CA1191103A - Production of gamma-decalactone - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Optically active .gamma.-decalactone is produced by culturing a microorganism capable of hydrolyzing castor oil and effecting .beta.-oxidation of the resulting hydrolysate in the presence of castor oil or castor oil hydrolysate and a co-oxidant. The resulting .gamma.-hydroxydecanoic acid is lactonized to form .gamma.-decalactone.

Description

l'RODUCri'ION Ofi' y--Di~Ci~T,ACTONE:

s~ KGROUND OF_rL'HE IN-vENrrIo-N

This invention is concerned with a microbial process for the production of optically active y-decalactone.
Considerable time and effort have been expended by microbiologists in the search for be-tter processess for the production of optically active lactones. U.S.
Patent 3,076,~50 discloses a method of preparing cer-tain optically active lactones and the corresponding hydroxycarboxylic acids by microbial reduction,of keto-carboxylic acids. The me-tabolism of ricinoleic acid by some Candida s-trains was inves-tigated by Okui et al.
IJ. Biochemistry, 54,536-540, 1963) who showed that y-hydroxydecanoic acid was an intermediate in the oxi-dative degradation of ricinoleic acid. However, only trace amounts of y-hydroxydecanoic acid were recovered from the fermenta-tion medium due to the rnetabolysis f r-hydroxydecanoic acid upon completion of the fer-mentation,and the toxicity of ricinoleic acid to the microorganism,which limits the amount of substrate that can be used.

SUMMP.RY OF THE INVENTION
_ _ . _ _ This invention provides a method of producing optically active y-hydroxydecanoic acid which comprises culturing or incubating a microorganism capable of hydrolyzing castor oil,and effec-ting ~-oxidation of the resulting hydrolysate in the presence of castor oil,to produce ~-hydroxydecanoi,c acid.

3 ~ 3 rn ano~her clllboclilnellt, ~he invcntion provi,des a mcthod of producillg op-tical,ly active ~--hydroxydecanoic acid which comprise~s enzymatically hyclrolyzillg castor oiL
using l;pase -to form an enzymatic hydrolysate and culturing ox ;ncubati,ng a microorganism capable of effecting ~-oxida-tion of the enzymatic hydrolysate in the presence of said hydrolysa-te to produce ~-hydroxy-decanoic acid.

In still another embodiment, the invention provides a method of producing optically active y-hydroxy-decanoic acid which comprises culturing or incubatirg a microorganism capable of hydrolyzing castor oil and a microorganism capable of effecting ~-oxida-tion of castor oil hydrolysate in -the presence of castor oil to produce y-hydroxydecanoic acid.

DETAILED I:)E CRIPTION _F TI~E_INV NTION

The invention provides a fermen-tation process for -the production of optically active y-hydroxydecanoic acid which may optionally be converted by lactonization to y-decalactone. Depending on the embodiment of the invention employed, the fermentation process involves culturing or incubating a microorganism capable of hydrolyzing castor oil and effecting ~-oxidation of the resulting hydrolysate, or a microorganism capable of effecting ~-oxidation of hydrolysate of castor oil,or a,microorganism capable oE effecting ~-oxidation of an enzymatic hydrolysate of castor oil~ in a suitable medium in the presence of the castor oil or cas-tor oil hydrolysate substrate. The use of cas-tor oil or castor oil hydrolysate as the substrate is deter-mined by -the microorganism(s) employed in the process.
A co-oxidant may ~e added to the culture medium in order to increase the yield of the process.

The selection of the appropriate microorganism for the process is crucial depending on the embodiment of the invention employed, the yield of product required, and the resistance to the toxicity of the fatty acids found in -the castor oil hydrolysate.
The microoryanisms in the inven-tion may be bacteria, yeast or filamentous fungi. Where a microorganism is employed to hydrolyze the castor oil substrate and ~-oxidize the resulting hydrolysate, the preEerred microorganisms are~ erc~_llus oryzae, Candida rUc3Osa~
Geotrichum klebahn~l or _arrow~a llpolytica, (formerly known as Saccharomycopsis lipolytica and previously __ _ Candida _ipolytica), more preferably Yarrowia ~ olytica.
Where the microorganism is employed to only ~-oxidize castor oil hydrolysate, the preferred microorganisms are- Hansenula saturnus, Candida guilliermondii, Candida _bicans r andida krusei, Candida parakrusei, Candida pseudotropicals, Candida stellatoidea,Candida -tropicalis, Asperyi lus oryzae, Candida rugosa, Geot.richum klebahnii or Yarrowia lipoly ica, more __ _ ___ __ __ preferably Candida guilliermondii. Where the 3~ )3 n:icLoor~lLln:ism is used in colllb; nat:ion wi th a 1 :ipase and with castor oi.l, the p:re:Ferrcd Inicroor(J.lnisrns are:
rl lnsellu]a sat-lrn-ls, Ca~ da yu~]llermond:L.L, C~.~nclida alb C~llS, C_ndida ?s~.use , Ca~ da ~aL-akrusel, Candlda ~_udotro~icalis, Cand;da s-te]latoidea, Candida _ . ., ~
tr_e~cal:Ls, As~ .us oryza_, C_ndida _u~Losa, Geo ri.chum kl_bah_il or Yar_ow a ~ lk~t ca, more preferably Candida q~ ermondllO Generally, any type of llpase enzyme may be used to hydrolyze the castor oil, including microbial, pancreatic, fungi or yeast.

I~lerea lipase is used with -the microorganism in the process of the invention, the formation of -the enzymatic hydrolysate may be controlled by limiting the amount of lipase used in the process. This will avold toxicity resulting from the presence of excessive amounts of hydrolysate. The appropria-te amount of lipase require~ may be conveniently found by experimen-tation and will depend upon the lipase and microor-ganism used and the culturing conditions. The hydrolysis using li.pase .is most preferably carried out concur-ren-tly with the fermentation in the same reaction vessel. However, the hydrolysis may be carried out prior to fermentatlon if appropriate measures are taken to avoid the toxic effect of the hydrolysate.
When castor oil is used in the invention, the concern for toxicity is eliminated because triglycerides are not toxic to the organisms. Additionally, the use of castor oil and castor oil hydrolysates as the substrate 3~6 provide co-oxidants to t~e pLocess wh;ch :inc~c.lse efflcierlcy due to the presence of other Eatty acids upon hydrolysis of the castor oil.
s The form in which the microorgan:isms are used is not critlcal. They can be used as -the culture (suspen-sion), i.e., including the cells and the corresponding nutrient solution, or in the form of cells suspended in a buffer solution. The cells or an enzyme extrac-t thereof may be immobilized on a suitable solid support which may then be used to efEec-t the trans-forma-tions .

The cul-ture susperlsion is prepared by inocula-tion of a suitable medium with the m;croorganism. ~ suitable medium is one which contains carbon sources, nitrogen sources, inor~anic salts and growth factors~ Among -the suitable carbon sources are Eor example, glucose, galactose, L-sorbose, rnaltose, sucrose, celloblose, trehalose, L-arabinose, L.-rhamnose, ethanol,glycerol, L-erythri-thol, D-mannitol, lactose, melibiose, raf-finose, meleritose, starch, ~-xylose, D-sorbitol, ~-me-thyl-D-glucoside, lactic acid, ci-tric acid and succinic acidO Among the suitable nitro~en sources are, for example, nitroc3en-containing oryanic substan-ces such as peptone, meat extract, yeast extract, corn steep liquor,and casein, urea, amino acids,or nitro~en COIItailli.11(3 :inorg.~ ic colllpound.s such as nilr.ltes, nitri--les,and inorc3an:ic amnlonium salL-s. ~rnony tllc suitabLe inorganic salts are/ for ex~ ple, phosp}lates, magnes:iuin, potassium, ca]clum~ sodium. The above mentioned nutrients in -the culture ~ned:ium may be supplemented with, for example, one or rnore vitam;ns of the B Group and~or one or more trace minerals such as Fe, Mo, Cu, Mn, B as desired. I-lowever, the process can be per-formedinavitamin--free medium, for example, when a small amount of yeast extract is added to the medium there is no need for vitamins or -trace minerals.

The cultivat:ion of the microorganism can be carried out as a stationary culture or as a submersed culture (e.g., shaking culture, fermentors) preLerably under aerobic conditions. One suitably may work in the pH ranye of Erom about 3.5 to abou-t 8.0, and preferably in the range of from about 4.0 to about 7.5. The pH may be regulated by the addition of inorganic or organic bases, such as sodium hydrox:ide, potassium hydroxide, calcium hydroxide, calcium carbon ate, by ion-exchange resins, or by the addition of a l~uffer such as phosphate or phthalate. The incubation -tem-perature is suitably maintained at between about 15C
and about 33C, with a range from about 20C to about 30~C being preferred.

The process in accordance with the invention is con~
veniently carried out by adding castor oil or castor oil hydrolysate, as the substrate, to the culture medium a~ the onset of cultivation, as the sole carbon source. Alternatively, the substrate may be added in combination with another carbon source, such as c~c~ ose, eitller du~ g c~ll.t.iv.ll-ion, or when cul-tivatioll is comple-te~ 'he amount, level,or conccn(-r.lt:i.on of the subs-t:ra-te in ~he m-di.um may vary. For cxample, in the case of hydro1yzed casl:or oil,levels of f:rom about 0.3~-to abou-t 5~ may make up -the med;um i.nitially or be added during -the course of the fermenta-tion, whereas substantially any level of castor oil may be used.
The reaction time may vary depending on the composition of the culture medium and the substrate concentration.
In general, shaking flask cultures require Erom between about 2h. and about 240h. depending upon the microorganism and the composition oE the cultllre medium. However, when a fermentor is used the Eermen-tation time may be :reduced to ahout 100 h- or less.

The ferMentation may be carr:ied out using the cells of the mlcroorcJanism isolated from the culture solution, or with an enzyme extract isolated from the cells in a manner known per se. In this case, the fermenta-tion can be conveniently carried out in aqueous solution, for example in a buffer solution, in a physiological salt solution, in a fresh nutrient solution~or in water. The isolated cells or enzyme extract may be immobilized on a solid support and the desired trans-formation effected in the absence of the live micro-oxganism~ The transformation of the substrate may be effected by mutants of the microorganism. Such mutants can be obtained readily by methods well known in the art, for example, by exposing the cells to W or X-rays, or customary mutagenic substances such as for example, acridine orange.

The substrate is genercllLy added cl:irectLy to the medium A surface-actlve agent or dispersLon agent, such as Tween ~0*(polyoxyethylenesorbitarl monostearate), can also be added to an aqueous suspenslon of the substrate. Conventional 5 an-tifoam agents, such as silicone oils (e.g. ~CON*), polyalkylenegLycol derivatives, maize oil, or soya oi] can be used to control foaming.

The transformation of the substrate can be monitored using standard analytical techniques such as GLC, TLC, HPLC, IR
and NMR. If a rapid disappearance of the subs-trate is observed, more substrate can then be added in order to maximize the transformation capacity of the microorganisms. The incubation is generally -terminated when all the substrate has disappeared from the culture medium.

After the fermentation process is complete, the ~-hydroxydecanoic acid can either be lactonized in the medium to form ~-decalactone or isolated and purified by conventional -techniques including solvent extraction and distillation. When ln .situ lactonization is desired, the pH of the medium is adjusted to between about 1 and about 5, preferably between about 1 and about 3, by the addition of a suitable acid, such as hydrochloric acid, and the resulting mixture heated to between about 50 C and about 100 C, preferably between ahout 70 C and about 100 C
for about ten minutes, depending upon -the temperature, to convert the ~-hydroxydecanoic acid to y-decalactone. The ~-decalactone is then recovered and purified by standard techniques. If the r-hydroxydecanoic acid is recovered, it may be lactonized according to known procedures ~see, for example, I.L. Finar, Organic Chemis-try, 6th ed.~ Vol.l, p 469 (1973)].
* -trade mark 'L`lle foll.(>w:iT~cl ~'X~llllp~ S S(?:l.VC,' I:.o :i.~ sL::r~ rnbod:irn(~nts oE the :irlvelllion as it is now prcferred l:o practice it ~u-t ;n no way are Inea!lt to ];mit the scope thereof.
Unless othe1-wise stated, we:iyhts are in grams, tempera-tures are in deyrees cen-tic3rade and pressure in mm l~g.

EXAMPLE I
__ _ __ _ A flask containing 100 ml of 2% beef extract and 0.02%
Tween 80 was autoclaved at 120C. for 20 minutes. The medium was then inoculated with 107 cells Yarrowia lipolytica ~Saccharomyco~sls l:L~ol~_i a)/ml of medium, and lOg of castor oil added. The culture was incubated at 26C. on a rotary shaker (200 rpm) for one week The pH of the medium was occasiona]ly adjusted to 6.5-7Ø A-t the end of the Eerrnentation period -the pH of the medium was adjusted to 1.5 by the addition of mineral acid, and the mixture heated at 100C. for 10 minu-tes. AE-ter cooling, the organic products were extracted w;th hexane, the hexane evaporated, and the resiclue dis-tilled to provide 0.61g ~-deca]actone having a GLC purity of 90%.

EXAMPI,E II

The procedures and-rnaterials similar to those descrihed ln Example I were followed~except 0.05g decanoic acid was added each day. There was obtained 0.69g y-decalactone having a GLC purity of 92%.

3~
-- 1.0 --EXAMPLE :r 1 i _ . _ _ . .

The procedures arld mater:ials similaL to those described in Example I, except that Candida guillie mondil was used and 3y were Eollowed of caster oil hydroLysate was added. There was obtained the deslred product-~ -decalactone in 34~ yield.

EXAMPLE IV

By employing the procedures and materials similar to those described in Example I except that lipase is added in conjunction with castor oil, there may be obtained the desired product,'~-decalactone.

EXAMPLE V

By employing the procedures and materials similar to -those described in Examples I, I:[, and III except that other members of the genus Candida such as C. albicans, C krusei lS C. parakrusei, C. pseudotropicalis, C. stellatoldea, C. tropicalis, etc., are used, there may be obtained the desired ~-decalactone.

EXAMPLE VI

By employing the procedures and materials similar to -those described in Example I, except that as a microorganism asperg,illus oryzae is used and 3g of castor oil is added, there is obtained the desired product ~-decalactone (0.3~g/L).

j/~

1~"3~

EX~MPI,E VII
. _ . _, . . _ _ . ... . _ By elllpl.oying lhe procedures and materia].s sim;lar to those descr;bed .in Exalnp].e I, except -that as a micro-organism Geotrichum klebahllii is used alld 3g oE castor oil is added, -there is obtained the desired product y-deca]ac~one (0.2g/L).

EXA~1PLE VIII

By employing the procedures and materials si.milar to those described in Example I, except that as a mi.cro-organism Candida gui.lliernond.ii is used and to each 1,00 ml of medium 100 mg of a lipase (steapsin, Nutritional Biochem Corp.) is added, the des;.red product y-decalactone may be obtai.ned.

The invention being thus described, i.t will be obvious that the sarne may be varied in many ways. Such vari-ations are not to be regarded as a departure from the spirit and scope of the inven-ti.on and all such modlfi-cations are intended to be included within the scope of the fol].owing claims:

Claims (14)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of producing optically active .gamma.-hydroxy-decanoic acid which comprises adding a lipase or a microorganism capable of hydrolyzing castor oil to a medium containing castor oil, and a microorganism capable of .gamma.- oxidizing the resulting hydrolysate to produce .gamma.-hydroxydecanoic acid.

2. A method as in claim 1, wherein said oxidizing and said hydrolyzing microorganism are the same.

3. A method in accordance with claim 2, wherein said microorganism is Aspergillus oryzae, Candida rugosa, Geotrichum klebahnii or Yarrowia lipolytica.

4. A method in accordance with claim 1, wherein the .gamma.-hydroxydecanoic acid is lactonized in situ to form .gamma.-decalactone and the resulting .gamma.-decalactone is recovered.

5. A method in accordance with claim 1, wherein the .gamma.-hydroxydecanoic acid is recovered and lactonized to form .gamma.-decalactone.

6. A method in accordance with claim 1, wherein a co-oxidant is added to, or present during the culturing or incubating of the microorganism.

7. A method of producing optically active .gamma.-hydroxydecanoic acid which comprises culturing or incubating a microorganism capable of effectinh .beta.-oxidation of castor oil hydrolysate in the presence of castor oil hydrolysate to produce .gamma.-hydroxydecanoic acid.

8. A method in accordance with claim 7, wherein said microorganism is Hansenula saturnus, Candida guilliermondii, Candida albicans, Candida krusei, Candida parakrusei, Candida pseudotropicalis, Candida stellatoidea, Candida tropicalis, Aspergillus oryzae, Candida rugosa, Geotrichum klebahnii or Yarrowia lipolytica.

9. A method in accordance with claim 7, wherein the .gamma.-hydroxydecanoic acid is lactonized in situ to form .gamma.-decal-actone and the resulting .gamma.-decalactone is recovered.

10. A method in accordance with claim 7, wherein the .gamma.-hydroxydecanoic acid is recovered and lactonized to form .gamma.-decalactone.

11. A method in accordance with claim 7, wherein a co-oxidant is added to, or present during the culturing or incubating of the microorganism.

12. A method in accordance with claim 2, wherein said microorganism is Hansenula saturnus, Candida guilliermondii, Candida albicans, Candida krusei, Candida parakrusei, Candida pseudotropicalis, Candida stellatoidea, Candida tropicalis, Aspergillus oryzae, Candida rugosa, Geotrichum klebahnii or Yarrowia lipolytica.

13. A method in accordance with claim 1 in which the lipase enzyme is microbial, pancreatic, fungi or yeast.

14. A method of producing optically active .gamma.-hydroxydecanoic acid which comprises fermenting Yarrowia lipolytica in the presence of castor oil and a co-oxidant to produce .gamma.-hydroxy-decanoic acid and lactonizing the .gamma.-hydroxydecanoic acid in situ to form .gamma.-decalactone by adjusting the pH of the fermentation media and recovering said .gamma.-decalactone.