dry substance content of 0.1% Glutaraldehyde was added to a
final concentration of 0.2% w/v. After one hour with thorough
mixing polyazetidine prepolymer was added, and finally the mixture
was flocculated with a cationic flocculent Filtrafloc. The
cross-linked enzyme was recovered by filtration, formed into
particles by extrusion through a 0.6 mm screen and dried at room
temperature.
Enzyme activity was measured by Novo analysis AF186, and the
physical stability determined as pressure drop and resistance to
grinding (see Example 6).
______________________________________ Kymene .RTM. Polyazetidine
type None Polycup .RTM. 2002 557H
______________________________________ Polyazetidine (%) 0 1 3 1
activity* (PVU/g) 72 57 42 35 10 pressure drop (mm 6 4 3 2 liquid)
grinding 0.563 0.305 0.140 0.103
______________________________________ *450-710 .mu.m fraction
As can be seen from the above Table polyazetidine increases
physical stability, i.e., gives increased resistance to grinding
and gives highly improved pressure stability. However, this
improvement is obtained at the expense of an activity loss which
partly is due to diffusion limitation, which again is believed to
be due to a more dense preparation when polyazetidine is
present.
EXAMPLE 9
Fusarium sp. was immobilized as described in Example 5, but at
different pH-values and with 1% Kymene.RTM. 557H in all
preparations.
Results are seen in the table below:
______________________________________ pH 6 7 8 25 activity (PVU/g)
33.3 36.1 41.5 pressure drop (mm liquid) 2 3 3 grinding 0.171 0.154
0.188 ______________________________________
The physical stability is good and independent of pH in the tested
range (6-8).
EXAMPLE 10
The significance of time of polyazetidine prepolymer addition was
examined with cell paste of Fusarium sp. Immobilization was done as
explained in Example 5, but without PEI and with polyazetidine
prepolymer (Polycup.RTM. 2002) addition to a final concentration of
0.3% both before and after the addition of glutaraldehyde.
______________________________________ Time for polyazetidine not
before after addition added glutaraldehyde glutaraldehyde activity
(PVU/g) 58 32 31 10 pressure drop 27 6 4 (mm liquid)
______________________________________
The physical stability is increased both when polyazetidine is
added before and after the glutaraldehyde.
EXAMPLE 11
Cell sludge of Rhodococcus erythropolis having ability to hydroyze
nitrile (prepared according to EP No. 188,316) was diluted to 2%
dry substance with water. 10% polyethyleneimine (Sedipur) (based on
dry substance of cell sludge) was added, and pH was adjusted to
7.0. The mixture was cross-linked by addition of 5% active
glutaraldehyde (based on cell sludge dry substance plus
polyethyleneimine dry substance). After 1 hour, 2% of polyacetidine
prepolymer (Kymene) (based on total dry substance) was added.
The mixture was then flocculated by addition of an anionic
flocculent, Superloc A 130. The cross-linked enzyme was then
recovered by filtration, formed into particles by extrusion through
a 0.8 mm screen and dried at room temperature.
For reference, the same cell sludge was cross-linked without
polyazeteidine, but otherwise in the same way as above.
For comparison, the same cell sludge was immobilized by a prior-art
method, viz. entrapment in polyacrylamide gel according to U.S.
Pat. No. 4,248,968. More specifically, the cell sludge was diluted
to 12.5% dry substance and immobilized as described in Example 12
thereof. Finally, the gel was sieved with 1 mm mesh sieve and
washed with saline until the supernatant became clear.
______________________________________ 11% glucose syrup
______________________________________ Flow rate: 40 g/min
Temperature: 35.degree. C.
______________________________________
Pressure drop was measured after 25 hours.
5 g (dry substance) of immobilized enzyme was applied for pressure
drop measurement.
______________________________________ Results (pressure drop in
g/cm.sup.2): ______________________________________ Invention
cross-linking with polyazetidine 13 Reference cross-linking without
polyazetidine >200 gel entrapment >200
______________________________________
The practical effect of physical stability of a product is
illustrated by the fact that a product with a pressure drop (mm
liquid) of 2-4 can be used in a fixed bed column reactor with a
height of 1 meter and a diameter of 1 meter with a holding time for
the liquid passing therethrough of 1 minute. The packed bed may be
functioning with a constant pressure drop over the bed for more
than 6 months. A product with a pressure drop of more than 10 is
not able to withstand these conditions.
The same situation is also seen when the process takes place in a
continuous stirred tank system. Products with low grinding
properties are significantly more stable in e.g. 4 m.sup.2 reactor
systems. Practical lifetimes of optimal products of more than 4
months can be expected without more loss than 50% activity.
* * * * *