CN104120156A - Applications of immobilized double enzymes in catalyzing esterification reaction of oleic acid in reverse micelle system - Google Patents
Applications of immobilized double enzymes in catalyzing esterification reaction of oleic acid in reverse micelle system Download PDFInfo
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Abstract
The invention relates to applications of immobilized double enzymes in catalyzing esterification reaction of oleic acid in a reverse micelle system. The applications comprise the following steps: co-immobilizing cellulase and lipase to obtain the immobilized double enzymes; mixing the immobilized double enzymes with the reverse micelle system, and adding oleic acid and organic alcohol for performing water bath oscillation to obtain an esterified product. Co-immobilizing cellulase and lipase can be applied to the esterification reaction of oleic acid, so that the esterification reaction rate of oleic acid can be obviously improved, the yield of the esterified product can be promoted, and the applications have the advantages of being mild in reaction conditions, higher in recovery of enzyme activity, long in the half-life period of the immobilized enzyme, simple and convenient to operate, low in cost, clean and safe and the like.
Description
Technical field
The present invention relates to environmental protection technical field, relate in particular to the application in catalyzed oil acid esterification in reverse micelle system of the two enzymes of a kind of immobilization.
Background technology
At present, a large amount of discarded oleic-acid biomass resources have had a strong impact on ecotope, oleic acid is carried out to effective esterification and generate the recycling that ethyl oleate can be realized waste resource, have higher application prospect.Ethyl oleate is a kind of higher fatty acid alcohol ester, is widely used in chemical industry, weaving, medicine and other fields.The ethyl oleate content of occurring in nature is few, collect difficulty, is difficult to meet the needs of human being's production and life.The preparation method of traditional ethyl oleate mainly adopts acid-base catalysis synthetic, need under the environment of high temperature, high pressure and strong acid, highly basic, carry out, and has the problems such as side reaction is many, production cost is high and oleic acid carbochain is large compared with length, reaction difficulty, and esterification rate is low.
A kind of method that adopts enzyme catalysis oleic acid esterification has been proposed both at home and abroad, enzyme is placed in to reverse micelle system, because reverse micelle system is the water-in-oil system that profit two-phase material forms by tensio-active agent, enzyme and other hydrophilic substances can enter in water by sequestering action, avoided preferably enzyme to contact the loss that causes enzymic activity with organism, for enzyme creativeness suitable microenvironment.This catalysis process is easy and simple to handle, and production cost is low, has improved largely the speed of oleic acid esterification.Due to enzyme in water in unbound state, and feed ethanol in the building-up reactions of ethyl oleate is profit parents materials, can see through Surfactant Films and enter water, contact and make zymoprotein sex change with enzyme, cause the character of enzyme stable not, can not guarantee the esterification process of oleic acid.Resolvase cannot reclaim environment sensitive, easily inactivation, and existence simultaneously, and reaction cost is high, secondary pollution problems.
The research of immobilized enzyme and application are the effective means with improved stability of reusing that realizes enzyme, and can only compare by expendable resolvase, and immobilized enzyme has the catalysis characteristics of resolvase, the advantage such as have again good stability, can recycle.Within 1970, Mosbach has set up immobilized multienzyme system, has realized the fixing and coreaction altogether of immobilized enzyme system.In recent years, development along with enzyme engineering technology, on the basis of single enzyme immobilization technology, set up again multienzyme co-immobilization technology, multienzyme co-immobilization system has high stability and the operate continuously ability of immobilized enzyme, the synergy again with multienzyme, this technology has represented very wide application prospect.In reverse micelle system, the esterification that is embodied as enzyme of effective maintenance of enzymic activity and efficient enzymic catalytic reaction provides another platform.But existing enzyme immobilization material is mainly silica gel, chitosan etc., also there is in various degree in actual use defect in these immobilization materials, as using the inorganic materials such as silica gel as fixation support, exist, surface separated with water body with the defects such as immobilized enzyme bonding is not tight; And there is the defects such as processed complex, character be unstable in chitosan etc.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provide a kind of reaction conditions gentle, enzyme activity reclaims higher, catalytic rate is fast, immobilized enzyme long half time, easy and simple to handle, with low cost, the method for the catalyzed oil acid esters of clean and safe, specifically provides the application of the two enzymes of a kind of immobilization catalyzed oil acid esterification in reverse micelle system.
For solving the problems of the technologies described above, provide the two enzymes of a kind of immobilization in reverse micelle system to the application in oleic acid esterification, comprise the following steps:
S1, cellulase and lipase are through the two enzymes of being fixed of co-immobilization;
S2, the two enzymes of aforementioned immobilization are mixed with reverse micelle system, add oleic acid and Organic Alcohol to carry out water-bath and vibrate and obtain esterification products.
Further, S1 step is specifically further comprising the steps of:
S1.1, aforementioned fibers element enzyme is fixed to being fixed cellulase in biological carbon;
S1.2, aforementioned lipase is fixed in aforementioned immobilized cellulase to the two enzymes of being fixed.
Further, S1.1 step is specially: cellulase is added to the damping fluid that obtains cellulase in damping fluid, biological carbon is placed in to the aforementioned damping fluid that contains cellulase and carries out being fixed of water-bath vibration step cellulase.Preferably, cellulase is added to the damping fluid that obtains cellulase in Sodium phosphate dibasic-citrate buffer solution.Aforementioned Sodium phosphate dibasic-citrate buffer solution pH is 2～8.Further preferred, Sodium phosphate dibasic-citrate buffer solution pH is 3.
Further, S1.2 step is specially: lipase is added to the damping fluid that obtains fatty enzyme in damping fluid; Aforementioned immobilized cellulase is placed in to the aforementioned damping fluid that contains lipase to carry out water-bath vibration step and obtains the two enzymes of aforementioned immobilization.Preferably, cellulase is added to the damping fluid that obtains cellulase in Sodium phosphate dibasic-citrate buffer solution.Aforesaid Sodium phosphate dibasic-citrate buffer solution pH is 2～8.Further preferred, Sodium phosphate dibasic-citrate buffer solution pH is 7.
Further, the weightmeasurement ratio of cellulase and damping fluid is preferably 0.1～0.4mg/ml; The weightmeasurement ratio of lipase and damping fluid is preferably 0.1～0.4mg/ml; The weightmeasurement ratio of the damping fluid of biological carbon and cellulase is preferably 0.25: 5～100g/ml; The weightmeasurement ratio of the damping fluid of immobilized cellulase and fatty enzyme is preferably 0.25～5: 5～100g/ml.
Further, the preparation method of aforementioned biological carbon is: with corn cob pyrolysis under vacuum environment, obtain.The biological carbon that aforementioned preparation method prepares also comprised activation step before for cellulase immobilization, aforementioned activation step specifically comprises: biological carbon is used to 95% ethanol, 5%HCL and 5%NaOH solution soaking successively, then suction filtration, distilled water flushing is to neutral, freeze-drying lyophilization obtains activating biological carbon, the preparation by activation biological carbon for the two enzymes of aforementioned immobilization.
Further, aforementioned reverse micelle system comprises rhamnolipid, octane-iso and n-hexyl alcohol.
Further, the micelle-forming concentration of aforementioned reverse micelle system is 20～100CMC.
Further, aforementioned Organic Alcohol is preferably the monobasic Organic Alcohol with C1～C10 carbon chain lengths; More preferably ethanol, n-propyl alcohol or butanols.
Further, the molar concentration rate of oleic acid and Organic Alcohol is preferably 2～7: 1.
Further, the volume proportion of oleic acid and reverse micelle system is preferably 0.001～0.002: 1.
Further, the temperature of water-bath vibration is preferably 25～65 ℃, is further preferably 55 ℃.
Compared with prior art, the invention has the advantages that:
(1) the present invention adopts cellulase and lipase to carry out co-immobilization and makes the two enzymes of immobilization, and in reverse micelle system, oleic acid is carried out to esterification.Due to by being fixed of enzyme, than resolvase, its stability is better, harsh unlike resolvase, reaction conditions being required; On the other hand, immobilized enzyme can be separated from reverse micelle system, recycles, and recyclable number of times reaches more than ten times, greatly reduces production cost.Adopt cellulase and lipase combined action to carry out catalyzed reaction to oleic acid simultaneously, utilize the intermolecular reactive force of enzyme, first cellulase is fixed on to being fixed cellulase in biological carbon, then on the basis of immobilized cellulase, carry out again the fixing of lipase, lipase plays katalysis to oleic acid, and cellulase can improve the immobilization degree of lipase, by largeizationr of the activity utilization of lipase, improved largely catalytic efficiency, esterification rate reaches as high as 0.43mmol/L ﹒ min ﹒ mg, and product yield can reach 60%; There is reaction efficiency high, the advantage such as working condition is gentle, and cost is low, and production unit is simple.
(2) using biological carbon that corn cob is that raw material forms as the fixation support of cellulase and lipase, there is good biocompatibility.Meanwhile, biological carbon can degradablely be carbonic acid gas and water at occurring in nature, can't cause secondary pollution to environment.
(3) reverse micelle that the present invention adopts adopts the reverse micelle system consisting of rhamnolipid, octane-iso and n-hexyl alcohol, to select bio-surfactant (Biosurfactant, be called for short BS) prepare, can be issued to micelle-forming concentration at lower concentration, pardon to material is larger, non-environmental-pollution problem.
Accompanying drawing explanation
For making object, technical scheme and the advantage of the embodiment of the present invention clearer, below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out to clear, complete description.
Fig. 1 is the schema of esterification process in the embodiment of the present invention 1.
Fig. 2 is the enzyme activity detected result figure of the embodiment of the present invention 1, comparative example 1 to 5 cellulase and lipase.
Fig. 3 is the suitableeest water-bath concussion temperature analysis figure of the two enzymes of immobilization in the embodiment of the present invention 1.
Fig. 4 is the suitableeest micelle-forming concentration analysis chart of the two enzymes of immobilization in the embodiment of the present invention 1.
Fig. 5 is the two enzymes of immobilization comparative analysis figure in reverse micelle and organic solvent respectively in the embodiment of the present invention 1.
Fig. 6 is the two enzyme Analyzsis of Operational Stability figure of immobilization in the embodiment of the present invention 1.
Embodiment
Below in conjunction with Figure of description, the invention will be further described with concrete preferred embodiment, but protection domain not thereby limiting the invention.
The material adopting in following examples and instrument are commercially available.
Embodiment 1
Referring to Fig. 1, the application of the two enzymes of a kind of immobilization catalyzed oil acid esterification in reverse micelle system, comprises the following steps:
(1) preparation of biological carbon: get corn cob 100g, put into drying oven with 105 ℃ of dry 18h.Taking out dried corn cob, to be cut into volume be 2 * 3 * 3cm
3corn pellet, corn pellet is put into vacuum atmosphere tube type electric furnace, with the speed of 10 ℃/min, be warmed up to 450 ℃, then at 450 ℃, maintain 45min, then be cooled to room temperature and obtain biological carbon.Biological carbon is ground, cross 100 mesh sieves and obtain biological carbon dust.
(2) biological carbon activation: take the biological carbon dust for preparing in 10g step (1) in Erlenmeyer flask, the alcohol immersion 24h that is 95% by volume fraction, vacuum filtration is got filter residue, uses 1L distilled water flushing.The HCL that is 5% by 25ml volume fraction by the filter residue after rinsing soaks 4h.The NaOH solution soaking 4h that biological carbon dust after HCL is soaked is 5% by 25ml volume fraction again after vacuum filtration, distilled water flushing, then carry out vacuum filtration, filter residue is extremely neutral with distilled water flushing, and at 4 ℃, dry 4h obtains activating biological carbon.
(3) cellulase immobilization: get 10mg cellulase and join 100ml pH and make the damping fluid of cellulase in Sodium phosphate dibasic-citrate buffer solution of 3; The damping fluid (cellulase concentration is 0.1mg/ml) of getting 5ml cellulase adds the activation biological carbon obtaining in 0.25g step (2), in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 3 with 100ml pH-citrate buffer solution rinses being fixed cellulase.
(4) lipase immobilization: get 10mg lipase and join 100ml pH and make the damping fluid of fatty enzyme in Sodium phosphate dibasic-citrate buffer solution of 7; Getting damping fluid (lipase concentration is 0.1mg/ml) that 5ml contains lipase adds in the immobilized cellulase that 0.25g step (3) prepares, in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 7 with 100ml pH-citrate buffer solution rinses being fixed pair enzymes.
Getting the two enzymes of the aforesaid immobilization of 1.5mg, to be dissolved in volume be that 10ml, CMC are in 60 the reverse micelle system being comprised of rhamnolipid-octane-iso-n-hexyl alcohol; Wherein, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding oleic acid and the volume that 20 μ l concentration are 20mmol/L in reverse micelle enzyme system, be the ethanol that 10 μ l concentration are 10mmol/L, after mixing, put into water-bath vibration case, with 55 ℃, under the condition of 150rpm, carry out esterification one hour.
After esterification completes, reaction product is carried out to vacuum filtration, with the two enzymes of distilled water flushing immobilization, until neutral.Rinse the two enzyme kept dry of complete immobilization and also can continue on for next batch esterification.
Embodiment 2
An application for the two enzymes of immobilization catalyzed oil acid esterification in reverse micelle system, comprises the following steps:
(1) preparation of biological carbon: get corn cob 100g, put into drying oven with 105 ℃ of dry 18h.Taking out dried corn cob, to be cut into volume be 2 * 3 * 3cm
3corn pellet, corn pellet is put into vacuum atmosphere tube type electric furnace, with the speed of 10 ℃/min, be warmed up to 450 ℃, then at 450 ℃, maintain 45min, then be cooled to room temperature and obtain biological carbon.Biological carbon is ground, cross 100 mesh sieves and obtain biological carbon dust.
(2) biological carbon activation: take the biological carbon dust for preparing in 10g step (1) in Erlenmeyer flask, the alcohol immersion 24h that is 95% by volume fraction, vacuum filtration is got filter residue, uses 1L distilled water flushing.The HCL that is 5% by 25ml volume fraction by the filter residue after rinsing soaks 4h.The NaOH solution soaking 4h that biological carbon dust after HCL is soaked is 5% by 25ml volume fraction again after vacuum filtration, distilled water flushing, then carry out vacuum filtration, filter residue is extremely neutral with distilled water flushing, and at 4 ℃, dry 4h obtains activating biological carbon.
(3) cellulase immobilization: get 20mg cellulase and join 100ml pH and make the damping fluid of cellulase in Sodium phosphate dibasic-citrate buffer solution of 3; The damping fluid (cellulase concentration is 0.2mg/ml) of getting 50ml cellulase adds the activation biological carbon obtaining in 0.25g step (2), in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 3 with 100ml pH-citrate buffer solution rinses being fixed cellulase.
(4) lipase immobilization: get 20mg lipase and join 100ml pH and make the damping fluid of fatty enzyme in Sodium phosphate dibasic-citrate buffer solution of 7; Getting damping fluid (lipase concentration is 0.2mg/ml) that 100ml contains lipase adds in the immobilized cellulase that 0.25g step (3) prepares, in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 7 with 100ml pH-citrate buffer solution rinses being fixed pair enzymes.
Getting the two enzymes of the aforesaid immobilization of 1.5mg, to be dissolved in volume be that 10ml, CMC are in 20 the reverse micelle system being comprised of rhamnolipid-octane-iso-n-hexyl alcohol; Wherein, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding oleic acid and the volume that 10 μ l concentration are 70mmol/L in reverse micelle enzyme system, be the ethanol that 10 μ l concentration are 10mmol/L, after mixing, put into water-bath vibration case, with 55 ℃, under the condition of 150rpm, carry out esterification one hour.
Embodiment 3
An application for the two enzymes of immobilization catalyzed oil acid esterification in reverse micelle system, comprises the following steps:
(1) preparation of biological carbon: get corn cob 100g, put into drying oven with 105 ℃ of dry 18h.Taking out dried corn cob, to be cut into volume be 2 * 3 * 3cm
3corn pellet, corn pellet is put into vacuum atmosphere tube type electric furnace, with the speed of 10 ℃/min, be warmed up to 450 ℃, then at 450 ℃, maintain 45min, then be cooled to room temperature and obtain biological carbon.Biological carbon is ground, cross 100 object sieves and obtain biological carbon dust.
(2) biological carbon activation: take the biological carbon dust for preparing in 10g step (1) in Erlenmeyer flask, the alcohol immersion 24h that is 95% by volume fraction, vacuum filtration is got filter residue, uses 1L distilled water flushing.The HCL that is 5% by 25ml volume fraction by the filter residue after rinsing soaks 4h.The NaOH solution soaking 4h that biological carbon dust after HCL is soaked is 5% by 25ml volume fraction again after vacuum filtration, distilled water flushing, then carry out vacuum filtration, filter residue is extremely neutral with distilled water flushing, and at 4 ℃, dry 4h obtains activating biological carbon.
(3) cellulase immobilization: get 40mg cellulase and join 100ml pH and make the damping fluid of cellulase in Sodium phosphate dibasic-citrate buffer solution of 2; The damping fluid (cellulase concentration is 0.4mg/ml) of getting 100ml cellulase adds the activation biological carbon obtaining in 0.25g step (2), in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 2 with 100ml pH-citrate buffer solution rinses being fixed cellulase.
(4) lipase immobilization: get 40mg lipase and join 100ml pH and make the damping fluid of fatty enzyme in Sodium phosphate dibasic-citrate buffer solution of 8; Getting damping fluid (lipase concentration is 0.4mg/ml) that 5ml contains lipase adds in the immobilized cellulase that 5g step (3) prepares, in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 7 with 100ml pH-citrate buffer solution rinses being fixed pair enzymes.
Getting the two enzymes of the aforesaid immobilization of 1.5mg, to be dissolved in volume be that 10ml, CMC are in 100 the reverse micelle system being comprised of rhamnolipid-octane-iso-n-hexyl alcohol; Wherein, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding oleic acid and the volume that 20 μ l concentration are 40mmol/L in reverse micelle enzyme system, be the ethanol that 10 μ l concentration are 10mmol/L, after mixing, put into water-bath vibration case, with 45 ℃, under the condition of 150rpm, carry out esterification one hour.
After esterification completes, reaction product is carried out to vacuum filtration, with the two enzymes of distilled water flushing immobilization, until neutral.Rinse the two enzyme kept dry of complete immobilization and also can proceed next batch esterification.
Comparative example 1
An application for the two enzymes of immobilization catalyzed oil acid esterification in reverse micelle system, comprises the following steps:
(1) getting 10mg lipase joins 100ml pH and in Sodium phosphate dibasic-citrate buffer solution of 7, makes the damping fluid of fatty enzyme; Get damping fluid (lipase concentration is 0.1mg/ml) that 5ml contains lipase and add the activation biological carbon of 0.25g embodiment 1 preparation, in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 7 with 100ml pH-citrate buffer solution rinses being fixed lipase.
(2) getting 10mg cellulase joins 100ml pH and in Sodium phosphate dibasic-citrate buffer solution of 3, makes the damping fluid of cellulase; The damping fluid (cellulase concentration is 0.1mg/ml) of getting 5ml cellulase adds the immobilized lipase of 0.25g step (1), in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 3 with 100ml pH-citrate buffer solution rinses being fixed pair enzymes.
(3) getting the two enzymes of the aforesaid immobilization of 1.5mg, to be dissolved in volume be that 10ml, CMC are in 60 the reverse micelle system being comprised of rhamnolipid-octane-iso-n-hexyl alcohol; Wherein, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding oleic acid and the volume that 20 μ l concentration are 20mmol/L in reverse micelle enzyme system, be the ethanol that 10 μ l concentration are 10mmol/L, after mixing, put into water-bath vibration case, with 55 ℃, under the condition of 150rpm, carry out esterification one hour.
Comparative example 2
An application for the two enzymes of immobilization catalyzed oil acid esterification in reverse micelle system, comprises the following steps:
(1) getting 10mg lipase joins 100ml pH and in Sodium phosphate dibasic-citrate buffer solution of 7, makes the damping fluid of fatty enzyme; Get damping fluid (lipase concentration is 0.1mg/ml) that 5ml contains lipase and add the activation biological carbon of 0.25g embodiment 1 preparation, in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 7 with 100ml pH-citrate buffer solution rinses being fixed lipase.
(2) getting 10mg cellulase joins 100ml pH and in Sodium phosphate dibasic-citrate buffer solution of 7, makes the damping fluid of cellulase; The damping fluid (cellulase concentration is 0.1mg/ml) of getting 5ml cellulase adds the immobilized lipase of 0.25g step (1), in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 7 with 100ml pH-citrate buffer solution rinses being fixed pair enzymes.
(3) getting the two enzymes of the aforesaid immobilization of 1.5mg, to be dissolved in volume be that 10ml, CMC are in 60 the reverse micelle system being comprised of rhamnolipid-octane-iso-n-hexyl alcohol; Wherein, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding oleic acid and the volume that 20 μ l concentration are 20mmol/L in reverse micelle enzyme system, be the ethanol that 10 μ l concentration are 10mmol/L, after mixing, put into water-bath vibration case, with 55 ℃, under the condition of 150rpm, carry out esterification one hour.
Comparative example 3
An application for the two enzymes of immobilization catalyzed oil acid esterification in reverse micelle system, comprises the following steps:
(1) getting 10mg cellulase joins 100ml pH and in Sodium phosphate dibasic-citrate buffer solution of 3, makes the damping fluid of cellulase; The damping fluid (cellulase concentration is 0.1mg/ml) of getting 5ml cellulase adds the activation biological carbon of 0.25g embodiment 1 preparation, in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 3 with 100ml pH-citrate buffer solution rinses being fixed cellulase.
(2) getting 10mg lipase joins 100ml pH and in Sodium phosphate dibasic-citrate buffer solution of 3, makes the damping fluid of fatty enzyme; Get the immobilized cellulase that damping fluid (lipase concentration is 0.1mg/ml) that 5ml contains lipase adds 0.25g step (1), be placed in, in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 3 with 100ml pH-citrate buffer solution rinses being fixed pair enzymes.
(3) getting the two enzymes of the aforesaid immobilization of 1.5mg, to be dissolved in volume be that 10ml, CMC are in 60 the reverse micelle system being comprised of rhamnolipid-octane-iso-n-hexyl alcohol; Wherein, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding oleic acid and the volume that 20 μ l concentration are 20mmol/L in reverse micelle enzyme system, be the ethanol that 10 μ l concentration are 10mmol/L, after mixing, put into water-bath vibration case, with 55 ℃, under the condition of 150rpm, carry out esterification one hour.
Comparative example 4
An application for the two enzymes of immobilization catalyzed oil acid esterification in reverse micelle system, comprises the following steps:
(1) getting 10mg lipase joins 100ml pH and in Sodium phosphate dibasic-citrate buffer solution of 7, makes the damping fluid of fatty enzyme; Get damping fluid (lipase concentration is 0.1mg/ml) that 5ml contains lipase and add the activation biological carbon of 0.25g embodiment 1, in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 7 with 100ml pH-citrate buffer solution rinses being fixed lipase.
(2) getting the aforesaid immobilized lipase of 1.5mg, to be dissolved in volume be that 10ml, CMC are in 60 the reverse micelle system being comprised of rhamnolipid-octane-iso-n-hexyl alcohol; Wherein, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding oleic acid and the volume that 20 μ l concentration are 20mmol/L in reverse micelle enzyme system, be the ethanol that 10 μ l concentration are 10mmol/L, after mixing, put into water-bath vibration case, with 35 ℃, under the condition of 150rpm, carry out esterification one hour.
Comparative example 5
An application for the two enzymes of immobilization catalyzed oil acid esterification in reverse micelle system, comprises the following steps:
(1) get the rhamnolipid 60ml of 100cmc, add the octane-iso of 20ml and the n-hexyl alcohol of 20ml, the reverse micelle system being formed by rhamnolipid (RL)-octane-iso-n-hexyl alcohol solution that to obtain micelle-forming concentration (CMC) be 60.
(2) getting 10mg cellulase joins 100ml pH and in Sodium phosphate dibasic-citrate buffer solution of 3, makes the damping fluid of cellulase; The damping fluid (cellulase concentration is 0.1mg/ml) of getting 5ml cellulase adds the activation biological carbon of 0.25g embodiment 1 to be placed in, in the water bath with thermostatic control vibrators of 30 ℃ (oscillation frequency is 150r/min), vibration absorption is two hours, then carry out vacuum filtration, the Sodium phosphate dibasic that to get filter residue be 3 with 100ml pH-citrate buffer solution rinses being fixed cellulase.
(3) getting the aforesaid immobilized cellulase of 1.5mg, to be dissolved in volume be that 10ml, CMC are in 60 the reverse micelle system being comprised of rhamnolipid-octane-iso-n-hexyl alcohol; Wherein, the mol ratio (W of water/rhamnolipid
0) be 40.Then to adding oleic acid and the volume that 20 μ l concentration are 20mmol/L in reverse micelle enzyme system, be the ethanol that 10 μ l concentration are 10mmol/L, after mixing, put into water-bath vibration case, with 45 ℃, under the condition of 150rpm, carry out esterification one hour.
The two enzymes of the reacted immobilization of oleic acid esterification, immobilized cellulase, immobilized lipase after filtration, washing, drying treatment, can continue on for oleic acid esterification.The micelle-forming concentration of aforesaid reverse micelle system can also be 20～100CMC, and the molar concentration rate of oleic acid and C1～C10 Organic Alcohol is 2～7: 1; The volume proportion of oleic acid and reverse micelle system is 0.001～0.002: 1.The monobasic Organic Alcohol of the replaceable one-tenth of the aforementioned ethanol adding arbitrary C1 of having～C10 carbon chain lengths, in order to improve esterification reaction rate, ethanol can replace to n-propyl alcohol or butanols.
The mensuration of the growing amount of oleic acid esterification rate and ethyl oleate:
In aforementioned esterification reaction process, after esterification starts, every 10 minutes, get 1ml esterification reaction product in Erlenmeyer flask, add the neutralized verdigris (it is 6.1 that neutralized verdigris regulates pH by pyridine) of 1ml and the benzene of 5ml, after mixing with 3000r/min rotating speed centrifugal 5 minutes, centrifugal rear stratification, got upper organic phase and in ultraviolet spectrophotometer (548nm wavelength), measures the esterification rate of oleic acid.
Esterification was carried out after 1 hour, in Erlenmeyer flask, added ethanol and acetone mixed solution (volume ratio of ethanol and acetone was by 1: the 1) termination reaction of 5ml, then added the diethyl phthalate of 0.0375g, evenly vibration, stratification.With the syringe of 5ml, get the oil phase in Erlenmeyer flask, the organic phase filtering membrane by 0.22 μ l filters and obtains solution to be measured.Solution to be measured is contained in to 1ml sample injection bottle and carries out gas chromatographic analysis.The peak area obtaining according to gas chromatographic analysis is determined the growing amount of ethyl oleate.Measuring result is listed in table 1.
Table 1: esterification rate and product yield result table
The mensuration of enzyme activity:
Respectively by the immobilization of embodiment 1 and comparative example 1 to 3 two enzyme and the immobilized cellulases of comparative example 4, the immobilized lipase of comparative example 5 carries out enzyme activity detection, detected result as shown in Figure 2:
The vigor of immobilized lipase (being comparative example 4) is 900U/g; The vigor of immobilized cellulase (being comparative example 5) is 700000U/g; Successively cellulase and lipase are fixed on to the two enzymes (being embodiment 1) of the immobilization preparing in biological carbon, its lipase activity is that the vigor of 950U/g, cellulase is 700000U/g; If prepare the two enzymes of immobilization not according to the method for embodiment 1, the vigor of lipase and cellulase be inhibited (referring to the enzyme activity detected result of comparative example in Fig. 21,2,3), this is mainly because cellulase of the present invention is firmly fixed on carrier by lipase, the enzyme exposing is more, and vigor increases.
The analytical test of the suitableeest water-bath concussion temperature:
Getting respectively the two enzymes of the immobilization that makes in the 1.5mg embodiment of the present invention 1, free-fat enzyme and wandering fibre element, to be dissolved in volume be in 10ml, micelle-forming concentration (CMC) the reverse micelle enzyme system consisting of rhamnolipid (RL)-octane-iso-n-hexyl alcohol solution that is 60; Wherein, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding 20 μ l concentration in reverse micelle enzyme system, be that 20mmol/L oleic acid and 10 μ l concentration are that 10mmol/L ethanol obtains mixing solutions.Mixing solutions is equally divided into five parts, puts into successively water-bath vibration case, it is 25 ℃, 35 ℃, 45 ℃, 55 ℃, 65 ℃, 75 ℃ that water-bath vibration case temperature is set respectively, with 150rpm rotating speed, carries out esterification one hour.
In aforementioned esterification reaction process, after esterification starts, every 10 minutes, get 1ml esterification reaction product in Erlenmeyer flask, add the neutralized verdigris (it is 6.1 that neutralized verdigris regulates pH by pyridine) of 1ml and the benzene of 5ml, after mixing with 3000r/min rotating speed centrifugal 5 minutes, centrifugal rear stratification, got upper organic phase and in ultraviolet spectrophotometer (548nm wavelength), measures the esterification rate of oleic acid.
Esterification was carried out after 1 hour, in Erlenmeyer flask, added ethanol and acetone mixed solution (volume ratio of ethanol and acetone was by 1: the 1) termination reaction of 5ml, then added the diethyl phthalate of 0.0375g, evenly vibration, stratification.With the syringe of 5ml, get the oil phase in Erlenmeyer flask, the organic phase filtering membrane by 0.22 μ l filters and obtains solution to be measured.Solution to be measured is contained in to 1ml sample injection bottle and carries out gas chromatographic analysis.The peak area obtaining according to gas chromatographic analysis is determined the growing amount of ethyl oleate, and detected result is referring to table 2 and Fig. 3.
Table 2: the data of differing temps to ethyl oleate turnout
Known from table 2 and Fig. 3: than resolvase, the two enzymes of immobilization of the present invention are high temperature resistant, and at high temperature can have good enzyme activity, can produce more ethyl oleate; Value at 55 ℃ is the highest, the effect that reaction system can obtain in the time of 55 ℃.
The analytical test of suitable micelle-forming concentration:
Get the two enzymes of the immobilization that makes in five parts of 1.5mg embodiment of the present invention 1, be dissolved in respectively in the reverse micelle enzyme system that rhamnolipid (RL)-octane-iso-n-hexyl alcohol solution of 20CMC, 40CMC, 60CMC, 80CMC, 100CMC forms; Wherein, the volume of reverse micelle enzyme system is 10ml, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding 20 μ l concentration in reverse micelle enzyme system, be that 20mmol/L oleic acid and 10 μ l concentration are that 10mmol/L ethanol obtains mixing solutions.Mixing solutions is put into water-bath vibration case, and it is 55 ℃ that water-bath vibration case temperature is set, and with 150rpm rotating speed, carries out esterification one hour.
Get five parts of lipase that 1.5mg is free, be dissolved in respectively in the reverse micelle enzyme system that rhamnolipid (RL)-octane-iso-n-hexyl alcohol solution of 20CMC, 40CMC, 60CMC, 80CMC, 100CMC forms; According to aforesaid method, carry out esterification 1 hour.
Get five parts of cellulases that 1.5mg is free, be dissolved in respectively in the reverse micelle enzyme system that rhamnolipid (RL)-octane-iso-n-hexyl alcohol solution of 20CMC, 40CMC, 60CMC, 80CMC, 100CMC forms; According to aforesaid method, carry out esterification 1 hour.
In aforementioned esterification reaction process, after esterification starts, every 10 minutes, get 1ml esterification reaction product in Erlenmeyer flask, add the neutralized verdigris (pH containing pyridine is 6.1) of 1ml and the benzene of 5ml, after mixing with 3000r/min rotating speed centrifugal 5 minutes, centrifugal rear stratification, got upper organic phase and in ultraviolet spectrophotometer (548nm wavelength), measures the esterification rate of oleic acid.
Esterification was carried out after 1 hour, in Erlenmeyer flask, added ethanol and acetone mixed solution (volume ratio of ethanol and acetone was by 1: the 1) termination reaction of 5ml, then added the diethyl phthalate of 0.0375g, evenly vibration, stratification.With the syringe of 5ml, get the oil phase in Erlenmeyer flask, the organic phase filtering membrane by 0.22 μ l filters and obtains solution to be measured.Solution to be measured is contained in to 1ml sample injection bottle and carries out gas chromatographic analysis.The peak area obtaining according to gas chromatographic analysis is determined the growing amount of ethyl oleate, and detected result is referring to table 3 and Fig. 4.
Table 3: the data of different critical micellar concentration to ethyl oleate turnout
As shown in table 3 and Fig. 4, than resolvase, the two enzymes of immobilization of the present invention have good enzyme activity, and enzyme stability is better; The value of 60CMC is the highest, the effect that reaction system can obtain when 60CMC.
The analysis simultaneous test of the two enzymes of immobilization in reverse micelle and organic solvent:
Getting the two enzymes of the immobilization that makes in the 1.5mg embodiment of the present invention 1, to be dissolved in respectively volume be in 10ml, micelle-forming concentration (CMC) the reverse micelle enzyme system consisting of rhamnolipid (RL)-octane-iso-n-hexyl alcohol solution that is 60 and in the organic solvent (heptane) of 10ml; Wherein, the mol ratio (w of water/rhamnolipid in reverse micelle enzyme system
0) be 40.Then to respectively adding 20 μ l concentration in two individual system, be that 20mmol/L oleic acid and 10 μ l concentration are that 10mmol/L ethanol obtains mixing solutions.Mixing solutions is put into water-bath vibration case, and water-bath vibration case temperature is 40 ℃, with 150rpm rotating speed, carries out esterification one hour.
In aforementioned esterification reaction process, after esterification starts, every 10 minutes, get 1ml esterification reaction product in Erlenmeyer flask, add the neutralized verdigris (it is 6.1 that neutralized verdigris regulates pH by pyridine) of 1ml and the benzene of 5ml, after mixing with 3000r/min rotating speed centrifugal 5 minutes, centrifugal rear stratification, got upper organic phase and in ultraviolet spectrophotometer (548nm wavelength), measures the esterification rate of oleic acid.
Esterification was carried out after 1 hour, in Erlenmeyer flask, added ethanol and acetone mixed solution (volume ratio of ethanol and acetone was by 1: the 1) termination reaction of 5ml, then added the diethyl phthalate of 0.0375g, evenly vibration, stratification.With the syringe of 5ml, get the oil phase in Erlenmeyer flask, the organic phase filtering membrane by 0.22 μ l filters and obtains solution to be measured.Solution to be measured is contained in to 1ml sample injection bottle and carries out gas chromatographic analysis.The peak area obtaining according to gas chromatographic analysis is determined the growing amount of ethyl oleate, and detected result is referring to table 4 and Fig. 5.
Table 4: the result table of the two enzyme catalyzed oil acetoacetic esters in organic solvent and reverse micelle system of immobilization
As shown in table 4 and Fig. 5, than at organic solvent, the two enzymes of immobilization of the present invention can have good enzyme activity under reverse micelle, and the esterification effect in reverse micelle system is better than the esterification effect of organic solvent, can produce more ethyl oleate.
The analytical test of the operational stability of the two enzymes of immobilization:
Getting the two enzymes of the immobilization that makes in the 1.5mg embodiment of the present invention 1, to be dissolved in volume be in 10ml, micelle-forming concentration (CMC) the reverse micelle enzyme system consisting of rhamnolipid (RL)-octane-iso-n-hexyl alcohol solution that is 60; Wherein, the mol ratio (w of water/rhamnolipid
0) be 40.Then to adding 20 μ l concentration in reverse micelle enzyme system, be that 20mmol/L oleic acid and 10 μ l concentration are that 10mmol/L ethanol obtains mixing solutions.Mixing solutions is equally divided into five parts, puts into successively water-bath vibration case, it is 55 ℃ that water-bath vibration case temperature is set, and with 150rpm rotating speed, carries out esterification one hour.
Getting the two enzymes of the immobilization that makes in the 1.5mg embodiment of the present invention 1 and be dissolved in the organic solvent that volume is 10ml (heptane), is then that 20mmol/L oleic acid and 10 μ l concentration are that 10mmol/L ethanol obtains mixing solutions to adding 20 μ l concentration in organic solvent.Mixing solutions is equally divided into five parts, puts into successively water-bath vibration case, it is 55 ℃ that water-bath vibration case temperature is set, and with 150rpm rotating speed, carries out esterification one hour.
After having reacted, reaction product is carried out to vacuum filtration, with the two enzymes of distilled water flushing immobilization, until neutral.Rinse the two enzyme kept dry of complete immobilization and carry out next batch experiment.With the two enzymes of a collection of immobilization, at 55 ℃, operate repeatedly, as shown in Figure 6, in organic solvent and reverse micelle system, the vigor of usining while using is for the first time as 100%, while using for the second time respectively, system remnant enzyme activity force rate has declined approximately 6% for the first time, it is not very large using loss for several times afterwards, and through 9 uses, enzyme activity still remains on 60%, until while using for the 12 time, its remaining vigor drops to less than 30%.By contrast, in reverse micelle system, it is better that the relative activity of enzyme will keep.
The above, be only preferred embodiment of the present invention, not the present invention done to any pro forma restriction.Although the present invention discloses as above with preferred embodiment, yet not in order to limit the present invention.Any those of ordinary skill in the art, in the situation that not departing from spirit of the present invention and technical scheme, all can utilize method and the technology contents of above-mentioned announcement to make many possible changes and modification to technical solution of the present invention, or be revised as the equivalent embodiment of equivalent variations.Therefore, every content that does not depart from technical solution of the present invention, according to technical spirit of the present invention to any simple modification made for any of the above embodiments, be equal to replacements, equivalence changes and modify, all still belong in the scope that technical solution of the present invention protects.
Claims (10)
1. an application for the two enzymes of immobilization catalyzed oil acid esterification in reverse micelle system, is characterized in that, comprises the following steps:
S1: cellulase and lipase are through the two enzymes of being fixed of co-immobilization;
S2: the two enzymes of described immobilization are mixed with reverse micelle system, add oleic acid and Organic Alcohol to carry out water-bath vibration and obtain esterification products.
2. application according to claim 1, is characterized in that, described S1 step is specifically further comprising the steps of:
S1-1: described cellulase is fixed to being fixed cellulase in biological carbon;
S1-2: described lipase is fixed on and obtains the two enzymes of described immobilization in described immobilized cellulase.
3. application according to claim 2, it is characterized in that, described S1-1 step is specially: described cellulase is added to the damping fluid that obtains cellulase in damping fluid, the damping fluid that contains cellulase described in biological carbon is placed in carries out water-bath vibration step and obtains described immobilized cellulase.
4. application according to claim 2, is characterized in that, described S1-2 step is specially: described lipase is added to the damping fluid that obtains fatty enzyme in damping fluid; The damping fluid that contains lipase described in described immobilized cellulase is placed in carries out water-bath vibration step and obtains the two enzymes of described immobilization.
5. according to the application described in claim 3 or 4, it is characterized in that, described damping fluid is pH2～8 Sodium phosphate dibasic-citrate buffer solution; The weightmeasurement ratio of described cellulase and described damping fluid is 0.1～0.4mg/ml; The weightmeasurement ratio of described lipase and damping fluid is 0.1～0.4mg/ml; The weightmeasurement ratio of the damping fluid of described biological carbon and cellulase is 0.25: 5～100g/ml; The weightmeasurement ratio of the damping fluid of described immobilized cellulase and fatty enzyme is 0.25～5: 5～100g/ml.
6. according to the application described in any one in claim 2 to 4, it is characterized in that, the preparation method of described biological carbon is: corn cob pyrolysis under vacuum environment is obtained to described biological carbon.
7. application according to claim 6, it is characterized in that, the preparation method of described biological carbon also comprises activation step: described biological carbon is used to 95% ethanol, 5%HCL and 5%NaOH solution soaking successively, and then suction filtration, distilled water flushing to neutral, freeze-drying lyophilization obtain activating biological carbon.
8. according to the application described in any one in claim 1 to 4, it is characterized in that, described reverse micelle system comprises rhamnolipid, octane-iso and n-hexyl alcohol, and described Organic Alcohol is the monobasic Organic Alcohol with C1～C10 carbon chain lengths.
9. application according to claim 8, is characterized in that, the micelle-forming concentration of described reverse micelle system is 20～100CMC, and described Organic Alcohol is a kind of in ethanol, n-propyl alcohol, butanols.
10. according to the application described in any one in claim 1 to 4, it is characterized in that, the molar concentration rate of described oleic acid and Organic Alcohol is 2～7: 1; The volume proportion of oleic acid and reverse micelle system is 0.001～0.002: 1; The temperature of described water-bath vibration is 25～65 ℃.
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