CN102250871A - Preparation method of bentonite-immobilized lipase - Google Patents
Preparation method of bentonite-immobilized lipase Download PDFInfo
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- CN102250871A CN102250871A CN 201110184511 CN201110184511A CN102250871A CN 102250871 A CN102250871 A CN 102250871A CN 201110184511 CN201110184511 CN 201110184511 CN 201110184511 A CN201110184511 A CN 201110184511A CN 102250871 A CN102250871 A CN 102250871A
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Abstract
The invention discloses a preparation method of a bentonite-immobilized lipase, and the preparation method comprises the following steps: immersing a certain amount of calcium bentonite in a NaCl solution, dissolving the treated bentonite in a buffer solution with the pH of 3-9, and adding a certain amount of an organic surfactant for modifying the bentonite carrier; then directly adding a certain amount of lipase powder for immobilization reaction, and enabling the carrier surface modification and immobilization reaction to continuously carry out in the same reaction system; centrifuging, discarding the supernatant, drying the obtained carrier in a vacuum oven, and grinding into powder to obtain the bentonite-immobilized lipase. The bentonite-immobilized lipase prepared by the preparation method disclosed in the invention not only has good stability and good repeat performance, but also has catalytic activity better than that of free lipase.
Description
Technical field
The present invention relates to a kind of preparation method of wilkinite immobilized lipase.
Background technology
Lipase (EC 3.1.3.3) can be used for the different catalyzed reactions such as hydrolysis, esterification and transesterificationization of multiple water-insoluble ester class, be the enzyme that a class has multiple catalysis, be widely used in important industrial circles such as medicine, food, daily use chemicals and grease processing.Because the free-fat enzyme can not reuse, unstable, not easily separated and in organic phase " reunions " and the shortcoming of inactivation is difficult to realization serialization production easily with product, restricted it in industrial application.And utilize the method for physics or chemistry that lipase is fixed on the water insoluble carrier, and be prepared into the immobilized lipase that stability is strong, can reuse, can overcome the above shortcoming of free-fat enzyme, kept the catalysis characteristics of lipase simultaneously again.Therefore, immobilized lipase has promoted lipase greatly in industrial using value, has widened its range of application.
Resolvase is after immobilization, and the microenvironment of its carrier surface has significant effects to catalysis activity and the stability that improves immobilized enzyme.At present, lipase immobilization carrier commonly used mainly contains natural organic high-molecular materials such as chitosan, sodium alginate, organic synthesis macromolecular materials such as polyacrylic acid, ion exchange resin, and inorganic materials such as sintered glass, silica gel.These materials have characteristics such as biocompatibility is better, mass-transfer performance is good mostly, but also exist certain limitation.As being that its physical strength of immobilized enzyme of carrier has much room for improvement with the chitosan, carrier causes the leakage of enzyme than being easier to disintegration; And the organic polymer molecule also can not be ignored " murder by poisoning " of zymoprotein effect, causes the decline of lipase-catalyzed vigor easily; Inorganic materials such as silica gel both had been unfavorable for correct fold of lipase on carrier because its hydrophobicity is very poor, also were unfavorable for mass transfer and the catalysis of water-insoluble ester class on carrier, thereby made catalytic rate and productive rate occur descending; In addition, along with constantly riseing of industrial chemicals and product price, the higher acquisition cost of carrier also is unfavorable for lipase-catalyzed in industrial application.Therefore, it is very very difficult finding a kind of desirable fixation support, and the immobilized lipase for the preparation high catalytic performance is again vital simultaneously.
Wilkinite is abundant at China's reserves, and it is cheap, unreactiveness, structure-function is adjustable, thermostability and Environmental compatibility are good.And the abundant reactive group of carrier surface has been created good condition for its carrier modification, and we can realize the adjusting to the wilkinite 26S Proteasome Structure and Function by selecting different modifier and ratio thereof.At present, the existing report that utilizes wilkinite alpha-amylase immobilized, alkaline phosphatase etc., but decline has in various degree all appearred in its catalysis activity after the immobilization.And in wilkinite immobilized lipase enzyme process, introduce the modification of organic surface active agent, the process for fixation that carrier modification and lipase load are coupled yet there are no report.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of wilkinite immobilized lipase, the immobilized lipase that the present invention makes not only good stability, to repeat performance good, has the better catalytic activity of specific ionization lipase simultaneously again.
In order to reach above-mentioned purpose, solution of the present invention is:
A kind of preparation method of wilkinite immobilized lipase, may further comprise the steps: (Linan, Zhejiang north of the city wilkinite company limited) impregnated in the NaCl solution with a certain amount of calcium-base bentonite, wilkinite after handling is dissolved in the buffered soln of pH 3～9, and adds a certain amount of organic surface active agent the wilkinite carrier is carried out modification; Add a certain amount of lipase powder then and carry out immobilized reactant, carrier surface modification and immobilized reactant are carried out in same reaction system continuously; Centrifugal abandoning supernatant, the carrier that obtains places the vacuum drying oven inner drying, after pulverizing promptly.
Described NaCl strength of solution is 0.1 mol/L, and the mass volume ratio of calcium-base bentonite and NaCl solution is 1: 5～1: 20(w/v), and stir 0～5 h, leave standstill 36 h after, solid precipitation is with distilled water wash 3～6 times, until through the no Cl of check
-Till.
The wilkinite after the described processing and the mass volume ratio of buffered soln are 1: 2～1: 10.
Described buffered soln adopts a kind of in phosphate buffer soln, the acetate buffer solution.
Described organic surface active agent adopts cetyl trimethylammonium bromide, palmityl trimethyl ammonium chloride, octadecyl trimethylammonium bromide, a kind of in the octadecyl trimethyl ammonium chloride; (above organic surface active agent is available from Alfa Aesar company) add-on is 20%～200% of a cation exchange capacity, and at 40～80 ℃ of following constant temperature stirring reaction 0～15 h.
Mass concentration after described lipase powder adds is 1～20mg/ml.(the lipase powder is available from Sigma-Aldrich company).
Described lipase solution adjusts the temperature to 0～50 ℃ before adding, and wants stirring reaction 2-4h after the adding.
The described immobilized reactant time is 1～10 h, and rotating speed is 0～500 rpm.
Described vacuum-drying temperature is 10～50 ℃, and vacuum tightness is≤0.1 MPa.
Beneficial effect of the present invention is: the present invention utilizes sodium chloride solution that original soil is carried out pre-treatment back loading free-fat enzyme, in the buffered soln of pH3～9, the surface tissue and the hydrophobic performance of carrier are regulated by adding different organic cation tensio-active agent (as cetyl trimethylammonium bromide etc.).After reaction for some time, reaction solution adds a certain amount of free-fat enzyme again without any processing, and carrier modification and zymoprotein load serialization are carried out.Discard loose zymoprotein at last, being fixed lipase after vacuum-drying, grinding.The invention carrier modification and lipase immobilization are coupled carried out in same reactor continuously, saved post-processing step and time greatly.After the immobilized lipase that the present invention is made was used for the biphasic catalysis hydrolysis reaction of organic phase-water, experimental result showed: its catalysis activity is compared resolvase all in various degree raising, and maximum reaches more than 130%; Thermostability and pH stability also are greatly improved; Immobilized lipase is after repeatedly recycling, and the enzyme catalysis vigor still keeps higher level, and operational stability is greatly improved.Compare traditional process for fixation, the immobilized enzyme that utilizes the present invention to prepare has following advantage:
1) lipase-catalyzed vigor height.Distinctive laminate structure of wilkinite and bigger specific surface area well disperse the lipase molecule on carrier, avoided the enzyme molecule in the reunion of carrier and substrate molecule can't fully be contacted.Wilkinite has structure and the adjustable characteristic of function, the water repellent surface that forms after organic surface active agent is modified helps the correct folding of lipase protein-active center conformation, and the abundant active group in organic bentonite surface is fully fixing by reactive force such as hydrophobic interaction power, hydrogen bond and Van der Waals force and zymoprotein molecule, significantly strengthen the rigidity of enzyme active center, reduced the variation of zymoprotein conformation in the reaction process.The hydrophobic carrier surface also helps fully contacting and enrichment of hydrophobicity ester class substrate and enzyme active center, has reduced the resistance to mass transfer in the reaction process, thereby the enzyme catalysis vigor is significantly strengthened.
2) stability of immobilized lipase is high.Because the distinctive rigidity laminate structure of wilkinite makes fixation support dissolved possibility in the recycling process reduce greatly, avoid the destruction of carrier and caused the leakage of enzyme, improved the repeat performance of immobilized lipase.Have strong inflexible zymoprotein structure and make immobilized enzyme in reaction process, resist the ability enhancing of acid, alkali and heat, help the active centre conformation of stabilized enzyme, thereby the stability of immobilized lipase is improved.
3) immobilization efficiency height.Utilize the powerful adsorptive power of wilkinite, and modify carrier surface that the back forms and the hydrophobic interaction power between the lipase, make the carrier surface that is adsorbed on that most free-fat enzyme can be firm.Compare not modified wilkinite, its immobilization efficiency is improved.
4) the used carrier wilkinite is abundant, cheap and easy to get at China's storage, helps reducing the production cost of immobilized lipase.
5) process for fixation that carrier modification and lipase load are coupled is simple, and mild condition does not need special equipment, and aftertreatment technology is simple, is suitable for industrial scale operation.
Description of drawings
The comparison diagram of immobilized lipase that Fig. 1 makes for the present invention and the fixed lipase catalyzed vigor of unmodified wilkinite soil;
The stable comparison diagram of immobilized lipase that Fig. 2 makes for the present invention and unmodified wilkinite soil immobilized lipase.
Embodiment
Below in conjunction with concrete detailed example process for fixation of the present invention and the technique effect that brings thereof are further described, but protection scope of the present invention is not limited in this:
Cetyl trimethylammonium bromide is modified the preparation and the enzyme activity determination of wilkinite immobilized lipase
1) take by weighing the 5g calcium-base bentonite and join in the NaCl solution that 50 mL concentration are 0.1 mol/L, stir earlier 2 h, leave standstill 36 h then, during change three fresh NaCl solution.The centrifugal supernatant liquor that inclines, the wilkinite that obtains 200mL distilled water wash, suction filtration obtains carrier.
2) getting the carrier that the 25g step 1) obtains joins in the phosphate buffered saline buffer of 100 mL pH 6, (add-on is equivalent to 100% of cation exchange capacity to add a certain amount of cetyl trimethylammonium bromide, cation exchange capacity is the wet wilkinites of 8.25 mmol/100g), 60 ℃ of following constant temperature oscillatory reaction 12h carry out modification to the wilkinite carrier.Temperature is transferred to 25 ℃ then, directly adding lipase powder, to make its mass concentration be 5 mg/mL, and carrier surface modification and immobilized reactant are carried out in same reaction system continuously.Behind oscillatory reaction 4 h, solution is centrifugal 15 min under 15000 rpm, obtain solid precipitation under the 200rpm.This precipitation is with 100 mL distilled water washs 5 times, and vacuum-drying is 3 days under 30 ℃, 0.08 MPa condition, being fixed of pulverizing lipase.
3) immobilized lipase enzyme activity determination
The sweet oil of certain volume is mixed (volume ratio is 1: 3) with 4% polyvinyl alcohol solution, mix with magnetic stirrer and stir twice, each 3 min, midfeather 5 min make the sweet oil emulsion.(pH 7.0 with this sweet oil emulsion and phosphate buffered saline buffer, 20 mM) mix, get this mixed solution of 10 mL and in 30 ℃ of water-baths, be incubated 10 min, adding step 2) immobilized lipase 0.15 g that makes, behind constant temperature vibration reaction 20 min, add the ethanolic soln termination reaction of 15 mL 95% under 180 rpm.With the NaOH solution titration of this reaction solution with 0.02 mol/L, blank solution is the substrate solution that 10 mL do not add lipase, and similarity condition is reaction 20 min down, adds the ethanolic soln of 15 mL 95% equally.The difference that consumes the amount of alkali lye according to both is calculated the catalysis activity of lipase.Lipase-catalyzed vigor is defined as: 30 ℃, under the pH7.0 condition, it is 1 enzyme activity unit that every min produces the needed enzyme amount of 1 μ mol lipase, is designated as U/g.
Experimental result is as shown in table 1 below, and table 1 is modified the catalysis activity comparison sheet of wilkinite immobilized lipase and free-fat enzyme for cetyl trimethylammonium bromide.From following table 1 as can be seen, the catalysis activity of this immobilized lipase resolvase of comparing same amount has improved more than 35%.
Palmityl trimethyl ammonium chloride is modified the preparation and the enzyme activity determination of wilkinite immobilized lipase
1) take by weighing the 5g calcium-base bentonite and join in the NaCl solution that 25 mL concentration are 0.1 mol/L, stir earlier 2 h, leave standstill 36 h then, during change three fresh NaCl solution.The centrifugal supernatant liquor that inclines, the wilkinite that obtains 200mL distilled water wash, suction filtration obtains carrier.
2) getting the carrier that 30 g step 1) obtain joins in the phosphate buffered saline buffer of 200 mL pH 7, add a certain amount of palmityl trimethyl ammonium chloride (add-on be equivalent to cation exchange capacity 50%), at 80 ℃ of following constant temperature oscillatory reaction 15 h, the wilkinite carrier is carried out modification.Temperature is transferred to 25 ℃ then, directly adding lipase powder, to make its mass concentration be 10 mg/mL, and carrier surface modification and immobilized reactant are carried out in same reaction system continuously.Behind oscillatory reaction 3 h, centrifugal 15 min obtain solid precipitation under 15000 rpm under the 200rpm.This precipitation is with 100 mL distilled water washs 5 times, and vacuum-drying is 3 days under 30 ℃, 0.08 MPa condition, being fixed of pulverizing lipase.
3) immobilized enzyme enzyme activity determination
The enzyme activity determination method is identical with step 3) among the embodiment 1.
Experimental result is as shown in table 2 below, and table 2 is modified the catalysis activity comparison sheet of wilkinite immobilized lipase and free-fat enzyme for palmityl trimethyl ammonium chloride.From following table 2 as can be seen, the catalysis activity of this immobilized lipase resolvase of comparing same amount has improved more than 10%.
The octadecyl trimethylammonium bromide is modified the preparation and the enzyme activity determination of wilkinite immobilized lipase
1) take by weighing the 5g calcium-base bentonite and join in the NaCl solution that 75 mL concentration are 0.1 mol/L, stir earlier 2 h, leave standstill 36 h then, during change three fresh NaCl solution.The centrifugal supernatant liquor that inclines, the wilkinite that obtains 200mL distilled water wash, suction filtration obtains carrier.
2) getting the carrier that 20 g step 1) obtain joins in the phosphate buffered saline buffer of 100 mL pH 6, add a certain amount of octadecyl trimethylammonium bromide (add-on be equivalent to cation exchange capacity 80%), at 60 ℃ of following constant temperature oscillatory reaction 14 h, the wilkinite carrier is carried out modification.Temperature is transferred to 25 ℃ then, directly adding lipase powder, to make its mass concentration be 15 mg/mL, and carrier surface modification and immobilized reactant are carried out in same reaction system continuously.Behind oscillatory reaction 4 h, solution is centrifugal 15 min under 15000 rpm, obtain solid precipitation under the 200rpm.This precipitation is with 100 mL distilled water washs 5 times, and vacuum-drying is 3 days under 30 ℃, 0.08 MPa condition, being fixed of pulverizing lipase.
3) immobilized lipase enzyme activity determination
The enzyme activity determination method is identical with step 3) among the embodiment 1.
Experimental result is as shown in table 3 below, and table 3 is modified the catalysis activity comparison sheet of wilkinite immobilized lipase and free-fat enzyme for the octadecyl trimethylammonium bromide.From following table 3 as can be seen, the catalysis activity of this immobilized lipase resolvase of comparing same amount has improved more than 20%.
Octadecyl trimethyl ammonium chloride is modified the preparation and the enzyme activity determination of wilkinite immobilized lipase
1) 1) take by weighing the 5g calcium-base bentonite and join in the NaCl solution that 250 mL concentration are 0.1 mol/L, stir earlier 2 h, leave standstill 36 h then, during change three fresh NaCl solution.The centrifugal supernatant liquor that inclines, the wilkinite that obtains 200mL distilled water wash, suction filtration obtains carrier.
2) getting the carrier that 25 g step 1) obtain joins in the acetate buffer of 200 mL pH 4, add a certain amount of octadecyl trimethyl ammonium chloride (add-on be equivalent to cation exchange capacity 150%), at 80 ℃ of following constant temperature oscillatory reaction 15 h, the wilkinite carrier is carried out modification.Temperature is transferred to 25 ℃ then, directly adding lipase powder, to make its mass concentration be 8 mg/mL, and carrier surface modification and immobilized reactant are carried out in same reaction system continuously.Behind oscillatory reaction 2 h, centrifugal 15 min obtain solid precipitation under 15000 rpm under the 200rpm.This precipitation is with 200 mL distilled water washs 5 times, and vacuum-drying is 3 days under 30 ℃, 0.08 MPa condition, being fixed of pulverizing lipase.
3) immobilized lipase enzyme activity determination
The enzyme activity determination method is identical with step 3) among the embodiment 1.
Experimental result is as shown in table 4 below, and table 4 is modified the catalysis activity comparison sheet of wilkinite immobilized lipase and free-fat enzyme for the octadecyl trimethylammonium bromide.As can be seen, the catalysis activity of this immobilized lipase resolvase of comparing same amount has improved more than 25%.
Employed lipase solution in the foregoing description also can replace to a kind of in the outer yielding lipase of born of the same parents, the cell wall breaking fermented liquid, can reach same effect.
Below by the comparative example advantage of the present invention than unmodified wilkinite immobilized lipase is described
The comparative example 1
The comparison of immobilized lipase that the embodiment of the invention 1 makes and the fixed lipase catalyzed vigor of unmodified wilkinite
1) immobilized lipase that makes of the present invention makes according to the method for embodiment 1.The preparation method of original soil immobilized lipase also carries out according to the method for embodiment 1, but does not add cetyl trimethylammonium bromide in the method and do not carry out the modification reaction step.
2) carry out enzyme activity determination according to the method in the step 3) among the embodiment 1.
The comparative result of immobilized lipase that the present invention makes and the fixed lipase catalyzed vigor of original soil as shown in Figure 1.As seen the catalysis activity of the wilkinite immobilized lipase after organic surface active agent is modified is apparently higher than unmodified wilkinite immobilized lipase, this result has proved that further introducing hydrophobic interfaces on carrier helps the correct folding of lipase protein-active division center, help fully contacting of hydrophobic substrate and enzyme active center, avoided lipase the situation that enzyme is lived and descended after immobilization, to occur.
The comparative example 2
The stability of immobilized lipase that the embodiment of the invention 1 makes and unmodified wilkinite soil immobilized lipase relatively
1) makes organic decoration wilkinite immobilized lipase and unmodified wilkinite soil immobilized lipase respectively according to the method in the comparing embodiment 1.
3) two kinds of immobilized lipases that step 1) made carry out a batch reaction according to the method for embodiment 1 step 3).After last batch reaction finishes, immobilized lipase put into carry out the next batch reaction in the fresh substrate solution.After every batch reaction finishes, participate in the mensuration of enzyme work according to the method for embodiment 1 step 3).
Experimental result as shown in Figure 2.As seen the catalytic stability of the immobilized lipase that makes of the present invention will be apparently higher than unmodified wilkinite immobilized lipase, this result has proved that further the hydrophobic interaction power of fixation support helps the conformation of stabilised fat enzyme active center, thereby its stability is improved.
Above embodiment discloses organic surface active agents such as utilizing cetyl trimethylammonium bromide and has modified the process for fixation that is coupled with the lipase load.This carrier modification method equally also is suitable for other organic surface active agents, as Dodecyl trimethyl ammonium chloride, Trimethyllaurylammonium bromide and trimethylchlorosilane etc., this tired no longer one by one stating.
Claims (9)
1. the preparation method of a wilkinite immobilized lipase, it is characterized in that may further comprise the steps: a certain amount of calcium-base bentonite be impregnated in the NaCl solution, wilkinite after handling is dissolved in the buffered soln of pH 3～9, and adds a certain amount of organic surface active agent the wilkinite carrier is carried out modification; Directly add a certain amount of lipase powder then and carry out immobilized reactant, carrier surface modification and immobilized reactant are carried out in same reaction system continuously; Centrifugal abandoning supernatant, the carrier that obtains places the vacuum drying oven inner drying, after pulverizing promptly.
2. the preparation method of a kind of wilkinite immobilized lipase as claimed in claim 1, it is characterized in that: described NaCl strength of solution is 0.1 mol/L, and the mass volume ratio of calcium-base bentonite and NaCl solution is 1: 5～1: 20, and stir 0～5 h, after leaving standstill 36 h, solid precipitation distilled water wash 3～6 times.
3. the preparation method of a kind of wilkinite immobilized lipase as claimed in claim 1 is characterized in that: the wilkinite after the described processing and the mass volume ratio of buffered soln are 1: 2～1: 10.
4. the preparation method of a kind of wilkinite immobilized lipase as claimed in claim 3 is characterized in that: described buffered soln adopts a kind of in phosphate buffer soln, the acetate buffer solution.
5. the preparation method of a kind of wilkinite immobilized lipase as claimed in claim 1, it is characterized in that: described organic surface active agent adopts cetyl trimethylammonium bromide, palmityl trimethyl ammonium chloride, the octadecyl trimethylammonium bromide, a kind of in the octadecyl trimethyl ammonium chloride; Add-on is 20%～200% of a cation exchange capacity, and at 40～80 ℃ of following constant temperature stirring reaction 0～15 h.
6. the preparation method of a kind of wilkinite immobilized lipase as claimed in claim 1 is characterized in that: the mass concentration after described lipase powder adds is 1～20mg/ml.
7. the preparation method of a kind of wilkinite immobilized lipase as claimed in claim 6 is characterized in that: described lipase solution adjusts the temperature to 0～50 ℃ before adding, and wants stirring reaction 2-4h after the adding.
8. the preparation method of a kind of wilkinite immobilized lipase as claimed in claim 1 is characterized in that: the described immobilized reactant time is 1～10 h, and rotating speed is 0～500 rpm.
9. the preparation method of a kind of wilkinite immobilized lipase as claimed in claim 1 is characterized in that: described vacuum-drying temperature is 10～50 ℃, and vacuum tightness is≤0.1 MPa.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131691A (en) * | 2013-03-06 | 2013-06-05 | 昆明理工大学 | Preparation method of immobilized lipase using clay as carrier |
| CN103627694A (en) * | 2013-11-25 | 2014-03-12 | 绍兴文理学院 | Bentonite single-layer stripping nano-sheet assembled enzyme and preparation method thereof |
| WO2024082328A1 (en) * | 2022-10-21 | 2024-04-25 | 湖南祥民制药有限公司 | Biological catalysis method for preparing acetyl phosphoric acid |
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| WO1989008695A1 (en) * | 1988-03-14 | 1989-09-21 | Novo-Nordisk A/S | Stabilized particulate composition |
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| WO1989008695A1 (en) * | 1988-03-14 | 1989-09-21 | Novo-Nordisk A/S | Stabilized particulate composition |
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| 《Applied Clay Science》 20080923 M. Ghiaci, et al. Enzyme immobilization Part 1. Modified bentonite as a new and efficient support for immobilization of Candida rugosa lipase 289-295 1-9 第43卷, * |
| 《Chemical Engineering Journal》 20121231 Huaping Dong ，et al. Improvement of catalytic activity and stability of lipase by immobilization on organobentonite 590-596 1-9 , * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131691A (en) * | 2013-03-06 | 2013-06-05 | 昆明理工大学 | Preparation method of immobilized lipase using clay as carrier |
| CN103131691B (en) * | 2013-03-06 | 2015-03-11 | 昆明理工大学 | Preparation method of immobilized lipase using clay as carrier |
| CN103627694A (en) * | 2013-11-25 | 2014-03-12 | 绍兴文理学院 | Bentonite single-layer stripping nano-sheet assembled enzyme and preparation method thereof |
| CN103627694B (en) * | 2013-11-25 | 2017-06-27 | 绍兴文理学院 | A kind of Bentonite single-layer stripping nano piece assembling enzyme and preparation method thereof |
| WO2024082328A1 (en) * | 2022-10-21 | 2024-04-25 | 湖南祥民制药有限公司 | Biological catalysis method for preparing acetyl phosphoric acid |
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Application publication date: 20111123 |