Note: Descriptions are shown in the official language in which they were submitted.
2000721 -
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~ACRGROUND OF THE INV~NTION
This invention relates to a method of making phenyl acetyl
carbinol (PAC), which is useful as an intermediate in the manu-
facture of l-ephedrine and d-pseudoephedrine, This invention
also relates to immobilized cells especially adapted for use in
the manufacture of PAC and to a method for providing the immobi-
lized cells.
Pseudoephedrine and ephedrine are two major medicinal chemi-
cals. Pseudoephedrine i~ useful as a nasal decongestant and is
found as an ingredient in cough and cold capsules, sinus medica-
tions, nose sprays, nose drops and allergy and hay fever medica-
tions. Ephedrine is useful as a topical nasal decongestant, as a
treatment for mild forms of shock (CNS stimulant) and as a bron-
chodilator.
L-ephedrine is a natural product found in various species of
plants. L-ephedrine is obtained from dried plant material by an
initial treatment with alkali followed by extraction with organic
solvent. While d-pseudoephedrine is also found in nature, it iS
more easily obtained in high yield from l-ephedrine by Welsh re-
arrangement.
L-phenyl acetyl carbinol (PAC) is the key intermediate in
the synthe~is of l-ephedrine. The transformation of benzaldehyde
to L-(-)phenyl acetyl carbinol by 8rewer's yeast was first
described by Newberg and ~irsch. 3iochem. Z., 115:282-310
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20QQ72~.
(1921). More particularly~ benzaldehyde can be transformed by a
ferm~nting yeast into L-(-)phenyl acetyl carbinol as follows:
- a
R~S
L-(-)PAC ~ .
The combination of yeast transformation of benzaldehyde to
produce PAC and chemical conversion of the PAC to make 1-
ephedrine is described in U.S. Patent 1,956,950. The PAC can be
converted by a chemical reductive amisation with methylamine to
. optically pure l-ephedrine as follows:
C
,. I
L-(-)PAC L-(-)EPHEDRINE (R,S)
. .
.... . . . ~ . . ~ ` -
Z000~21
The l-ephedrine can then be converted in high yield to d-pseudo-
ephedrine as follows:
C~3
~ U C~3
i~ VE~
. .
:
L-(-)EPHEDRINE(R,S) D-(+)PSEUDOEP~EDRINE (S,S)
It is apparent from this reaction scheme that microbial transfor-
mation of benzaldehyde by yeast to form L-(-)phenyl acetyl
carbinol in high yield and purity is of prime importance for suc-
cessful commercial operation of the s~nthetic route.
Prior processes for the production of PAC from yeast involve
the addition of the yeast to a medium containing molasses, beer
wort, MgS04 and other salts at a pH o~ 5.5-6Ø After an initial
short period of stirring and aeration, a mixture of acetaldehyde
and benzaldehyde is added in portions. A final concentration of
PAC of a~cut 7.5 g/L is obtained in 5-10 hours of continued stir-
ring and aeration. With brewer's or baker's yeast, benzyl alco-
hol is always observed as a co-product. The highest reported
yield of PAC based on benzaldehyde is about 73%. The remaining
benzaldehyde is conver~ed to the alcohol. Acetaldehyde is rot
~ssential for PAC production, but addition of this compound is
required in order to achieve the highest yields of PAC.
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2~0721
Most of the literature concerning the synthesis of PAC by
fermenting yeast deals with yield optimization. There is a gen-
eral concensus that high levels of yeast are needed to obtain
relatively low levels of PAC. The available literature suggests
that the current yeast transformation of benzaldehyde to PAC is
inefficient and yeast productivity is low. The yeast cannot be
used for multiple batches because PAC production drops with in-
creased exposure to the substrates and to the end product.
In addition, current yeast transformation provides only low
concentrations of PAC in the fermentation liquor. This requires
large process volumes and consequently large volumes of extrac-
tion solvent, which adversely impact on labor and utility costs
in commercial operations.
In addition, the yield of PAC fr~m benzaldehyde is decreased
as a result of the catalytic reduction of benzaldehyde by an
alcohol dehydrogenase to form benzyl a!cohol, which is an unwant-
ed by-product. All of the PAC-producing strains that have been
examined produce benzyl alcohol.
Accordingly, there exists a need in the art for an improved
method of making PAC by yeast transformation of benzaldehyde.
The method should provide a higher yeast productivity and higher
maximum concentrations of PAC in the fermentation liquor than
heretofore yossible. In addition, the catalytic reduction of
benzaldehyde to benzyl alcohol should be minimized.
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SUMMARY OF THE INVENTION
Thi, invention aids in fulfilling these needs in the art by
provid ng an improved process for the production of PAC by con-
versi n of benzaldehyde. The process of this invention makes it
pos~ ble to obtain higher yeast productivity and to obtain higher
cor:entrations of PAC in the fermentatlon liquor than in present
p ocesses. In addition, the process of this invention makes it
?ossible to produce PAC while obtaining a lower or essentially
undectable amount of benzyl alcohol as a by-product.
More particularly, this invention provides a process for the
production of L-phenyl acetyl carbinol (PAC), which comprises
providing an immobilized cell mass consisting essentially of non-
viable cells of a mutant yeast strain that exhibits resistance to
aldehyde inhibition during PAC produc- on. The cells contain
endogenous pyruvate decarboxylase. T~e cells in the cell mass
have cell walls, and the walls of adjacent cells are chemically
crosslinked. At least a portion of the cell walls in the cell
mass is modified in order to increase permeability of the cell
mass to reactants. 8enzaldehyde and a source of pyruvate are re-
acted in an aqueous medium in the presence of the immobilized
cell mass to produce L-phenyl acetyl carbinol. The aqueous medi-
um contains a cosolvent, which is a non-inhibitory, water
miscible, organic solvent for the benzaldehyde. The cosolvent is
employed in an amount sufficient to increase the rate of PAC for-
mation and to increase ~he concentration of PAC in the aqueous
~edium of a fermentation reaction over a similar reaction car~ied
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2CI1007Zl.
out without a cosolvent. The product can be separated from thecell mass if desired.
This invention provides a similar process for the production
of PAC in which the mutant yeast strain in the immobilized cell
mass is SaccharomYces cerevisiae P-2180-lA-8pa. Benzaldehyde and
a source of pyruvate in an aqueous medium are reacted in the
presence of the immobilized cell mass containing the mutant
yeast. PAC can be separated as the product from the conversion
of the benzaldehyde.
In addition, this invention provides a cell mas,s for the
conversion of benzaldehyde to L-phenyl acetyl carbinol. The cell
mass consists esssentially of non-viable cells of a mutant yeast
strain that exhibits resistance to aldehyde inhibition during PAC
production. The cells contain endoger~us pyruvate decarboxylase.
The cells in the cell mass have cell ~alls, and the walls of
adjacent cells are chemically crosslinked with polyazetidine. At
least a portion of the cell walls is modified in order to in-
crease permeability of the cell mass to reactants.
Finally, this invention provides a process for preparing the
cell mass of the invention. The process comprises providing
cells of a mutant yeast strain that exhibits resistance to
aldehyde inhibition during PAC production. The cells have celi
walls and contain endogenous pyruvate decarboxylase. The cells
are mixed with polyazetidine in an amount and under conditions
adequate to chemically crosslink walls of adjacent cells to form
a self-supporting mass of crosslinked cells. The amount of
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~007Zl
polyazetidine and the condition of crosslinking are such that the
cell mass is permeable to substrates and products.
BRI~P DESCRIPTION OF TH~ DRAWINGS
This invention will be more fully understood by reference .o
the drawings in which:
Figure lA is a scanning electron micrograph (360X) of
S. cerevisiae yeast cells that have been crosslinked with
polyazetidine and Figure lB is an enlargment (1800X) of a portion
of the cells;
Figures 2 and 3 show the effects of a cosolvent on PAC for-
mation using immobilized cells according to the invention; and
Figure 4 depicts schematically batch and column production
metr.ods of PAC.
D~SCRIPTION OF THE PR~FER~ED EM~ODIMENTS
The process of this invention is especially adapted to pro-
duce L-phenyl acetyl carbinol (PAC) in high yield. The abbrev.a-
tion "PAC" is used herein to refer to the stereospecif iG form oÇ
phenyl acetyl carbinol identified as L-(-)phenyl acetyl carbinol.
The designation L-(-)phenyl acetyl carbinol is uced intercrange-
ably with the designation L-phenyl acetyl carbinol and both des-
`~ ignations are abbreviated as "PACn.
' PAC is prepared by the transformation of benzaldehyde ard
pyruvate. The expression "pyruvate~ is used in its convent.onal
sense as referring to the moiety
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,
The transformation of benzaldehyde and pyruvate is carried
out in the process of this invention with a mutant strain of a
microorganism. The term ~mutant" as used herein is intended tO
include all pro~eny of a parent microorganism in which there is a
difference in genotype between the parent strain and its progeny.
The term is also intended to include progeny in which there is a
phenotypic difference from the parent strain without a difference
in genotype. Of course, the term additionally includes progeny
that exhibit differences in both genotype and phenotype from the
parent strain.
More particularly, the method of the inveneion is carried
out with mutants of yeast microorgani~ms that efficiently convert
pyruvic acid and benzaldehyde to L-ph~nyl acetyl carbinol. The
species of microorganisms employed in the conversion contain -
endogenous pyruvate decarboxylase. The abbreviation "PDCase~'
when used herein means pyruvate decarboxylase enzyme. Exa~ples
of suitable microor~anisms are mutants of SaccharomYces
cerevisiae and Candida flareri.
Pyruvate decarboxylase catalyzes the conversion of
benzaldehyde to PAC. This enzyme is also capable of convertin~
the pyruvate to acetaldehyde. The formation of acetaldehyde is
believed to inhibit the enzyme, which is the apparent cause o a
~ecrease in the yield of PAC ~rom benzaldehyde.
.. ... .. . .
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200072~
This invention utilizes m~ltant strains with resistance to
aldehy~e inhibition during PAC production. By this it .s meant
that, in comparison to the parent strain, there is a reduction in
the inhibition of activity of the mutant strain in the conversion
of benzaldehyde as evidenced by the concentration-of PAC in the
reaction medium. The concentration of PAC in the reaction medium
is higher with the mutant strain than with the parent strain when
fermentations carried out under otherwise identical conditions
are compared. In the preferred embodiments of this invention,
the mutant strains also produce less acetaldehyde and less benzyl
alcohol from the benzaldehyde than the parent strain.
The mutant yeast cells are crosslinked to form a mass of im-
mobilized cells,~which can be added to a reaction vessel to form
a bioreactor. Techniques for prepar;:g the immobilized cells and
for the use of the immobilized cells n a method of producing PAC
will now be described. A descriptior of methods for making the
mutant strains will then be provided.
1. Preparation of Immobilized Cell Mass
The conventional PA~ synthesis process involves the use of
freshly grown cells for each batch reaction. Upon completion of
the reaction (e.g., synthesis cessation due to product-
inhibition, etc.) the spent cells are discarded and the PAC prod-
uct is puri-fied. In addition, when using the conventional pro-
cess, the cells must be speciaily treated to maintain their
viability. For example, temperature control, pH control and
~aintenance of low benzaldehyde concentrations are usuaily
ritical.
007Z~.
In this invention, mutant yeast cells containing endogeneous
pyruvate decarboxylase are crosslinked to form a mass of immobi-
lized cells. Immobilizins whole cells containing endogeneous
pyruvate decarboxylase as a catalyst for PAC production allows
for extended and continuous use of the catalyst. In addition,
lar~e batches of the immobilized cells housing intact PDCase can
be prepared and stored for future use. Immobilizing and using
non-viable whole cells, instead of viable cells or isolated
PDCase, also saves considerable expense because the immobilized
cells do not have to be disrupted and the enzyme need not be
purified for use.
By crosslinking the cells to each other, no costly inert
. ....
physical support is required. Furthermore, the catalyst
crosslinked in this fashion has adequlte handling properties
(i.e., a density of 7 1, flowability, and a lack of caking), and
is also large enough to be easily retained in both batch and col-
umn operations.
Many classes of yeast cell lines containing endogeneous
pyruvate decarboxylase can be employed in practicing this inven-
tion. A number of mutant species of Saccharomyces cerevisiae (S.
cerevisiae) and Candida flareri (C. flareri) have been cuLtured
as highly active PAC producers. These mutants are described in
greater detail below. Use of these mutants is a preferred
embodiment of the present invention. In addition, a number of
bacterial cell lines have been examined, many of which are rea-
sonable wild type PAC producers for the purposes of the presen-
invention.
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The cells can be crosslinked with polyazetidine, which is
commarcially available under trade name Polycup from Hercules,
Inc. A preferred form of polyazetidine suitable for use in the
immobilization process has the structure shown below:
-C~ -N~-cH2-cH2 ~ C~2-UH-
CH~ /H2
Cl
OH n .
This mul~i-functional polymer is available as Polycup 172.
Other forms of polyazetidine exist, e.g., Polycup 2002 and
Polycup 1883, which are believed to react similarly to Polycup
172. In general, any chemically reac-.ve polymer that can
covalently bind to functional groups .n the cell outer envelope
is acceptable. While other crosslinking agents, such as
glutaraLdehyde, have been suggested and are satisfactory,
polyazetid;ne is preferred because polyazetidine generally pro-
vides superior biomass retention and is more resistant to abra-
sion. The use of polyazetidine and other polymeric materials for
crosslinking cells i5 described in U.S. Patent 4,436,813 the
entire disclosure of which is relied upon and incorporated by
reference herein.
Cells can be immobilized by preparing a cell paste with
polyazetidine. The cel1 paste generally contains about 5% by
~eight to about 25~ by weight polyaze;idine, and preferably about
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~00072~
8% to about 15% by weight, on a dry basis, Typically, the cell
paste contains about 2% by weight to about 40% by weight of the
cells on a dry basis, and preferably about 10% to about 30% by
weight dry basis. The cells and the polyazetidine are combined
in a weight ratio of about 1:0.2 to about 1:3, preferably about
1:0.5 to about 1:1, in the cell paste. The resulting mixture can
be dried.
The preferred immobilization technique of this invention in-
volves mixing a cell pas~e, typically at 24% dry weight, with
polyazetidine at 12% dry weight in a ratio of 1:0.5 (cell
paste:polyazetidine) and allowing the resulting polymeric mixture
to air dry on trays at room temperature overnight.
In an alternate embodiment of the invention, the cells can
be crosslinked with polyazetidine to ~arious physical supports,
such as sand, crushed brick, ion exch~nge resins, ceramic beads,
glass beads, zeolites and diatomateous earth. Due to cost con-
siderations, the cell-to-cell crosslinking is preferred over
cell-to-inert physical support crosslinking.
The resulting self-supporting mass of crosslinked cei;s -an
be subjected to size reduction to form freely flowing part c'es
comprising the crosslinked cells. More particularly, the
crosslinked cells can be ground in a knife-mill, and sieve C`~tS
between 0.5 and 1.0 mm taken. Larger particles can be recyc.~c
; through the mill. The resulting preparation is in the form OI
catalyst beads. The catalyst beads prepared in this manrer _ar.
be stored at a temperature of about -20C to about 4C.
-12-
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In an alternate embodiment, a slurry comprising cells, water
and polyazetidine can be poured to form a thicker film and then
ground to size. This procedure will usually expose more of the
porous interior, thereby improving diffusion of both reactants
and products into and out of the immobilized cells.
Scanning electron microscopy of a catalyst bead preparation
shows that the yeast cells are tightly packed by polymeric
crosslinking with the polyazetidine. The immobilization proce-
dure results in crosslinking of the cells in the form of a thin
film, which can be cut int:o sub-millimeter squares. Fig. lA
shows the packing at 360 X magnification. In Fig. lB, individuaL
cells are shown at a magnification of 180Q X. The arrangement of
cells appears to be different on the surface of the plate as com-
pared to the edge of the plate where ~ piece was chipped-off the
corner. The interior portion of the ^atalyst (as viewed at the
chip edge) seems to be more reticulated and porous. This charac-
teristic is believed to be due to differences in drying bet~een
the surface and the interior portions of the film.
once the mass of immobilized celis has been prepared, the
mass can be added to a reaction vessel. If the vessel is no'
filled, suitable retainins means can be inserted in the vessel in
order to hold the cells in a fixed bed.
2. Method of Makinq PAC Usinq the Immobilized Cell Mass
In the conventional process of making PAC using living
cells, pyruvate is generated from exogenous glucose. The conven-
tional process is characterized by:
-13-
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(1) A decreased reaction rate;
(2) Poor conversion efficiency of substrate to product;
(3) Formation of undesirable products due to competing re- -
actions;
(4) The inclusion of multiple media components necessary
for cell growth, which may hinder ease of production
and purification; and
(5) Maintainance of cell viability, while the benzaldehyde
- is toxic to the cell.
These problems are avoided in the process of the present inven-
tion. By using non-viable, immobilized cells housing intact
pyruvate decarboxylase, the immediate precursors of PAC, namely,
pyruvate and benz~ldehyde, can be easily supplied to the cata-
lyst.
The cell mass containing immobilized cells prepared as
described above can be used to produce PAC in an aqueous reaction
mediu~. ~he process of the invention can be carried out in a
conventional bioreactor with submerged ceils of the mutant strain
; and under substantially oxygen deficient or anaerobic conditions.
Since the cells in the cell mass are non-viable, it is not neces-
sary to add a nutrient medlum or an assimilable source of carbon
to the reactor. Simplified procedures for preparing PAC are
shown in Fig. 4.
With reference to Fig. 4, a batch PAC reaction is shown in
the upper portion of the figure. A conventional laboratory
'lask 2 can be provided with a reaction medium, such as a medium
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~~~ 200072 ~
~ontaining 20 cc of reaction buffer and suitable substrate5 for
biomas~ and product production- About 0.2 g cells (dry weight)
or an equivalent cell dry weight of the immobilized cell mass of
the invention can be included in the reaction medium, The flask
can be rapidly shaken at room temperature and PAC recovered from
the reaction medium.
With reference to the lower portion of Fig. 4, a column PAC
method is described. A column 6 can be packed with about 1 g of
the immobilized cell mass 8 of the invention. The resulting
packed bed column is supplied by a pump 10 with a reaction medium
12 from a reservoir 14. ~he flow rate of the reaction medium
from the reservoir 14 to the packed bed 6 is typically about 7-10
ml/hr for a packed bed containing 1 g of the immobilized cell
mass 8. The reaction medium 12 in re~ervoir 14 contains suitable
buffers and substrates for reaction. rhe reservoir 1~ can be
positioned over a magnetic stir plate 16, which rotates a magnet-
ic stirrer 18 in the reservoir. The PAC product from the column
6 can be collected in fraction collectors 20, ~2, 24 and 26.
Typically, 5 ml fractions will be collected.
More generally, PAC production can be carried out with an
immobilized cell mass of the invention or under conditions in
which the cell mass of the invention is mobile. Reaction can be
carried out in a batch reactor or a continuous reactor. When a
batch reactor is employed, the reaction can be carried out in a
true batch or fed batch system. A mechanically agitated fer-
;enter or a fixed bed or fluidized bed containing the cell mass
.. : ~ : . :
X000721.
of the in~ention`can be employed for batch fermentations~ Con-
tinuous fer~entation can be carried out in an immobili2ed cell
reactor, such as a fixed bed reactor, or in a fluidized bed re-
actor. A chemostat, tower fermenter or continuous stirred tank
reactor can also be employed as a continuous bioreactor.
The benzaldehyde employed in practicing the process of this
invention is generally a technical or pharmaceutical grade of
commercially available material. The pyruvate i5 usually derived
from a technical or pharmaceutical grade of pyruvic acid or a
non-toxic, water soluble salt thereof. A non-toxic alkali metal
salt, such as sodium pyruvate, is preferred. ~ -
The pyruvate and benzaldehyde can be individually added to
the reactor if there is sufficient turbulence to ensure uniform
dispersion throughout the cell mass. rn the preferred embodiment
of the invention, the pyruvate and be zaldehyde are mixed togeth-
er in an aqueous medium and the resulting composition is added to
the reactor.
At the start of the reaction, the concentration of
benzaldehyde in the reaction medium is generally about 5 g/L to
about 20 g/L, preferably about 12 g/L to about 15 g/L. Simi-
larly, the concentration of pyruvate in the reaction medium at
the start of the reaction is about 5 g/L to about 20 g/L, prefer-
ably about }2 g/L to about 15 g/L.
The weight ratio of benzaldehyde to pyruvate in the reactior.
medium at the start of the reaction will generally be about 0.5:1
:o about 2:1, preferabl~ about 1:1 to about 1.2:1.
-16-
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~ Z00()72~.
Primary goals of most chemical conversions, including the
present PAC reaCtiOn, are to achieve high rates of product forma-
tion and as high a final concentration of product as possible.
It has been found that higher conversion rates and higher final
product concentrations can be achieved by incorporating certain
organic solvents n the reaction medium. The organic solvent is
a water miscible compound or mixture of compounds in which the
benzaldehyde is soluble to an extent of more than 25mM at the
fermentation temperature. The organic solvent is also
non-inhibitory that is, the organic solvent does not adversely
affect the rate or extent of PAC formation or the stability of
PAC in the fermentation medium. An organic solvent or mixture of
solvents meeting these criteria is referred to herein as a
"cosolvent." A number of different a -ohols can be employed for
this purpose. For example, aliphatic ~lcohols, such as methanol,
ethanol, propanal and butanol can be employed as cosolvents.
More particularly, Fig. 2 shows the rate and extent of 2AC
production with increasing levels of benzaldehyde, with and ~ th-
out a cosolvent. A comparison of curve A, which resulted ~r~r~ a
fermentation using only 25 mM benzaldehyde and no cosolven~, w :h
curve B, which resulted from a fermentation carried out wi~h 2,
mM benzaldehyde and 20% by weight ethanol as a cosolvent, sho~s
that the rate of PAC formation substantially increased with ~e
cosolvent. The amount of PAC produced showed a similar i^c.e~se
at the end of the reaction period. A comparison of curve B ~ -h
-urve C shows that further increases in the rate and amounr o~
.
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-
PAC formation can be achieved by increasing benzaldehyde concen-
tration to 100 mM fr~m 25 mM while using 20~ by weight ethanol as
cosolvent. The cosolvent effect as shown in Fig. 2 is one of
stimulatory action. Twenty percent ethanol stimulated PAC pro-
duction even at concentrations as low as 25 mM benzaldehyde.
In addition to ethanol and other monohydric solvents, short
chain and long chain polyols can also be employed as cosolvents.
Typical short chain polyols are ethylene glycol and glycerol. AS
shown in Fig. 3, both of these solvents have been found to stimu-
late PAC production. Specifically, Fig. 3 depicts the rate and
extent of PAC formation in separate batch fermentations carried
out with fresh cells of the microorganism S, cerevisiae
P2180-lA-8pa in an aqueous reaction medium at 22C and pH 6 using
220 mM benzaldehyde and 37.5% (w/v) c-sol~ent and without
dimethyl sulfoxide (DMSO). There was a substantial increase in
the rate and amount of PAC production when ethylene glycol and
glycerol were used as cosolvents as compared with the fermenta-
tion in which no cosolvent was used.
Typical examples of long chain polyols that are suitabie as
cosolvents are polyethyleneglycol polymers with molecular weishts
of 200 - 8000. More particularly, as shown in Fig. 3, polyethyl-
ene glycol (PEG) cosolvents having molecular weights of approxi-
mately 200 (PEG 200), 600 (PEG 600), 1000 (PEG 1000), 3350 (PEG
3350) and 8000 (PEG 8000) all stimulated PAC production as com-
pared with the fermenta~ion carried out without a cosolvent. It
will be understood that polyethyleneglycol cosolvents of other
molecular weights can be employed.
. '
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Dimethyl sulfoxide and short chain alcohols, such as ethanol
and metha~ol, can show detrimental effects on the stability of
the PDCase enzyme, even though the rate and extent of PAC forma-
tion are increased over a fermentation conducted without these
cosolvents. PEG-1000 and other polyethyleneglycol- Cosolvents do
not inhibit PDCase activity. While glycerol is a suitable
cosolvent, PEG-1000 is less expensive than glycerol and is re-
quired in smaller quantities.
The cosolvent is employed in an amount sufficient to in-
crease the rate of formation of PAC and to increase the concen-
tration of PAC in the aqueous medium of a batch fermentation re-
action as compared with a batch fermentation reaction carried out
without a cosolvent, but under otherwise identical conditions.
The cosolvent generally comprises abo t 2% by weight to about 50%
(w/v) of the aqueous medium, preferab:y about 10% to about 30%
(w/v). These concentrations will enaure that the concentration
of the benzaldehyde in the aqueous fermentation medium will
exceed the solubility limit of benzaldehyde in water at the fer-
mentation temperature. Polyethyleneglycol cosolvents of lower
molecular weight are required in greater quantities to reach com-
parabl~ results. Higher molecular weight polymers allow for
-19-
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greater solubility, but they are usually more costly.
Polyethyleneglycol-1000 at 20~ w/v in solution is the COSolvent
of choice.
The PAC production process of the invention can be carried
out over a moderate range of reaction temperatures. The tempera-
ture will generally be about 15~C to about 30C, preferably about
20C to about 22C. The optimum reaction temperature will depend
upon the microorganism that is employed, and the optimum tempera-
ture can be determined with a minimum of experimentation.
The process of the invention can also be carried out over a
moderate range of pH values in the reaction medium. The pH will
generally be about 5 to about 8, preferably about 6 to about 6.5,
and in any case will be such as to avoid denaturing the PDCase or
otherwise inhibiting conversion of th- benzaldehyde.
The fermenter can be operated ov~r a range of cell concen-
trations and the optimum concentratio can be determined without
undue experimentation. Cell concentration does not appear to
have a major bearing on the reaction. The practical range of
values will generally depend upon process economics.
The concentration of PAC in the reaction medium should be
maximized in order to reduce the cost of product recovery. The
process of this invention can be carried out at PAC concentra-
tions of at least 10 g/L, and preferably at PAC concentra~ions of
about 12 g/L to about lS g/L, in the reaction medium.
The process of this invention can be carried out on a batch
or a continuous basis. For the production of PAC by continuous
-20-
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-`` XQ~10721
reaction, the microor9anism can be placed in a column and a stan-
dard reaction mixture can be pumped over the column Thiamine
pyrophosphate (TPP), a cofactor for the enzyme PDCase, can be in-
cluded in the standard reaction mixture, e.g. at a concentration
of 0.1 mM. Fractions of column effluent can be collected and
sampled quantitively for the presence of PAC.
The PAC can be recovered from the reaction medium using con-
ventional techniques. For example, suspended cells can be re-
moved from the liquid phase in the reaction medium by filtration,
centrifugation or settling. The resulting liquid phase can be
further processed to concentrate the PAC solution. PAC can then
be removed from the solution by solvent e~traction.
PAC can be purified according to the method of Neuberg,
Biochim. Z. 12~:610 (1922). The GC/Ma profile of the purified
product shows a parent peak with a mo:ecular weight of 150. The
product of the reaction can also be de~ermined to be the correct
optical isomer, namely L-phenyl acetyl carbinol, by polarimetry.
Conversion to ephedrine by reaction with methylamine has been
performed and the results substantiated. -
The process of this invention maKes it possible to ob~ain
higher concentrations of PAC in the fermentation medium while
obtaining less benzyl alcohol as a by-product. For example, the
process of this invention yields about 0% by weisht to about 1%
by weight, and preferably about 0% to about 0.2% by weight benzyl
alcohol. The lower concentrations of benzyl alcohol are of
-ourse preferred because PAC yield in increased and cost of
:ecovery of PAC from the fermentation medium is reduced.
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It has also been found that when the process of the inven-
tion is carried out with the cell mass of the invention~ higher
yeast productivity is obtained- Specifically, yeast productivity
is typically about 1 to about 2 9 PAC-g cells~l-hr~l, and prefer-
ably about 1.5 to about 2 9 PAC- ~ cells~1-hr~l, with the cell
mass of the invention.
3. Pre~aration of Microorqanisms with Induced Mutations
Mutations can be induced in a yeast microorganism containing
endogeneous pyruvate decarboxylase, such as a microorganism se-
lected from the species SaccharomYces cerevisiae or species of
Candida flareri. The resulting mutants are cultured in the pres-
ence of acetaldehyde under conditions to form colonies having re-
sistance to acetaldehyde inhibition.
Cells from the colonies that res !t when the mutated organ-
isms are cultured in the presence of ~cetaldehyde are isolated
and tested for yield of PAC in a fermentation with benzaldehyde
and pyruvate. The cells can also be tested for yield of
acetaldehyde or benzyl alcohol or both. It is thus possible tO .
select yeast cells that produce PAC at elevated levels ar.d ?ro-
duce acetaldehyde or benzyl alcohol at reduced levels, and tO use
these organisms for the production of ?AC with improved yie.~ as
compared to the parent strains. The production of PAC in :~is:~er
yields in a commercial operation is es?ecially advantageous sirce
the cost of production will be reduced.
More particularly, muta;ions were induced in S! cere~;siae
~-21~0-lA, a wild-type (WT) haploid st.ain, using
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.
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methylnitrosoguanidine. A number of mutants were selected for
resi~tance to pyruvate aldehyde using the procedure descrihed
above. A mutant strain which has been identified as
S cerevisiae P-21~0-lA-8pa was substantially more effective in
the production of PAC than the wild-type (WT) strain P-2180-lA.
The improved performance of the mutant ~8pa is believed to be the
result of a reduction in inhibition of PDCase by aldehyde.
A detailed description of the procedures used to provide mu-
tant P-21a0-lA-8pa and other mutant strains is included in com-
monly owned U.S. Patent Application Serial No. , filed
, (Attorney Docket No. SYNE-030), by Donald L.
Heefner, Robert J. Seely, Robert V. Hageman, Michael J. Yarus and
Sally A. Sullivan, and entitled PROCESS FOR MAXING PHENY~ ACETYL
CARBINOL (PAC), MICROORGANISMS FOR US_ IN THE PROCESS, AND A
METHOD OP PREPARING THE MICROORGANISMS. The entire disclosure of
the commonly owned application is relied upon and incorporated by
reference herein.
This invention will be more clearly understood by reference
to the following Example, in which all parts, proportions, per-
centages and ratios are by weight unless otherwise indicated.
Exam~le
Mutant S. cerevisiae P-2180-lA-8pa cells were grown in a 250
liter fermentor with rapid stirring on complex medium containing
50 g/l glucose. At OD 30 the aeration was turned down to 8 l/min
and the media was allowed to become oxygen depleted. OD and glu-
cose were monitored, and the glucose was allowed to fall to 25
-23-
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g/l before harvest. The cells must be allowed to metabolize
anaerobically for several hours (2-6) before harvest, and the
qlucose must not be allowed to fall below about 1-2 g/l.
To harvest, the culture was pumped through a cooling coil
submersed in ice and the cells were removed by centrifugation.
The cell paste (24% dry wt.) was mixed with polyazetidine (12%
dry wt.) at a ratio of 1:0.5 (cell paste:polyazetidine) and
allowed to dry on Teflon coated trays at room temperature over-
night. Cell/polyazetidine slurries were poured on ~he drying
trays at a rate of 0.2-0.3 g/cm2 area. This gave an appropriate
thickness to the final dried sheet which, when chopped-up by the
knife mill, exposed a sufficient amount of core or edge as op-
posed to the top ~and bottom drying surfaces. The edges appeared
to be considerably more porous than t e top or bottom.
The dried polymer sheets were pe~led off the trays and
ground in a knife-mill. Sieve cuts between 0.5 - 1.0 mm were
taken using standard sieving trays. Larger particles were
recycled through the knife mill. A total of 1.6 kg catalyst was
recovered. The resulting catalyst beads were stored dry at 4C.
In a single pass through a column of catalyst, the material
showed an initial specific activity (SA)o of 0.075 kg PAC-kg
cells 1-hr 1, an initial PAC concentration of 25 mM, and a
half-life of about 11 days.
Batch studies were also conducted using 1 g of catalyst
(I-86193) in 20cc of reaction buffer. A batch progression curve
ndicated that the maximum product concentration was 56 m.~ in
approximately 2 hours.
:
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* *
rn summary, this invention provides an efficient procesS for
~he conversion of benzaldehyde to PAC. Productivity of the cell
mass of the invention for PAC is high. In addition, it is possi-
ble to obtain a relatively high concentration of PAC in the reac-
tion medium while reducing the formation of acetaldehyde during
the transformation. In addition, the process of this invention
produces less benzyl alcohol as an unwanted by-product.
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