CN109943551A - A kind of lipase compositions and its application - Google Patents
A kind of lipase compositions and its application Download PDFInfo
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- CN109943551A CN109943551A CN201910332001.9A CN201910332001A CN109943551A CN 109943551 A CN109943551 A CN 109943551A CN 201910332001 A CN201910332001 A CN 201910332001A CN 109943551 A CN109943551 A CN 109943551A
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- 108090001060 Lipase Proteins 0.000 title claims abstract description 144
- 102000004882 Lipase Human genes 0.000 title claims abstract description 144
- 239000004367 Lipase Substances 0.000 title claims abstract description 144
- 235000019421 lipase Nutrition 0.000 title claims abstract description 144
- 239000000203 mixture Substances 0.000 title claims abstract description 78
- 238000006243 chemical reaction Methods 0.000 claims abstract description 58
- 102000004190 Enzymes Human genes 0.000 claims abstract description 38
- 108090000790 Enzymes Proteins 0.000 claims abstract description 38
- 238000005886 esterification reaction Methods 0.000 claims abstract description 31
- 230000032050 esterification Effects 0.000 claims abstract description 29
- 238000002360 preparation method Methods 0.000 claims abstract description 24
- 239000003599 detergent Substances 0.000 claims abstract description 18
- 241000223258 Thermomyces lanuginosus Species 0.000 claims abstract description 17
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 16
- 239000004519 grease Substances 0.000 claims abstract description 14
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 53
- 235000019387 fatty acid methyl ester Nutrition 0.000 claims description 33
- 239000003921 oil Substances 0.000 claims description 26
- 235000019198 oils Nutrition 0.000 claims description 25
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 239000000758 substrate Substances 0.000 claims description 13
- 239000003549 soybean oil Substances 0.000 claims description 12
- 235000012424 soybean oil Nutrition 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 235000019441 ethanol Nutrition 0.000 claims description 8
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 claims description 6
- 239000010806 kitchen waste Substances 0.000 claims description 6
- 230000020477 pH reduction Effects 0.000 claims description 4
- 239000008399 tap water Substances 0.000 claims description 4
- 235000020679 tap water Nutrition 0.000 claims description 4
- 229930182558 Sterol Natural products 0.000 claims description 3
- 229930003427 Vitamin E Natural products 0.000 claims description 3
- 239000010775 animal oil Substances 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 claims description 3
- 238000004332 deodorization Methods 0.000 claims description 3
- 230000001877 deodorizing effect Effects 0.000 claims description 3
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims description 3
- 239000010773 plant oil Substances 0.000 claims description 3
- 235000003702 sterols Nutrition 0.000 claims description 3
- 150000003432 sterols Chemical class 0.000 claims description 3
- 235000015112 vegetable and seed oil Nutrition 0.000 claims description 3
- 239000008158 vegetable oil Substances 0.000 claims description 3
- 239000011709 vitamin E Substances 0.000 claims description 3
- 235000019165 vitamin E Nutrition 0.000 claims description 3
- 229940046009 vitamin E Drugs 0.000 claims description 3
- 230000007062 hydrolysis Effects 0.000 abstract description 13
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 13
- 238000006555 catalytic reaction Methods 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 230000036632 reaction speed Effects 0.000 abstract description 8
- 230000035484 reaction time Effects 0.000 abstract description 7
- 230000003301 hydrolyzing effect Effects 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 5
- 230000002195 synergetic effect Effects 0.000 abstract description 5
- 150000003626 triacylglycerols Chemical class 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 19
- 239000002253 acid Substances 0.000 description 15
- 238000005119 centrifugation Methods 0.000 description 11
- 239000000654 additive Substances 0.000 description 10
- 230000000996 additive effect Effects 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
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- 238000010586 diagram Methods 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
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- 229960004756 ethanol Drugs 0.000 description 7
- 235000014113 dietary fatty acids Nutrition 0.000 description 6
- 229930195729 fatty acid Natural products 0.000 description 6
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- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 6
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- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 5
- 239000003225 biodiesel Substances 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- NWFNSTOSIVLCJA-UHFFFAOYSA-L copper;diacetate;hydrate Chemical compound O.[Cu+2].CC([O-])=O.CC([O-])=O NWFNSTOSIVLCJA-UHFFFAOYSA-L 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- -1 feed addictive Substances 0.000 description 4
- 239000004006 olive oil Substances 0.000 description 4
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- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 108090000371 Esterases Proteins 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
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- 239000012086 standard solution Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 235000019482 Palm oil Nutrition 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 150000004665 fatty acids Chemical group 0.000 description 2
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- 238000002156 mixing Methods 0.000 description 2
- 239000002540 palm oil Substances 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000011295 pitch Substances 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 235000010358 acesulfame potassium Nutrition 0.000 description 1
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- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
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- 230000001427 coherent effect Effects 0.000 description 1
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- 229960000935 dehydrated alcohol Drugs 0.000 description 1
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- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019256 formaldehyde Nutrition 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
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- 239000002798 polar solvent Substances 0.000 description 1
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- 150000004666 short chain fatty acids Chemical class 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Fats And Perfumes (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of lipase compositions and its applications, belong to technical field of enzyme engineering.Lipase compositions of the invention are made of the lipase from pseudomonas aeruginosa and the lipase from Thermomyces lanuginosus, by protein content, concrete composition is as follows: the lipase 50%~75% of the lipase 25%~50% of pseudomonas aeruginosa, Thermomyces lanuginosus.Lipase compositions of the invention have synergistic function to the hydrolysis and esterification of catalysis grease, can fast hydrolyzing triglycerides, accelerate esterification initial reaction speed, reaction process is gentler, fluctuation is few, reaction can generate more esterification products when ending, reduce enzyme preparation dosage, reaction time and production cost are effectively saved, there is excellent application value in the fields such as oil and fat chemical, feed addictive and detergent.
Description
Technical field
The present invention relates to technical field of enzyme engineering, and in particular to a kind of lipase compositions and its application.
Background technique
Enzyme preparation is as biocatalyst, specificity and specificity with height.The essence of enzyme preparation is protein, it
Catalysis and specificity be to be determined by the higher structure of protein.The protein being made of long chain amino acid, due to not
With the difference of difference group polarity possessed by amino acid residue, for example, due to intermolecular hydrogen bond, Van der Waals force, ionization
Power and the sometimes covalent bonds such as formation disulfide bond, passing through in the cell is folded and is modified, in being total to for these intermolecular forces
It is ultimately formed under same-action with functional higher structure.Zymoprotein by folding often has at least one catalysis reaction
Activated centre and a catching reaction substrate integrated structure.Integrated structure determine zymoprotein to the affinity of substrate, and
Reaction center is the position that true positive catalysis occurs.
Due to different zymoproteins primary structure (amino acid sequence) and higher structure (different folding modes) no
Together, different protease has different reaction centers and combines center.This namely different enzyme preparation height turns a property
And the reason of high efficiency.Enzyme preparation, due to the complexity of substrate, generally requires compound to reach during being catalyzed biochemical reaction
To effect.
Lipase is important one of industrial enzyme preparation kind, can be catalyzed the reactions such as solution rouge, transesterification, Lipase absobed, extensively
It is general to be applied to the industry such as fats and oils processing, food, medicine, daily use chemicals.In the existing lipase having been found that, triglyceride is acted on
Lipase be divided into lipase and esterase.The fatty acid chain length that the two difference is that they are acted on is different: esterase effect
In short chain fatty acids, and lipase acts on long chain fatty acids.The combination center of esterase and lipase is different, so to difference
The affinity of fatty acid is different.
Compared with general enzymatic structure, lipase is had the following characteristics that
1) interface is activated: when substrate is dissolved in water with monomeric form, lipase does not show catalysis activity or vigor
It is low, and concentration of substrate be more than solubility and with coherent condition occur (i.e. interface appearance) when, the vigor of lipase is sharply promoted.Rouge
For the substrate of fat enzyme based on long-chain fat acid glyceride not soluble in water, catalytic kinetics follow the rule of " interface activation ".
2) " cap structure ": the activated centre of lipase is covered by one section of peptide chain on surface, and substrate can not be directly entered.Cause
This lipase will exercise catalysis and need to undergo a conformation change, be exposed in closing structure and be covered on above catalytic site
Peptide fragment, this section of peptide chain by people visually be known as lipase " lid " structure.The cap structure of most of lipase is by one
A or multiple amphipathic α-helixstructures are constituted, and the hydrophobic region of lid and hydrophobic catalytic pocket interact, and hydrophilic segment is then
It is exposed to protein surface, is interacted with polar solvent molecule, the conformation of enzyme is stablized.After the lid of lipase is opened, lid
The hydrophobic amino acid exposure of son, plays an important role to the combination of Interfacial Adsorption and substrate.Therefore, the specific ammonia in cap structure
The arrangement of base acid and amphipathic substrate selectivity and catalysis activity important in inhibiting to lipase.
Since presently found lipase active is lower, so needing to develop the compound lipases group that can be improved enzymatic activity
Close object.It is fewer for the document and patent of lipase compatibility at present, it is also relatively easy, often only according to most basic most suitable
PH, optimum temperature etc. carry out compound, seldom start with compatibility from the affinity and activity of its structure and catalysis substrate.
Summary of the invention
For overcome the deficiencies in the prior art, the purpose of the present invention is to provide a kind of lipase compositions and its application,
The lipase compositions can accelerate initial reaction speed, fast hydrolyzing triglyceride, and reaction can generate more when ending
Esterification products greatly reduce enzyme preparation dosage, effectively save the reaction time.
To solve the above problems, the technical solution adopted in the present invention is as follows:
A kind of lipase compositions, the lipase compositions are continuous by the lipase from pseudomonas aeruginosa and from dredging
The lipase composition of shape is thermophilic hyphomycete.
As a preferred embodiment of the present invention, the lipase compositions are by following ingredient by mass percentage
Composition: the lipase 50%~75% of the lipase 25%~50% of pseudomonas aeruginosa, Thermomyces lanuginosus.
As a preferred embodiment of the present invention, the lipase compositions are by following ingredient by mass percentage
Composition: the lipase 50% of the lipase 50% of pseudomonas aeruginosa, Thermomyces lanuginosus.
The present invention should provide lipase compositions as described above in the application of esterification.
As a preferred embodiment of the present invention, the reaction substrate of the esterification is vegetable oil, animal oil, kitchen are given up
Abandon one of grease, deodorization distillate, acidification oil；The acry radical donor of the esterification is methanol, ethyl alcohol, one in glycerol
Kind；The reaction product of the esterification is one of fatty acid methyl ester, glycerol, pitch, vitamin E, sterol.
It is using lipase compositions as described above the present invention also provides a kind of preparation method of fatty acid methyl ester
Prepare fatty acid methyl ester, the specific steps are as follows:
1) pick-up kitchen waste grease or plant oil deodorizing distillate are placed in reaction unit, adjust pH to 5~6.5, stirring
It is uniformly spare；
2) it weighs in mass ratio in 0.1%~0.6% lipase compositions addition pure water or tap water, mixing is equal
It is even, it adds in the soybean oil deodorizer distillate in step 1), the reaction was continued；
3) methanol of 1.5~2 equivalents is repeatedly added in reaction process, continues to be stirred to react with the revolving speed of 10~300r/min
2~for 24 hours, it after completion of the reaction, draws oil sample, fatty acid methyl ester is made.
As a preferred embodiment of the present invention, the step 1) further includes the steps that lye is added, and the lye is 0-
The NaOH of 560ppm.The addition of lye forms a small amount of soap, to create bigger water-oil interface, enables lipase in water oil
Interfacial catalysis esterification or hydrolysis.
As a preferred embodiment of the present invention, before drawing oil sample in the step 3), by with 3000~12000r/min
Revolving speed centrifugation or stand 6~be layered oil sample for 24 hours.
As a preferred embodiment of the present invention, the temperature reacted in the step 1) is 30~45 DEG C；Before reaction starts
Moisture total moisture content is 1.5%-10% in system.
The present invention also provides lipase compositions as described above prepare feed addictive, the application in detergent.
Compared with prior art, the beneficial effects of the present invention are:
Lipase compositions of the invention will be by that will derive from pseudomonas aeruginosa (Pseudomonas aeruginosa)
Lipase (hereinafter referred to as PAL) and derive from Thermomyces lanuginosus (Thermomyces lanuginosus) rouge
Fat enzyme (hereinafter referred to as TLL) is applied in combination, and the hydrolysis and esterification reflection to catalysis grease have synergistic function, together
The lipase compositions hydrolysate oil enzyme activity of sample albumen additive amount is more than single enzyme same dose of 20% or more, can reacted
Fast hydrolyzing triglyceride obtains free fatty acid when beginning, to effectively accelerate initial reaction speed, reaction process is gentler,
Fluctuation is few, and reaction can generate more such as fatty acid methyl esters, ethyl ester esterification products when ending, so that lipase compositions exist
The collaboration that 1+1 > 2 occur in enzyme activity, hydrolysis efficiency, catalyzing esterification efficiency, feed addictive application, detergent application etc. is made
With, the additive amount of enzyme preparation in reaction time or the reaction when production can be saved, so that the production cost of enterprise is reduced,
Have in the industries such as oil and fat chemical industry, medicine intermediate synthesis, detergent, feed addictive, biodiesel and promotes well
Application value.
In same albumen additive amount, the raising of enzyme activity is 20% or more.Enzyme activity goes out emblem mark as enzyme preparation
Standard, the raising for measuring enzyme activity can save albumen additive amount for enterprise to save the cost.
Detailed description of the invention
Fig. 1 is the protein structure schematic diagram of the lipase PAL of the present invention from pseudomonas aeruginosa；
Fig. 2 is the protein structure schematic diagram of the lipase TLL of the present invention from Thermomyces lanuginosus；
Fig. 3 is the coordinate diagram that lipase compositions of the present invention measure enzyme activity three times；
Fig. 4 is the acid value comparison diagram of fatty acid methyl ester made from the lipase of different content；
The acid value comparison diagram for each group that Fig. 5 is the reaction time when being 6h；
The acid value comparison diagram for each group that Fig. 6 is the reaction time when being 8h；
Fig. 7 is the grease stain removal rate comparison diagram that lipase compositions of the invention are applied to detergent；
Fig. 8 is the hydrolysis efficiency comparison diagram that lipase compositions of the invention are applied to feed.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and detailed description.
A kind of lipase compositions, the lipase compositions are by the lipase PAL from pseudomonas aeruginosa and from thin
The lipase TTL of lanuginosus spore bacterium is formed.Preferably, lipase compositions of the invention are by below by albumen quality hundred
Score meter at being grouped as: the lipase of the lipase 25%~50% of pseudomonas aeruginosa, Thermomyces lanuginosus
50%~75%.It is highly preferred that the lipase compositions be by it is following by mass percentage at being grouped as: verdigris is false
The lipase 50% of the lipase 50% of monad, Thermomyces lanuginosus.Preferably, the lipase selected by the present invention
Biodiesel specific enzyme of the PAL from grass company, the Eversa Transform that lipase TTL is produced from Novozymes Company
1.0 and Eversa Transform 2.0.
The protein structure of lipase PAL and lipase TLL difference are as depicted in figs. 1 and 2, and both the above lipase all has
The typical activated centre cap structure of lipase, but the difference of " ditch " the shape structure in its lid and activated centre makes it have difference
Catalytic activity and specificity.In the esterification reaction, PAL starts three rouge hydrolysis rate of catalyzing glycerol quickly, but reacts close to eventually
When esterification ability it is slightly worse；TLL starts that hydrolysis ability is slightly weak, reaction is slower, and it is strong to be finally esterified ability, can generate more
Esterification products.The present invention can effectively accelerate initial reaction speed, fast hydrolyzing glycerol by the way that PAL and TLL to be applied in combination
Three rouge, reaction can generate more esterification products when ending, so that lipase compositions are urged in enzyme activity, hydrolysis efficiency, esterification
Change efficiency, feed addictive application, detergent application etc. and the synergistic effect of 1+1 > 2 occurs, it is anti-when can save production
The additive amount of enzyme preparation between seasonable or in reaction, so that the production cost of enterprise is reduced, among oil and fat chemical industry, medicine
There is good application value in the industries such as body synthesis, detergent, feed addictive, biodiesel.
The present invention should provide lipase compositions as described above in the application of esterification.Specifically, the esterification
The reaction substrate of reaction is one of vegetable oil, animal oil, kitchen waste grease, deodorization distillate, acidification oil；The esterification
The acry radical donor of reaction is one of methanol, ethyl alcohol, glycerol；The reaction product of the esterification is fatty acid methyl ester, sweet
One of oil, pitch, vitamin E, sterol.The present invention also provides lipase compositions as described above to add preparing feed
Add agent, the application in detergent.Lipase compositions of the invention are applied to reaction temperature when esterification or hydrolysis
It is 30~45 DEG C, the additive amount of enzyme is 0.1% or more (mass ratio)；When applied to detergent, reaction temperature is 15~40 DEG C.
It is using lipase compositions as described above the present invention also provides a kind of preparation method of fatty acid methyl ester
Prepare fatty acid methyl ester, the specific steps are as follows:
1) pick-up kitchen waste grease or plant oil deodorizing distillate are placed in reaction unit, and lye is added, and adjust pH to 5
~6.5, it is stirred evenly at 30~45 DEG C spare；Wherein, lye is the NaOH of 0-560ppm；
2) it weighs in mass ratio in 0.1%~0.6% lipase compositions addition pure water or tap water, mixing is equal
It is even, it adds in the soybean oil deodorizer distillate in step 1), the reaction was continued；
3) methanol of 1.5~2 equivalents is repeatedly added in reaction process, continues to be stirred to react with the revolving speed of 10~300r/min
2~for 24 hours, after completion of the reaction, by be centrifuged or stand 6 with the revolving speed of 3000~12000r/min~be layered oil sample for 24 hours, take
Fatty acid methyl ester is made in oil sample.
Embodiment 1:
A kind of lipase compositions, the lipase compositions by it is following by mass percentage at being grouped as: verdigris
The lipase 25% of the lipase 75% of pseudomonad, Thermomyces lanuginosus.
A kind of preparation method of fatty acid methyl ester is to prepare fatty acid methyl using lipase compositions as described above
Ester, the specific steps are as follows:
1) pick-up kitchen waste grease is placed in triangular flask, and the lye NaOH that 240-560ppm is added adjusts pH to 5~6, in
It is shaken up in the shaking table that temperature is 30 DEG C spare；
2) it takes the lipase compositions that mass ratio is 0.3% to be added in pure water, is uniformly mixed, adds in step 1)
Soybean oil deodorizer distillate in, be placed in shaking table and react；
3) methanol of 1.5 equivalents is added in 3h several times before reacting, and for 24 hours with the revolving speed centrifugation reaction of 300r/min, when 0h adds
Add the methanol of 0.25 equivalent, when 1h adds the methanol of 0.25 equivalent, and 2h adds the methanol of 0.5 equivalent, and when 3h adds 0.5
The methanol of equivalent；After completion of the reaction, it is drawn oil sample with being layered after the revolving speed centrifugation of 10000r/min, fatty acid methyl ester is made.
Embodiment 2:
A kind of lipase compositions, the lipase compositions by below based on the albumen quality percentage at being grouped as:
The lipase 50% of the lipase 50% of pseudomonas aeruginosa, Thermomyces lanuginosus.
A kind of preparation method of fatty acid methyl ester is to prepare fatty acid methyl using lipase compositions as described above
Ester, the specific steps are as follows:
1) it takes soybean oil deodorizer distillate to be placed in the reaction kettle that the volume with agitating paddle is 6 cubic metres, 200- is added
The lye NaOH of 300ppm adjusts pH to 5~6, spare for 38 DEG C in temperature under appropriate stirring；
2) it takes the lipase compositions that mass ratio is 0.2% to be added in tap water, is uniformly mixed, adds in step 1)
Soybean oil deodorizer distillate in, adjust entire reaction system moisture content be 3%, be placed in reaction kettle and react；
3) methanol of stream plus 1.5 equivalents in reaction 8h, with the revolving speed centrifugation reaction 8h of 50r/min；After completion of the reaction
10000rpm centrifugation layering, is made fatty acid methyl ester.
Embodiment 3:
A kind of lipase compositions, the lipase compositions by it is following by mass percentage at being grouped as: verdigris
The lipase 50% of the lipase 50% of pseudomonad, Thermomyces lanuginosus.
A kind of preparation method of fatty acid methyl ester is to prepare fatty acid methyl using lipase compositions as described above
Ester, the specific steps are as follows:
1) acidification oil is taken to be placed in triangular flask, the lye NaOH that 240-560ppm is added adjusts pH to 5~6, is in temperature
It is shaken up in 36 DEG C of shaking table spare；
2) it takes the lipase compositions that mass ratio is 0.3% to be added in pure water, is uniformly mixed, adds in step 1)
Soybean oil deodorizer distillate in, be placed in shaking table and react；
3) methanol of 1.5 equivalents is added in 3h several times before reacting, and for 24 hours with the revolving speed centrifugation reaction of 300r/min, when 0h adds
Add the methanol of 0.25 equivalent, when 1h adds the methanol of 0.25 equivalent, and 2h adds the methanol of 0.5 equivalent, and when 3h adds 0.5
The methanol of equivalent；After completion of the reaction, 24 hours are stood, fatty acid methyl ester is made.
Embodiment 4:
A kind of lipase compositions, the lipase compositions by it is following by mass percentage at being grouped as: verdigris
The lipase 75% of the lipase 25% of pseudomonad, Thermomyces lanuginosus.
A kind of preparation method of fatty acid methyl ester is to prepare fatty acid methyl using lipase compositions as described above
Ester, the specific steps are as follows:
1) soybean oil is taken to be placed in triangular flask, the lye NaOH that 240-560ppm is added adjusts pH to 5~6, is in temperature
It is shaken up in 40 DEG C of shaking table spare；
2) it takes the lipase compositions that mass ratio is 0.3% to be added in pure water, is uniformly mixed, adds in step 1)
Soybean oil deodorizer distillate in, be placed in shaking table and react；
3) ethyl alcohol of 1.5 equivalents is added in 3h several times before reacting, and for 24 hours with the revolving speed centrifugation reaction of 300r/min, when 0h adds
Add the ethyl alcohol of 0.25 equivalent, when 1h adds the ethyl alcohol of 0.25 equivalent, and 2h adds the methanol of 0.5 equivalent, and when 3h adds 0.5
The ethyl alcohol of equivalent；After completion of the reaction, to draw oil sample after the revolving speed centrifugation of 3000r/min, obtained fatty-acid ethyl ester=.
Comparative example 1:
A kind of preparation method of fatty acid methyl ester is using lipase PAL system for fatty acid methyl ester, and specific steps are such as
Under:
1) take soybean oil deodorizer distillate to be placed in triangular flask, be added 240-560ppm lye NaOH adjust pH to 5~
6, it is shaken up in the shaking table that temperature is 36 DEG C spare；
2) it takes the lipase compositions that mass ratio is 0.3% to be added in pure water, is uniformly mixed, adds in step 1)
Soybean oil deodorizer distillate in, be placed in shaking table and react；
3) methanol of 1.5 equivalents is added in 3h several times before reacting, and for 24 hours with the revolving speed centrifugation reaction of 300r/min, when 0h adds
Add the methanol of 0.25 equivalent, when 1h adds the methanol of 0.25 equivalent, and 2h adds the methanol of 0.5 equivalent, and when 3h adds 0.5
The methanol of equivalent；After completion of the reaction, to draw oil sample after the revolving speed centrifugation of 5000r/min, fatty acid methyl ester is made.
Comparative example 2:
A kind of preparation method of fatty acid methyl ester is that fatty acid methyl ester is prepared using lipase TTL, and specific steps are such as
Under:
1) take soybean oil deodorizer distillate to be placed in triangular flask, be added 240-560ppm lye NaOH adjust pH to 5~
6, it is shaken up in the shaking table that temperature is 36 DEG C spare；
2) it takes the lipase TTL that mass ratio is 0.3% to be added in pure water, is uniformly mixed, adds big in step 1)
In soybean deodorized sludge, it is placed in shaking table and reacts；
3) methanol of 1.5 equivalents is added in 3h several times before reacting, and for 24 hours with the revolving speed centrifugation reaction of 300r/min, when 0h adds
Add the methanol of 0.25 equivalent, when 1h adds the methanol of 0.25 equivalent, and 2h adds the methanol of 0.5 equivalent, and when 3h adds 0.5
The methanol of equivalent；After completion of the reaction, 8 hours are stood, fatty acid methyl ester is made.
One, enzyme activity determination
The enzyme activity of lipase compositions of the present invention is measured referring to National Standard Method, as a result as shown in Figure 3.From the figure 3, it may be seen that of the invention
The enzyme activity of lipase compositions be respectively more than theoretical value 40%, 23% and 14%, (note: theoretical value be single enzyme according to its
The calculated enzyme activity of adding proportion in composition) therefore lipase PAL from pseudomonas aeruginosa and thermophilic from continuous shape is dredged
The lipase TTL of hyphomycete is applied in combination, and has significant synergistic effect.
Two, the acid value determination of fatty acid methyl ester
Acid value determination, measuring method ginseng are carried out to the fatty acid methyl ester in 1~2 preparation process of Examples 1 to 3 and comparative example
According to GB5009.229-2016, the specific method is as follows:
Accurately weigh respectively each group in 0,1,2,3,4,6, the parallel oil sample of 1g in 8h reaction time in 250ml triangular flask,
It is each that 20ml petroleum ether-dehydrated alcohol (mixed volume ratio 2:1) is added, sufficiently shake up.Then 2 drop phenolphthalein indicators are added
(1000ml95% ethyl alcohol is added in 10g/L, i.e. 10g phenolphthalein), is added dropwise 0.05mol/L KOH standard solution, until solution is in light red
Color, and colour-fast after 30 seconds is terminal.Acid value calculation formula is as follows:
Acid value (mgKOH/g)=(V × M × 56.11)/W
In formula, V is volume needed for titration KOH standard solution, unit ml；M is the concentration of KOH standard solution, unit
For mol/L；W is the quality for weighing oil sample, unit g.
Measurement record each group is in experimental result is as shown in table 1 and Fig. 4 respectively.
The acid value of fatty acid methyl ester made from the lipase of 1 different content of table
| Time | 100%PAL | 75%PAL+25%TTL | 50%PAL+50%TTL | 25%PAL+75%TTL | 100%TTL |
| 0h | 29.2840 | 29.3958 | 30.6253 | 30.6812 | 31.2121 |
| 1h | 29.5914 | 27.4957 | 28.6413 | 28.7811 | 32.1063 |
| 2h | 24.9529 | 28.9767 | 27.1324 | 27.5516 | 25.4279 |
| 3h | 23.4160 | 23.1925 | 22.9131 | 23.6675 | 26.7971 |
| 4h | 17.0451 | 18.5819 | 17.2407 | 17.9952 | 22.6895 |
| 6h | 10.5100 | 12.0154 | 6.5107 | 8.9417 | 12.6860 |
| 8h | 7.2300 | 6.0077 | 5.0297 | 5.0297 | 5.5886 |
As shown in table 1 and Fig. 4, the 1 initial reaction speed of comparative example for individually adding PAL is fast, but last stage reaction (6~8h) is urged
The ability decline for changing esterification, cannot further decrease acid value；Individually 2 reaction speed of comparative example of addition TLL is slower, but most
Acid value can be lowered eventually；And it is added to the Examples 1 to 3 of lipase compositions of the invention, it can effectively accelerate initial reaction speed
Degree, fast hydrolyzing triglyceride, and final acid value can be reduced, reaction can generate more esterification products when ending, and thus may be used
See, the reaction time of 2 hours can be saved by adding lipase compositions of the invention, huge to the practical significance of production.
As shown in figure 5, the sequence of the acid value of each group from low to high is successively when reaction 6h: 2 (50%PAL+ of embodiment
50%TTL), embodiment 3 (25%PAL+75%TTL), comparative example 1 (100%PAL), embodiment 1 (75%PAL+25%TTL),
Comparative example 2 (100%TTL).As shown in fig. 6, the sequence of the acid value of each group from low to high is successively when reaction 8h: embodiment 2
(50%PAL+50%TTL), embodiment 3 (25%PAL+75%TTL), comparative example 2 (100%TTL), 1 (75%PAL of embodiment
+ 25%TTL), comparative example 1 (100%PAL).It can be seen that the optimum proportioning of two lipase is 50%PAL+50%TTL, rouge
The optimum ratio range of fat enzyme PAL is 25%~50%, for most when the optimum ratio range of lipase TTL is 50%~75%
Good, the above ratio is the additive amount of protein.
Three, lipase compositions are applied to the compliance test result of detergent
Lipase compositions (PAL+TTL) of the invention, PAL, TTL are conventionally made containing enzyme washing respectively
Agent washs the soiled cotton containing olive oil spot using each group detergent, grease stain removal rate is measured respectively, as a result such as table 2 and Fig. 7 institute
Show.
Specific assay method is as follows:
1, soiled cotton is made on cotton with the olive oil solution drop that 100mg/ml is dissolved in benzene；
2, soiled cotton is put into the detergent of ready fatty enzyme and is divided at 37 DEG C with 100rpm revolving speed shaking flask 30
Clock；Soiled cotton is air-dried after flushing；
3, soiled cotton is extracted with petroleum ether and measure remaining olive oil mass, calculate olive oil removal rate on soiled cotton.
The grease stain removal rate of detergent of the table 2 containing different content lipase
| Enzyme additive amount | PAL | PAL+TLL | TLL |
| 0.10% | 49% | 51% | 51% |
| 0.20% | 57% | 62% | 57% |
| 0.30% | 63% | 68% | 64% |
| 0.40% | 68% | 73% | 69% |
| 0.50% | 70% | 75% | 70% |
As shown in table 2 and Fig. 7, it is added to oil of the detergent of lipase compositions of the invention in different enzyme additive amounts
Stain removal rate is superior to individually add the grease stain removal rate of PAL lipase or TTL lipase, it can be seen that, fat of the invention
Enzymatic compositions collaboration enhances the grease stain removal efficiency of detergent.
Four, lipase compositions are applied to the effect Validation in vitro of feed
Animal simple stomach intestinal physiology condition is simulated, reaction is hydrolyzed in the lipase that 10% is added in palm oil, reacts
Condition is as follows: pH value 7.5, and reaction temperature is 37 DEG C, hydrolysis time 2h；Compare PAL, the palm oil of PAL+TTL, TTL
Hydrolysis, as a result as shown in Figure 8.As shown in Figure 8, in vitro digestion is tested, the hydrolysis of lipase compositions of the invention
Efficiency can be improved to 123%.
In conclusion lipase compositions of the invention are by will be from lipase (and the source of pseudomonas aeruginosa
It is applied in combination in the lipase of Thermomyces lanuginosus, can effectively accelerate initial reaction speed, fast hydrolyzing triglyceride,
Reaction can generate more esterification products when ending, so that lipase compositions are in enzyme activity, hydrolysis efficiency, catalyzing esterification effect
There is the synergistic effect of 1+1 > 2 in rate, feed addictive application, detergent application etc., when can save reaction when production
Between or reaction in the additive amount of enzyme preparation closed in oil and fat chemical industry, medicine intermediate to reduce the production cost of enterprise
At there is good application value in, industries such as detergent, feed addictive, biodiesel.
The above embodiment is only the preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto,
The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed range.
Claims (10)
1. a kind of lipase compositions, it is characterised in that: the lipase compositions are by the lipase from pseudomonas aeruginosa
With the lipase composition from Thermomyces lanuginosus.
2. lipase compositions according to claim 1, it is characterised in that: the lipase compositions are by below by matter
Amount percentage meter at being grouped as: the fat of the lipase 25%~50% of pseudomonas aeruginosa, Thermomyces lanuginosus
Enzyme 50%~75%.
3. lipase compositions according to claim 1, it is characterised in that: the lipase compositions are by below by matter
Amount percentage meter at being grouped as: the lipase 50% of the lipase 50% of pseudomonas aeruginosa, Thermomyces lanuginosus.
4. lipase compositions as described in claim 1 are in the application of esterification.
5. lipase compositions according to claim 4 are in the application of esterification, it is characterised in that: the esterification
Reaction substrate be one of vegetable oil, animal oil, kitchen waste grease, deodorization distillate, acidification oil；The esterification
Acry radical donor be one of methanol, ethyl alcohol, glycerol；The reaction product of the esterification is fatty acid methyl ester, glycerol, drip
One of blueness, vitamin E, sterol.
6. a kind of preparation method of fatty acid methyl ester, it is characterised in that: use lipase compositions system as described in claim 1
Standby fatty acid methyl ester, the specific steps are as follows:
1) pick-up kitchen waste grease or plant oil deodorizing distillate are placed in reaction unit, are adjusted pH to 5~6.5, are stirred evenly
It is spare；
2) it weighs in 0.1%~0.6% lipase compositions addition pure water or tap water, is uniformly mixed in mass ratio, then
It is added in the soybean oil deodorizer distillate in step 1), the reaction was continued；
3) methanol of 1.5~2 equivalents is repeatedly added in reaction process, continue to be stirred to react 2 with the revolving speed of 10~300r/min~
For 24 hours, after completion of the reaction, it draws oil sample, fatty acid methyl ester is made.
7. the preparation method of fatty acid methyl ester according to claim 6, it is characterised in that: the step 1) further includes being added
The step of lye, the lye are the NaOH of 0-560ppm.
8. the preparation method of fatty acid methyl ester according to claim 6, it is characterised in that: draw oil sample in the step 3)
Before, by be centrifuged or stand 6 with the revolving speed of 3000~12000r/min~be layered oil sample for 24 hours.
9. the preparation method of fatty acid methyl ester according to claim 6, it is characterised in that: the temperature reacted in the step 1)
Degree is 30~45 DEG C；Moisture total moisture content is 1.5%-10% in system before reaction starts.
10. lipase compositions according to claim 1 prepare feed addictive, the application in detergent.
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