Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-1-
TITLE:
An enzymatic oil-degumming process
FIELD OF INVENTION
The present invention relates to an improved process for
enzymatic reducing the content of phosphorus containing
components in an edible oil.
BACRGRODND OF THE INVENTION
Oils obtained from the usual oil and fat production
processes by compressing oil-bearing materials or by extracting
oil from the materials and removing the extraction solvent
contain impurities such as polar lipids mainly composed of
phospholipids, as well as fatty acids, pigments, odor components
and the like. Thus it is necessary to remove these impurities by
a refining process. Such a process may require a degumming step.
In the art it is known to use phospholipase for
enzymatic degumming of an edible oil (US 5,264,367; JP-A-
2153997; and EP 622446), to reduce the phosphorus content of
said water degummed edible oil.
However those references do not specifically suggest to
use low amount of water in the enzymatic degumming process.
In contrary EP 622446 suggest to use high amount of
2s water in the enzymatic degumming process. See page 3, line 33-44
and claim 4 in said document, which suggest to use more than 30
percent of water by weight of the oil in said process.
SUMMARY OF THE INVENTION
3o The problem, to be solved, by the present invention is to
provide a simplified and economically cheaper process for
enzymatic degumming of edible oils.
The solution is to perform said process using low amounts of
water.
3s Accordingly, the present invention relates to a process for
reducing the content of phosphorus containing components in an
edible oil, having from 50 to 10.000 part per million (ppm) of
phosphorous content, which method comprises contacting said oil
at a pH from 1.5 to 8 with an aqueous solution of a
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99100202
-2-
phospholipase A1 (PLA1), phospholipase A2 (PLA2), or
phospholipase B (PLB) which is emulsified in the oil until the
phosphorous content of the oil is reduced to less than 12 ppm,
and then separating the aqueous phase from the treated oil, -
s and wherein said process is characterized by that said
emulsified condition is formed using from 0.01 to 1.5 percent of
water by weight of the oil, preferably from 0.01 to 1.0 percent
of water by weight of the oil, more preferably from 0.01 to 0.75
percent of water by weight of the oil, even more preferably from
l0 0.01 to 0.5 percent of water by weight of the oil, and most
preferably from 0.01 to 0.4 percent of water by weight of the
oil.
Further, the lower range above of 0.01 percent of water by
weight of the oil, may preferably be 0.1 percent of water by
is weight of the oil.
An advantage of the process described herein is that costs
for water and waste water treatment may be reduced. Furthermore,
oil recovery yields may be increased because less amount of oil
will be wasted to the aqueous phase.
2o Further, an advantage of the process described herein may be
that an oil-mill using this process may skip sludge recycling of
the polluted water used in the process.
The in the art known enzymatic degumming processes give rise
to a high amount of polluted water, which is expensive to clean
2s up. This is of course an economically burden.
Further oil-mills traditionally have been forced to
implement recycling of the water processes in order to save cost
in said purifying of the polluted water.
Said recycling step may be saved by the low amount of water
3o used in the process described herein.
In enzymatic degumming carried out according to the art
(e.g. US 5,264,367) a heat treatment to e.g. 65-75 °C of the
water in oil emulsion is usually carried out in order to
facilitate separation of the oil and aqueous phases by e.g.
35 centrifugation. When using the thermostable phospholipase
LecitaseT"' (Novo Nordisk A/S, Denmark) in the oil degumming
process, the aqueous phase containing the enzyme can
advantageously be reused several times (with or without addition
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-3-
of fresh enzyme solution).
However, for the oil mill it may be advantageous if the
recycling of the aqueous phase could be totally omitted. This
would in the normal case mean that overall water consumption
would be increased with increased costs. If only a low amount of
' water is used in the enzymatic degumming process, recycling of
the sometimes problematic sludge phase could be omitted.
Embodiments) of the present invention is described below,
by way of examples) only.
DETAINED DESCRIPTION OF THE INVENTION
Edible oils:
In principle any edible oil may be degummed according to
is a process of the invention. Example of oils are crude oils and
water degummed oils.
A crude oil (also called a non-degummed oil) may be a
pressed or extracted oil or a mixture thereof from e.g.
rapeseed, soybean, or sunflower. The phosphatide content in a
2o crude oil may vary from 0.5-3% w/w corresponding to a phosphorus
content in the range of 200-10.000 ppm, more preferably in the
range of 250-1200 ppm. Apart from the phosphatides the crude oil
also contains small concentrations of carbohydrates, sugar
compounds and metal/phosphatide acid complexes of Ca, Mg and Fe.
2s Preferably, said edible oil is an oil from which
mucilage has previously been removed and which has a phosphorus
content from 50 to 250 ppm.
Such an oil is generally obtained by a water-degumming
process and termed "a water-degummed oil".
3o A water-degummed oil is typically obtained by mixing 1-3%
w/w of hot water with warm (60-90°C) crude oil. Usual treatment
periods are 30-60 minutes. The water-degumming step removes the
phosphatides and mucilaginous gums which become insoluble in the
oil when hydrated. The hydrated phosphatides and gums can be
s5 separated from the oil by settling, filtering or centrifuging -
centrifuging being the more prevalent practice.
Alternatively, the process here termed "water-degumming"
may be called "wet refining to remove mucilage" (see US
5,264,367).
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-4=
Further, an edible is preferably an vegetable oil.
A Phos~holipase used in the process:
Preferably, a phospholipase used in the process of the
s invention is a phospholipase obtained from a microorganism,
preferably a filamentous fungus, a yeast, or a bacterium.
For the purpose of the present invention the term
"obtained from", as used herein in connection with a specific
microbial source, means that the enzyme and consequently the DNA
1o sequence encoding said enzyme is produced by the specific source.
The enzyme is then obtained from said specif is source by
standard known methods enabling the skilled person to obtain a
sample comprising the enzyme and capable of being used in a
process of the invention. Said standard methods may be direct
is purification from said specific source or cloning of a DNA
sequence encoding the enzyme followed by recombinant expression
either in the same source (homologous recombinant expression) or
in a different source (heterologous recombinant expression).
More preferably, a phospholipase used in a process of the
2o invention is obtained from a filamentous fungal species within
the genus Fusarium, such as a strain of F. culmorum, F.
heterosporum, F. solani, or in particular a strain of~F.
oxysporum; or
a filamentous fungal species within the genus Aspergillus,
2s such as a strain of ~lspergillus awamori, Aspergillus foetidus,
Aspergillus japonicas, Aspergillus niger or in particular
~lspergillus oryzae.
Examples of suitable Fusarium phospholipases are
disclosed in
i) Tsung-Che et al. (Phytopathological notes 58:1437-38
(1968)) (a phospholipase from Fusarium solani); and
ii) EP Patent Application No. 97610056.0 disclosing a
suitable F. culmorum PL (see example 18 in said doc.)
3s and a suitable F, oxysporum PL (see example 1-17).
Suitable Aspergillus phospholipases are diclosed in
i) EP 575133 disclosing numerous different Aspergillus PL's
(see claim 14) and in particular a PL from A. oryzae(Claim
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-5-
17 or 18) and a PL from A. niger (claim 19); and
ii) DE 19527274 A1 dicloses a suitable Aspergillus preparation
(see examples).
Further the commercial available phospholipase preparation
s Degomma VOD (Roehm, Germany), which is believed to comprise an
Aspergillus phospholipase is suitable to be used in a process of
the invention.
Further, it is preferred that a phospholipase used in a
process of the invention exhibits certain properties.
io Accordingly, embodiment of the invention relates to
i) a process according to the invention, wherein the
phospholipase is a phospholipase which is substantively
independent of Ca2+ concentration measured as,
relative phospholipase activity at 5 mM EDTA and 5mM Ca2+ in
i5 a phospholipase activity assay measuring release of free fatty
acids from lecithin in a buffer comprising 2% lecithin, 2%
Triton X-100, 20 mM citrate, pH 5; incubated for l0 min. at 37°C
followed by stop of reaction at 95°C for 5 min.;
wherein the ratio of relative phospholipase activity at 5mM
2o EDTA/5 mM Ca2+ is greater than 0.25, more preferably greater
than 0.5; and/or
ii) a process according to the invention, wherein the
phospholipase is a phospholipase which has a phospholipase
activity which is capable of releasing at least 7 ~mol of free
2s fatty acid/min./mg enzyme; more preferably at least 15 ~uaol of
free fatty acid/min./mg enzyme; measured as,
phospholipase activity is measured in an assay measuring
release of free fatty acids from lecithin in a buffer comprising
2% lecithin, 2% Triton X-100, 20 mM citrate, pH 5; incubated for
30 10 min. at 37°C followed by stop of reaction at 95°C for 5
min..
A detailed description of above mentioned assays is
disclosed in a working example herein (vide infra). For even
further details reference is made to EP Patent Application No.
97610056.0 (see example 9 in said document).
35 Further it has been demonstrated that a phospholipase
special suited for enzymatic oil degumming in general and in
CA 02324653 2000-09-26
WO 99!53001 PCT/DK99/00202
-6-
particular for the improved process described herein is
characterized by having a certain primary amino acid sequence.
Accordingly, in an even further embodiment the invention
relates to a process according to the invention, wherein the
phospholipase is a phospholipase having an polypeptide sequence
selected from the group comprising of:
(a) polypeptide having an amino acid sequence as shown in
positions 31-346 of SEQ ID NO 1;
(b} a polypeptide having an amino acid sequence as shown in
1o position 31-303 of SEQ ID NO 1;
(c) a polypeptide which is at least 70 % homologous with said
polypeptide defined in (a), or (b); and
a fragment of (a), (b) or (c).
is For a detailed description of cloning and purification
of a phospholipase having the above mentioned polypeptide
sequence reference is made to EP Patent Application No.
97610056Ø
In this document it can further be seen that a
2o phospholipase obtained from F. oxysporum and having the
polypeptide sequence shown in (b) above exhibits both of the
above mentioned functional characteristic. Accordingly, this
phospholipase is the most preferred phospholipase to be used in
a process of the invention. A working example herein
2s demonstrates the use of this phospholipase (vide infra).
Finally an example of a suitable non-microbial
phospholipase is the commercial available PL (LecitaseTM, Novo
Nordisk A/S, Denmark) obtained from porcine pancreas.
3o Standard process parameters of the process of the inventio
Besides the specific use of low amount of water in the
process of the invention, any of the other process parameters
may be done according to the art. See Background section above
for references to the art known processes.
35 The enzymatic treatment is conducted by dispersing an
aqueous solution of the phospholipase, preferably as droplets
with an average diameter below 10 ~(micro)m.
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-
According to the process of the invention the amount of
water is from 0.01 to 1.5% by weight in relation to the oil.
An emulsifier may optionally be added. Mechanical
agitation may be applied to maintain the emulsion.
s The enzymatic treatment can be conducted at any pH in the
range 1.5-8, preferably from pH 3-6. The pH may be adjusted by
adding citric acid, a citrate buffer, NaOH or HC1.
A suitable temperature is generally 30-75°C (particularly
40-60°C). The reaction time will typically be 0.5-12 hours (e. g.
2-6 hours), and a suitable enzyme dosage will usually be 100-
5000 IU per liter of oil, particularly 200-2000 IU/1.
The enzymatic treatment may be conducted batchwise, e.g.
in a tank with stirring, or it may be continuous, e.g, a series
of stirred tank reactors.
1s The enzymatic treatment is followed by separation of an
aqueous phase and an oil phase. This separation may be performed
by conventional means, e.g. centrifugation. The process of the
invention can reduce this value to below 12 ppm, more preferably
below 10, and even more preferably below 5 ppm.
MATEItIALB AND METHODB
EXAMPLES
EXAMPLE 1
General description of assay for enzymatic degumming of edible
oil
Eauipment for carrying' out enzymatic deaumminq
The equipment consists of a 1 1 jacketed steel reactor fitted
so with a steel lid, a propeller (about 600 rpm), baffles, a
temperature sensor, an inlet tube at the top, a reflux condenser
(about 4°C) at the top, and an outlet tube at the bottom. The
reactor jacket is connected to a thermostat bath. The outlet
tube is connected via silicone tubing to a Silverson in-line
3s mixer head equipped with a "square hole high shear screen",
driven by a Silverson L4RT high shear lab mixer (about 8500 rpm,
flow ca. 1.1 1/minute). The mixer head is fitted with a cooling
coil (5-10 °C) and an outlet tube, which is connected to the
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-g-
inlet tube of the reactor via silicone tubing. A temperature
sensor is inserted in the silicone tubing just after the mixer
head. The only connection from the reactor/mixer head system to
the atmosphere is through the reflux condenser.
General procedure for carrying out enzymatic deqummina
All cooling and thermostat equipment is turned on. Then 0.6 1
(ca. 560 g) of oil is loaded in the reactor, which is kept at
about the temperature needed for the specific experiment. The
io lab mixer is turned on, whereby the oil starts to circulate from
the reactor to the mixer head and back to the reactor. The
system is allowed to equilibrate for about 10 minutes, during
which period the temperature is fine tuned. The pre-treatment
period starts with addition of 0.6 g (2.86 mmol) citric acid
i5 monohydrate in the appropriate amount of water or the appropri-
ate amount of a mixture of citric acid and trisodium citrate
(see Tables 1 and 7 below; [citric acid] in water/oil emulsion =
4.6 mM), which sets t = 0. At t = 30 minutes the appropriate
amount of 4 M NaOH solution is added (see Tables 1 and 7).
Table Z. Water content in Experiments A-D; wdg rape seed oil.
Experi- ~;": Water Water Water Total
"~A'~' ~~~Water
.
~,
~
ment y ~ added in NaOH in water
~ in 560
g oil at t=0 solutio enzyme
~....;~.: .
a.>
.
n solutio
sf;7:u,
.
:;.y~ii i
.~ :6o v n
~;~
'
.~
;
~
:::
:
? :?%o>..... ... ... . .
.c . ~.
A ~ :'~> ~;: ~ 0 . 5 6 g 2 7 g 1.1 g 1. 0 g 2 9 .
7 g
:,::::~.-..~.. ~.: ;:. ..>..
T
B w-~ ~~.' 5 . 0 g 0 . 7 1. 0 g 7 . 3
0 . 5 6 g g g
~
~
C ~~~;:iN' 0 . 05 0 g 1. 0 g 1. 6
;,fi; g* g
:; ~
~' 0 . 56 g
_
;
','.,~,.u'~::Yt'b:.-..~~t~.~
... i::i
D ~:~::'.' 0.56 g 0.07 0 g 1.0 g 1.6 g
.
::vt~.,:.
>:..i.:::: .
:~,:_ .. . g
<4.y : ~ 3rr.;
::i;:C":?f...
>.... ".".l:z.vv
.v;:,;~:jI: ~i'T.i.'
* Water contribution from o.6 g citric acid monohydrate.
** Water contribution from mixt. of 0.5 g citric acid monohy-
drate and 0.14 g trisodium citrate dihydrate.
At t = 35 minutes samples are drawn for P-analysis and pH
determination. Just after this the required amount of enzyme
CA 02324653 2000-09-26
WO 99/53001 PCTlDK99/00202
_g-
solution is added (end of pre-treatment period). Samples for P-
analysis and pH determination are drawn at t = 1, 2, 3.5, 5, 6
hours, and then the reaction is stopped.
The reactor/mixer system is emptied and rinsed with 2x500
ml 10% Deconex/DI water solution followed by minimum 3x500 ml of
DI water. Table 2 is a presentation of the various additions and
samplings during the reaction.
Table 2. Schedule for enzymatic degumming
Sampling
Time Addition of
P-analysis pH determi-
nation
X
0 Citric acid
5 min. X
30 min. X X
30 + b min. NaOH
35 min. X X
35 + 8 min. Enzyme
1 hour X X
2 hours X X
3.5 hours X X
5 hours X X
6 hours X X
Phosphorus analysis:
Sampling for P-analysis:
is Take 10 ml of water in oil emulsion in a glass centrifuge
tube. Heat the emulsion in a boiling water bath for 30 minutes.
Centrifuge at 5000 rpm for 10 minutes. Transfer about 8 ml of
upper (oil) phase to a 12 ml polystyrene tube and leave it (to
settle) for 12-24 hours. After settling draw about 1-2 g from
2o the upper clear phase for P-analysis.
P-analysis was carried out according to procedure 2.421 in
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-10-
"Standard Methods for the Analysis of Oils, Fats, and Deriva-
tives, 7th ed. (1987)":
Weigh 100 mg of Mgo (leicht, Merck #5862) in a porcelain
dish and heat with a gas burner. Add 1-2 g of oil and ignite
s with a gas burner to give a black, hard mass. Heat in a Vecstar
furnace at 850°C for 2 hours to give white ashes. Dissolve the
ashes in 5 ml of 6 M HN03 and add 20 ml of reagent mix. Leave
for 20 minutes. Measure absorbance at 460 nm (use a blank (5 ml
HN03 + 20 ml reagent mix) for zero adjustment).~Calculate by
to using calibration curve.
pH determination
Take 2 ml of water in oil emulsion and mix with 2 ml of MilliQ
water. After phase separation, pipette off top oil layer.
15 Measure pH in aqueous phase with pH electrode Orion. Measure-
ments are transformed to "real" pH values by the formula
PHreal - P~neasured - 0.38.
2o A calibration curve was obtained by dissolving 0.6 g of
citric acid monohydrate in 27 g of DI water; pH of this solution
was measured by pH electrode Orion (pHreal)~ 100 ~tl were mixed
with 2 ml MilliQ water, and pH of this solution was measured by
pH electrode Orion (pH~,ea~ured) ~ PH of the citric acid solution
25 was changed gradually by adding NaOH solution, and for each
adjustment dilution and pH measurements were carried out as
described above.)
EXAMPLE 2
3o Degumming of hater-degummed rape seed oil (I)
Experiments were carried out according to the "General
procedure for carrying out enzymatic degumming" as described in
example 1 above.
Oil:
Water-degummed rape seed oil from $.rhus Oliefabrik (AOM),
Denmark. Batches 000730/801700 and C00730/B01702, P-content 231-
236 ppm. Water content <_ 0.1 % w/w.
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-11-
Enzyme:
PL from Fusarium oxysporum having the amino acid sequence shown
in SEQ NO 1.
Batch F-9702027, estimated conc. 0.75 mg/ml.
The enzyme Was recombinantly expressed and purified as described
in EP Patent application number 97610056Ø
Experiment A (water content 5.3 %)
0.6 1 (560 g) of wdg rape seed oil is loaded in the equipment
and heated to 40°C. At t = 0 min. a solution of 0.6 g of citric
acid monohydrate in 27 g of water was added. At t = 30 min. 1.07
ml (4.3 mmoles) of 4 M NaOH solution were added, which yield a
pH of about 5. At t = 35 min., 1 ml (0.75 mg) of a purified
solution of phospholipase from F. oxysporum is added. The
measured phosphorus content in the oil phase after centrifuga-
tion as well as the pH values in the aqueous phase is shown in
Table 3.
Table 3. Results from degumming of wdg rape seed oil with
phospholipase from F. oxysporum, water content 5.3 %.
Time (hours) Phosphorus pH
content in oil
phase
0 243
0.50 215 4.7
0.58 216 5.5
1.0 66 4.9
2.0 10 . 4.9
3.5 8 5.4
5.0 9 5.0
Experiment B water content 1.3 %L
As in Experiment A above except that at t = 0 min. 0.6 g of
CA 02324653 2000-09-26
WO 99/53001 PC'T/DK99/00202
-12-
citric acid monohydrate in 5.0 g of water was added, and at t =
30 min. 0.71 ml (2.86 mmoles) of 4 M NaOH solution were added
which yield a pH of about 5. The measured phosphorus content in
the oil phase after centrifugation as well as the pH values in
the aqueous phase is shown in Table 4.
Table 4. Results from degumming of wdg rape seed oil with
phospholipase from F. oxysporum, water content 1.3 %.
io
Time (hours) Phosphorus pH
content in oil
phase
0 237
0.50 213 4.7
0.58 197 5.7
1.0 78 4.9
2.0 9 4.9
3.5 10 5.0
5.0 12 5.1
6.0 ~ 10 ~ 5.0 r
Experiment C !water content 0.3 %Z
As in Experiment A above except that at t = 0 min. 0.6 g of
citric acid monohydrate powder was added, and at t = 30 min. no
NaOH solution was added, which yield a pH of about 5. The
measured phosphorus content in the oil phase after
centrifugation as well as the pH values in the aqueous phase is
2o shown in Table 5.
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-13-
Table 5. Results from degumming of wdg rape seed oil with
phospholipase from F. oxysporum, water content 0.3 %.
Time (hours) Phosphorus
content in oil
phase
0 246 4.9
0.50 234 5.1
0.58
1.0 101 4.8
2.0 18 5.2
3.5 11 5.2
Ext~eriment D water content 0.3 %)
As in Experiment C above except that at t = 0 min. a mixture of
0.5 g of citric acid monohydrate and 0.14 g trisodium citrate
dihydrate powder was added, which yield a pH of about 5. The
io measured phosphorus content in the oil phase after
centrifugation as well as the pH values in the aqueous phase is
shown in Table 6.
Table 6. Results from degumming of wdg rape seed oil with
phospholipase from F. oxysporum, water content 0.3 %.
Time (hours) Phosphorus pH
content in oil
phase
0 243
0.50 244 5.5
0.58
1.0 101 5.1
2.0 8 4.9
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-14-
~ EXAMPLE 3
Degumming of crude (mixture of pressed and extracted) rape seed
oil (II)
s Experiments were carried out according to the "General
procedure for carrying out enzymatic degumming" as described in
example 1 above.
oil:
to Crude rape seed oil from MILD Olomouk, Czech rep. Batch
C00745/B02042, P-content 263 ppm. Water content 0.17 % w/w.
is Table 7. Water content in Experiments E and F; crude rape seed
oil.
Experi- ~at~.t~k'~x>; Water Water Water in Water Total
r,.:..:.xqy_. .
ment ""':e:~: ;in 560 added NaOH in en- water
i~>~~w'~'~~ oil at t=0 solution z a
>: Ym
~
x
>
g
~
.
.;: solu-
:::
,,.>
.,
:...::~;;.,~:.x:
f
:~- ~~
.
y~;:: .~~~:~~~~
:v.12:
'.~,~':::C
' t l~n
~"f::iuii4i i~~~.$r~fi.
.:.i:.,:.........v.t.:::~.',!y...:
!k:
E ~:;'3~k~~~~ ". 0 . 9 5 2 7 g 1. 1 g 1. 0 g 3 0
g .1
-
'.~i::S:':n:~V:E:\i-f-~ !y
...~:i.:.iIy1'
: T
.
:?
.
<
R 9
NC: a
. ..,; ,~'- .
.:h.>~~
.
S
:
F ''.xr >;.': 0 . 9 5 g 5 . 0 0 . 7 g 1. 0 g 7 .
g 7 g
.....,..t... s
2o Experiment E (water content 5.4 %)
0.6 1 (560 g) of crude rape seed oil is loaded in the equipment
and heated to 40°C. At t = 0 min. a solution of 0.6 g of citric
acid monohydrate in 27 g of water was added. At t = 30 min. 1.07
2s ml (4.3 mmoles) of 4 M NaOH solution were added, which yield a
pH of about 5. At t = 35 min., 1 ml (0.75 mg) of a purified
solution of phospholipase from F. oxysporum is added. The
measured phosphorus content in the oil phase after centrifuga-
tion as well as the pH values in the aqueous phase is shown in
3o Table 8.
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-15-
Table 8. Results from degumming of crude rape seed oil with
phospholipase from F. oxysporum, water content 5.4 %.
Time (hours) Phosphorus con- pH
tent in oil phase
0 222
0.50 165
0.58 136 4.8
1.0 38 5.1
2.0 10 5.0
3.5 11 5.0
5.0 11 5.0
6 . 0 -- ~ _ 20 - ~ _ 5: 3
Experiment F (water content 1.4 %)
As in Experiment E above except that at t = 0 min. 0.6 g of
citric acid monohydrate in 5.0 g of water was added, and at t =
30 min. 0.71 ml (2.86 mmoles) of 4 M NaOH solution were added
which yield a pH of about 5. The measured phosphorus content in
the oil phase after centrifugation as well as the pH values in
the aqueous phase is shown in Table 9.
Table 9. Results from degumming of crude rape seed oil with
phospholipase from F. oxysporum, water content 1.4 %.
Time (hours) Phosphorus con- pH
tent in oil phase
0 223
0.50 119
0.58 92 5.1
1.0 31 5.1
2.0 12 5.0
3.5 11 5.1
5.0 9 4.8
6.0 8 4.3
CA 02324653 2000-09-26
WO 99/53001 PCf/DK99/00202
-16=
EXAMPLE 9
Assays used for characterization of a phospholipase suitable to
s be used in an oil degumming process of the invention.
Phospholipase activity assays:
Phospholipase activity (PHLU) was measured as the release of
free fatty acids from lecithin. 50 ~1 4% L-alpha
1o phosphatidylcholine (plant lecithin from Avanti, USA), 4% Triton
X-100, 5 mM CaCl2 in 50 mM HEPES, pH 7 was added, 50 ~C1 enzyme
solution diluted to an appropriate concentration in 50 mM HEPES,
pH 7. The samples were incubated for 10 min at 30°C and the
reaction stopped at 95°C for 5 min prior to centrifugation (5
is min at 7000 rpm). Free fatty acids were determined using the
NEFA C kit from Wako Chemicals GmbH; 25 ~1 reaction mixture was
added to 250 ul reagent A and incubated for 10 min at 37°C. Then
500 ~.1 Reagent B was added and the sample was incubated again,
min at 37°C. The absorption at 550 nm was measured using an
2o HP 8452A diode array spectrophotometer. Samples were run at
least in duplicates. Substrate and enzyme blinds {preheated
enzyme samples (10 min at 95°C) + substrate) were included.
Oleic acid was used as a fatty acid standard. 1 PHLU equals the
amount of enzyme capable of releasing 1 ~mol of free fatty
25 acid/min under these conditions.
Alternatively, the assay was run at 37°C 'in 20 mM citrate
buffer, pH 5 (Ca2+-dependence) or 20 mM Britton-Robinson buffer
(pH-profile/temperature-profile/stability).
Phospholipase A1 activity (PLA1) was measured using 1-(S
3o decanoyl)-2-decanoyl-1-thio-sn-glycero-3-phosphocholine (D3761
Molecular Probes) as a substrate. 190 ~,1 substrate (100 ~C1 D3761
(2 mg/ml in ethanol) + 50 ~,1 1 % Triton X-100 + 1.85 ml 50 mM
HEPES, 0.3 mM DTNB, 2 mM CaCl2, pH 7) in a 200 ~1 cuvette were
added to 10 ~,1 enzyme, and the absorption at 410 nm was measured
3s as a function of time on the HP 8452A diode array spectropho-
tometer at room temperature. Activity was calculated as the
slope of the curve in the linear range. PLA1 equals the amount
of enzyme capable of releasing 1 ~mol of free fatty acid
(thiol)/min at these conditions.
CA 02324653 2000-09-26
WO 99/53001 PCT/DK99/00202
-17-
Phospholipase A2 activity (PLA2) was measured at 40°C using 1-
hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine
(H361 Molecular Probes). 2 ml substrate (50 ~l 1°s Triton X-100 +
25 ~C1 0.1% H361 in methanol + 10 ml 50mM HEPES, pH 7) in a 2 ml
cuvette with stirring was added to 10 ~cl enzyme, and the pyrene
fluorescence emission was measured at 376 nm (excitation at 340
nm) as a function of time (1 sec. intervals) using the Perkin
Elmer LS50 apparatus. In the Triton X-100/phospholipid micelles
the concentration of phospholipid was adjusted to have excimer
io formation (emits at 480 nm). Upon cleavage the fatty acid in the
2-position containing the pyrene group is released into the
aqueous phase resulting in an increase in the monomer emission.
PLA2 was taken as the slope of the curve in the linear range at
equal conditions.
CA 02324653 2000-09-26
WO 99/53001 ~ PCT/DK99/00202
SEQUENCE LISTING
<110> NOVO NORDISK A/S
<120> AN ENZYMATIC OIL-DEGUMMING PROCESS
<130> 5570-WO
<190>
<141>
<160> 1
<170> PatentIn Ver. 2.0
<210> 1
<211> 346
<212> PRT
<213> Fusarium oxysporum
<900> 1
Met Leu Leu Leu Pro Leu Leu Ser Ala Ile Thr Leu Ala Val Ala Ser
1 5 10 15
Pro Val Ala Leu Asp Asp Tyr Val Asn Ser Leu Glu Glu Arg Ala Val
20 25 30
Gly Val Thr Thr Thr Asp Phe Ser Asn Phe Lys Phe Tyr Ile Gln His
35 40 45
Gly Ala Ala Ala Tyr Cys Asn Ser Glu A1a Ala Ala Gly Ser Lys Ile
50 55 60
Thr Cys Ser Asn Asn Gly Cys Pro Thr Val Gln Gly Asn Gly Ala Thr
65 70 75 80
Ile Val Thr Ser Phe Val Gly Ser Lys Thr Gly Ile Gly Gly Tyr Val
85 90 95
Ala Thr Asp Ser Ala Arg Lys Glu Ile Val Val Ser Phe Arg Gly Ser
100 105 110
Ile Asn Ile Arg Asn Trp Leu Thr Asn Leu Asp Phe Gly Gln Glu Asp
115 120 125
Cys Ser Leu Val Ser Gly Cys Gly Val His Ser Gly Phe Gln Arg A1a
130 135 140
CA 02324653 2000-09-26
WO 99/53001 2 PCT/DK99/00202
Trp Asn Glu Ile Ser Ser Gln Ala Thr Ala Ala Val Ala Ser Ala Arg
145 150 155 160
Lys Ala Asn Pro Ser Phe Asn Val Ile Ser Thr Gly His Ser Leu Gly
165 170 175
Gly Ala Val Ala Val Leu Ala Rla Ala Asn Leu Arg Val Gly Gly Thr
180 185 190
Pro Val Asp Ile Tyr Thr Tyr Gly Ser Pro Arg Val Gly Asn Ala Gln
195 200 205
Leu Ser Ala Phe Val Ser Asn Gln Ala Gly Gly Glu Tyr Arg Val Thr
210 215 220
His Ala Asp Asp Pro Val Pro Arg Leu Pro Pro Leu Ile Phe Gly Tyr
225 230 235 240
Arg His Thr Thr Pro Glu Phe Trp Leu Ser Gly Gly Gly Gly Asp Lys
245 250 255
val Asp Tyr Thr Ile Ser Asp Val Lys Val Cys Glu Gly Ala Ala Asn
260 265 270
Leu Gly Cys Asn Gly Gly Thr Leu Gly Leu Asp Ile Ala Ala His Leu
275 280 285
His Tyr Phe Gln Ala Thr Asp Ala Cys Asn Ala Gly Gly Phe Ser Trp
290 295 300
Arg Arg Tyr Arg Ser Ala Glu Ser Val Asp Lys Arg Ala Thr Met Thr
305 310 315 320
Asp Ala Glu Leu Glu Lys Lys Leu Asn Ser Tyr Val Gln Met Asp Lys
325 330 335
Glu Tyr Val Lys Asn Asn Gln Ala Arg Ser
340 345
