Note: Descriptions are shown in the official language in which they were submitted.
Lf~ r~ 3~
IMMOBILIZED MICROBIAL CELLS AND
PROCESSES FOR PREPARING AND US ING THE SAME
_
The present invention is concerned with
the immobilization of microbial cells and processes
5 for preparing and using the same. The invention is
particularly concerned with an improved process for
manufacturing L-aspartic acid using immobilized
microbial cells, notably Escherichia coli (~. coli)
cells, which contain L-aspartase activity. However,
lo the immobilization and use of other cells are also
contemplated.
Backqround to The Invention
There is a considerable amount of prior
art regarding the immobilization of E. coli or
other microbial cells for use in the preparation
of L-aspartic acid. For example, U.S. patent
3,791,926 (Chibata et al) describes a process for
the production of L-aspartic acid which involves
polymerizing a monomer selected from acrylamide,
20 N,N'-lower alkylene-bis (ac--ylamide) and
bis(acrylamidomethyl) ether in an aqueous
suspension containing an aspartase-producing
microorganism such as E. coli ATCC no. 11303. The
resultant immobilized aspartase-producing micro-
organism is treated with ammonium fumarate or amixture of fumaric acid or its salt and an
inorganic ammonium salt whicil by enzymatic
reaction gives L-aspartic acid.
The immobilization of E~ coli cells
containing aspartase activity and use of the
resulting immobilized cells for the production of
L-aspartic acid are also described by Fusee et al,
Applied and Environmental Microbioloqy, Vol. 42,
~.~g~$5~3
No. 4, pages 672-676 (October 1981). According to
Fusee et al, the cells are immobilized by mixing a
suspension of the cells with a liquid isocyanate-
capped polyurethane prepolymer tHYPOL ~ ) so as to
s form a "Eoam" containing the immob.ilized cells.
Sato et al (Biochimica et Biophysica
Acta, 570tl979~ pages 179-186) have disclosed the
immobilization of E. coli cells containing
aspartase activity with K carrageenan, and use of
the immobilized preparation for the production of
L-aspartic acid.
Additional literature disclosures
describing the immobilization of microbial cells
in urethane prepolymers or polyurethanes or the
like include the following:
(a) Immobilization of MicrobiaL Cells in
Polyurethane Matrices, by Klein et al,
Biotechnolo~ Lettes, Vol. 3, No. 2,
pages 65-70 (1981);
(b) Hyrophilic Urethane Prepolymers:
Convenient Materials for Enzyme
Entrapment, Biotechnoloqy_and
Bioenqineering, Vol. XX, pages 1465-1469
(1978),
(c) Transformation of Steroids by Gel-
Entrapped Cells in Organic Solvent, by
Omata et al, European J. Applied
Microbioloqy and Blotechnoloqy, 8, pages
143-155 (1979); and
(d) Entrapment of Microbial Cells and
Organelles With Hydrophilic Urethane
Prepolymers, by Tanaka et al, European
J. Applied Microbioloqy and Biotech_
nology, 7, 351-354 (1979j~
`~ ~2~$~
The above-noted processes for preparing
L~aspartic acid using immobilized microbial cells
~uffer from various dis advantages. For example,
g-carrageenan gum and polyurethane "foam" as
5 disclosed by Fusee et al and Sato et al are
relativel~ soft and compressible. Hence when
these immobilized cell CGmpOSitiOnS are used, in a
column through which ammonium fumarate is pa~sed
for conversion to ammonium aspartase, they tend to
be compressed and plug up, particularly where high
flow rates and/or relatively tall column heights
are involved.
Objects of the Invention
The principal object of the present
invention is to provide an improved process for
preparing L aspartic acid using L-aspartase active
microbial cells, preferably E. coli cells, which
have been immobilized in a special way whereby the
resulting composition is highly effective for the
20 preparation of L-aspartic acid batchwise or in
continuous fashion.
A more specific object includes the
provision of an improved process for preparing L-
aspartic acid from ammonium fumarate using
25 immobilized E. coli cells which maintain optimum
L-aspartase activity for relatively long periods
of time. Another specific object of the invention
is to provide novel immobilized microbial systems
suitable for use in making L-aspartic acid which
obviate problems encountered in prior procedures
involving the use of immobilized cells. 0ther
objects will also be hereinafter apparent.
5V;~
Summar~L_f ~ ~ veAtion
According to one aspect of the invention,
there is provided a process for preparing L-
aspartic acid by contacting ammonium fumarate or
its equivalent with an immobilized microbial cell
composition comprising E. coli ATCC 11303 cells or
equivalent cells containing h-aspartase activity,
the cells being immobilized by means of a fixed~
insoluble, crosslinked polymer obtained by curing
o a curable prepolymer material selected from the
group consisting of polyazetidine prepolymers,
carboxymethyl cellulose (CMC), polyurethane
hydrogel prepolymers and polymethylene isocyanates
cured at a temperature below the temperature at
which the L-aspartase activity of the microbial
cells is significantly reduced, the cell/cross-
linked polymer composition constituting a coating
on a solid inert carrier. The application of the
immobilized cell/crosslinked polymer coating on
the carrier provideq a highly advantageous form of
the cell/polymer composition for use in preparing
L-aspartic acid. It is a particularly advantageous
feature that the cell/polymer systems of the
invention can be provided as a dry coating on the
carrier without significant loss of the L-
aspartase activity.
An especially preferred a~pect of the
invention contemplates the provision of
immobilized E. coli cells having L-aspartase
activity, the cells being immobilized by means of
a crosslinked, water-insoluble polymer obta~ned by
curing a polyazetidine prepolymer.
Broadly speaking, the invention i~
dependent on binding whole cells of E. coli,
notably E. coli ATCC 11303, or the equivalent,
s~
which are known to have L-aspartase activity and
therefore are capable of producing L-aspartic acid
(or ammonium salt thereof~ by conversion sf
ammonium fumarate/ in a novel and useful configur-
a~ion using a special crosslinked polymer systemas set forth above to bind the cells.
The indicated prepolymer systems can be
crossLinked at temperature below 40C and in the
presence of relatively large volumes of water
lo containing E. coli cells without signiEicantly
deteriorating the L-aspartase activity of the
cells. It is a surprising aspect of the invention
that such aqueous crosslinking conditions leave
the E. coli cells with their L-aspartase activity
even though the cells are immobilized in
insoluble, crosslinked polymer networks.
Another unique aspect of the invention is
that the wet dispersion of the E. coli cells iQ
the aqueous polymer solutions can be taken to
dryness while the immobilized cells still
surprisingly retain most of their original L-
aspartase activity. The drying process has the
advantage of providing strong, weLl-crosslinked,
insoluble compositions in the form of coatings,
25 membranes, particles, etc. having high concentra-
tions of cells retaining L-aspartase activity~
The novel immobilized L-asparta~e cell
compositions disclosed herein have been found to
outperform the immobilized L-aspartase active
E. oli cell compositions previously reported by,
fcr example, Fusee et al and Sato et al in the
references noted above.
Prepolymer materials suitable for use to
provide the crosslinked polymer net~ork for
immobilizing th~ E. coli cells according to the
invention include:
(1) polyazetidine prepolymers which may
be crosslinked in aqueous solution by reaction
with =NH, -SH, -UH, -COOH; or other polyaze-
tidines which may be crosslinked by H20 removal,
heat, or by changing to a more basic pH. The
lo following is an idealiæed structure of a
representative polyazetidine such as Polycup ~172
(Hercules, Inc.) which is useful for present
purposes:
o O Cl-
----C - R--CNEICH2 CH2~ N--CH2 - CH2NH--
CH~ CH2
OH
where R is typically tCH2t4
(2~ carboxymethyl cellulose which can be
crosslinked in aqueous solution by reaction with
polyvalent ions such as Fe+~, Al++~, Ca++, Mg+~.
The following is an idealized structure of a
carboxymethyl cellulose polymer such as CMC7HF
(Hercules, Inc.):
___ O
CH20CH2CONa OH
H / CH - O O CH CH H
\ / OH \ / \ / OH \ /
C / H H \ / C
/ CH- CH CH O
OH CH20CH2CONa
(3) polyurethane hydrogel prepolymers
which can be crosslinked in aqueous solution by
reaction with -NH, -SH, -OH, -COOH or H20. The
following is an idealized structure of a repre-
sentative polyurethane hydrogel prepolymer which
is made by taking a water soluble polyether polyol
having greater than two hydroxyl groups per
molecule and a moleculal weight greater than 3,000
lo and capping each hydroxyl group with a low
molecular weight polyisocyanate:
R ~ (ocH2~H23 ~(~cEl2--cH~l ~cE~2cH ~ OCHNR2N(~;
where Rl typically ls a low molecular weight
polyol fragment
(from glyceroll pentaerithritol,
sorbitolj etc.);
S~3
a and b are integers, eOg. a is typically>
10 and the ratio of a/b is generally
from 2 to 5, the sequence of aerangement
of the a and b segments being random or
in blocks;
n is also an integer typically > 2; and
R2 is typically a diisocyanate such as a
toluene diisocyanate, methylene
di(phenylisocyanate)) m-xylylene
diisocyanate, isophorone-diisocyanate,
hexamethylene diisocyanate, hexahydro-m-
xylylene diisocyanate, dodecahydro-
methylene di(phenylisocyanate) and the
like; and
(4) polymethylene isocyanates which can
be cured by mixing with water such as the
following:
[~L CE2 ~L C 2 [~
where n=0 to 4
Such polyisocyanates are commercially available
and are commonly called "crude polymeric methylene
dilphenylisocyanates)~. Other polyisocyanates
which are convertible into crosslinked insoluble
polymers with water are also thought to be useful,
e.g. toluene dlisocyanates, methylene di(phenyl-
isocyanate), m-xylylene diisocyanate, isophorone-
diisocyanate, hexamethylene diisocyanate,
hexahydro-m-xylylene diisocyanate, dodecahydro-
methylene di(phenylisocyanate) and the like
z~5~3
The isocyanates are usefully employed by
coating an appropriate particulate substrate and
then mixing with an aqueous slurry of E. coli
cells~
Any of the above-noted polymer systems
may be used to immobilize L-aspartase active
E. coli cells to provide cell/polymer compositions
having a variety of different forms and shapes~
For example, the immobilized cell/polymer composi-
lo tion may be prepared in the form of membranes,
filaments, fibers, tubes, beads or the like. A
particuldrly important embodiment oE the invention
involves peoviding the immobilized cell/polymer
composition as a coating on an appropriately
shaped support which advantageously, although not
necessarily, is in the form of solid or reticu-
lated beads or particles composed of an inert
solid organic or inorganic, porous or non~porous
materialO As noted earlier, the composition of
the invention can be cured and dried without
substantively afecting the L-aspartase activity
of the cells and, as a consequence, the composi
tion can be made up in the form of dried coated
beads or other particulate material and stored
until needed fcr use. Typically, useful supports
or carriers for the immobilized cell/crosslinked
polymer compositions of the invention include the
following in bead or particle form:
Molecular sieves
Ion exchange resins
Alumina
Silica and silica ~el
Foraminifera skeletons
Polymer la~exes
Metals
9S~3
It is a useful property of the polymer systems
used in the present invention that they can be
contacted or mixed in water wlth E. coli cells
containing L~aspartase and then crosslinked (or
cured) to form insoluble and relatively fixed
polymer matrices which hold or otherwise
immobilize the E. coli cells. It is particularly
useful that the crosslinking conditions which need
to be used are mild enough so that the L-aspartase
lo activity oE the E. coli mutant cells is
main~ained.
As indicated, a particularly desirable
CompGsition according to the invention is obtained
by applying the E. coli cells containing L-
aspartase activity and the immobilizing polymer asa coating on hard inorganic or organic polymer
beads~ Using these relatively noncompressible
compositions an L-aspartase active catalyst bed
capable of high throughput in a fixed bed or
20 fluidized bed is possible. As noted earlier, the
L-aspartase active immobilized cell compositions
previously described in the literature involving
the use of relatively soft, compressible
k-carrageenan gum or polyurathane foam are
2s compressible and therefore tend to plug under high
flow rates, and/or tall column (bed) heights.
Various methods may be used to immobilize
the E. coli ATCC 11303 cells~ while retaining
their L-aspartase activity, by combination with
cured or crosslinked forms of prepolymers
according to the invention. The preferred method
used in any specific situation will depend, at
least to some extent, on the prepolymer involved.
Thus:
9Sj~3
a) In the case of the preferred poly-
azetidine prepolymers, E. coli ATCC
11303 cells are advantageously mixed
with an aqueous solution of the prepoly-
mer so as to obtain a homogeneous
mixture after which the polyazetidine
may be cured or crosslinked to give an
insoluble L-aspartase active composition
by any of the following means:
lo Removal of part or nearly all of the
water at temperatures below 60C
~usually between 40 and 0C) and
pressures of 760 to 1~0 Torr;
raising the pH above 7.5; exposing
the E. coli/polyazetidine mixture to
polyamines (using, for example, 100
parts of the composition for each
1-2 parts/wt. of polyethylene imine,
polyamino ion exchange re~ins,
diethylene triamine, ethylene
diamine, etc.)
b) For carboxymethyl cellulose (CMC) gums,
homogeneous mixtures with E. coli ATCC
11303 may be crosslinked (cured) to
insoluble, L-aspartase active coatings,
membranes, ibers, beads, etc.~ by
contacting an aqueous mixture of, Eor
example, 1.0 part/wt. of cell past/CMC
mixture with an aqueous solution of
0.001 to 1.0 parts/wtO of polyvalent
cation salt or, ideally 0.01 to 0 4
parts/wt. of polyvalent cation salt per
1.0 part/wt. of the cell paste/CMC
mixture.
5V3
12
The resultant water insoluble
immobilized cells/polymer network
composition can be thus cured in the
form of membranes, fibers or beads, but
most efficiently as a coating around
high sur~ace area particles. As an
illustration lO to 1000 parts/wt. of the
high surface area particles may be
coated with 1.0 part/wt. of cell
lo paste/CMC mixture and ideally 50 to 500
par~sJwt. of the particles per 1.0
partJw~. of cell paste/CMC mixture are
used. Removal of 75% or more of the
water contained in the cured coatings
may be desirable to improve strength and
bonding of the cell/polymer coating.
c) When polyurethane hydrogels are used~
homogeneous aqueous dispersions of
E. coli ATCC 11303 (typically l.0 9 of
cells per l.0 to 1000 ml H2O broadly and
10 to 190 ml H2O preferably) are mixed
with the hydrogel prepolymer and the
prepolymer is cured into a water-
insoluble L-aspartase active composition
by allowing the polyisocyanate to react
with water or in a more rapid manner by
either removing 50 to 100% of the
available water 1000 to 0.1 Torr and
below 50C (preferably 760 to 5.0 Torr
and 0 to 30C) or by exposing the
undried compositions to aqueous solu-
tions o~ molecules having more than two
primary or secondary amine groups.
Typically these polyamine molecules can
13
range from hydrazine or ethylene diamine
to polyethyleneimine. Generally for
each l.0 g of hydrogel prepolymer used,
from 0.01 to lO.0 g of the polyamine in
from 1.0 to 500 ml of water could be
used. Ideally 0 1 to l.0 9 of the
polyamine and 10 to lO0 ml of water are
employed per gram of hydrogel prepolymer
used.
The above E. ~ hydrogel compositions
can be fabricated into membranesp
fibers, beads, etc., but a preferred
configuration is to use the compositions
as coatings onto high surface area
lS particles, as discussed before.
Depending on the surface area of the
particle employed from 0.1 to 100.0 g of
the E. col~/hydrogel composition can be
coated onto lO0 g of the particle.
d~ In the case where polyisocyanates are
used, these are generally first applied
to a surface and the cells of E. coli
ATCC 11303 thereafter added thereto.
Usually, from 0.1 to 20.0 g of the
polyisocyanate per lO.0 9 of the surface
material can be employed, preferably 0.5
to 10.0 g of polyisocyanate per lO.0 9
of surface material. The polyisocyan-
ates, if liquid, can ~e added undiluted
to the substrate with good mixing
(agitator or tumbling~. However, the
preferable method is to use solutions of
~he polyisocyanates in inert, readily
evaporated solvents such as toluene,
~ 2q:119S~3
14
acetone, chloroform, tetrahydrofuran~
dimethoxyethane, ethyl acetate, and the
like. These solutions are added to the
substrate, prefeeably dry particles of
hi~h surface area, with good mixing and
the solvents removed at their boiling
points at 760 Torr or below while
continuing good mixing to insure
homogeneously coated, mobile substrate
lo particles~ The amounts of solvents used
will usually ran~e from 1.0 to 100
parts/wt. per 1.0 parts/wt. of
polyisocyanate.
The immobilized cell/polymer compositions
1~ of the invention may be used to make aspartic acid
by either a batch or continuous process. However,
it is noted that these compositions, particularly
when coated onto a bead or other particulate
support, are especially effective for the
continuous conversion of aqueous solutions of
ammonium fumarate to ammonium L-aspartate
according to the following equation:
O O
Il 11
--O--C ~ CO--
\ / bed of immobilized
C 3' CHNE~2 H~
NH4~ ll L-aspartase active
/~ ~. coli compositions CH2
H 90 1 o NH4
O O
pH 8.5 pH 8.5
51503
As illustrative of processes which may be
used to prepare L-aspartic acid or ammonium L-
aspartate according to the invention are the
following:
(i) A batch type process wherein the
catalyst compositions are stirred in
from 0.1 to 5.0 molar (preferably 0.5 to
2.0 molar3 solutions of ammonium
fumarate in water at 5.0 to 10.0 pH
lo (preferably 7.5 to 9.5 pH) for periods
of 1.0 to 100 hours (preEerably B to 48
hours) at temperatures below 50C
(preferably 20 to 40C). Broadly from
0.05 to 50 g of immobilized cells,
preferably from 1.0 to 15 9, are used
per 1.0 mole of starting ammonium
fumarate. After the conversiont the
catalyst compositions may be removed by
filtration or the equivalent for reuse
in converting fresh batches o fumarate
solutions. The produ~t solutions are
obtained in a form suitable for conven-
tional processing to isolate the L-
aspartic acid (acidification,
precipitation, filtration, washing,
recrystallization, drying).
(ii) A continuous process wherein the
catalyst compositions, e.g. coated
beads, are placed in columns and the
solutions of ammonium umarate (concen-
trations, pH's, and temperatures are the
same as described above for the batch
processes) are passed through the
catalyst beds either from above or from
5~3
16
below (fluidized bPd mode). The rates
of passage of these fumarate solutions
may range from 0.1 to 1000 space
velocities/hour. For example~ 5.0 liters
of solution per hour may be passed
through 1.0 liter of catalyst bed repre-
senting 5.0 space velocities (S.V.) per
hour. Preferably the fumarate solution
flow rates which yield essentially 100%
lo conversion of the fumarate to the L-
aspartate fall in the range of 0.5 to
20.0 S.V./hour. The effluent from these
columns of catalyst beds is suitable for
conventional processing to isolate L-
aspartic acid (as outlined above in the
batch processes).
The invention is illustrated by the
following Examples:
Example 1.
Preparation of E. coli Containing L-
Aspartase Immobilized in CMC Gum.
To 10 9 of a 1% aqueous solution of high
molecular weight carboxymethyl cellulose (Hercules
7 HF CMC gum, approximate molecular weight of
2s 70D,000 and 0.65 to 0.85 carboxymethyl yroups per
anhydroglucose units~ was added 0.5 g of a paste
of the E. coli cells, ATCC 11303. A thoroughly
homogeneous dispersion was made by hand mixing
with a wooden stick . A 2.0 9 aliquot of this
dispersion was placed onto 10 9 of hydrated 5
molecular sieve beads (8-12 mesh, previously
hydrated by mixing 100 g of the beads with.200 9
of water following by decanting the excess water
and allowing the beads to dry at 25 C in air).
After a thorough mixing by hand for five to ten
minutes, uniformly coated, damp, but free flowing,
beads were obtained. These coated beads were then
poured in~o 40 ml of a 0.1% solution of FeSO~.nH20
in water. After 4 hours of periodic gentle
stirring at 25C the liquid was decanted
(considerable gel microparticles were noted in the
liquid~ and the beads washed four times with 0.1 M
lo sodium phosphate buffer solution. A total of 22.2
of wet coated beads were obtained. The beads
were stored at 5C in damp air. For L-aspartase
activity (see Table I at 5.0 ml aliquot of these
wet beads was taken. This aliquot weighed 6.3 and
was estimated to contain d.027 9 of the E. coli
cells
(0 5 g X 2 . 0 9 X 26 2 g a 0 . 027 g)
Example 2
Preparation of E. coli Cells Containing
L-Aspartase Immobiliæed In a Polyurethane
Hydrogel.
a) A polyurethane hydrogel prepolymer
was prepared by capping a 7,000 molecular weight
water soluble polyether (high level of polyoxy-
ethylene segments) triol, Plueacol ~V 7 from BASF
Wyandotte, with three moles of dodecahydro-
methylene di(phenylisocyanate), Desmodur ~ W from
Mobay Chemical Company. A solution of 1.0 9 of
this hydrogel prepolymer in 9.0 g of water was
thoroughly mixed with 1.0 9 of E. coli ATCC 11303
cell paste at 25C. To 20 g oE hydrated 5A, 8-12
mesh, molecular sieve beads was added a 2.0 g
aliquot of the E. coli-hydrogel prepolymer aqueous
- `~2~5C~3
18
dispersion. The resultant mixture ~as stirred
ovet a period of 10 minutes by hand with a wooden
stick at 25C to give 22.0 9 of wet but free
flowing coated beads~ Preliminary experiments
showed that 10% solutions of this hydrogel
prepolymer in water formed water soluble gels
upon drying at 25C in air for 8 to 24 hours.
Thus, the wet coated beads were allowed to dry in
the open air at 25C for 16 hours to give 20.1 9
lo of free flowing cured coated beads. A 4.3 9 aliquot
of these coated beads had a volume of 5O0 ml and
an estimated E~ coli cell content of 0.039 9
(1 0 ~ X 2.0 9 X 4 3 q = 0.039 g) -
This 5.0 ml sample was used for L-aspartase
activity measurements (see Table 1).
b) A 1.0 9 aliquot of the aqueous
E~ coll-hydrogel prepolymer dispersion described
above was also coated onto 10.0 g of 5A, 8-12
mesh, hydrated molecular sieve beads at 25C~
After stirring for S to 10 minutes the resultant
free flowing wet beads were poured with good
mixing into 40 ml of a 0.1% solution o poly-
ethylene imine (Aldrich Chemical Co. ~181g7-8) in
water. Preliminary experiments showed that a
fresh, less than 15 minute old, solution of 1.0 9
of hydrogel prepolymer in 10 ml H2O formed a water
insoluble gel immediately upon contacting a 0.1%
aqueous solution oE the polyethylene imine. The
slurry of coated beads in the polyethylene imine
solution were gently stirred for 30 minutes at 25C
and then washed four times with 40 ml portions of
0.1 M sodium phosphate buffer solution (pH 7.5) to
give 1105 g damp, free Elowing beads. A 5.0 ml
ali~uot of those beads weighed 5.1 g and was
g503
19
estimated to contain 0~040 g of the E. coli cells
02 9 X 1.0 g X 15 l q = 0.041 ~ -
This 5~0 ml aliquot was used for L-aspartase
measurements (see Table I).
c) A polyurethane hydrogel membrane
having E. coli ATCC 11303 containing L-aspartase
was prepared in the Eollowing manner:
A sLurry of 10.0 9 of E. coli ATCC 11303
cell paste ~n 10.0 9 of phosphate buffered saline
lo water (pH 7.4) was mixed for 30 minutes at 25C to
insure a homo~eneous dispersion. A solution of
5.0 9 of the above-mentioned hydrogel prepolymer
(7,000 molecular weight triol capped with three
moles of Desmodur ~W) in 10.0 g of deionized
1S water was formed by vigorous stirring Eor one
minute at 25C. This solution was mixed with the
homogeneous cell paste slurry. Within 20 seconds
of vigorous stirring the resultant mixture became
quite thick and was immediately poured onto a
Teflon ~sheet. Within a few seconds at 25C the
mixture became an elastomeric gel. After 45
minutes in air at 25C the resultant elastomeric
membrane composite (1/16" to 1/4" thick by 4"
diameter) was stripped off the Teflon ~surface.
2s The membrane was placed in 500 ml of 105
M ammonium fumarate solution and shaken for 30
minutes. Considerable turbidity developed in the
aqueous solution indicating cells and/or gel were
washed out of the membrane. This washing process
was repeated with one liter of the 1.5 M ammonium
fumarate solution at 25C for 64 hour~. The solu-
tion was only very slightly hazy and its optical
density at 280 nm (1/1000 dilution3 was 0.005
"` ~Z~ 3
2~
compared to 0.43 (1/1000 dilution) at the start.
This indicates that essentially all the ammonium
fumarate had been consumed.
The membrane was removed intact and
s placed in 750 ml fresh 1.5 M ammonium fumarate.
After 16 hours of gentle stirring at 25C the
clear product liquid over the membrane had an
optical density at 280 nm (1/1000 dilution) of
0.005. This process was repeated with 750 ml more
lo of the ammonium Eumarate for 20 hours at 25C.
The combined 15,000 ml oE product solution was
clear and had an optical density at 280 nm of
0.~05.
A 1000 ml aliquot of the combined product
solution was acidified to a pH of 2.3 using 145 9 of
concentrated hydrochloric acid. The resultant
slurry of white crystals was kept at 5C for 16
hours and then filtered. The damp filter cake was
then stirred in 1000 ml oE deion;zed water at 80 to
90C Eor 2 hours and then allowed to cool to 30C.
The white crystalline solids were collected by
filtration and washed with 250 ml deionized water.
The solids were dried at 25C to 100C and 5 to 20
Torr for 16 hours to a constant weight of 178.0 g
which represents an 89.4% yield of L-aspartic acid
(78.0 X 133 = 0.894) The optical rotation of a
solution of 2.04 9 of the white solid in 100 ml of
6.0 N HCl was measured on a Perkin-Elmer polari-
meter at 25C and found to be +0.497. On the same
instrument a solution of 2.04 9 of an authentic
sample of L-aspartic acid (Sigma Chemical Co~ ~A-
9256) had a rotation of ~0.495.
5~3
21
Preparation of E. coli Containing
L-Aspartase Immobilized On Surfaces Pretreated
with Polyisocyanates.
a) A 10.0 9 sample of activated
anhydrous 5A, 8-12 mesh molecular sieve beads was
mixed in a rotary evaporation apparatus with a
solution of 2.0 9 oE a crude polymerîc aromatic
polyisocyanate (Upjohn Chemical Company PAPI ~320,
7.1 meg NCO/g) in 50.0 9 of dry toluene. The
toluene was removed at 85C and 10 to 20 Torr over
a period of an hour of continued tumbling~ ~he
free flowing impregnated beads were cooled to 25C
and a slurry of 1.0 g E. coli ATCC 11303 paste in
1.0 9 of 0.1 M sodium phosphate bufEer (pH 7.5)
was added. The resultant bead mixture was tumbled
slowly Eor four hours at 25~C and one atmosphere
to give 12.9 9 dry free flowing beads. A 5.0 ml
aliquot of the dry beads weighed 4.9 g. This
volume did not change after soaking in 1.5 M
ammonium fumarate solution. An estimated 0~38 g
of ~. coli cells were on this 5.0 ml aliquot
(4 9 X 1.0 9 = 0.38 ~ which was used to measure
5 L-aspartase activity (see Table I).
b) In a similar manner 10.0 g of
anhydrous, weakly basic, =N-H form, ion exchange
beads (Rohm & Haas, Amberlite ~ IRA 45, 0.42 mm
diameter dried at 85 to 90C and 5 to 20 Torr for
several hours~ were treated with a solution of
2.0 g P~PI ~ 20 in 50 ml dry toluene. Removal of
the toluene at 85 to 90C and 5 to 20 ~orr over a
period of an hour gave a somewhat sticky but still
mobile mass of coated beads. To this tumbled
mixture was added a slurry of 1.0 9 E. coli ATCC
3;~ 3
11303 paste in 1.0 ml of 0.1 M sodium phosphate
(pH 7.5) buffer solution. To aid in achieving a
homogeneous coating of this rather gummy mass,
sixty 0025" diameter Teflon ~balls were added.
s The resultant mixture was tumbled at 25C and 5 to
20 Torr for two hours to yive, after removal of
the Teflon ~ balls, 15.6 9 of dry free flowing
coated beads. A 5.0 ml aliquot of these coated
beads weighed 2.B g and did not change volume
lo after soaking in 1.5 M ammonium fumarate
solution. This 5.0 ml aliquot was used for
L-aspartase measurements (see Table I) and was
estimated to contain 0.18 9 of E. Coli
(15 0 q X 2.8 g = ~.18 ~ -
Example 4
Preparation of E coli Containing L-
Aspartase Immobilized With Polyazetidine Gels.
a) A mixture of 2~0 g of an aqueous
polyazetidine solution (Hercules Polycup ~ 172, as
received 12~ solids in H20) and 2.0 g of E. coli
ATCC 11303 paste was stirred rapidly with a
magnetic stirring bar for five minutes at 25C, to
insure a homogeneous dispersion. This mixture was
then poured out onto a polystyrene surface to dry
at 16 hours at 25C. The resultant 2" X 2" X
1/16" thick flexible membrane was stripped from
the surface and found to weight 0.7 9. ~his film
was used in L-aspartase measurements (see Table I3
and was estimated to contain 2.0 g of ~. coli0 cells (hydrated state).
b) A homogeneous mixture of 2.0 g
Polycup ~ 172 and 2.0 9 of E. coli ATCC 11303 was
disteib~ted onto 30 g of hydrated 5A, 8-12 mesh
~2a~95(?3
23
molecular sieve beads by means of tumbling at 25C
and 5 to 20 Torr for 45 minutes. After 16 more
hours drying at 25~C in open air the resultant
coated beads weighed 30.4 9. A 5.0 ml aliquot of
these coated beads weighed 3.9 9. This sample was
usPd for L-aspartase ~ctivity (see Table I) and
was estimated to have 0.26 g of E. coli cells
(3 9 X 2.0 g = 0.2~ g) -
lo c) A homogeneous dispersion of 2.0 9
E. coli ATCC 11303 in 2.0 g of Polycup ~ 172 at
25C was dispersed onto 30.0 9 of Amberlite R IRA
45 ion exchange beads (as received, 42% H20, =N-H
form~ by hand mixing with a wooden stisk for 30
minutes at 25C. A thin layer of the somewhat
sticky mixture was allowed to stand in air at 25C
for 16 hours. The resultant free flowing beads
weighed 25.8 9. ~ 3.0 9 sample of these dry coated
beads had a volume of 5.0 ml. Upon soaking in 1.5
M ammoniwm fumarate solution the volume expanded to
5.5 ml. A 5.0 ml aliquot of these wet coated beads
was taken for L-aspartase measurements ~see Table
I) and the estimated E. coli content was 0.21 9
(3 0 9 X 200 9 X 55-~ = 0.21 9) -
d) A homogeneous dispersion of 2.0 g
E. coli ATCC 11303 in 4.0 g deionized water and 2.0
g Polycup @9 172 was added over a period of 3.5
hours in three equal portions to 10.0 g of
Amberlite ~IRA 45 (as received, 42~ H20, free =N-H
form) containing sixty 0.25 diameter ~eflon ~
balls. This mixture was tumbled at 25C at 5 to 20
Torr. After a total of five hours of drying at
reduced pressure the resultant dry~ coated beads
(Teflon ~ balls removed) weighed 7.8 g, A 5.0
~20956~3
24
aliquot of these dry, free ~lowing coated beads
weighed 2.9 g. After saturating with 1.5 M ammonium
fumarate solution the volume swelled to 6.25 ml7 A
5.0 ml aliquot of these wet, coated beads was taken
for L-aspartase measurements (see Table I)o The
estimated E. coli content o this aliquot was
~( C_~ X 2.9 9 X 56 5 = 0-59 9) -
e) A homogeneous dispersion of 3.0 9
lo E. coli ATCC 11303 in 6.0 ml deionized water plus
3.0 g of Polycup ~ 172 was spread onto 10.0 g of
Amberlite ~IRA 45 (as received, 42% H20; free =N-~
form) in the same manner of drying at reduced
pressure described above. A total of 10.79 dry,
coated, free flowng beads were obtained. A 5.0 ml
aliquot oE dry coated beads weighed 3.0 g. After
soaking in 1.5 M ammonium fumarate solution this
volume swelled to 5~75 ml~ A 5.0 ml aliquot of the
wet, coated beads was taken for L-aspartAse
measurement (see Table 1)~ The estima~ed E. coli
content of this 5.0 ml aliquot was 0.73 g
3O~ g X 5 -5- 0-73 9) -
f) A homogeneous dlspersion of 4.0 9
E. coli ATCC 11303 in 4.0 9 of Polycup ~ 172 and
8.0 g deionized water was added in approximatelysix equal portions over a 5.5 hour pericd at 25C
and 5 to 20 Torr to 10.0 9 of Amberlite ~ IR~ 938
beads (Rohm and Haas, 73% H20, 0.38 mm diameter,
tert. amine chloride salt form) containing eight
0.5 inch diameter Teflon ~ balls. A~ter 6 hours
total of tumbling at 25C and 5 to 20 Torr the
resultant dry, coated free flowing beads weighed
10.3 g and had a volume of 16~6 ml, Soaking ~hese
beads in excess 1.5 M ammonium fumarate solution
~L2'~ 3
(pH B.5) for 16 hours at 25C gave 17.5 ml wet
beads. A 5.0 ml aliquot of the wet beads,
estimated to contain 1.14 9 E. coli ATCC 11303
(7 gmlX S.0 ml = 1.14 9) ~ was taken for L-
aspartase activity measurements (see Table I).
Example 5
Coating Molecular Sieve Bead~ and Ion
Exchange Resin Beads with E. coli Cells Containing
L-Aspartase Using No Polymer Binders.
lo To demonstrate the utility of the above
described polymer systems used to immobilize the
Eo coli cells containing L-aspartase, the
_ _
following experiments were conducted to coat
materials with the cells containing L-aspartase
alone, i.e., with no polymer bindees.
a) A homogeneous slurry of 1.0 g E. coli
cells, ATCC 11303, containing L-aspartase in 5.0
ml of Na phosphate buffer (0.1 M, pH 7.5) was
added in four equal portions over a period of 3.5
hours to 10.0 9 of hydrated 5A molecular sieve
beads (8-12 mes~) also containing ten O.S inch
diameter TeElon @~ balls. This mixture was tumbled
at 25~C and 5 to 10 Torr. After a total of about
4.0 hours the resultant dry, coated, free flowing
beads weighed 9.9 9. A 5.0 ml aliquot of these
dry beads weighed 4.2 g. The volume did not
change upon soaking in 1.5 M ammonium fumarate
solution. The estimated E. coli cell content o
this aliquot of coated beads was 0~42 9
(1 902 9 X 4.2 = 0.42 ~ ~ A 5.0 ml aliquot of
the coated beads was gently stirred in 50 ml 1.5 M
ammonium fumarate solution at 25C for 24 hours~
09503
26
~he solution became very turbid indicating con-
siderable amounts of material were sloughing off
the beads. The beads were isolated by decantation
and again gently stirred in 50.0 ml fresh 1.5 M
ammonium fumarate at 25C for L-aspartase activity
measurement (see Table I). However, even after
one hour the solution became very turbid again.
On several more occasions the beads were isola~ed
and gently stirred in feesh l.S M ammonium
lo fumarate solution. The beads continued to give
ofE considerable amounts of material to make the
solutions very turbid. This behavior was not
observed in any of the preparations of L-aspartase
active cell coatings of beads where the polymer
binders were also added to the formula~ion. In all
the polymer examples the ammonium fumara~e
solutions over the coated beads remained clear.
b) In a similar wayy a slurry of 10.0 9
E. coli ATCC 11303 in 30 9 of aqueous phosphate
bufEer solution (O.i M, p~ 7.5)~ but no polymer
binders, was coated onto 50.0 g of Amberlite ~IRA
4~ (as received from Rohm and Haas, 42% H20, =N-H
form) over a period of 4.5 hours at 25C and 5 to
20 Torr. The resultant 47.6 g of free Elowing
dry, coated beads had a volume of 83.5 ml. Upon
soaking these beads in excess 1.5 M aqueous
ammonium fumarate the solution became thick and
opaque from the massive amount of material
detached from the beads. All attempts to Eilter
(cotton wadding, fiberglass mat, 43 mesh nylon
screen, medium porosity sintered glass3 off the
beads from this opaque mother liquid failed
because the thick gelatinous particles quickly
plugged the filters. This behavior was not
observed in any of the examples where the polymer
~2~5~
27
binders were incorporated into the formulation.
In these cases the aqueous solutions over the
beads remained clear at all times, and flowed
quickly through filters.
ExamPle 6
L~Aspartase Activity Measurements
Shown in Table I are the L-aspartase
activities of the immobilized Eo coli ATCC 11303
compositions described in the preceding examples.
In each case the samples were preconditioned by
gently shaking in 50 ml of a 1.5 M ammonium
fumarate solution in deionized water (pH 8.5)
containing 0.002 M MgSO4 for a period of 8 to 16
hours at 25C. The solution was then drained from
each sample and 50 ml fresh fumarate solution
added p~ior to gentle shaking again for 8 to 16
hours at 25C. At this point all the samples
prepared with the polymer binders discussed in the
previous examples showed no evidence of material
sloughiny off the solid supports; i.e., their
supernatant solutions were all clear. 5amples
prepared using no polymer binders (Example S)
continued to give very turbid supernatant
solutions even after several washings. This
indicate~ the cell material was not securely bound
to the solid supports.
The next step in each example was to take
a 5.0 ml aliquot ~or in the case of the membrane,
a 2 inch X 2 inch X 0.125 inch section3 of the
washed and well drained composite~ and gently
shake in a fresh 50.0 ml of the above mentioned
ammonium fumarate solutin at 25C. Samples were
removed after 60 minutes and diluted 1/1000 with
deionized water. The decrease in optical density
5~;~
28
at 280 nm from the starting 1.5 M ammonium fumarate
solution (observed to be 0.43 at 1/1030 dilution~
indicated the L-aspartase activity. The optical
density o a 1 5 M L-aspartic acid ammonium salt
~pH 8.5) at 280 nm is essentially zero O0.005).
The observed conversion of the fumarate
to the L-aspartate is shown in Table I as moles of
aspartic acid/hr/liter of catalyst bed and as
moles oE aspartic acid/hr/kg we~ E. coli. For
lo example in experiment ~4d using polyazetidines,
cell paste and ion exchange beads, the optical
density at 280 nm dropped from 0.43 to 0.32 after
60 minutes at 25C. The ¢alculations are:
Q-43-0-32 X 1.5 mol~s X 50 ml X 1000 ml
0.43 1000 ml 5.0 ml
= 3.8 moles ASP/hr/liter catalyst bed
0.43-0~32 X 1.5 moles X 1000 g/kq
0.43 0.59 g wet cells
1000 ml 32t5 mlles ASP/hr~kg
Also shown in Table I are values calculated erom
data in the literature showing the L-aspartase
20 performance at 37C of a polyurethane foam
(HYPOL ~ containing E~ coli ATCC 11303. Here a
0.777 liter bed having 126 9 o cells required 32
minutes to convert 2.0 liters of 1.0 M fumarate
100% to L-aspartate at 37C. These workers also
report the average rate of conversion of 1.5 M
fumarate to be 8% lower than 1.0 M fumarate
solution. They also observed rates at 37C to be
1.67 times rates at 25C. Thus a fair estimate of
their L-aspartic acid production rate for
comparison to our data (25Ct 1.5 M fumarate) is:
5~3
29
_ 2.0 1 - X 1.0 moles/l X 0.92 X
32 hrs X 0.77 1 bed 1.67
= 2.69 moles L-ASP
hr X 1 cat. bed
_ 2.0 1 X 1.0 moles/l X 0.92 X
0.126 k~ cell X 32 hrs 1.67
= 16 4 moles L-ASP
hr X kg ~ells
Comparing these values to the data obtained repre-
sentative of the present invent;on shows the
superiority of the present process over the
polyurethane foam system.
~IL;2~5~51~3
TABLE 1
ASPARTASE ACTIVITY OF BOU~D
Moles of _SP
5 EX~MPLE ~ B INDING MF.THt)D lhr X li ter cat . bed )
CMC + Fe~++ on M.S. beads0.87
2a P.U. Hydrogel via drying on 0.45
M . S O beads
2b POU. Hydrogel via poly EI0 . 27
lo on M.S. bead~
3a PAPI 20 on M.5. beads 2.5
3b PAPI 20 e)n I .E . beads1. 2
4a Polycup 6~) 172 film
4b Polycup (~) 172 on M.S. beads 0.6
4c Polycup ~) 172 on I.E. beads 1.0
4d Polycup ~)172 on I ~E. beads 3.8
4e Polycup g) 172 on I.E. beads 3.9
4f Polycup ~)172 on I.G. beads 12.75
5a E. coli alone (No binder)2.6*
on M.S. beads
-~ HYPOL ~ foam ~estimate from 2.69
data of Fusee et al) 2
* Massive amounts of cells sloughed of f the
beads, thus much of the activity shown i5
2 s caused by unbound cells .
-
5(~3
31
TABLE 1 (Continued)
ASPARTASE ACTIVITY ~F BOUND
E COLI (ATCC 11303) AT 25C
Est. g of E. Coli
Moles sf ASPused (per 5.0 ml
EXAMPLE # (hr X kg wet E. coli) bed sample)
1 185 ~.027
2a 57.7 0.039
2b 33.8 00040
3a 32.9 0038
3b 33.3 0.18
4~ 65.0 0.30
4b 11.5 0.26
4c 23~8 0.21
4d 32.5 0.59
4~ 2Ç~7 0.73
4f 55.9 lo 14
5a 30.9* 0~42*
~~ 16.~
20 * Ma~sive amounts of cells sloughed off the
beads, thus much of the activity shown is
caused by unbound cells.
~2~9~3
32
Example 7
Continuous Produc~ion of L-Aspart.i~ Acid
~as ammonium L~aspartate).
A 5.0 ml aliquot of the wet beads from
Example ~4d were placed in a 0.7 cm diameter X 15
cm jacketed glass column. The column temperatu~e
was maintained at 37C. A fresh 1.5 M ammonium
fumarate solution in deionized water containin~
0.002 m MgSO4 (all at pH B.5) was continuously
lo passed up thorugh the bed of beads at a rate of
0.5 ml~min. Samples of the effluent product
solution were taken periodically and analyzed for
L-aspartate contenk as 1/1000 dilutions in
deionized water as described in Example 6. The
bulk of the efEluent was collected but not
returned to the column. The Eollowing data shows
that over a period of 50 days of continuous
running the L-aspartase activity of the coated
beads remained essentially unchanged.
L-aspartate content
Time~ day~L f effluent (Percent
5g .6-)
61~5 ~ 62~5 average
6~.5~
At the 62.5% conversion rate or a flow of 6.0 bed
volumes/hr ~0.5 ml/min through 5.0 ml of catalyst
bed) the production rate o L-aspartic acid iso
6.n 1 X 0.625 X 1 5 mol~s _ 5.6 moles L-ASP
hr X 1 cat bed 1 hr X 1 cat bed
';~
33
For a period of one hour at 37C the same column
was run at the rate of 4.0 bed volumes/hr (0O33
ml/min through 5.0 ml of catalyst bed) and the
average rate of Eumarate (1.5 M) conversion was
80.0~. This corresponds to:
~ X 5.6 - 7-2 moles L-ASP
62.5 hr X 1 cat. bed
Based on these conversion rates of 6205% and 80.0%
at 6.0 and 4.0 ~ed vol/hr respectively, a
conservative estimate by graphical methods, of the
flow rate required to achieve 100% conversion is
1.5 bed volume~. At 1.5 bed volumeS/hr the L-~SP
hr
production rate is:
_ 1.5 1 X l.5 moles L-~SP = 2.25 moles L~ASP
hr X l bed 1 hr X l cat bed
These values (100% conversion at 1.5 bed vol/hr,
and 2.25 moles h-ASP are significantly better than
hr X 1 cat bed
values of 1.1 bed vol/hr for 100% conversion and
1.65 moles L-ASP repor~ed for the continuous
hr % l cat bed
operation of a column of E. coli ATCC 11303 cells
immobilized on k -carrageenan gel.
In a fuether variation of the invention,
it is contemplated that the aqueous ammonium L-
aspartate solution obtained in Example 7 may be
used directly, i.e. without recovery or separation
of dry, solid aspartic acid, for reaction with
benzyl chloroformate to form the benzyloxy
carbamate of the amine oE aspartic acid, this
carbamate being an important intermediate in the
preparation of the commercially important
34
sweetener, L-aspartyl-L-phenylalanine methyl
ester. Presently this benzyloxy carbamate i~ made
by starting with dry, crystalline solid L-aspartic
acid. The acid is dissolved in aqueous sodium
hydroxide prior to reaction with benzyl
chloroformate according to the following
reactions:
Q O
Il ll
HOCCH2-CH-COH + 2 NaOH
~H2
dry solid aqueous
~'
O O
~1 11
NaOCCH2-CH~CONa ~ 2H20
NH2
0cH
O O
Il 11
NaOCCH2fHCOH + NaCl (aq solu)
NHCOCH2125
The immobilized whole cell enzymatic process for
converting ammonium Eumarate to ammonium L-
lo aspartate according to the invention provides
~3
directly ~ basic aqueous produc~ stream of, for
example, 1.5 molar 98.5% ammonium L aspar~ate
solution which, after removal of ammonia, is
suitable for direct use in the reaction of enzyl
chlo~oformate (or other chloroformate) to make the
desired aqueous solution of benzyl (or other)
oxycarbamates. This shsuld make it possible to
avoid the need to isolate dry aspartic acid
crystals and the time and expense incident
lo thereto. For example, the isolation of dry
aspartic acid crystals from the ammonium L-
aspar~ate solution of Example 7 would generally
r~quire the following:
(1) acidification of the 1.5 molar ammonium
L aspartate product (L-ASP) stream using
approximately 0.7 to 0.8 moles of ~2S04
to precipitate th L~ASP at pH of 2.8.
This offers a prospective corrosion
problem and the expense involved in
disposing of the ammonium sulfate by-
product;
(2) although the L-ASP product stream may
contain 98.5~ L-ASP (the rest is
ammonium fumarate), the precipitate of
L-ASP and washing with water gives only
an 89-93% yield of solid L-ASP. The
retention of 5-9% of the 1-ASP in the
mother liquor is an appreciable loss,
considering the value of the L-ASP.
(3) subsequent drying of the precipitated L-
A5P crystals is also costly (estimated
at about 5-10 cents per pound3.
Use of the aqueous ammonium L-ASP product
directly, according to the invention, eliminatQs
the disadvantages inherent in steps ~1) (3~.
503
3~
Ad~ustment of the ammonium L-ASP product stream is
appropriate to obtain optimum results. For
example, the addition of sodium hydroxide (in the
order of 0.5 to 2.0 moles/mole ammonium L-ASP)
results in displacement o~ the NH4~ as NH3 which
can be boiled off and used to make additonal
ammonium fumarate. Most of the NH4~ ion should be
removed to facilitate the chloroformate reactionO
The invention is not limited to the
lo immobil~zation oE _ coli cells or the use of such
immobilized cells for the production of L-aspartic
acid. Thus, other cells may be immobilized in
generally similar fashion using the indicated
curable polymeric mater;als. Such further immo-
bilized cell variations, and uses thereof, areexemplified in ~i~e following examples:
Example 8
Production of L-Alanine Via Immobiliza-
tion of a Pseudomonas dacunhae Immobilized with
Polyazetidine Gels.
Pseudomonas dacunh _ ATCC 21192 was
cultured on a medium of peptone (Bacto--Difco),
9.0 g/liter~ casein hydrolysate (Casamino acids;
Difco), 2.0 g/liter; KH2P04, 0.5 g/liter;
MgS0~-7H~0, 0.1 g/liter; and L-glutamic acid
sodium salt, 15.0 g/liter, pH adjusted to 7.2 with
NH40H. The Eermentation was carried out under
aerobic conditions for 23 hours at 30C, 300
rotations/min. A mixture of 3.62 grams ~wet
weight) of cell paste and 3.6 grams uf aqueous
polyazetidine solution (Hercules Polycup ~172)
was stirred to homogenity at 25C by hand mixing
with a wooden stick. This mixture was dispersed
on 3.6 9 of Amberlite ~IRA 938 ion exchange bead
5a!3
37
(Rohm & 8aas) which had ~een previously air dried
at 25-30C for 60 hours to remove greater than
85% of the moisture associated with the beads.
The thin film of the mixture on the beads wa~
allowed to air dry at 25C for 24 hour~. The
resulting free flowing beads occupied a volume of
20 ml. These beads were washed with 5 volumes of
normal saline and an 18 ml. ali~uot was placed in
a flask containing .5 liters oE 1.5 M ammonium
aspartate solution, pH 5.5 (adjusted with ~2SO4)
which was held at 37C. The entiee mixture was
stirred for a period of 48 hours while the pH was
maintained by the addition of sulfuric acid as
needed.
Analysis of the liquid at the end of this
period on HP~C (uNH2 Bondapak column, Waters
Assoc.; eluant CH3CN, 25~, 5 mM KHPO4 pH 4.4, 75%3
showed that L-aspartic a~id had been decarboxy-
lated to form L-alanine in 98% yield.
Example 9
Preparation of Penicillin-G Acylase
Containing Bacillus meqaterium Immobilized With
Polyazetidine Gels.
Bacillus meqaterium ATCC 14945 was
cultured under aerobic conditions as previously
described (U.S. Patent 3,446,705). A mixture of
10.5 9 (wet weight) of the cell material and 10.5
g of an aqueous polyazetidine solution (Hercules
Polycup ~172) was stirred to homogeneity at 25C
by hand mixing with a wooden stick. This mixture
was dispersed onto 9.8 9 of Amberlite ~IRA 938
ion exchange beads (Rohm and Haas) which had been
previously air-drled at 25-30C for 60 hou,rs
which removed> 85% of the moisture from the beads.
~3
38
A thin layer of the mixture was allowed to air dry
at 25C for 24 hours. The resultant free flowing
beads weighed 13.1 g and occupied a volume of 53
ml. A 1247 mg aliquot of these beads corres-
ponding to 1 gram of ree cells was assayed forpenicillin acylase activity. 6-Aminopenicillanic
acid production was quantitated by high pressure
liquid chromatography (HPLC). The activity of the
immobilized preparation was 19.7 ~ moles hr 1 9
lo cell paste 1. This value was 67% of the free cell
activity.
Ex amPl e 10
Preparation of Penicillin-G Acylase
Containing Escherichia coli Immobilized With
Polyazetidine Gels.
Escherichia coli ATCC 9637 was cultured
aerobically as described by 5ato et al, Eur. J.
A~pl. Microbiol. 2:153-160 (1976). A mixture o
20.7 9 (wet weight) of cell paste and 20.7 g of
~o aqueous polyazetidine solution ~Hercules Polycup
172) was stirred to homogeneity at 25C by hand
mixing with a wooden stick. This mixture was
dispersed onto 19.3 g of Amberlite ~IRA 938 ion
exchange beads (Rohm and Haas) which had been
previously air-dried at 25-30C for 60 hours to
remove >85% of the moisture associated with the
beads. A thin film o~ the mixture was allowed to
air dry at 25C for 24 hours. The resultant free
flowing beads weighed 27.4 9 and occupied a volume
of 95 ml. A 1325 mg aliquot of these ~eads
corresponding to 1 gram of free cells was assayed
for penicillin acylase activity. 6-Amino
penicillanic acid production was quantitate~ by
high-pressure li~uid chromatography (HPLC). The
39
activity of the immobilized preparation was
53.6 ~ moles hr~l gram cell paste~l which was 99%
of the free cell activity.
ExamPle 11
Immobilization of Arthrobacter species
Containing Glucose Isomerase Activity for Produc-
tion of ~igh Fructose Corn Syrup.
Arthrobacter species ATCC 21748 was grown
under submerged aerobic conditions and the cells
lo were harvested. The cells had a glucose isomerase
activity of 420~ moles hr~lgm~l cell paste (wet
weight) at 60~C.
To 47 gm of Polycup ~ 172 was added 47 gm
of Arthrobacter species ATCC 21748 (wet weight~
and the mixture was stirred vigorously for 5-10
minutes. The Polycup ~/cell mixture was then
added slowly to 46.4 gm of air dried IRA @9938
beads with mixing and the resultant beads were
dried at rsom temperature for 24 hours.
To a flask containing 5 ml oE the
Arthrobacter species ATCC 21748 immobilized beads
was added 50 ml of 0.2 M phosphate buffer, pH 7.5,
10 mM MgS04, 1 mM CoC12 and the solution was
heated to 60C with stir~ing. 450 mg ofO~-D-
glucose was added to the flask and the flask was
placed in an incubated shaker at 60C for 1 hour
at 150 rpm. The glucose isomerase activity of the
immobilized Arthrobacter species ATCC 21748 was
530~ moles-hr l gm 1 cell paste (wet weight) at
60C as determined by the cysteine/H2SO4 reaction.
[(Dische, Biochim. Biophys. Acta, 39, 140 (1960)].
10 Ml of the Arthrobacter species ATCC
21748 immobilized beads were added to a 0.9 x 30
cm jacketed glass column which was maintained at
~g~ 3
60C by a circulating waterba~h. Prewarmed
substrate solution containing 250 mM glucose, 5 mM
MgS04 and 0.5 mM CoC12 in 50 mM Tris-HCl buffer,
p~ 7.5, was passed through the column. After 6
days of continuous operation the glucose isomerase
activity of the immobilized Arthrobacter species
ATCC 21748 was 280 ~ moles-hr l gm 1 cell paste
(wet weight) at 60C and after 37 days the glucose
isomerase activi~y was 18 ~ moles hr~l-gm~~ cell
lo paste (wet weight) at 60C.
Example 12
Immobilization of Arthrobacter simplex
Having Steroid Dehydrogenase Activity for The
Production of Prednisolone or Related Steroids.
Arthrobacter simplex ATCC 6946 was grown
under submerged aerobic conditions and the cells
were harvested. The cells have a ~ -l-dehydro-
genase activity of 980 ~ moles-hr l-gm~~ cell
paste ~wet weight) at 34C.
To 20 gm of Polycup ~172 was added 20 gm
of A. ~ ATCC 6946 and the mixture was
stirred vigorously for 5-10 minutes. The
Polycup ~/cell mixture was then added slowly ~o
18.6 gm of air dried IRA ~938 beads with mixing
and the resultant beads were dried at room
temperature for 24 hours.
To a Elask containing 5 Ml of the A ~
simplex ATCC 6946 immobilized heads was added 36
Ml of 20 mM phosphate buffer (pH 7.0) followed by
4 Ml of ethanol containing 60 mg of hydrocortisone.
The flask was placed in an incubated shaker at
34C for 1 hr at 150 rpm~ The~ dehydrogenase
activity of the immobilied A. simplex ATCC 6946
~Z(~95C~3
41
was 440 ~ moles hr~l gm ~ cell paste (wet weight)
at 34C as determined by absorbance at 285 nm.
Example 13
Immobilization of a StrePtomyces species
Having Glucose Isomerase Activity for the Produc~
tion of High Fructose Corn Syrup.
Streptomyces phaeochromogenes NRRL B3559
was grown under submerged ae~obic conditions and
the cells were harvested. The cells had a glucose
lo isomerase activity of 7.7 ~ moles min~l-gm~l cell
paste (wet weight) at 60C.
To 64.5 gm of Polycup ~ 172 was added
64.5 gm of S. ~haeochromogenes NRR~ B35S9 and the
mixture was vigorously stirred 5-10 minutes. The
Polycup ~/cell mixture was then slowly added to
60.0 gm of air dried IRA ~938 beads with mixing
and the resultant beads were dried at room
temperature or 24 hours.
10 Ml of the S. phaeochromo~enes NRRL
B3559 immobilized beads were added to a 0.9 x 30
cm jacketed glass column which was maintained at
60C by a circulating waterbath. Prewarmed
substrate solution containing 250 mM glucose, 100
mM Na2S03, 10 mM Mg504 and 1 mM CoC12 adjusted to
pH 7.0 with HCl was passed through the column at a
flowrate of 28 ml/hr~ The glucose isomerase
activity of the immobilized S. phaeochromo~enes
NRRL B3559 was 7.7~ moles-min l~gm 1 cell paste
(wet weight) at 60 C as determined by the
cysteine/H2SO4 reaction (Dische et al, supra~.
~.~09~03
42
Example 14
Immobiliztion of a Rhodosporidium Species
High In Phenylalanine ~mmonia Lyase for the
Production of Phenylalanine~
Rhodos~or dium toruloides ATCC 10788 was
grown under submerged aerobic conditions and the
cells were harvested. The cells had an L-
phenylalanine ammonia-lyase activity of 112~x
moles~hr~l~gm~l cell paste (wet weight) at 30C.
To 14.9 gm of Polycup ~ 172 was added
14.9 gm of R7 toruloides ATCC 10788 and the cells
mixture was vigorously stirred for 5-10 minutes.
The Polycup ~/cell mixture was then added slowly
to 13.8 gm of air dried IRA ~938 beads with
lS mixing and the resultant beads were dried at room
temperature for 24 hours7
To a flask containing 1 Ml of the R_
toruloides ATCC 10788 immobilized beads was added
5 Ml of 25 mM Tris-HCl bufEer (pH 808), 25 mM ~-
phenylalanine, 0.005% cetyl pysîdinium chloride.
The flask was placed in a Dubnoff shaking
incubator at 30C for 1 hour at 100 rpm. The
phenylalanine ammonia-lyase activity of the
immobilized R. toruloides ATCC 10788 was 10~
moles~hr l gm 1 cell paste ~wet weight) at 30~C as
determined by absorbance at 278 nm,
Those in the art will appreciate from the
prior literature how the immobilized cell/polymer
compositions disclosed in Examples 8-14 can be
used to make the products indicated. Thus, with
respect to Example 8, it is known that the
production of L-alanine via decarboxylation of
aspartic acid is mediated by the enzymP, L-
aspartic-~ -decarboxylase. The presence of this
enzyme i5 well known in many genera of micro~
~L2~5Q3
~3
organisms, as reported by Chibata et al, Applied
Microbioloq~, Vol. 13V No. 5, pages 638-645
(1965). It is produced by Clostridium
perfri~ens, Desulfovibrio desulfuricans, Nocardia
,
globerula, P _ domonas eeptilivora, Acetobacter
species, Achromobacter species, and Alcaliqenes
faecalisc
=
Chibata et al determined that alanine
forming strains were fairly common in the genera
lo Acetobacter, Achromobacter, Pseudomonas, Torula,
Torulopsis, Absidia, ~ Mucor, and
Oospora. Chibata et al increased the knowledge
p~eviously ob~ained by Meister and coworkers and
determined tha~ L-aspartic acid was converted
stoichiometrically to alanine. Isolated yields of
L-alanine over 90% from L-aspartic acid were
easily obtained. This process is described with
respe~t to the organisms, Pseudomonas dacunhae and
Achromobacter Pestifer in U.S. Patent No.
3,458,4~0, which claims a process for production
oE L-alanine in an aqueous nutrient medium, by
dried living cells, or by cell free extracts. A
modification of this process was patented by
Chlbata et al, 1975, U.S. Patent No. 3,898,1285 by
im~obilization of the microorganism in acrylamide
polymers. A further patent on the use oE the
enzyme from Pseudomonas dacunhae was obtained in
1969, U.9. Patent No. 3,463,704. Continued
research by Shibatani et al, Applied ~ Environ-
mental Microbiology, Vol 38, No~ 3, pages 359 364
(1~79) has shown that additional species have been
found by other workers to possess L-aspartic-~ -
decarboxylase and shows that production of this
enzyme can be stimulated by the addition of
certain amino acids, such as glutamate. An
44
improved continuous method of production was
published by ~amamoto et al, Biotechnoloqy ~
Bioenqineerin~l, Yol. XXII, pages 2045-2054 ~1980),
when the whole OrganiSM was immobilized in
s carrageenan gel. Additionally, others have worked
on the production of alanine from Pseudomonas
.
dacunhae and Alcaligenes faecalis immobilized in
HYPOL ~foam lFusee et al, American SocietY for
Microbiology Abstracts, Dallas (March 1980~].
The present invention contemplates using
the exemplified cell/polymer composition to
produce L-alanine from aspartic acid in lieu of
the previously used cell compositions.
A great deal of work has also previously
been done with respect to the immobilization of
the enzyme penicillin acylase~ Seet for example,
Biochimie, 62: 317-321 (1980). Marconi et al have
immobiliæed penicillin acylase in cellulose
triacetate fibers [Biotechnolo~ and
Bioen~ineerinq 22:735~756 ~1980); Biotechnoloqy
and Bioen~ineerin~ _:1057-1073 (1979)], and a
numbee of U.S. patents have issued on the enzyme
immobilization: U.S. patents 3,278,391; 3,116,218;
3,190,~86; 3,446,705; 3,622,46~; 3,736,230;
2s 3,766,00~; 3,801,962; 3,~83,394; 3,900,488;
3,499,909; 3,736,230; 4,001,264; 4,113,566; and
4,230,804.
It appears that relatively few papers on
whole cell immobilization are available. Tanabe
30 Seiyaku has published a report of cells entrapped
in polyacrylimide gel. Chîbata et al (1974),
German Patent 2 9 414,128, and Mandel et al,
published on entrapment in polyacrylamide gel
[Prikl. Blkhim. Mikrobiol. 11-219-225 (1975)3.
Immobilized whole cells of Bacillu~ meqaterium or
9L;~ Q~
Achromobacter adsorbed on DE~E cellulose have been
used by Toyo Jozo, Fujii et al, Japanese patent
73 99393 and Sato et al, 1976, Eur. J. A~plied
Microbiolo~y 2:153-160 ~1976), have repo~ted on
the production oE 6-APA from pencillin-G by using
immobilized E. coli having high penicillin acylase
activity.
It will be appreciated that various other
modifications may also be made without deviating
from the invention, the scope of which is defined
in the following claims wherein:
