CA2000721A1 - Process for producing l-phenyl acetyl carbinol (pac), an immobilized cell mass for use in the process and a method for preparing the cell mass - Google Patents

Abstract

ABSTRACT
A process for the production of L-phenyl acetyl carbinol (PAC) is carried out with an immobilized cell mass of non-viable cells of a mutant yeast strain that exhibits resistance to aldehyde inhibition during PAC production. Benzaldehyde and a source of pyruvate are reacted in an aqueous medium in the pres-ence of the immobilized cell mass. Cells in the cell mass con-tain endogenous pyruvate decarboxylase. Cell walls are chemi-cally crosslinked with polyazetidine and modified to increase permeability to the reactants. A cosolvent for benzaldehyde can be employed to increase PAC production. PAC is useful as an in-termediate in the preparation of 1-ephedrine and d-pseudoephedrine, two well-known medicinal chemicals.

Description

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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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(1921). More particularly~ benzaldehyde can be transformed by a ferm~nting yeast into L-(-)phenyl acetyl carbinol as follows:

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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:
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L-(-)PAC L-(-)EPHEDRINE (R,S) . .

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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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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 ~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 ` R ~-ii CH3 - C - C~

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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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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.

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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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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.

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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:

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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 -~4-~' .

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~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 .. : ~ : . :

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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.

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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 . . :

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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 - ,. ~ , . . .

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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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- z~oo7~1 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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Z0~07~1 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 , , : ' ' ' ; ~ " ~ , -, ~, , - : , ~, ., :
, ,~ , ,. ~ . - ~, . . ,. ~
. ~ . : . :, 200072~.

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.

:

200072~.

* *
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.

Claims (28)

1. A process for the production of L-phenyl acetyl carbinol (PAC), which comprises, (A) providing an immobilized cell mass consisting essen-tially of non-viable cells of a mutant yeast strain that exhibits resistance to aldehyde inhibition during PAC production, wherein the cells contain endogenous pyruvate decarboxylase, and wherein cells in the cell mass have cell walls and walls of adjacent cells are chemically crosslinked, and at least a portion of the cell walls in the cell mass is modified in order to increase per-meability of the cell mass to reactants and (B) reacting benzaldehyde and a source of pyruvate in an aqueous medium in the presence of the immobilized cell mass to produce PAC;
wherein the aqueous medium contains as a cosolvent which is a non-inhibiting, water mixable, organic solvent for the benzaldehyde in an amount sufficient to increase the rate of for-mation of PAC and the concentration of PAC in the aqueous medium over a similar reaction carried out without a cosolvent.

2. Process as claimed in claim 1, wherein the cell mass consists essentially of freely flowing particles comprised of cells cross linked with polyazetidine.

3. Process as claimed in claim 2, wherein the particles pass through a screen having mesh openings of about 1 mm and the particles are retained on a screen having mesh openings of about 0.5 mm.

4. Process as claimed in claim 1, wherein the cosolvent is a polyol.

5. Process as claimed in claim 4, wherein the polyol is a polyethyleneglycol having a molecular weight of about 200 to about 8000.

6. Process as claimed in claim 1, wherein the cosolvent comprises about 2% by weight to about 50% w/v of the aqueous me-dium.

7. Process as claimed in claim 1, wherein the cosolvent comprises about 10% by weight to about 30% w/v of the aqueous me-dium.

8. Process as claimed in claim 1, wherein the cosolvent is a polyethyleneglycol having a molecular weight of about 1000.

9. A process for the production of L-phenyl acetyl carbinol (PAC), which comprises, (A) providing an immobilized cell mass consisting essen-tially of non-viable cells of mutant yeast strain Saccharomyces cerevisiae P-2180-1A-8pa, which exhibits resistance to aldehyde inhibition during PAC production, wherein the cells contain endogenous pyruvate decarboxylase, and wherein cells in the cell mass have cell walls and walls of adjacent cells are chemically crosslinked, and a portion of the cell walls are disrupted to expose the pyrvuate decarboxylase in the cells; and (B) reacting benzaldehyde and a source of pyruvate in an aqueous medium in the presence of the immobilized cell mass to produce PAC.

10. Process as claimed in claim 9, wherein the aqueous me-dium contains a cosolvent for the benzaldehyde in an amount suf-ficient to increase the rate of PAC formation and the concentra-tion of PAC in the aqueous medium over a similar reaction carried out without a cosolvent.

11. Process as claimed in claim 10, wherein the cell mass consists essentially of freely flowing particles comprised of cells crosslinked with polyazetidine.

12. Process as claimed in claim 11, wherein the particles pass through a screen having mesh openings of about 1 mm and the particles are retained on a screen having mesh openings of about 0.5 mm.

13. Process as claimed in claim 12, wherein the organic solvent is a polyethyleneglycol having a molecular weight of about 200 to about 8000.

14. A cell mass for the conversion of benzaldehyde to L-phenyl acetyl carbinol (PAC), which consists essentially of non-viable cells of a mutant yeast strain that exhibits re-sistance to aldehyde inhibition during PAC production, wherein the cells contain endogenous pyruvate decarboxylase, and wherein cells in the cell mass have cell walls and walls of adjacent cells are chemically crosslinked, and at least a portion of the cell walls is modified in order to increase permeability of the cell mass to reactants.

15. Cell mass as claimed in claim 14, wherein the cell mass consists essentially of freely flowing particles comprised of cells crosslinked with polyazetidine.

16. Cell mass as claimed in claim 15, wherein the particles pass through a screen having mesh openings of about 1 mm and the particles are retained on a screen having mesh openings of about 0.5 mm.

17. Cell mass as claimed in claim 16, wherein the yeast strain is mutant strain Saccharomyces cerevisiae P-2180-1A-8pa.

18. A process for preparing a cell mass for the conversion of benzaldehyde to L-phenyl acetyl carbinol (PAC), wherein the process comprises:
(A) providing cells of a mutant yeast strain that exhibits resistance to aldehyde inhibition during PAC production, wherein the cells have cell walls and contain endogenous pyruvate decarboxylase;
(B) mixing the cells with polyacetidine in an amount and under conditions adequate to chemically crosslink walls of adja-cent cells to form a self-supporting mass of crosslinked cells;
and (C) modifying a portion of the cell walls in the cell mass in order to increase permeability of the cell mass to reactants.

19. Process as claimed in claim 18, wherein a paste of the yeast cells is mixed with a paste of polyazetidine and the resulting mixture is dried.

20. Process as claimed in claim 18, wherein the polyazetidine paste contains about 5% by weight to about 25% by weight (dry basis) of the polyazetidine.

21. Process as claimed in claim 18, wherein the polyazetidine paste contains about 8% by weight to about 15% by weight (dry basis) of the polyazetidine.

22. Process as claimed in claim 21, wherein the cell paste contains the cells in an amount of about 2% by weight to about 40% by weight (dry basis).

23. Process as claimed in claim 21, wherein the cell paste contains the cells in an amount of about 10% by weight to about 30% by weight (dry basis).

24. Process as claimed in claim 23, wherein the cells and the polyazetidine are combined in a weight ratio of about 1:0.2 to about 1:3 in the paste.

25. Process as claimed in claim 23, wherein the cells and the polyazetidine are combined in a weight ratio of about 1:0.5 to about 1:1 in the paste.

26. Process as claimed in claim 18, wherein the self-supporting mass of crosslinked cells is subject to size reduction to form particles comprising the crosslinked cells.

27. Process as claimed in claim 26, wherein the particles pass through a screen having mesh openings of about 1 mm and the particles are retained on a screen having mesh openings of about 0.5 mm.

28. Process as claimed in claim 26, wherein the mutant yeast is strain Saccharomyces cerevisiae P-2180-1A-8pa.