CN1293706A - Enzymatic oil-degumming process - Google Patents

Enzymatic oil-degumming process Download PDF

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Publication number
CN1293706A
CN1293706A CN 99804005 CN99804005A CN1293706A CN 1293706 A CN1293706 A CN 1293706A CN 99804005 CN99804005 CN 99804005 CN 99804005 A CN99804005 A CN 99804005A CN 1293706 A CN1293706 A CN 1293706A
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oil
phospholipid hydrolase
water
aspergillus
phospholipase
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CN 99804005
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CN1139652C (en
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金·克劳森
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Novo Nordisk AS
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Novo Nordisk AS
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/003—Refining fats or fatty oils by enzymes or microorganisms, living or dead

Abstract

An improved process for enzymatic reducing the content of phosphorus containing components in an edible oil. The method comprises the use of phospholipase and a low amount of water.

Description

A kind of method of enzymatic degumming of oil
Invention field
The present invention relates to the improving one's methods of content that a kind of enzymatic reduces phosphorus containing components in the edible oil.
Background of invention
From common oil and fatty prod by the oil-containing raw material being squeezed or extracting solvent and process the oil of gained and comprise impurity such as polar lipid class (mainly forming), lipid acid, pigment, odour component or the like by phosphatide by from described raw material, extracting oil and removing.Therefore need remove these impurity by precision work.These class methods may need the step of coming unstuck.
Application Phospholipid hydrolase known in the art make the edible oil enzymatic degumming (US 5,264,367; JP-A-2153997; With EP 622446), to reduce the come unstuck phosphorus content of edible oil of above-mentioned water.
But these documents specifically do not indicate in the enzymatic degumming method and use less water.
On the contrary, big water gaging is used in EP 622446 suggestions in the enzymatic degumming method.Referring to the page 3 of the document, the capable and claim 4 of 33-44, its suggestion is used in aforesaid method and is accounted for the oily water that weighs more than 30%.
The invention summary
The present invention is that not only simplification but also the economic inexpensive method that makes the edible oil enzymatic degumming are provided with the problem that solves.
Described solution uses less water to implement aforesaid method.
Therefore, the present invention relates to reduce the method for phosphorus containing components content in the edible oil, the phosphorus content of described edible oil is 50-10,000ppm, this method comprises above-mentioned oil and the phospholipase A1 (PLA1) that makes pH 1.5-8, Phospholipase A2 (PLA2), or the aqueous solution that is emulsified in the oil of phospholipase B (PLB) contacts, phosphorus content reduces to below the 12ppm in oil, water phase separated from the oil of handling then, wherein aforesaid method is characterised in that, above-mentioned emulsification condition is to utilize the water that accounts for the heavy 0.01-1.5% of oil, preferably account for the water of the heavy 0.01-1.0% of oil, more preferably account for the water of the heavy 0.01-0.75% of oil, more preferably account for the water of the heavy 0.01-0.5% of oil, most preferably account for the water of the heavy 0.01-0.4% of oil and form.
And this may being preferably than the low dosage scope that accounts for that oil weighs 0.01% above water accounts for oily heavy 0.1% water.
The benefit of methods described herein is to have reduced the cost of water and wastewater treatment.In addition, owing to having only a spot of oil will waste aqueous phase the oil recovery amount is increased.
In addition, the benefit of methods described herein may be, adopts the oily factory of this method might omit mud process recycling to used sewage in this method.
Enzymatic degumming method known in the art produces large amount of sewage, and the cost of its purifying treatment is very high.Fortune obviously is a kind of economical load.
In addition, oily factory has to process water is carried out recirculation to reduce the cost of above-mentioned sewage purifying in the past.
Can omit above-mentioned recirculation step by less water used in the methods described herein.
According to this area (as US 5,264,367) when carrying out enzymatic degumming, usually with the water-in-oil emulsion heat treated to as 65-75 ℃ to promote oil phase and water by as centrifugal the separation.When in the degumming of oil method, using thermostability Phospholipid hydrolase Lecitase TM(Novo Nordisk A/S, in the time of Denmark), the water that contains this enzyme can advantageously be reused repeatedly (add or do not add fresh enzyme solution all can).
Yet, for oily factory, if can avoid the recirculation of water more favourable fully.Generally speaking, recirculation means that the overall consumption of water will increase, and cost also will increase.If in the enzymatic degumming method, only used less water, can save quite debatable sometimes mud phase process recycling.
Embodiment of the present invention are described below, only for illustrating usefulness.
Detailed Description Of The Invention
Edible oil:
In principle, any edible oil can come unstuck by method of the present invention.The oil that the example of oil such as crude oil and water come unstuck.
Crude oil (also claiming not degummed oil) may be from as squeezing Semen Brassicae campestris, soybean or the Sunflower Receptacle or oil that extracts or their mixture.Phospholipids content in the crude oil may change between 0.5-3%w/w, is 200-10 corresponding to phosphorus content, 000ppm, more preferably 250-1200ppm.Except phosphatide, also comprise the metal/phosphatidic acid mixture of carbohydrate, sucrate and calcium, magnesium and the iron of lower concentration in the crude oil.
Preferably, above-mentioned edible oil is to remove viscose glue in advance and phosphorus content is the oil of 50-250ppm.
This class oil obtains and is referred to as " water degummed oil " by the water method for degumming usually.
The water degummed oil hot water by making 1-3%w/w usually mixes with temperature (60-90 ℃) crude oil and obtains.The general treatment time is 30-60 minute.When the step that water comes unstuck is removed hydration in oil insoluble phosphatide and cement natural gum.Hydration phosphatide and natural gum can be main method by sedimentation, filtration or centrifugal separation from oil-centrifugal.
Alternatively, also can be described as " hygrometric state is refining to remove viscose glue " (referring to US 5,264,367) in this method that is called " water comes unstuck ".
In addition, edible oil is preferably vegetables oil.
Used Phospholipid hydrolase in present method:
Preferably, used Phospholipid hydrolase is the Phospholipid hydrolase that derives from microorganism in the method for the present invention, and preferred microorganism is filamentous fungus, yeast or bacterium.
For the purposes of the present invention, " deriving from " dna sequence dna that refers to described enzyme and coding this kind of enzyme at this term with concrete microbe-derived use produces from described concrete source.
Thereby described enzyme is concrete to obtain microbe-derived from this by known standard method, and wherein said standard method can make the technician obtain to contain described enzyme also can be applied to sample in the method for the present invention.Above-mentioned standard method may be from the above-mentioned concrete dna sequence dna that carries out direct purification or the described enzyme of clones coding microbe-derived, recombinant expressed in same source then (homologous recombination expression) or recombinant expressed in different sources (allos is recombinant expressed).
More preferably, used Phospholipid hydrolase derives from the filamentous fungus of fusarium in the method for the present invention, as the bacterial strain of machete sickle spore, different spore sickle spore, fusariun solani, or the bacterial strain of fusarium oxysporum especially; Or
The filamentous fungus of Aspergillus, as the bacterial strain of Aspergillus awamori, smelly aspergillus, aspergillus japonicus, aspergillus niger, or the bacterial strain of aspergillus oryzae especially.
The example of suitable fusarium Phospholipid hydrolase is disclosed in:
I) people (Phytopathological notes 58:1437-38 (1968)) (a kind of fusariun solani bacterium Phospholipid hydrolase) such as Tsung-Che; With
Ii) EP number of patent application 97610056.0 discloses a kind of suitable machete sickle spore bacterium Phospholipid hydrolase (referring to the embodiment 18 of the document) and a kind of fusarium oxysporum bacterium Phospholipid hydrolase (referring to embodiment 1-17).
Suitable means of aspergillus phospholipase example is disclosed in:
I) EP 575133 discloses the Phospholipid hydrolase (claim 17 or 18) of multiple different aspergillus tubigensis Phospholipid hydrolase (referring to claim 14), especially aspergillus oryzae and the Phospholipid hydrolase (claim 19) of aspergillus niger; With
Ii) DE 19527274 A1 disclose a kind of suitable aspergillus goods (referring to embodiment).
In addition, (Roehm Germany) also is applicable in the method for the present invention commercially available Phospholipid hydrolase goods Degomma VOD, it is believed that this goods comprise the aspergillus tubigensis Phospholipid hydrolase.
In addition, used Phospholipid hydrolase has specific characteristic in the preferred method of the present invention.
Therefore, embodiment of the present invention relate to:
I) a kind of the method according to this invention, wherein said Phospholipid hydrolase are not rely on Ca in fact 2+The Phospholipid hydrolase of concentration, this Phospholipid hydrolase are at 5mM EDTA and 5mM Ca in the phospholipase activity test 2+The relative phospholipase activity that records down, this test are to comprise 2% phosphatidylcholine, 2% TritonX-100,20mM Citrate trianion, in the slow middle liquid of pH 5; 37 ℃ of incubations 10 minutes are measured the free fatty acids that discharges in the phosphatidylcholine then in 95 ℃ of termination reactions 5 minutes; 5mMEDTA/5mM Ca wherein 2+The time the ratio of relative phospholipase activity greater than 0.25, more preferably greater than 0.5; And/or
Ii) a kind of the method according to this invention, wherein said Phospholipid hydrolase be have can discharge at least 7 μ mol free fatty acidies/minute/the mg enzyme; More preferably at least 15 μ mol free fatty acidies/minute/Phospholipid hydrolase of the phospholipase activity of mg enzyme, this Phospholipid hydrolase is measured in test with the form of its phospholipase activity, this test is to comprise 2% phosphatidylcholine, 2% Triton X-100,20mM Citrate trianion, in the damping fluid of pH 5; 37 ℃ of incubations are measured the free fatty acids that discharges in the phosphatidylcholine in 95 ℃ of termination reactions 5 minutes after 10 minutes.
The detailed description of above-mentioned test is disclosed in the specific embodiment of this paper (vide infra).EP number of patent application 97610056.0 (referring to the embodiment in the document 9) is seen in more detailed reference.
In addition, confirmed that the feature that specificity is suitable for the general method of enzymatic degumming of oil and is particularly suited for Phospholipid hydrolase of improving one's methods described herein is to have specific one-level aminoacid sequence.
Therefore, in another embodiment of the present invention, the present invention relates to a method according to the present present invention, wherein said Phospholipid hydrolase is the Phospholipid hydrolase with the peptide sequence that is selected from following group:
(a) has the polypeptide that is shown in the aminoacid sequence of 31-346 position among the SEQ ID NO 1;
(b) has the polypeptide that is shown in the aminoacid sequence of 31-303 position among the SEQ ID NO 1;
(c) with (a), or the polypeptide that defined aforementioned polypeptides has at least 70% homology (b); And (a), (b) or fragment (c).
To the clone of Phospholipid hydrolase and the detailed description of purifying, referring to EP number of patent application 97610056.0 with aforementioned polypeptides sequence.
In the document, the Phospholipid hydrolase that also derives from the fusarium oxysporum bacterium as can be known and have above-mentioned (b) described peptide sequence shows above-mentioned two functional characteristics.Therefore, this Phospholipid hydrolase is a most preferred Phospholipid hydrolase used in the method for the present invention.The embodiment of this paper has confirmed the application (vide infra) of this Phospholipid hydrolase.
At last, an example of suitable non-microorganism Phospholipid hydrolase is the commercially available Phospholipid hydrolase (Lecitase that derives from the pig pancreas TM, Novo Nordisk A/S, Denmark).
The standard technology parameter of method of the present invention
Except special use less water, can use any other processing parameter in the method for the present invention according to this area.Background parts referring to the reference methods known in the art.
Enzymically treat is by disperseing the Phospholipid hydrolase aqueous solution to carry out, preferably the described aqueous solution being separated into the droplet of mean diameter less than 10 μ (little) m.
The method according to this invention, the consumption of water are the 0.01-1.5% with respect to weight of oil.
Can choose the adding emulsifying agent wantonly.Can utilize mechanical oscillation to keep this emulsion.
Enzymically treat can be carried out under the arbitrary pH in the scope of pH 1.5-8, preferred pH 3-6.Can adjust pH by adding citric acid, citrate buffer, NaOH or HCl.
Temperature is generally at 30-75 ℃ suit (particularly 40-60 ℃).Reaction times is generally 0.5-12 hour (as 2-6 hour), and suitable enzyme dosage is generally every liter of oily 100-5000IU, particularly 200-2000IU/l.
Enzymically treat can stir in as jar in batches carries out, or can carry out continuously in the retort that stirs as a group.
Water phase separated and oil phase after the enzymically treat.This separation is by conventional methods as centrifugal carrying out.But this value of method of the present invention is reduced to below the 12ppm, more preferably below the 10ppm, below the further preferred 5ppm.
Material and method
Embodiment
Embodiment 1
Summation to the test of edible oil enzymatic degumming
Be used to carry out the equipment of enzymatic degumming
This equipment is by 1L jacketed type steel reactor (being furnished with the steel lid), thruster (about 600rpm), traverse baffle, temperature sensor, top inhalent siphon, top reflux exchanger (about 4 ℃) and bottom rising pipe composition.Reactor jacket communicates with water bath with thermostatic control.Rising pipe leads to the online stirring-head of Silverson that has been equipped with " square opening high-shear isolated screen (high shear screen) " through the polysiloxane pipeline, this stirring-head is driven by SilversonL4RT high-shear laboratory stirrer (about 8500rpm, about 1.1 liters/minute of flow velocity).Stirring-head is equipped with cooling worm (5-10 ℃) and rising pipe and communicates with the inhalent siphon pipeline of this reactor through the polysiloxane pipeline.Temperature sensor is inserted in the polysiloxane pipeline, just after stirring-head.Reactor/stirring-head system is carried out unique being connected with external environment by reflux exchanger.
Carry out the general method of enzymatic degumming
Start all coolings and thermostatic equipment.Then 0.6L (about 560g) oil is added in the reactor, this reactor remains near the required temperature of concrete experiment.Start laboratory stirrer, oil begins to return reactor from reactor cycles to stirring-head whereby.Made this system balancing about 10 minutes, accurate during this period base measuring temperature.Add an amount of mixture of the citric acid monohydrate compound 0.6g (2.86mmol) be dissolved in the suitable quantity of water or citric acid and trisodium citrate and begin pretreatment stage and (see table 1 and 7; Water/the fat liquor of [citric acid]=4.6mM), t=0 is set this moment.In the time of t=30 minute, add an amount of 4MNaOH solution (referring to table 1 and 7).
Water content among the table 1. experiment A-D; Wdg (water comes unstuck) rapeseed oil
Experiment Water content Water in the 560g oil The water that adds during t=0 Water in the NaOH solution Water in the enzyme solution Total Water
A 5.3％ 0.56g 27g 1.1g 1.0g 29.7g
B 1.3％ 0.56g 5.0g 0.7g 1.0g 7.3g
C 0.3％ 0.56g 0.05g * 0g 1.0g 1.6g
D 0.3％ 0.56g 0.07g ** 0g 1.0g 1.6g
* the water that provides of 0.6g citric acid monohydrate compound.
The water that the mixture of * 0.5g citric acid monohydrate compound and 0.14g citrate trisodium dihydrate provides.
In the time of t=35 minute, the P-analysis is carried out in sampling and pH measures.And then, the enzyme solution (pretreatment stage end) that adds aequum.At t=1, take out the sample that is used for P (phosphorus) analysis and pH mensuration in the time of 2,3.5,5,6 hours, then termination reaction.
Emptying reactor/agitator system with 500ml 10% Deconex/DI (deionization) aqueous solution rinsing 2 times, is washed 3 times at least with 500ml DI water again.Respectively add step and sampling procedure during table 2 Indicator Reaction.
Table 2. enzyme processing time table
Figure 9980400500101
Phosphorus is analyzed:
The P analytical sampling:
Get the 10ml water-in-oil emulsion, add in the glass centrifuge tube.This emulsion was heated in boiling water bath 30 minutes.Under 5000rpm centrifugal 10 minutes.The upper strata (oil phase) of about 8ml is transferred in the polystyrene test tube of a 12ml, left standstill (with sedimentation) 12-24 hour.Transparently after the sedimentation get about 1-2g mutually and carry out P and analyze from the upper strata.
P analyzes by 2.421 schemes in " standard method that oil, fat and derivative thereof are analyzed, the 7th edition (1987) " and is undertaken:
Claim 100mg Mgo (leicht, Merck #5862) to be placed in the porcelain dish, heat with Gas blowtorch.Add 1-2g oil, light, produce black, hard material with Gas blowtorch.In the Vecstar process furnace,, form lime in 850 ℃ of heating 2 hours.This ash is dissolved in the 5ml 6M nitric acid, adds 20ml mix reagent (reagent mix).Left standstill 20 minutes.Measure the absorbancy at 460nm place and (use blank (5ml HNO 3+ 20ml mix reagent) zeroing).Utilize calibration curve to calculate.
Get the 2ml water-in-oil emulsion, mix with 2ml MilliQ water.After waiting to be separated, remove the top oil reservoir with the transfer pipet suction.Measure the pH of aqueous phase with pH electrode Orion.Observed value is converted to " truly " pH value with following formula
PH Truly=pH Measured value-0.38.
Calibration curve obtains by 0.6g citric acid monohydrate compound is dissolved in the 27g DI water; Measure the pH (pH of this solution with pH electrode Orion Truly).Get 100 μ l and mix, measure the pH (pH of this solution with pH electrode Orion with 2ml MilliQ water Measured value).Change the pH of citric acid solution by adding NaOH solution gradually,, as above dilute and pH mensuration for each adjustment.
Embodiment 2
The vegetable seed oil degumming that water comes unstuck is handled (I)
Experiment is all undertaken by above embodiment 1 described " carrying out the general method of enzymatic degumming ".
Oil:
The rapeseed oil that water comes unstuck is from the Arhus Oliefabrik (AOM) of Denmark.Lot number C00730/B01700 and C00730/B01702, phosphorus content 231-236ppm.Water content≤0.1%w/w.
Enzyme:
PL (Phospholipid hydrolase) with fusarium oxysporum bacterium of aminoacid sequence shown in the SEQ NO 1.Lot number F-9702027, estimated concentration 0.75mg/ml.
This enzyme carries out recombinant expressed and purifying as described in EP number of patent application 97610056.0.
Experiment A (water content 5.3%)
With in the rapeseed oil adding equipment of 0.6L (560g) wdg (water comes unstuck) and be heated to 40 ℃.In the time of t=0 minute, adding is dissolved in the solution that forms in the 27g water by 0.6g citric acid monohydrate compound.In the time of t=30 minute, add 1.07ml (4.3 mmole) 4M NaOH solution, making pH is about 5.In the time of t=35 minute, add the purification solution of 1ml (0.75mg) fusarium oxysporum bacterium Phospholipid hydrolase.The oil phase phosphorus content and the aqueous pH values that record after centrifugal are shown in Table 3.
The result that table 3.wdg rapeseed oil comes unstuck with fusarium oxysporum bacterium Phospholipid hydrolase, water content 5.3%.
Time (hour) The oil phase phosphorus content pH
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%)
As above experiment A, different is, in the time of t=0 minute, adds and is dissolved in the solution that forms in the 5.0g water by 0.6g citric acid monohydrate compound, in the time of t=30 minute, adds 0.71ml (2.86 mmole) 4M NaOH solution, and making pH is about 5.The oil phase phosphorus content and the aqueous pH values that record after centrifugal are shown in Table 4.
The result that table 4.wdg rapeseed oil comes unstuck with fusarium oxysporum bacterium Phospholipid hydrolase, water content 1.3%.
Time (hour) The oil phase phosphorus content pH
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
Experiment C (water content 0.3%)
As above experiment A, different is, in the time of t=0 minute, adds 0.6g citric acid monohydrate compound powder, in the time of t=30 minute, does not add NaOH solution, and making pH is about 5.The oil phase phosphorus content and the aqueous pH values that record after centrifugal are shown in Table 5.
The result that table 5.wdg rapeseed oil comes unstuck with fusarium oxysporum bacterium Phospholipid hydrolase, water content 0.3%.
Time (hour) The oil phase phosphorus content pH
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
Experiment D (water content 0.3%) is as described in the above experiment C, and different is, in the time of t=0 minute, adding is by the mixture of 0.5g citric acid monohydrate compound and 0.14g citrate trisodium dihydrate powder constituent, and making pH is about 5.The oil phase phosphorus content and the aqueous pH values that record after centrifugal are shown in Table 6.
The result that table 6.wdg rapeseed oil comes unstuck with fusarium oxysporum bacterium Phospholipid hydrolase, water content 0.3%.
Time (hour) The oil phase phosphorus content pH
0 243
0.50 244 5.5
0.58
1.0 101 5.1
2.0 8 4.9
Embodiment 3
The processing (II) of coming unstuck to thick rapeseed oil (squeezing with mixture that extract)
Experiment is all undertaken by above embodiment 1 described " general method of enzymatic degumming ".
Oil:
Thick rapeseed oil is from MILO Olomouk, Czechrep.Lot number C00745/B02042, phosphorus content 263ppm.Water content 0.17%w/w.
Water-content among table 7. experiment E and the F; Thick rapeseed oil
Experiment Water-content Water in the 560g oil The water that adds during t=0 Water in the NaOH solution Water in the enzyme solution Total Water
E 5.4％ 0.95g 27g 1.1g 1.0g 30.1g
F 1.4％ 0.95g 5.0g 0.7g 1.0g 7.7g
Experiment E (water content 5.4%)
With in the thick rapeseed oil adding equipment of 0.6L (560g) and be heated to 40 ℃.In the time of t=0 minute, adding is dissolved in the solution that forms in the 27g water by 0.6g citric acid monohydrate compound.In the time of t=30 minute, add 1.07ml (4.3 mmole) 4M NaOH solution, making pH is about 5.In the time of t=35 minute, add the purification solution of 1ml (0.75mg) fusarium oxysporum bacterium Phospholipid hydrolase.The oil phase phosphorus content and the aqueous pH values that record after centrifugal are shown in Table 8.
The result that the thick rapeseed oil of table 8. comes unstuck with fusarium oxysporum bacterium Phospholipid hydrolase, water content 5.4%.
Time (hour) The oil phase phosphorus content pH
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 10 5.3
Experiment F (water content 1.4%)
As above experiment E, different is, in the time of t=0 minute, adds and is dissolved in the solution that forms in the 5.0g water by 0.6g citric acid monohydrate compound, in the time of t=30 minute, adds 0.71ml (2.86 mmole) 4MNaOH solution, and making pH is about 5.The oil phase phosphorus content and the aqueous pH values that record after centrifugal are shown in Table 9.
The result that the thick rapeseed oil of table 9. comes unstuck with fusarium oxysporum bacterium Phospholipid hydrolase, water content 1.4%.
Time (hour) The oil phase phosphorus content pH
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
Embodiment 4
Be applicable to the qualification test of the Phospholipid hydrolase in the degumming of oil method of the present invention
The phospholipase activity test:
Phospholipase activity (PHLU) is to measure by measuring the free fatty acids that phosphatidylcholine discharged.Add 4% L-α-phosphatidylcholine (deriving from Avanti, the plant phosphatidylcholine of USA), 4%Triton X-100,5mM CaCl 250mM HEPES solution (pH 7), make 50 μ l enzyme solution in 50mM HEPES solution (pH 7), be diluted to proper concn.Sample is in 30 ℃ of incubations 10 minutes, centrifugal (under 7000rpm 5 minutes) preceding elder generation was 95 ℃ of termination reactions 5 minutes.NEFA C kit measurement free fatty acids with Wako ChemicalsGmbH; 25 μ l reaction mixtures are added in the 250 μ l reagent A and 37 ℃ of incubations 10 minutes.Add 500 μ l reagent B then, make sample once more 37 ℃ of incubations 10 minutes.Measure the absorbancy at 550nm place with HP 8452A diode array spectrophotometer.Sample all carries out at least in duplicate.The blind test of substrate and enzyme (pretreated enzyme sample (95 ℃ 10 minutes)+substrate) be also included within.Oleic acid is made the lipid acid standard substance.1 PHLU equals the enzyme amount that per minute under these conditions discharges 1 μ mol free fatty acids.
Alternatively, liquid during this test can be delayed at 37 ℃ 20mM Citrate trianion, pH 5 (Ca 2+Dependency) or carry out (pH distribution/temperature distribution/stability) in the 20mM Britton-Robinson damping fluid.
Phospholipase A1 activity (PLA1) is measured for substrate with 1-(sulphur-decanoyl)-2-decanoyl-1-sulfo--Xi-glyceryl-3-phosphocholine (D3761 molecular probe).In the cuvette of 200 μ l, to 190 μ l substrates (100 μ l D3761 (2mg/ml is dissolved in the ethanol)+50 μ l, 1% Triton X-100+1.85ml 50mM HEPES, 0.3mM DTNB, 2mM CaCl 2, add 10 μ l enzymes in pH7), on the HP8452A diode array spectrophotometer, at room temperature measure absorbancy as the 4l0nm place of the function of time.Calculate the interior rate of curve of linearity range as activity.PLA1 equals the enzyme amount that per minute under these conditions discharges 1 μ mol free fatty acids.
Phospholipase A2 activity (PLA2) is to measure with 1-palmitoyl-2-(1-pyrene decanoyl)-Xi-glyceryl-3-phosphocholine (H361 molecular probe).2ml substrate in the 2ml cuvette (methanol solution+10ml 50mM HEPES of 50 μ l 1%Triton X-100+25 μ l 0.1%H361, pH 7) under agitation add 10 μ l enzymes, with the pyrene fluorescent emission amount (at 340nm place excite) of Perkin Elmer LS50 measurement device as the 376nm place of the function (with 1 second interval) of time.In Triton X-100/ phosphatide micella, the concentration of adjusting phosphatide makes it to have the structure of excimer (in the emission of 480nm place).Through cracking, make the lipid acid of the 2-position that contains pyrenyl group be released into aqueous phase, cause the monomer emission measure to increase.PLA2 is the rate of curve in the isoconcentration lower linear scope.
Xu Liebiao ﹠amp; #60110﹠amp; #62NOVO NORDISK A/S﹠amp; #60120﹠amp; #62 enzymatic degumming of oil method; #60130﹠amp; #625570-WO﹠amp; #60140﹠amp; #62﹠amp; #60141﹠amp; #62﹠amp; #60160﹠amp; #621﹠amp; #60170﹠amp; #62PatentIn 2.0 ,1 ﹠amp; #60210﹠amp; #621﹠amp; #60211﹠amp; #62346﹠amp; #60212﹠amp; #62PRT﹠amp; #60213﹠amp; #62 fusarium oxysporum Jun ﹠amp; #60400﹠amp; #621Met Leu Leu Leu Pro Leu Leu Ser Ala Ile Thr Leu Ala Val Ala Ser 15 10 15Pro Val Ala Leu Asp Asp Tyr Val Asn Ser Leu Glu Glu Arg Ala Val
20??????????????????25??????????????????30Gly?Val?Thr?Thr?Thr?Asp?Phe?Ser?Asn?Phe?Lys?Phe?Tyr?Ile?Gln?His
35??????????????????40??????????????????45Gly?Ala?Ala?Ala?Tyr?Cys?Asn?Ser?Glu?Ala?Ala?Ala?Gly?Ser?Lys?Ile
50??????????????????55??????????????????60Thr?Cys?Ser?Asn?Asn?Gly?Cys?Pro?Thr?Val?Gln?Gly?Asn?Gly?Ala?Thr?65??????????????????70??????????????????75??????????????????80Ile?Val?Thr?Ser?Phe?Val?Gly?Ser?Lys?Thr?Gly?Ile?Gly?Gly?Tyr?Val
85??????????????????90??????????????????95Ala?Thr?Asp?Ser?Ala?Arg?Lys?Glu?Ile?Val?Val?Ser?Phe?Arg?Gly?Ser
100?????????????????105?????????????????110Ile?Asn?Ile?Arg?Asn?Trp?Leu?Thr?Asn?Leu?Asp?Phe?Gly?Gln?Glu?Asp
115?????????????????120?????????????????125Cys?Ser?Leu?Val?Ser?Gly?Cys?Gly?Val?His?Ser?Gly?Phe?Gln?Arg?Ala
130?????????????????135?????????????????140Trp?Asn?Glu?Ile?Ser?Ser?Gln?Ala?Thr?Ala?Ala?Val?Ala?Ser?Ala?Arg145?????????????????150?????????????????155?????????????????160Lys?Ala?Asn?Pro?Ser?Phe?Ash?Val?Ile?Ser?Thr?Gly?His?Ser?Leu?Gly
165?????????????????170?????????????????175Gly?Ala?Val?Ala?Val?Leu?Ala?Ala?Ala?Asn?Leu?Arg?Val?Gly?Gly?Thr
180?????????????????185?????????????????190Pro?Val?Asp?Ile?Tyr?Thr?Tyr?Gly?Ser?Pro?Arg?Val?Gly?Asn?Ala?Gln
195?????????????????200?????????????????205Leu?Ser?Ala?Phe?Val?Ser?Asn?Gln?Ala?Gly?Gly?Glu?Tyr?Arg?Val?Thr
210?????????????????215?????????????????220His?Ala?Asp?Asp?Pro?Val?Pro?Arg?Leu?Pro?Pro?Leu?Ile?Phe?Gly?Tyr225?????????????????230?????????????????235?????????????????240Arg?His?Thr?Thr?Pro?Glu?Phe?Trp?Leu?Ser?Gly?Gly?Gly?Gly?Asp?Lys
245?????????????????250?????????????????255Val?Asp?Tyr?Thr?Ile?Ser?Asp?Val?Lys?Val?Cys?Glu?Gly?Ala?Ala?Asn
260?????????????????265?????????????????270Leu?Gly?Cys?Asn?Gly?Gly?Thr?Leu?Gly?Leu?Asp?Ile?Ala?Ala?His?Leu
275?????????????????280?????????????????285His?Tyr?Phe?Gln?Ala?Thr?Asp?Ala?Cys?Asn?Ala?Gly?Gly?Phe?Ser?Trp
290?????????????????295?????????????????300Arg?Arg?Tyr?Arg?Ser?Ala?Glu?Ser?Val?Asp?Lys?Arg?Ala?Thr?Met?Thr305?????????????????310?????????????????315?????????????????320Asp?Ala?Glu?Leu?Glu?Lys?Lys?Leu?Asn?Ser?Tyr?Val?Gln?Met?Asp?Lys
325?????????????????330?????????????????335Glu?Tyr?Val?Lys?Asn?Asn?Gln?Ala?Arg?Ser
340?????????????????345

Claims (8)

1. method that reduces the content of phosphorus containing components in the edible oil, phosphorus content is per 1,000,000 parts of 50-10 in this edible oil, 000 part (ppm), this method comprises above-mentioned oil and the phospholipase A1 (PLA1) that makes pH 1.5-8, the aqueous solution that is emulsified in the oil of Phospholipase A2 (PLA2) or phospholipase B (PLB) contacts, phosphorus content reduces to below the 12ppm in oil, water phase separated from the oil of handling then, wherein aforesaid method is characterised in that, above-mentioned emulsification condition is to utilize the water that accounts for the heavy 0.01-1.5% of oil, preferably account for the water of the heavy 0.01-1.0% of oil, the water that most preferably accounts for the heavy 0.01-0.5% of oil forms.
2. the process of claim 1 wherein that above-mentioned oil is to remove viscose glue in advance and phosphorus content is the oil of 50-250ppm.
3. claim 1 or 2 method, wherein said Phospholipid hydrolase is the Phospholipid hydrolase that derives from microorganism, preferably derives from filamentous fungus, yeast or bacterium.
4. the method for claim 3, wherein said filamentous fungus belongs to fusarium, as the bacterial strain of machete sickle spore, different spore sickle spore, fusariun solani, or the bacterial strain of fusarium oxysporum especially.
5. the method for claim 3, wherein said filamentous fungus belongs to Aspergillus, as the bacterial strain of Aspergillus awamori, smelly aspergillus, aspergillus japonicus, aspergillus niger, or the bacterial strain of aspergillus oryzae especially.
6. each method in the aforementioned claim, wherein said Phospholipid hydrolase is not rely on Ca in fact 2+The Phospholipid hydrolase of concentration, this Phospholipid hydrolase be as relative phospholipase activity in the phospholipase activity test at 5mM EDTA and 5mM Ca 2+Record down, this test is to comprise 2% phosphatidylcholine, 2%Triton X-100,20mM Citrate trianion, in the damping fluid of pH 5; 37 ℃ of incubations 10 minutes in 95 ℃ of termination reactions 5 minutes, are measured the free fatty acids that discharges in the phosphatidylcholine then; 5mMEDTA/5mM Ca wherein 2+Under the ratio of relative phospholipase activity greater than 0.25, more preferably greater than 0.5.
7. each method in the aforementioned claim, wherein said Phospholipid hydrolase be have can discharge at least 7 μ mol free fatty acidies/minute/the mg enzyme; More preferably at least 15 μ mol free fatty acidies/minute/Phospholipid hydrolase of the phospholipase activity of mg enzyme, this Phospholipid hydrolase is measured in test with the form of its phospholipase activity, this test is to comprise 2% phosphatidylcholine, 2%Triton X-100,20mM Citrate trianion, in the slow middle liquid of pH 5; 37 ℃ of incubations 10 minutes in 95 ℃ of termination reactions 5 minutes, are measured the free fatty acids that discharges in the phosphatidylcholine then.
8. each method in the aforementioned claim, wherein said Phospholipid hydrolase is the Phospholipid hydrolase with the peptide sequence that is selected from following group:
(a) has the polypeptide that is shown in the aminoacid sequence of 31-346 position among the SEQ ID NO 1;
(b) has the polypeptide that is shown in the aminoacid sequence of 31-303 position among the SEQ ID NO 1;
(c) with (a) or the polypeptide that defined aforementioned polypeptides has at least 70% homology (b); And (a), (b) or fragment (c).
CNB998040053A 1998-04-08 1999-04-07 Enzymatic oil-degumming process Expired - Fee Related CN1139652C (en)

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