CN104109662A - Immobilized Burkholderia cepacia lipase and preparation method thereof - Google Patents
Immobilized Burkholderia cepacia lipase and preparation method thereof Download PDFInfo
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Abstract
The invention discloses immobilized Burkholderia cepacia lipase and a preparation method thereof. The immobilized Burkholderia cepacia lipase comprises a carbon nanotube substrate and Burkholderia cepacia lipase, wherein the Burkholderia cepacia lipase is bonded at the opening port, the surface and the inner wall of the carbon nanotube substrate in an affinity adsorption and/or covalent bonding mode. The preparation method comprises the following steps: (1) the carbon nanotube is processed through sulfuric acid ultrasound; (2) a mixed solution of the carbon nanotube and the Burkholderia cepacia lipase; (3) oscillatory reaction is carried out on the mixed solution of the carbon nanotube and the Burkholderia cepacia lipase at the temperature between 4 DEG C to 60 DEG C, and the immobilized Burkholderia cepacia lipase is obtained. According to the immobilized Burkholderia cepacia lipase provided by the invention, dispersion of apoenzyme in the reaction system is increased, high transesterification catalyzing enzyme activity is obtained, and the needed time of achieving reaction balance is shortened.
Description
Technical field
The invention belongs to the immobilization field of enzyme, more specifically, relate to a kind of immobilized Burkholderia cepacia lipase.
Background technology
Burkholderia cepacia lipase (BCL) is a kind of extracellular lipase, at water and nonaqueous phase, all has catalytic activity.BCL has higher resistance to multiple organic solvent, is therefore widely used in nonaqueous phase catalyzed reaction, especially in the mapping fractionation field of preparation biofuel and chiral drug, has more application.But because zymoprotein itself has stronger wetting ability, free enzyme powder is difficult for being scattered in solvent, the reaction interface of the relative efficiency that can form is less, therefore there is the shortcomings such as catalytic activity is low, long reaction time, has limited to a certain extent BCL in industrial application.Yet, enzyme immobilization technology is applied to the preparation of BCL, can improve the transesterification enzyme of BCL in nonaqueous phase solvent and live, Reaction time shorten.
Enzyme immobilization technology is applied and has been obtained certain realization in actual production, and current existing BCL process for fixation advocates to be divided into chemical method and Physical according to immobilization role.Chemical process mainly contains the use of carrier free crosslinking, Hara P, Hanefeld U, Kanerva L T has carried out immobilization with colloidal sol entrapping method and carrier free crosslinking to BCL respectively, more than the reaction times of two kinds of different process for fixation still needs 24h, the immobilized enzyme of chemical process mediation preparation has good stability and good recycling rate conventionally, because it is more firm firm that the chemical bond forming in preparation process connects counterpart reason method, but zymoprotein structure originally is also had largely and destroyed, cause its protein conformation to change, thereby cannot obtain higher enzyme lives.In view of this characteristic, the major part for the immobilized report of BCL is all the research work about physical method at present.Physical method, mainly by adsorptions such as Van der Waals force and hydrogen bonds, comprises the methods such as physisorphtion, microcapsule method, colloidal sol embedding, and physisorphtion major advantage is that preparation condition is gentle, enzyme active center space conformation is not destroyed etc.But current physics fixedly method catalyzed reaction time of BCL longer, transesterification yield is not high.
Summary of the invention
Above defect or Improvement requirement for prior art, the invention provides a kind of immobilized onion Burkholder lipase and preparation method thereof, its object is by selecting suitable matrix and method by onion Burkholder lipase immobilization, solve thus the lipase-catalyzed sound of ancient India of current immobilized onion Burkholder in a hurry longer, transesterification yield is hi-tech problem not.
For achieving the above object, according to one aspect of the present invention, a kind of immobilized Burkholderia cepacia lipase is provided, it is characterized in that, comprise carbon nano-tube matrix and Burkholderia cepacia lipase, described Burkholderia cepacia lipase is combined in open port, surface and the inwall of carbon nano-tube matrix by affine absorption and/or covalently bound mode.
Preferably, described immobilized Burkholderia cepacia lipase, its carbon nano-tube matrix is the carbon nanotube through sulfuric acid supersound process.
According to another aspect of the present invention, a kind of preparation method of immobilized Burkholderia cepacia lipase is provided, it is characterized in that, comprise the following steps:
(1) sulfuric acid supersound process carbon nanotube: every gram of carbon nanotube is evenly mixed with the vitriol oil of 100ml to 300ml, 200W to 250W supersound process 2 hours to 5 hours, dry after washing and filtering, grind the carbon nanotube that obtains sulfuric acid supersound process;
(2) prepare carbon nanotube and Burkholderia cepacia lipase mixed solution: the carbon nanotube of the sulfuric acid supersound process obtaining in step (1) is joined in Burkholderia cepacia lipase solution, make the mass ratio of described carbon nanotube and Burkholderia cepacia lipase between 1:0.5 to 1:6, form carbon nanotube and Burkholderia cepacia lipase mixed solution;
(3) prepare immobilized Burkholderia cepacia lipase: by the carbon nanotube obtaining in step (2) and Burkholderia cepacia lipase mixed solution at 4 ℃ at 60 ℃, oscillatory reaction 1 hour to 6 hours, the centrifugal supernatant liquor that removes obtains described immobilized Burkholderia cepacia lipase.
Preferably, described preparation method, also comprises step:
(4) prepare immobilized Burkholderia cepacia lipase dry powder: by the immobilized Burkholderia cepacia lipase obtaining in step (3), lyophilize, grinds to form powdery.
Preferably, described preparation method, its step (1) adopts cellulose acetate as filter membrane while filtering.
Preferably, described preparation method, the described Burkholderia cepacia lipase solution of its step (2), its pH value is between 5.0 to 8.0.
In general, the above technical scheme of conceiving by the present invention compared with prior art, due to the affinity interaction to bioprotein molecule by means of carbon nanotube six-membered ring structure, utilize vitriol oil supersound process carbon nanotube, its closed port is opened, carbon nanotube itself is by suitably brachymemma, increase its specific surface area, Burkholderia cepacia lipase can be adsorbed onto on carbon nanotube preferably, increased the dispersiveness of zymoprotein in reaction system, obtain higher transesterification katalaze enzyme and lived, greatly shortened the required time of molecular balance that reaches.
Accompanying drawing explanation
Fig. 1 is the carbon nanotube scanning electron microscope phenogram after embodiment 1 acid treatment;
Fig. 2 is the catalysis activity measurement result figure of embodiment 5 immobilization Burkholderia cepacia lipase;
Fig. 3 is the affect experimental result pictures of 6 times of embodiment on immobilization efficiency and immobilized enzyme work;
Fig. 4 is the affect experimental result picture of embodiment 7pH on immobilization efficiency and immobilized enzyme work.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.In addition,, in each embodiment of described the present invention, involved technical characterictic just can not combine mutually as long as do not form each other conflict.
Effective process for fixation of Reaction time shorten when improving BCL transesterification yield in order to explore, we have done screening to variety carrier and have attempted, find a kind of novel nano-material, it is carbon nanotube, can effectively complete above-mentioned requirements, for BCL has established solid technical foundation in the industrial applications of mapping chiral material fractionation.
Carbon nanotube (CNTs), has another name called Baji-tube, mainly by the carbon atom that is six-ring arrangement, forms the coaxial pipe of several layers to tens of layers.Carbon nanotube has the basic nano material feature that diameter is little, specific surface area is large, has good electroconductibility and heat-conductive characteristic, has stable chemical property and thermostability and good bio-compatibility simultaneously.Low for solving Burkholderia cepacia lipase immobilization enzyme transesterification yield, the problems such as the interior catalyzed reaction starting time of nonaqueous phase solvent is long, we attempt the affinity interaction to bioprotein molecule by means of carbon nanotube six-membered ring structure first, utilize vitriol oil supersound process carbon nanotube, its closed port is opened, carbon nanotube itself is by suitably brachymemma, increase its specific surface area, Burkholderia cepacia lipase can be adsorbed onto on carbon nanotube preferably, increased the dispersiveness of zymoprotein in reaction system, having obtained higher transesterification katalaze enzyme lives, greatly shortened the required time of molecular balance that reaches.
Immobilized Burkholderia cepacia lipase provided by the invention, comprise carbon nano-tube matrix and Burkholderia cepacia lipase, described Burkholderia cepacia lipase is combined in open port, surface and the inwall of carbon nano-tube matrix by affine absorption and/or covalently bound mode.Described carbon nano-tube matrix is preferably the carbon nanotube through sulfuric acid supersound process, its electron-microscope scanning figure, as shown in Figure 1.
Immobilized Burkholderia cepacia lipase provided by the invention, its preparation method, comprises the following steps:
(1) sulfuric acid supersound process carbon nanotube: every gram of carbon nanotube is evenly mixed with the vitriol oil of 100ml to 300ml, 200W to 250W supersound process 2 hours to 5 hours, dry after washing and filtering, grind the carbon nanotube that obtains sulfuric acid supersound process; Preferably cellulose acetate is as filter membrane.
(2) prepare carbon nanotube and Burkholderia cepacia lipase mixed solution: the carbon nanotube of the sulfuric acid supersound process obtaining in step (1) is joined in Burkholderia cepacia lipase solution, make the mass ratio of described carbon nanotube and Burkholderia cepacia lipase between 1:0.5 to 1:6, form carbon nanotube and Burkholderia cepacia lipase mixed solution; Described Burkholderia cepacia lipase solution, its preferred pH value is between 5.0 to 8.0, and preferably pH value is 7.0.
(3) prepare immobilized Burkholderia cepacia lipase: by the carbon nanotube obtaining in step (2) and Burkholderia cepacia lipase mixed solution at 4-60 ℃, oscillatory reaction 1-6 hour, the centrifugal supernatant liquor that removes obtains described immobilized Burkholderia cepacia lipase.
(4) prepare immobilized Burkholderia cepacia lipase dry powder: by the immobilized Burkholderia cepacia lipase obtaining in step (3), lyophilize, grinds to form powdery.
Be below embodiment:
Embodiment 1
A kind of immobilized Burkholderia cepacia lipase, comprise carbon nano-tube matrix and Burkholderia cepacia lipase, described Burkholderia cepacia lipase is combined in open port, surface and the inwall of carbon nano-tube matrix by affine absorption and/or covalently bound mode.
Described carbon nano-tube matrix is that preparation process is as follows through the carbon nanotube of sulfuric acid supersound process:
The vitriol oil of getting 1.5g carbon nanotube and 150ml98% adds respectively in a 200ml beaker, stirs it is mixed, 250W supersound process 4h.Ultrasonic completing, is slowly diluted to neutrality by mixed solution, filters, and more than 3 times, reduce as much as possible sulfate ion residual with clear water washing with acetate film.By filter thing to be placed in the baking oven of 60 ℃ dry, to complete drying (both having weighed quality no longer changes), be ground into powder, collect the carbon nanotube that powder is functionalization.Carbon nanotube scanning electron microscope phenogram after acid treatment as shown in Figure 1.
Embodiment 2
An immobilized Burkholderia cepacia lipase, its preparation method, comprises the following steps:
(1) sulfuric acid supersound process carbon nanotube: every gram of carbon nanotube is evenly mixed with the vitriol oil of 100ml, 200W supersound process 5 hours, dry after washing cellulose acetate membrane filtration, grind the carbon nanotube that obtains sulfuric acid supersound process.
(2) prepare carbon nanotube and Burkholderia cepacia lipase mixed solution: the carbon nanotube of the sulfuric acid supersound process obtaining in step (1) is joined in Burkholderia cepacia lipase solution, the mass ratio that makes described carbon nanotube and Burkholderia cepacia lipase is 1:0.5, forms carbon nanotube and Burkholderia cepacia lipase mixed solution; Described Burkholderia cepacia lipase solution, its pH value is 7.0.
(3) prepare immobilized Burkholderia cepacia lipase: by the carbon nanotube obtaining in step (2) and Burkholderia cepacia lipase mixed solution at 4 ℃, oscillatory reaction 6 hours, the centrifugal supernatant liquor that removes obtains described immobilized Burkholderia cepacia lipase.
(4) prepare immobilized Burkholderia cepacia lipase dry powder: by the immobilized Burkholderia cepacia lipase obtaining in step (3), lyophilize, grinds to form powdery.
Embodiment 3
An immobilized Burkholderia cepacia lipase, its preparation method, comprises the following steps:
(1) sulfuric acid supersound process carbon nanotube: every gram of carbon nanotube is evenly mixed with the vitriol oil of 300ml, 250W supersound process 2 hours, dry after washing cellulose acetate membrane filtration, grind the carbon nanotube that obtains sulfuric acid supersound process.
(2) prepare carbon nanotube and Burkholderia cepacia lipase mixed solution: the carbon nanotube of the sulfuric acid supersound process obtaining in step (1) is joined in Burkholderia cepacia lipase solution, the mass ratio that makes described carbon nanotube and Burkholderia cepacia lipase is 1:6, forms carbon nanotube and Burkholderia cepacia lipase mixed solution; Described Burkholderia cepacia lipase solution, its pH value is 5.0.
(3) prepare immobilized Burkholderia cepacia lipase: by the carbon nanotube obtaining in step (2) and Burkholderia cepacia lipase mixed solution at 60 ℃, oscillatory reaction 1 hour, the centrifugal supernatant liquor that removes obtains described immobilized Burkholderia cepacia lipase.
(4) prepare immobilized Burkholderia cepacia lipase dry powder: by the immobilized Burkholderia cepacia lipase obtaining in step (3), lyophilize, grinds to form powdery.
Embodiment 4
An immobilized Burkholderia cepacia lipase, its preparation method, comprises the following steps:
(1) sulfuric acid supersound process carbon nanotube: every gram of carbon nanotube is evenly mixed with the vitriol oil of 200ml, 225W supersound process 3.5 hours, dry after washing cellulose acetate membrane filtration, grind the carbon nanotube that obtains sulfuric acid supersound process.
(2) prepare carbon nanotube and Burkholderia cepacia lipase mixed solution: the carbon nanotube of the sulfuric acid supersound process obtaining in step (1) is joined in Burkholderia cepacia lipase solution, the mass ratio that makes described carbon nanotube and Burkholderia cepacia lipase is 1:4, forms carbon nanotube and Burkholderia cepacia lipase mixed solution; Described Burkholderia cepacia lipase solution, its preferred pH value is 8.0.
(3) prepare immobilized Burkholderia cepacia lipase: by the carbon nanotube obtaining in step (2) and Burkholderia cepacia lipase mixed solution at 35 ℃, oscillatory reaction 4 hours, the centrifugal supernatant liquor that removes obtains described immobilized Burkholderia cepacia lipase.
(4) prepare immobilized Burkholderia cepacia lipase dry powder: by the immobilized Burkholderia cepacia lipase obtaining in step (3), lyophilize, grinds to form powdery.
Embodiment 5
The catalysis activity of immobilization Burkholderia cepacia lipase is measured
Take 5ml normal heptane as solvent, getting 1-phenylethyl alcohol 1mmol and 4mmol vinyl-acetic ester is reaction substrate, reaction system is dissolved in 50ml ground triangular flask, after being placed in constant-temperature table (50 ℃) and mixing with plug sealing, add rapidly respectively immobilized Burkholderia cepacia lipase the encloses container in the embodiment 2 to 4 of 0.05g, prepared, continue to be placed in constant-temperature table oscillatory reaction, accurately after reaction 10min, take out reaction solution centrifugal, get supernatant liquid filtering, for liquid-phase chromatographic analysis, calculate respectively transesterification enzyme and live, result is as Fig. 2.As can be seen from Figure, above three examples all have more than 90% fixedly rate and enzyme work to reach 45, more than 000U/min/g protein, the more than 48 times of resolvase katalaze enzyme work, show that the related experiment condition of above example is all feasible in this patent is described scope, and can keep higher fixedly rate and katalaze enzyme to live.
The impact of 6 times of embodiment on immobilization efficiency and immobilized enzyme work:
The preparation of A, functionalized carbon nano-tube carrier:
The vitriol oil of getting 1.5g carbon nanotube and 150ml98% adds respectively in a 200ml beaker, stirs it is mixed, 250W supersound process 4h.Ultrasonic completing, is slowly diluted to neutrality by mixed solution, filters, and more than 3 times, reduce as much as possible sulfate ion residual with clear water washing with acetate film.By filter thing to be placed in the baking oven of 60 ℃ dry, to complete drying (both having weighed quality no longer changes), be ground into powder, collect the carbon nanotube that powder is functionalization.
The preparation of B, carbon nanotube immobilization Burkholderia cepacia lipase
0.2g functionalized carbon nano-tube carrier is joined in the lipase solution of 5ml120mg/ml pH=7, repeat to prepare 6 groups of tests, 37 ℃ of shaking tables are distinguished oscillatory reaction 1h, 2h, 3h, 4h, 5h, 6h; With 12,000rmp, 4 ℃ of centrifugal 10min, collecting precipitation thing is the immobilized Burkholderia cepacia lipase of carbon nanotube, and by its lyophilize, grinding to form powdery can be for catalyzed reaction.Collect its centrifuged supernatant and lipase solution and with Xylene Brilliant Cyanine G, measure the method for protein content, measure respectively its protein content, calculate respectively fixedly rate.
Result as shown in Figure 3, by figure, can be drawn, between immobilization time 1-4h, along with the increase of immobilization time, immobilized enzyme is lived and to be existed slowly and to raise with fixing rate, and after the immobilization time reaches 4 hours, it is constant that immobilized enzyme work and fixedly rate maintain higher level substantially, after representing 4h, immobilization has reached balance and stability state, therefore the immobilization time is condition more preferably more than 4h.
Embodiment 7pH is on immobilization efficiency and immobilized enzyme impact alive
A, according in embodiment 1 through the preparation method of the carbon nanotube of sulfuric acid supersound process, preparation:
Concrete steps are with embodiment 6 steps A.
The preparation of b, carbon nanotube immobilization Burkholderia cepacia lipase:
0.2g functionalized carbon nano-tube carrier is joined respectively in the lipase solution that pH is respectively 3,4,5,6,7,8,9,10 5ml120mg/ml to 37 ℃ of shaking table oscillatory reaction 4h; With 12000rmp, 4 ℃ of centrifugal 10min, collecting precipitation thing is the immobilized Burkholderia cepacia lipase of carbon nanotube, and by its lyophilize, grinding to form powdery can be for catalyzed reaction.Collect its centrifuged supernatant and lipase solution and with Xylene Brilliant Cyanine G, measure the method for protein content, measure respectively its protein content, calculate respectively fixedly rate.Other step parts embodiment 6 step B.
Result as shown in Figure 4, increase along with pH value, immobilization efficiency presents the trend of first increases and then decreases, it is to have maximum value at 7 o'clock in pH value that immobilization beautifies with fixing rate, but pH of buffer is significantly less than on the fixing impact of rate the impact that it is lived on immobilized enzyme, and fixedly rate all maintains more than 90%, illustrate that pH of buffer is not the principal element that affects immobilization efficiency, but it there is certain influence for the zymoprotein character being immobilized.
Those skilled in the art will readily understand; the foregoing is only preferred embodiment of the present invention; not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (6)
1. an immobilized Burkholderia cepacia lipase, it is characterized in that, comprise carbon nano-tube matrix and Burkholderia cepacia lipase, described Burkholderia cepacia lipase is combined in open port, surface and the inwall of carbon nano-tube matrix by affine absorption and/or covalently bound mode.
2. immobilized Burkholderia cepacia lipase as claimed in claim 1, is characterized in that, described carbon nano-tube matrix is the carbon nanotube through sulfuric acid supersound process.
3. the preparation method of immobilized Burkholderia cepacia lipase as claimed in claim 1 or 2, is characterized in that, comprises the following steps:
(1) sulfuric acid supersound process carbon nanotube: every gram of carbon nanotube is evenly mixed with the vitriol oil of 100ml to 300ml, 200W to 250W supersound process 2 hours to 5 hours, dry after washing and filtering, grind the carbon nanotube that obtains sulfuric acid supersound process;
(2) prepare carbon nanotube and Burkholderia cepacia lipase mixed solution: the carbon nanotube of the sulfuric acid supersound process obtaining in step (1) is joined in Burkholderia cepacia lipase solution, make the mass ratio of described carbon nanotube and Burkholderia cepacia lipase between 1:0.5 to 1:6, form carbon nanotube and Burkholderia cepacia lipase mixed solution;
(3) prepare immobilized Burkholderia cepacia lipase: by the carbon nanotube obtaining in step (2) and Burkholderia cepacia lipase mixed solution at 4 ℃ at 60 ℃, oscillatory reaction 1 hour to 6 hours, the centrifugal supernatant liquor that removes obtains described immobilized Burkholderia cepacia lipase.
4. preparation method as claimed in claim 3, is characterized in that, also comprises step:
(4) prepare immobilized Burkholderia cepacia lipase dry powder: by the immobilized Burkholderia cepacia lipase obtaining in step (3), lyophilize, grinds to form powdery.
5. preparation method as claimed in claim 3, is characterized in that, step (1) adopts cellulose acetate as filter membrane while filtering.
6. preparation method as claimed in claim 3, is characterized in that, the described Burkholderia cepacia lipase solution of step (2), and its pH value is between 5.0 to 8.0.
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| WO2018158459A1 (en) * | 2017-03-03 | 2018-09-07 | Nordmark Arzneimittel Gmbh & Co. Kg | Orodispersible tablet containing burlulipase and pharmaceutical composition produced therefrom |
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| CN104404027A (en) * | 2014-12-10 | 2015-03-11 | 南京工业大学 | Method for treating multi-walled carbon nanotube immobilized enzyme by plasma |
| CN104404027B (en) * | 2014-12-10 | 2018-01-05 | 南京工业大学 | Method for treating multi-walled carbon nanotube immobilized enzyme by plasma |
| WO2018158459A1 (en) * | 2017-03-03 | 2018-09-07 | Nordmark Arzneimittel Gmbh & Co. Kg | Orodispersible tablet containing burlulipase and pharmaceutical composition produced therefrom |
| US11464834B2 (en) | 2017-03-03 | 2022-10-11 | Nordmark Pharma Gmbh | Orodispersible tablet containing burlulipase and pharmaceutical composition produced therefrom |
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