CN104313010A - Preparation method of magnetic response composite lipase and application in biodiesel synthesis - Google Patents
Preparation method of magnetic response composite lipase and application in biodiesel synthesis Download PDFInfo
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- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention relates to a preparation method of magnetic response composite lipase, and preparation of biodiesel by using the magnetic response composite lipase, and belongs to the technical field of preparation of biodiesel by using immobilized enzyme. According to the preparation method, magnetic nano Fe3O4 is adopted to modify lipase A with 1,3-position specificity, an immobilization technique is adopted to prepare the lipase A with the 1,3-position specificity and lipase B without position specificity into a composite enzyme double-enzyme system, as a method of composite lipase concerted catalysis is adopted, the substrate specificity of single lipase is overcome, the transesterification efficiency in preparing biodiesel by using an enzyme method is improved, recycling and repeated use of enzyme are achieved, and the enzyme activity loss in the reaction process is reduced.
Description
Technical field
The present invention relates to process for preparing biodiesel by enzyme technical field, particularly utilize two kinds of lipase preparations with different catalysis characteristics to have the immobilization Composite Double enzyme system of magnetic responsiveness and the application in biofuel synthesis thereof.
Background technology
Biofuel has the good characteristics such as renewable, biodegradable, environmental friendliness and is subject to extensive concern compared with traditional petrifaction diesel.Biofuel take mainly vegetable and animals oils as raw material, the long chain fatty acid ester class material (fatty acid methyl ester or ethyl ester) be prepared into by transesterification.The preparation method of current biofuel mainly contains chemical method and biological enzyme.Chemical method, mainly base catalysis method, production technique is more ripe, is to prepare the conventional method of biofuel at present; But the shortcomings such as chemical method exists, and energy consumption is high, complex process, alcohol consumption large, contaminate environment.Biodiesel production by bio-enzymatic method is the katalysis utilizing lipase, realizes the transesterification of grease and short chain fatty alcohol, has the advantages such as reaction conditions gentleness, alcohol consumption are few, environmental protection; Thus Lipids Enzymatic prepares the friendly process that biofuel is considered to substituted chemistry method production biofuel.
Lipase (lipase, E.C.3.1.1.3), also known as acylglycerol lytic enzyme (acylglycerol hydrolases), is the enzyme of a class energy catalysis ester-type hydrolysis, synthesis or transesterification reaction; Extensively be present in prokaryotic organism and eukaryote, comparatively horn of plenty of originating.But different enzymes is in substrate glycerol three ester 1 and 3, i.e. Sn-1,3 and Sn-2 position ester bond there is preferences.1,3 specific lipase of ester bond are in the process of catalyzing acyl shift reaction, and the acyl group of the first catalysis 1,3 of lipase shifts, and then the acyl moiety of catalysis 2 moves to 1 or 3, continues to be carried out transesterification reaction by lipase-catalyzed.So this fermentoid the phase can be subject to the impact of acyl group transport velocity after the reaction, restriction transesterification reaction speed.Dredging cotton like thermophilic hyphomycete lipase (Lipozyme TLIM) as immobilization is a kind of tool 1,3 specific lipase of ester bond, and theoretical ester yield 67%, adds that acyl group shifts, and total ester yield is still less than 84%.Add the low-carbon alcohol such as enzyme preparation cost is higher, less stable, repeating utilization factor are low, methyl alcohol and the application of enzyme process in biofuel suitability for industrialized production be significantly limit to problems such as the inactivating effects of enzyme.
In order to overcome single fat enzyme in catalysis biological biodiesel synthesis process because the narrow spectrum difference of ester bond is on the impact of transesterification efficiency, develop the novel process by compound lipases production biofuel in recent years.Compound lipases can overcome the Substratspezifitaet of single fat enzyme effectively, improves the transesterification efficiency of process for preparing biodiesel by enzyme, quite shows stronger synergistic effect between the different lipase of location specific especially in transesterification ability.As (Huang Ying such as Huang Ying, Gao Huan, Zheng Hai etc. lipase concerted catalysis lard biodiesel synthesis technical study [J]. Chinese biological engineering magazine, 2008,28 (1): 30-35.) Novozym 435 and Lipozyme TLIM composite catalyzing lard are prepared biofuel, bio-diesel yield, up to 97%, all prepares the yield of biofuel higher than single enzyme catalysis, investigator thinks may there is certain positive coorperativity between Novozym 435 and Lipozyme TLIM.(the Ibrahim such as Ibrahim, N, Guo Z, Xu X. Enzymatic Interesterification of Palm Stearin and Coconut Oil by a Dual Lipase System [J]. Journal of the American Oil Chemists'Society, 2008.
85(1): 37-45.) utilize Novozym 435 and Lipozyme TL IM or Novozym 435 and immobilization rhizomucor miehei Lipozyme RM IM for the transesterification of cetylate and Oleum Cocois, find that these two kinds immobilized mixed enzyme systems all have positive coopertive effect to transesterification.Therefore by a certain percentage two kinds can be had the specific lipase of different catalysis and carry out common catalysis, can Reaction time shorten and improve yield, and to reduce costs.
But be all adopt existing immobilized enzyme for the compound enzyme technology of production of biodiesel at present, price is all higher, be the Novozym 435 of Novozymes Company as what be most widely used, per kilogram price is 2270 dollars.On the other hand, because lipid substrate is hydrophobic, lipase must could play catalyzed reaction with Binding Capacity, therefore lipase can only occur on oil-water interface the catalyzed reaction of ester class.So in reaction process, must constantly stir with the abundant contact ensureing enzyme-to-substrate, but mechanical stirring can produce certain influence to carrier or enzyme, causes the loss of enzyme or the reduction of enzyme activity.3rd, existing native lipase is in use gone back the low-carbon alcohol such as existence and stability difference, methyl alcohol and is easily caused the problems such as the inactivation of enzyme.If do not changed the character of native lipase itself and being only fixed, the demand of its suitability for industrialized production still cannot be solved.Therefore, find a kind of simple method, while meeting the high transesterification efficiency of substrate, can reduce production cost again, shorten the transesterification time, the lipase system prepared for biofuel developing a kind of high catalytic activity is significant.
Summary of the invention
The technical problem that the present invention will solve is to provide a kind of preparation method of magnetic responsiveness compound fat ester, and this magnetic responsiveness compound fat ester is applied to biofuel when preparing, and has the high transesterification efficiency of substrate, and can reduce production cost.
For solving above technical problem, the preparation method of a kind of magnetic responsiveness compound lipases of the present invention, comprises step:
(1) preparation of magnetic responsiveness lipase liquid A: with monodisperse water soluble nano-magnetic nano ferriferrous oxide granule for modifier, to having 1, the lipase A of 3 position specifics is modified, and is mixed with the phosphate buffer soln of pH 8.0 the magnetic responsiveness lipase liquid A that enzyme activity concentration is 5-25U/mL respectively;
(2) with the lipase liquid B without position specific of the phosphate buffer soln preparation 5-20U/mL of pH 8.0;
(3) be (0.5-10) by the volume ratio of magnetic responsiveness lipase A and fat liquor B: the proportions lipase mixed solution of 1;
(4) be the sodium alginate soln of 3-6% with the phosphate buffered saline massfraction of pH 8.0-10.0;
(5) by volume for lipase mixed solution joins in sodium alginate soln by the ratio of 2:1, stir 0.5-2 h, be configured to mixed solution of sodium alginate;
(6) CaCl that above-mentioned mixed solution of sodium alginate dropwise instills massfraction 1-5% is drawn
2in solution, form the microcapsule ball that diameter is 2-3 mm, leach gelled pill, change 1-5% CaCl
2add 1% glutaraldehyde solution after solution hardening 3-5 h, be placed in 4 DEG C of environment immobilization 3-12 h; Microcapsule are leached, after using distilled water, phosphate buffered saline buffer filtration washing several times respectively, is placed in 4 DEG C of Refrigerator stores for subsequent use.
Preferably have 1, the lipase A of 3 position specifics is for dredging cotton like thermophilic hyphomycete lipase Lipase from
thermomyces lanuginosus, TLL; Or Pseudomonas fluorecens lipase Lipase from
pseudomonas fluorescens, PFL.
The preferred lipase B without position specific is penicillium expansum lipase Lipase from
penicillium expansum, PEL.
The enzyme proportioning alive of preferred lipase A and lipase B is (2-10): 1.
Simultaneously, present invention also offers and a kind ofly utilize the above-mentioned method that there is magnetic responsiveness compound lipases and prepare biofuel: in reaction vessel, add short chain alcohol, grease and have magnetic responsiveness compound lipases, temperature of reaction is 30-50 DEG C, reaction 48-72 h, obtained biofuel; Wherein, short chain alcohol and grease mol ratio are (3-5): 1, and magnetic responsiveness compound fat enzyme dosage is 5-25 U/g grease; In biofuel building-up process, utilize the magnetic responsiveness of prozyme system, replace mechanical stirring by magnetic agitation.Decrease the vigor loss of prozyme, improve reusability and the activity of prozyme.
As preferred scheme, described grease is Vegetable oil lipoprotein.
As preferred scheme, described Vegetable oil lipoprotein is the combination of one or more in soybean oil, rapeseed oil, peanut oil and Semen Maydis oil.
As preferred scheme, described short chain alcohol is methyl alcohol, ethanol, n-propyl alcohol or Virahol.
As preferred scheme, the feed postition of described short chain alcohol is two steps or three steps.
Compared with prior art, tool of the present invention has the following advantages:
The first, the present invention adopts sodium alginate to carry out co-immobilization to two kinds of specific lipase of different catalysis, not only played natural macromolecular material sodium alginate have safety, environmental protection, can natural biology degraded advantage; Magnetic Nano material is applied to the embedding of lipase simultaneously, efficiently solves sodium alginate bad mechanical strength, the easy deliquescing of gel, causes the problems such as the loss of enzyme in use procedure.
The second, utilize magnetic Nano Fe
3o
4to having 1, the lipase A of 3 position specifics is modified, compound fat enzyme system magnetic is given while effective raising enzyme transesterification efficiency, utilize the magnetic of compound lipases self, traditional mechanical alr mode can be replaced by externally-applied magnetic field, realize the contact of compound lipases and lipid substrate, to eliminate in mechanical agitation process mechanical shear stress to the loss of enzyme, thus decrease the loss of enzyme activity in reaction process, and can rapidly enzyme be reclaimed and recycling after the completion of reaction, effectively cost-saving.
3rd, different for catalytic performance two kinds of lipase are combined, immobilization technology is adopted to have 1, the lipase A of 3-position specific is prepared into prozyme dual-enzyme system with the lipase B without position specific, utilize the method for compound lipases concerted catalysis, take the Substratspezifitaet of single fat enzyme, improve the transesterification efficiency (transesterification efficiency reaches more than 90%) of process for preparing biodiesel by enzyme, and use the method for compound fat enzyme catalysis, required enzyme amount reduces (reducing 30% than single fat enzyme dosage) compared with the enzyme dosage of single fat enzyme, thus greatly reduce production cost.This method has good practicality, can produce good economic benefit, has good industrial prospect.
4th, the whole preparation process technique of the present invention is simple, easy to operate, with low cost; Prepared magnetic responsiveness compound fat enzyme system still can keep higher reactive behavior through repeatedly recycling, and shows good operational stability and practical application feasibility.
Accompanying drawing explanation
The preparation of Fig. 1 magnetic responsiveness lipase immobilization Composite Double enzyme system.
The infrared spectrogram with the TLL lipase of magnetic response prepared by Fig. 2 comparative examples 1.
Embodiment
Illustrate the present invention further below in conjunction with specific embodiment, the present embodiment is implemented under premised on technical solution of the present invention, should understand these modes and only be not used in for illustration of the present invention and limit the scope of the invention.
Embodiment 1 has 1, the preparation of the magnetic responsiveness lipase A of 3 position specifics
The concrete nanometer Fe referring to employing 10nm
3o
4thermophilic hyphomycete lipase (the Lipase from of thin cotton like of particle modification
thermomyces lanuginosus, TLL) preparation: get 1mL surface containing the nanometer Fe of carboxyl functional group
3o
4solution (10nm, iron-holder is 155mg/L), add respectively carbodiimide (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide, EDC) solution that 10 μ L concentration are 10mmol/L and 25mmol/L N-hydroxy thiosuccinimide (
n-hydroxysulfosuccinimide, Sulfo-NHS) solution, oscillatory reaction 10min under room temperature, the nanometer Fe after activated carboxylic is collected in centrifugation
3o
4particle; By the nanometer Fe after activation
3o
4it is in the TLL lipase solution of 5mg/mL that particle adds 200 μ L concentration, oscillatory reaction 2h under room temperature, and Magneto separate, uses distilled water repetitive scrubbing, removes the free-fat enzyme in non-coupling, collects nanometer Fe
3o
4particle-lipase mixture.
Embodiment 2 has 1, the preparation of the magnetic responsiveness lipase A of 3 position specifics
The concrete nanometer Fe referring to employing 10nm
3o
4pseudomonas fluorecens lipase (the Lipase from of particle modification
pseudomonas fluorescens, PLL) preparation: get 1mL surface containing the nanometer Fe of carboxyl functional group
3o
4solution (10nm, iron-holder is 155mg/L), adding 10 μ L concentration is respectively the EDC solution of 10mmol/L and the Sulfo-NHS solution of 25mmol/L, oscillatory reaction 10min under room temperature, and the nanometer Fe after activated carboxylic is collected in centrifugation
3o
4particle; By the nanometer Fe after activation
3o
4it is in the PFL lipase solution of 1mg/mL that particle adds 200 μ L concentration, oscillatory reaction 2h under room temperature, and Magneto separate, uses distilled water repetitive scrubbing, removes the free-fat enzyme in non-coupling, collects nanometer Fe
3o
4particle-lipase mixture.
Embodiment 3 has 1, the preparation of the magnetic responsiveness lipase A of 3 position specifics
The concrete nanometer Fe referring to employing 15nm
3o
4thermophilic hyphomycete lipase (the Lipase from of thin cotton like of particle modification
thermomyces lanuginosus, TLL) preparation: get 1mL surface containing the nanometer Fe of carboxyl functional group
3o
4solution (15nm, iron-holder is 100mg/L), add the Sulfo-NHS solution that EDC solution that 10 μ L concentration are 10mmol/L and 20 μ L concentration are 25mmol/L respectively, oscillatory reaction 30min under room temperature, the nanometer Fe after activated carboxylic is collected in centrifugation
3o
4particle; By the nanometer Fe after activation
3o
4it is 5mg/mL's that particle adds 1mL concentration
thermomyces lanuginosusin lipase solution, oscillatory reaction 2h under room temperature, Magneto separate, uses distilled water repetitive scrubbing, removes the free-fat enzyme in non-coupling, collects nanometer Fe
3o
4particle-lipase mixture.
Embodiment 4 has 1, the preparation of the magnetic responsiveness lipase A of 3 position specifics
The concrete nanometer Fe referring to employing 20nm
3o
4thermophilic hyphomycete lipase (the Lipase from of thin cotton like of particle modification
thermomyces lanuginosus, TLL) preparation: get 1mL surface containing the nanometer Fe of carboxyl functional group
3o
4solution (20nm, iron-holder is 80mg/L), add the Sulfo-NHS solution that EDC solution that 5 μ L concentration are 10mmol/L and 10 μ L concentration are 10mmol/L respectively, oscillatory reaction 30min under room temperature, the nanometer Fe after activated carboxylic is collected in centrifugation
3o
4particle; By the nanometer Fe after activation
3o
4it is in the TLL lipase solution of 5mg/mL that particle adds 1mL concentration, oscillatory reaction 4h under room temperature, and Magneto separate, uses distilled water repetitive scrubbing, removes the free-fat enzyme in non-coupling, collects nanometer Fe
3o
4particle-lipase mixture.
Embodiment 5 has 1, the preparation of the magnetic responsiveness lipase A of 3 position specifics
The concrete nanometer Fe referring to employing 10nm
3o
4pseudomonas fluorecens lipase (the Lipase from of particle modification
pseudomonas fluorescens, PLL) preparation: get 1mL surface containing the nanometer Fe of carboxyl functional group
3o
4solution (10nm, iron-holder is 155mg/L), adding 10 μ L concentration is respectively the EDC solution of 10mmol/L and the Sulfo-NHS solution of 25mmol/L, oscillatory reaction 30min under room temperature, and the nanometer Fe after activated carboxylic is collected in centrifugation
3o
4particle; By the nanometer Fe after activation
3o
4it is 1mg/mL's that particle adds 100 μ L concentration
pseudomonas fluorescensin lipase solution, oscillatory reaction 2h under room temperature, Magneto separate, uses distilled water repetitive scrubbing, removes the free-fat enzyme in non-coupling, collects nanometer Fe
3o
4particle-lipase mixture.
The preparation of embodiment 6 magnetic responsiveness lipase immobilization dual-enzyme system
(1) prepare the lipase liquid A in above-described embodiment 1 with the phosphate buffer soln of pH 8.0, make its enzyme activity be 5U/mL;
(2) with the phosphate buffer soln preparation PEL lipase liquid B of pH 8.0, its enzyme activity is made to be 20U/mL;
(3) by the volume ratio of lipase A and B be the proportions lipase mixed solution of 10:1;
(4) be the sodium alginate soln of 3% with the phosphate buffered saline massfraction of pH 8.0;
(5) by volume for lipase mixed solution joins in sodium alginate soln by the ratio of 2:1,0.5h is stirred;
(6) CaCl of massfraction 1% is dropwise instilled with the mixing solutions that above-mentioned sodium alginate drawn by 7# syringe needle
2in solution, form the microcapsule ball that diameter is 2 ~ 3 mm, leach gelled pill, change massfraction 1%CaCl
2add massfraction 1% glutaraldehyde solution after solution hardening 3 h, be placed in 4 DEG C of environment immobilization 3 h; Microcapsule are leached, after using distilled water, phosphate buffered saline buffer filtration washing several times respectively, is placed in 4 DEG C of Refrigerator stores for subsequent use.
The preparation of embodiment 7 magnetic responsiveness lipase immobilization dual-enzyme system
(1) prepare the lipase liquid A in above-described embodiment 2 with the phosphate buffer soln of pH 8.0, make its enzyme activity be 20U/mL;
(2) with the phosphate buffer soln preparation PEL lipase liquid B of pH 8.0, its enzyme activity is made to be 20U/mL;
(3) by the volume ratio of lipase A and B be the proportions lipase mixed solution of 2:1;
(4) be the sodium alginate soln of 3% with the phosphate buffered saline massfraction of pH 9.5;
(5) by volume for lipase mixed solution joins in sodium alginate soln by the ratio of 2:1,2 h are stirred;
(6) CaCl of massfraction 2% is dropwise instilled with the mixing solutions that above-mentioned sodium alginate drawn by 7# syringe needle
2in solution, form the microcapsule ball that diameter is 2 ~ 3 mm, leach gelled pill, change 2% massfraction CaCl
2add massfraction 1% glutaraldehyde solution after solution hardening 5 h, be placed in 4 DEG C of environment immobilization 5 h; Microcapsule are leached, after using distilled water, phosphate buffered saline buffer filtration washing several times respectively, is placed in 4 DEG C of Refrigerator stores for subsequent use.
The preparation of embodiment 8 magnetic responsiveness lipase immobilization dual-enzyme system
(1) prepare the lipase liquid A in above-described embodiment 3 with the phosphate buffer soln of pH 8.0, make its enzyme activity be 25U/mL;
(2) with the phosphate buffer soln preparation PEL lipase liquid B of pH 8.0, its enzyme activity is made to be 20U/mL;
(3) by the volume ratio of lipase A and B be the proportions lipase mixed solution of 0.5:1;
(4) be the sodium alginate soln of 3% with the phosphate buffered saline massfraction of pH 9.5;
(5) by volume for lipase mixed solution joins in sodium alginate soln by the ratio of 2:1,2 h are stirred;
(6) CaCl of massfraction 5% is dropwise instilled with the mixing solutions that above-mentioned sodium alginate drawn by 7# syringe needle
2in solution, form the microcapsule ball that diameter is 2 ~ 3 mm, leach gelled pill, change massfraction 5% CaCl
2add massfraction 1% glutaraldehyde solution after solution hardening 5 h, be placed in 4 DEG C of environment immobilization 12h; Microcapsule are leached, after using distilled water, phosphate buffered saline buffer filtration washing several times respectively, is placed in 4 DEG C of Refrigerator stores for subsequent use.
The preparation of embodiment 9 magnetic responsiveness lipase immobilization dual-enzyme system
(1) prepare the lipase liquid A in above-described embodiment 4 with the phosphate buffer soln of pH 8.0, make its enzyme activity be 5U/mL;
(2) with the phosphate buffer soln preparation PEL lipase liquid B of pH 8.0, its enzyme activity is made to be 5U/mL;
(3) by the volume ratio of lipase A and B be the proportions lipase mixed solution of 5:1;
(4) be the sodium alginate soln of 3% with the phosphate buffered saline massfraction of pH 10.0;
(5) by volume for lipase mixed solution joins in sodium alginate soln by the ratio of 2:1,1 h is stirred;
(6) CaCl of massfraction 2% is dropwise instilled with the mixing solutions that above-mentioned sodium alginate drawn by 7# syringe needle
2in solution, form the microcapsule ball that diameter is 2 ~ 3 mm, leach gelled pill, change massfraction 2% CaCl
2add massfraction 1% glutaraldehyde solution after solution hardening 4h, be placed in 4 DEG C of environment immobilization 10h; Microcapsule are leached, after using distilled water, phosphate buffered saline buffer filtration washing several times respectively, is placed in 4 DEG C of Refrigerator stores for subsequent use.
Enzyme activity determination method: lipase activity adopts polyvinyl alcohol-sweet oil emulsion process to measure.Polyvinyl alcohol with 2%-sweet oil emulsion 5 mL is for substrate, add the phosphate buffered saline buffer of 4 mL pH8.0, enzyme liquid 1.0 mL to be measured is added after preheating 5min in 30 DEG C of water-baths, mix timing immediately, after reaction 10min, in sample bottle, add dehydrated alcohol 20mL make enzyme denaturation inactivation with termination reaction.In each bottle, add several phenolphthalein indicators, the lipid acid generated with the NaOH standardized solution titration of 0.05mol/L, thus calculate lipase activity.Lipase activity unit definition is enzyme amount per minute under experiment condition being produced 1 μm of ol lipid acid by lipase-catalyzed sweet oil.
Embodiment 10 magnetic responsiveness lipase immobilization dual-enzyme system is used for the preparation of biofuel
By methyl alcohol and soybean oil mol ratio be 1:1(methyl alcohol 0.18 g and soybean oil 5 g), the magnetic responsiveness immobilization prozyme (above-described embodiment 6) of 50 U, load in triangular flask and mix, controlling temperature of reaction is 40 DEG C, be placed in magnetic stirring apparatus reacts and add residue methyl alcohol 0.36 g to 12h, namely total molar ratio of methanol to oil is 3:1.The content of fatty acid methyl ester in product is measured and to calculate yield be 90% after continuing reaction 48h.
Embodiment 11 magnetic responsiveness lipase immobilization dual-enzyme system is used for the preparation of biofuel
By methyl alcohol and soybean oil mol ratio be 1:1(methyl alcohol 0.18 g and soybean oil 5 g), magnetic responsiveness immobilization prozyme (above-described embodiment 7 of 25 U, compound fat enzyme dosage is 5U/g soybean oil), load in triangular flask and mix, controlling temperature of reaction is 50 DEG C, be placed in magnetic stirring apparatus reacts and add methyl alcohol 0.18 g to 12h, add methyl alcohol 0.36 g after continuing reaction 12h, namely total molar ratio of methanol to oil is 5:1.The content of fatty acid methyl ester in product is measured and to calculate yield be 92% after continuing reaction 48h.
Embodiment 12 magnetic responsiveness lipase immobilization dual-enzyme system is used for the preparation of biofuel
By methyl alcohol and soybean oil mol ratio be 1:1(methyl alcohol 0.18 g and soybean oil 5 g), magnetic responsiveness immobilization prozyme (above-described embodiment 8 of 125 U, compound fat enzyme dosage is 25U/g soybean oil), load in triangular flask and mix, controlling temperature of reaction is 30 DEG C, be placed in magnetic stirring apparatus reacts and add methyl alcohol 0.18 g to 12h, add methyl alcohol 0.18 g after continuing reaction 12h, namely total molar ratio of methanol to oil is 3:1.The content of fatty acid methyl ester in product is measured and to calculate yield be 94% after continuing reaction 48h.
Short chain alcohol methyl alcohol in embodiment 10-12, can use methyl alcohol, ethanol, n-propyl alcohol or Virahol to replace, and soybean oil can use rapeseed oil, peanut oil and Semen Maydis oil and their combination to replace.
Claims (9)
1. a preparation method for magnetic responsiveness compound lipases, is characterized in that comprising step:
(1) preparation of magnetic responsiveness lipase liquid A: with monodisperse water soluble nano-magnetic nano ferriferrous oxide granule for modifier, to having 1, the lipase A of 3 position specifics is modified, and is mixed with the phosphate buffer soln of pH 8.0 the magnetic responsiveness lipase liquid A that enzyme activity concentration is 5-25U/mL respectively;
(2) with the lipase liquid B without position specific of the phosphate buffer soln preparation 5-20U/mL of pH 8.0;
(3) be (0.5-10) by the volume ratio of magnetic responsiveness lipase A and fat liquor B: the proportions lipase mixed solution of 1;
(4) be the sodium alginate soln of 3-6% with the phosphate buffered saline massfraction of pH 8.0-10.0;
(5) by volume for lipase mixed solution joins in sodium alginate soln by the ratio of 2:1, stir 0.5-2 h, be configured to mixed solution of sodium alginate;
(6) CaCl that above-mentioned mixed solution of sodium alginate dropwise instills massfraction 1-5% is drawn
2in solution, form the microcapsule ball that diameter is 2-3 mm, leach gelled pill, change massfraction 1-5% CaCl
2add massfraction 1% glutaraldehyde solution after solution hardening 3-5 h, be placed in 4 DEG C of environment immobilization 3-12 h; Microcapsule are leached, after using distilled water, phosphate buffered saline buffer filtration washing several times respectively, is placed in 4 DEG C of Refrigerator stores for subsequent use.
2. method according to claim 1, is characterized in that: have 1, and the lipase A of 3 position specifics is for dredging cotton like thermophilic hyphomycete lipase Lipase from
thermomyces lanuginosus, TLL; Or Pseudomonas fluorecens lipase Lipase from
pseudomonas fluorescens, PFL.
3. method according to claim 1 and 2, is characterized in that: the lipase B without position specific is penicillium expansum lipase Lipase from
penicillium expansum, PEL.
4. method according to claim 1, is characterized in that: the enzyme proportioning alive of lipase A and lipase B is (2-10): 1.
5. a utilization has the method that magnetic responsiveness compound lipases prepares biofuel, it is characterized in that: in reaction vessel, add short chain alcohol, grease and have magnetic responsiveness compound lipases, temperature of reaction is 30-50 DEG C, reaction 48-72 h, obtained biofuel; Wherein, short chain alcohol and grease mol ratio are (3-5): 1, and magnetic responsiveness compound fat enzyme dosage is 5-25 U/g grease; In biofuel building-up process, utilize the magnetic responsiveness of prozyme system, replace mechanical stirring by magnetic agitation.
6. method according to claim 5, is characterized in that: described grease is Vegetable oil lipoprotein.
7. method according to claim 6, is characterized in that: described Vegetable oil lipoprotein is the combination of one or more in soybean oil, rapeseed oil, peanut oil and Semen Maydis oil.
8. the method according to claim 5 or 6, is characterized in that: described short chain alcohol is methyl alcohol, ethanol, n-propyl alcohol or Virahol.
9. according to the method preparing biofuel described in claim 1, it is characterized in that: the feed postition of described short chain alcohol is two steps or three steps.
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| CN105274157A (en) * | 2015-12-01 | 2016-01-27 | 河北工业大学 | Method for producing biodiesel by utilizing immobilized lipase and adopting static emulsion method |
| CN108686575A (en) * | 2018-05-15 | 2018-10-23 | 江南大学 | A kind of compound emulsifying agent with magnetic responsiveness energy |
| CN110004138A (en) * | 2019-04-29 | 2019-07-12 | 中国检验检疫科学研究院 | Immobilised enzymes and preparation method thereof |
| CN110055242A (en) * | 2019-04-29 | 2019-07-26 | 中国检验检疫科学研究院 | Biodiesel and preparation method thereof |
| CN111593045A (en) * | 2020-04-16 | 2020-08-28 | 东北农业大学 | Immobilized magnetoenzyme preparation method of OPO grease in supercritical state |
| CN111593046A (en) * | 2020-04-16 | 2020-08-28 | 东北农业大学 | Immobilized magnetoenzyme preparation method of OPL grease |
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| CN105274157A (en) * | 2015-12-01 | 2016-01-27 | 河北工业大学 | Method for producing biodiesel by utilizing immobilized lipase and adopting static emulsion method |
| CN105274157B (en) * | 2015-12-01 | 2018-07-20 | 河北工业大学 | A kind of method of static state emulsion method immobilized lipase production biodiesel |
| CN108686575A (en) * | 2018-05-15 | 2018-10-23 | 江南大学 | A kind of compound emulsifying agent with magnetic responsiveness energy |
| CN110004138A (en) * | 2019-04-29 | 2019-07-12 | 中国检验检疫科学研究院 | Immobilised enzymes and preparation method thereof |
| CN110055242A (en) * | 2019-04-29 | 2019-07-26 | 中国检验检疫科学研究院 | Biodiesel and preparation method thereof |
| CN111593045A (en) * | 2020-04-16 | 2020-08-28 | 东北农业大学 | Immobilized magnetoenzyme preparation method of OPO grease in supercritical state |
| CN111593046A (en) * | 2020-04-16 | 2020-08-28 | 东北农业大学 | Immobilized magnetoenzyme preparation method of OPL grease |
| CN111849959A (en) * | 2020-08-14 | 2020-10-30 | 威海百合功能食品技术研究院有限公司 | Method for preparing cycloastragenol by catalyzing astragaloside by using co-immobilized double enzymes |
| CN111849959B (en) * | 2020-08-14 | 2023-10-27 | 威海百合生物技术股份有限公司 | Method for preparing cycloastragaloside by using co-immobilized double-enzyme catalytic astragaloside IV |
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