Note: Descriptions are shown in the official language in which they were submitted.
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
33
--7--
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:
