CN103320420B - A kind of magnetic cross-linked lipase aggregate and preparation method thereof and application - Google Patents
A kind of magnetic cross-linked lipase aggregate and preparation method thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of magnetic cross-linked lipase aggregate and preparation method thereof and application, described magnetic cross-linked lipase aggregate adopts the functionalizing magnetic nanoparticle of surface band dendroid polyamines base to prepare.Preparation method of the present invention is integrated with cross-linked enzyme aggregate technology and magnetic immobilized zymotechnic, not only increase immobilization efficiency and the enzyme rate of recovery alive of magnetic immobilized enzyme, but also substantially improve the problem of cross-linked enzyme aggregate operational stability difference, the magnetic cross-linked lipase aggregate obtained has high reactivity and good operational stability, can be used as biological catalyst widespread use.
Description
Technical field
The invention belongs to cross-linked enzyme aggregate field, relate to a kind of magnetic cross-linked lipase aggregate adopting the functionalizing magnetic nanoparticle of surface band dendroid amido to prepare and preparation method thereof and application particularly.
Background technology
Biological enzyme is as catalyzer, because having the features such as high efficiency, specificity, reaction conditions gentleness, environmental friendliness, and be widely used in the industry (Reetz such as medicine, food, spices, grease, makeup and agricultural chemicals, M.T., 2002.Lipasesaspracticalbiocatalysts.Curr.Opin.Chem.Biol. 6,145 – 150).Wherein especially lipase because of its substrate spectrum acted on extensive, demonstrate more wide application prospect.But natural lipase operational stability is poor, easy in inactivation, not easily reuse, be difficult to implementation procedure serialization and automatization, is mixed in product after reaction, often causes enzyme and product separation difficulty.Although free-fat enzyme technology is very ripe, real commercial application still receives very large restriction.Lipase immobilization can be overcome the shortcoming that above-mentioned free-fat enzyme exists, therefore the catalysis technique industrialization of lipase depends on fatty enzyme immobilizatio to a great extent.The performance of immobilized lipase depends primarily on again immobilized material and immobilized method.Use immobilized enzyme prepared by carrier, because carrier occupies very large ratio, be generally 99.9% to 90% (Cao of immobilized enzyme by mass, L.Q., Langen, L.V., Sheldon, R.A., 2003.Immobilisedenzymes:carrier-boundorcarrier-free Curr.Opin.Chem.Biol.14, 387-394), this makes the vigor of enzyme by " dilution ", greatly reduce its catalytic efficiency, cause the low (Tischer of space-time yield, W., Kasche, V., 1999.Immobilizedenzymes:crystalsorcarriers TrendsBiotechnol.17, 326-335).Therefore, immobilized enzyme starts to turn to carrier-free immobilization technology, as crosslinked enzyme crystal (Quiocho, F.A., Richards, F.M.1964.Intermolecularcross-linkingofaproteininthecryst allinestate:CarboxypeptidaseA.Proc.Natl.Acad.Sci.USA.52, 833 – 839) and the crosslinked enzyme (Jansen dissolved, E.F., Olson, A.C., 1969.Propertiesandenzymaticactivitiesofpapaininsolubiliz edwithglutaraldehyde.Arch.Biochem.Biophys.129, 221-228).But prepare crosslinked enzyme crystal, purifying enzyme molecule and want crystallization, the condition of crystallization is high, which limits its widespread use.Crosslinked its cross linking conditions of enzyme (as ionic strength, temperature, pH value, type of crosslinking agent and consumption) dissolved of preparation controls difficulty, the often very low and operational stability also very poor (Zhang Si of obtained its vigor of crosslinking enzyme, Zhang Yanan, Cui Jiandong, progress prepared by 2010. cross-linked enzyme aggregates. China brewages (8), 8-11).The people such as Cao proposed a kind of novel process for fixation-cross-linked enzyme aggregate technology (Cao in 2000 on the basis of the above, L.Q., Rantwijk, F.V., Sheldon, R.A., 2000.Cross-linkedenzymeaggregates:Asimpleandeffectivemet hodfortheimmobilizationofpenicillinacylase.Org.Lett.2,1361-1364).
The preparation of cross-linked enzyme aggregate is divided into precipitation and crosslinked two steps, first utilize the precipitation agent of enzyme to be carried out precipitating the aggressiveness obtaining enzyme by the enzyme dissolved in the solution, utilize linking agent to be cross-linked by the aggregate of enzyme subsequently thus obtained water-insoluble cross-linked enzyme aggregate.Cross-linked enzyme aggregate is without the need to introducing carrier, and be the immobilized enzyme of a kind of enzyme as carrier itself, the charge capacity of its enzyme is high, and catalytic activity is high, and production cost is low, has potential application prospect.Its enzyme of cross-linked enzyme aggregate of the report such as Schoevaart lives the rate of recovery unexpectedly more than 100% (Schoevaart, R., Wolbers, M.W., Golubovic, M., Ottens, M., 2004.Preparation, optimization, andstructuresofcross-linkedenzymeaggregates.Biotechnol.B ioeng.87,754-762).Although also clearly do not explain this phenomenon so far, but the enzyme immobilization method of cross-linked enzyme aggregate technology not still a kind of novelty as can be seen here, and very likely become a kind of modifying method (Wang Mengfan improving enzyme catalysis performance, Qi Wei, Su Rongxin, He Zhimin, 2010. cross-linked enzyme aggregate Research progress. chemical progress 22,173-178).
But, also there are some technical bottlenecks in cross-linked enzyme aggregate: first, not every enzyme effectively can make cross-linked enzyme aggregate, prepared by cross-linked enzyme aggregate success, depend on the content height (Sheldon of Methionin contained by enzyme surface to a great extent, R.A., 2007.Enzymeimmobilization:thequestforoptimumperformance. Adv.Synth.Catal.349,1289 – 1307), secondly, the high catalytic activity of cross-linked enzyme aggregate requires that its grain diameter is little as much as possible, but the lower size limit of immobilized enzyme industrial application is generally 0.1 ~ 0.2mm, if the particle diameter that therefore will increase cross-linked enzyme aggregate certainly will cause most of enzyme to be sealed in aggressiveness inside to meet industrial application demand, be difficult to play katalysis, again, in enzyme aggregate forming process, enzyme molecule is close to each other, subsequently this " intensive " state be crosslinked again agent be cross-linked, and in the process used, adopt method that is centrifugal or that filter to be separated from reaction system, this causes cross-linked enzyme aggregate to reunite, therefore for macromolecule substrate, as: high molecular polymer, protein, polysaccharide etc., due to sterically hindered effect, substrate cannot enter cross-linked enzyme aggregate inside to carry out reacting (Dalal, S., Kapoor, M., Gupta, M.N., 2007.Preparationandcharacterizationofcombi-CLEAscatalyzi ngmultiplenon-cascadereactions.J.Mol.Catal.B:Enzym.44, 128 – 132Wang, M., Jia, C., Qi, W., Yu, Q., Peng, X., Su, R., He, Z., 2011.Porous-CLEAsofpapain:applicationtoenzymatichydrolys isofmacromolecules.Bioresour.Technol.102, 3541 – 3545).
Low for solving enzyme lysine content, be difficult to the problem of obtained stable and firm enzyme aggregate, researcher is by enzyme and the superpolymer (Honda with amido, T., Miyazaki, M., Nakamura, H., Maeda, H., 2006.Facilepreparationofanenzyme-immobilizedmicroreactor usingacross-linkingenzymemembraneonamicrochannelsurface. Adv.Synth.Catal.348, 2163 – 2171Yamaguchi, H., Miyazaki, M., Asanomi, Y., Maeda, H., 2011.Poly-lysinesupportedcross-linkedenzymeaggregateswit hefficientenzymaticactivityandhighoperationalstability.C atal.Sci.Technol.1, 1256 – 1261), bovine serum albumin (the Cruz that lysine content is high, J., Barbosa, O., Rodrigues, R.C., Fernandez-Lafuentec, R., Torres, R., Ortiz, C., 2012.OptimizedpreparationofCALB-CLEAsbyresponsesurfaceme thodology:Thenecessitytoemployafeedertohaveaneffectivecr oss-linking.J.Mol.Catal.B:Enzym.80, 7 – 14) and Ovum Gallus domesticus album (Karimpil, J.J., Melo, J.S., Dsouza, S.F., 2011.Heneggwhiteasafeederproteinforlipaseimmobilization. J.Mol.Catal.B:Enzym.71, 113 – 118) co-precipitation prepares cross-linked enzyme aggregate.But this centrifugal or filtering separation that can't solve owing to using in use procedure causes the agglomeration traits of enzyme aggregate.
Recently, for solving agglomeration traits, by magnetic particle for the preparation of cross-linked enzyme aggregate, obtained magnetic cross-linked enzyme aggregate avoids the agglomeration traits caused with centrifugal separation from reaction system with the mode of filtration.The people such as Li Bing have prepared a kind of magnetic immobilized cross-linked lipase aggregate (Li Bing, Dong Shouli, Li Lin with chitosan magnetic micro-sphere, clock Nanjing, Li Xiaoxi, Chen Ling, Su Jianyu, a kind of magnetic immobilized cross-linked lipase aggregate and preparation method thereof and application, CN102505008A).Kim etc. are by business-like Dynabeads
m-270 micron order magnetic bead is for the preparation of magnetic immobilized CRL lipase aggregate enzyme-catalyzed change degraded polycaprolactone (Kim, M., Park, J.M., Um, H.J., Lee, D.H., Lee, K.H., Kobayashi, F., Iwasaka, Y., Hong, C.S., Min, J., Ki, Y.H., 2010.Immobilizationofcross-linkedlipaseaggregatesontomag neticbeadsforenzymaticdegradationofpolycaprolactone.J.Ba sic.Microbiol.50,218-226).Nano particle due to the specific surface area had large, surface energy wraps up a large amount of amido (Hu, B., Pan, J., Yu, H.-L., Liu, J.-W., Xu, J.-H., 2009.ImmobilizationofSerratiamarcescenslipaseontoamino-f unctionalizedmagneticnanoparticlesforrepeateduseinenzyma ticsynthesisofDiltiazemintermediate.ProcessBiochem.44, 1019 – 1024), such particle not only can be overcome cross-linked enzyme aggregate agglomeration traits for the preparation of magnetic cross-linked enzyme aggregate and can also guarantee that enzyme can be full cross-linked, improve its stability, it can be repeatedly used.The magnetic nanoparticle such as Talekar prepares magnetic α-amylase cross-linked aggregates (Talekar, S., Ghodake, V., Ghotage, T., Rathod, P., Deshmukh, P., Nadar, S., 2012.Novelmagneticcross-linkedenzymeaggregates (magneticCLEAs) ofalphaamylase.Bioresour.Technol.123,542-547).But magnetic nanoparticle is seldom used to preparation magnetic cross-linked lipase aggregate seldom has report, especially adopt the functionalizing magnetic nanoparticle of surface band dendroid polyamines base to prepare the method for magnetic cross-linked lipase aggregate so far there are no bibliographical information.
Summary of the invention
First object of the present invention is the deficiency overcoming existing cross-linked enzyme aggregate, provides a kind of method adopting the functionalizing magnetic nanoparticle of surface band dendroid amido to prepare magnetic cross-linked lipase aggregate.
Second object of the present invention is to provide a kind of magnetic cross-linked lipase aggregate adopting the functionalizing magnetic nanoparticle of surface band dendroid amido to prepare.
3rd object of the present invention is to provide the application of described magnetic cross-linked lipase aggregate.
Object of the present invention is realized by following technical scheme:
In in first, the invention provides a kind of preparation method of magnetic cross-linked lipase aggregate, it is characterized in that, described method comprises and being mixed with lipase solution by the dispersion system of the functionalizing magnetic nanoparticle of surface band dendroid polyamines base.
The preparation method of magnetic cross-linked lipase aggregate of the present invention can comprise the dispersion system of the functionalizing magnetic nanoparticle preparing described surface band dendroid polyamines base, the precipitation agent mixing described lipase with described dispersion system, add successively in the mixture of described lipase and described dispersion system described lipase and linking agent and by precipitate and crosslinked after mixture solid-liquid separation obtain described magnetic cross-linked lipase aggregate.
The preparation method of magnetic cross-linked lipase aggregate of the present invention can comprise the following steps:
(1) the functionalizing magnetic nanoparticle taking appropriate surface band dendroid polyamines base is dissolved in phosphate buffered saline buffer, and ice-bath ultrasonic makes it be uniformly dispersed, and obtains the dispersion system of magnetic nanoparticle;
(2) lipase solution being dissolved in phosphate buffered saline buffer is poured in the described dispersion system of step (1), shaken at room temperature 30 ~ 50 minutes, preferably 35 ~ 45 minutes, more preferably 40 minutes;
(3) add the precipitation agent of described lipase in the solution obtained in step (2), low temperature stirs 20 ~ 50 minutes, preferably 30 ~ 40 minutes, more preferably 45 minutes;
(4) linking agent is added in the solution obtained in step (3), crosslinked, shaken at room temperature 6 ~ 24 hours, preferably 10 ~ 20 hours, more preferably 15 hours;
(5) by the solution solid-liquid separation that step (4) obtains, solid is got, after distilled water repetitive scrubbing, lyophilize.
In some embodiments, in described dispersion system, the concentration of nano particle can be 1 ~ 10mg/ml, preferably 3 ~ 7mg/ml, most preferably 5mg/ml.
In some embodiments, the frequency of described ice-bath ultrasonic can be 20 ~ 40KHz, is preferably 30KHz.
In some embodiments, the power of described ice-bath ultrasonic can be 240w.
In some embodiments, ultrasonic time can be 15 ~ 30 minutes, is preferably 20 ~ 25 minutes.
In some embodiments, the concentration of described phosphate buffered saline buffer can be 0.05 ~ 0.2mol/L, is preferably 0.1 ~ 0.15mol/L.
In some embodiments, the pH value of described phosphate buffered saline buffer can be 6.0 ~ 10.0, is preferably 7.0 ~ 9.0, most preferably is 8.0.
In some embodiments, the concentration of described lipase solution can be 0.5 ~ 20mg/ml, is preferably 1 ~ 15mg/ml, most preferably is 5 ~ 10mg/ml.
In some embodiments, described low temperature whipping temp can be 0 ~ 4 degree Celsius, is preferably 2 ~ 3 degrees Celsius;
In some embodiments, described solid-liquid separation can be Magneto separate;
In some embodiments, described lyophilize can be vacuum lyophilization and freeze-drying time can be 36 ~ 72 hours, is more preferably 45 ~ 63 hours.
In the preparation method of magnetic cross-linked lipase aggregate of the present invention, described functionalizing magnetic nanoparticle can be the Fe of homogeneous, the superparamagnetism adopting chemical precipitation method synthesis
3o
4for magnetic core, its surface be with primary amine base unit weight>=0.5mmol/g.
In the preparation method of magnetic cross-linked lipase aggregate of the present invention, described functionalizing magnetic nanoparticle particle diameter can be 100 ~ 400nm, is preferably 150 ~ 350nm, is more preferably 200 ~ 250nm.
In the preparation method of magnetic cross-linked lipase aggregate of the present invention, described functionalizing magnetic nanoparticle consumption can be 1/100 ~ 1 times of zymoprotein, is preferably 1/10 ~ 1/80 times, is more preferably 1/30 ~ 1/50 times.
In the preparation method of magnetic cross-linked lipase aggregate of the present invention, described precipitation agent is saturated solution or the organic solvent of ammonium sulfate;
In some embodiments, the consumption of described ammonium sulfate saturated solution can be 0.8 ~ 2.5 times of enzyme solution by volume, more preferably 1.5 ~ 2.0 times, in more specifically embodiment, the consumption of described ammonium sulfate saturated solution can be 0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9 or 2.0 times of enzyme solution by volume.
In some embodiments, described organic solvent can elect a kind of of ethanol, Virahol, acetone, acetonitrile or the trimethyl carbinol or two kinds as and its consumption can be 0.8 ~ 10 times of enzyme solution by volume, preferably 2.0 ~ 8.0 times, most preferably 4.0 ~ 6.0 times, in more specifically embodiment, described consumption of organic solvent can be 0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0 or 10.0 times of enzyme solution by volume.
In some embodiments, the feed postition of described organic solution can for slowly dropwise to add.
In the preparation method of magnetic cross-linked lipase aggregate of the present invention, described linking agent can be glutaraldehyde and its consumption can for final concentration between 20 to 400mM, preferably between 50 to 300mM, most preferably between 100 to 200mM.
In second aspect, the invention provides the magnetic cross-linked lipase aggregate adopting the preparation method of the magnetic cross-linked lipase aggregate according to first aspect to obtain.
In a third aspect, the invention provides the purposes adopting the magnetic cross-linked lipase aggregate according to second aspect as biological catalyst.
Embodiment
Technical scheme of the present invention is further illustrated below in conjunction with by embodiment.
Embodiment 1
(1) the functionalizing magnetic nanoparticle (preparation method: with the magnetic Nano polymkeric substance of surface amination (see GuobinShan of 15mg homemade surface band dendroid amido is taken, HuizhouLiu, JianminXing, GuandongZhang, ZhiyaMa, XianqiaoLiu, JunguoLiu.SurfaceModificationofNano-magneticFe3O4Particl esanditsApplications.China-EUForumonNanosizedTechnology, 2002, Beijing, China.P68-76.) be core, with quadrol and methyl acrylate for monomer, employing well known to a person skilled in the art disperses synthesis method synthesis dendrimer materials.) be placed in the triangular flask of the 100mM phosphate buffered saline buffer filling 15mlpH=8, frequency be 40KHz, power be the ultrasound condition of 240w under ice-bath ultrasonic 20min, be uniformly dispersed;
(2) phosphate buffered saline buffer that 250mg lipase (in protein content) is placed in 250ml100mM is taken, room temperature lower magnetic force stirs 30min, make lipase fully dissolve formation homogeneous phase solution, pour in dispersed magnetic granular system obtained in (1), shaken at room temperature 40min;
(3) in (2), slowly add the saturated ammoniumsulphate soln of 300ml, then stir 30min at 4 DEG C;
(4) in (3), add the glutaraldehyde of 6ml25%, shaken at room temperature 18 hours, after Magneto separate, gets solid, fully washs with distilled water, and vacuum lyophilization 48 hours, obtains magnetic cross-linked lipase aggregate.
Enzyme work is according to document (Uyanik, A., Sen, N., Yilmaz, M., 2011.ImprovementofcatalyticactivityoflipasefromCandidaru gosaviasol-gelencapsulationinthepresenceofcalix (aza) crown.Bioresour.Technol.102,4313-4318) described mensuration, the enzyme yield alive of magnetic cross-linked lipase aggregate is 92%.Under the effect of externally-applied magnetic field, solid-liquid separation is carried out after every secondary response terminates, by magnetic cross-linked lipase aggregate with after distilled water repetitive scrubbing 5 times, rejoin the reaction that reactant carries out next round again, replication like this 10 times, the remnant enzyme activity (enzyme used relative to first time is lived) of magnetic cross-linked lipase aggregate is 95%.
Embodiment 2
(1) the functionalizing magnetic nanoparticle taking 12mg surface band dendroid amido is placed in the triangular flask of the 100mM phosphoric acid salt buffer filling 12mlpH=8, frequency be 40KHz, power be the ultrasound condition of 240w under ice-bath ultrasonic 20min, be uniformly dispersed;
(2) phosphate buffered saline buffer that 150mg lipase (in protein content) is placed in 150ml100mM is taken, room temperature lower magnetic force stirs 30min, make enzyme fully dissolve formation homogeneous phase solution, pour in dispersed magnetic granular system obtained in (1), shaken at room temperature 40min;
(3) in (2), dropwise add 150ml ethanol, then stir 30min at 4 DEG C;
(4) in (3), add the glutaraldehyde of 5ml25%, shaken at room temperature 8 hours, after Magneto separate, gets solid, fully washs with distilled water, and vacuum lyophilization 36 hours, obtains magnetic cross-linked lipase aggregate.
Enzyme work is according to document (Uyanik, A., Sen, N., Yilmaz, M., 2011.ImprovementofcatalyticactivityoflipasefromCandidaru gosaviasol-gelencapsulationinthepresenceofcalix (aza) crown.Bioresour.Technol.102,4313-4318) described mensuration, the enzyme yield alive of magnetic cross-linked lipase aggregate is 78%.Under the effect of externally-applied magnetic field, solid-liquid separation is carried out after every secondary response terminates, by magnetic cross-linked lipase aggregate with after distilled water repetitive scrubbing 5 times, rejoin the reaction that reactant carries out next round again, replication like this 10 times, the remnant enzyme activity (enzyme used relative to first time is lived) of magnetic cross-linked lipase aggregate is 85%.
Embodiment 3
(1) the functionalizing magnetic nanoparticle taking 10mg surface band dendroid amido is placed in the triangular flask of the 100mM phosphoric acid salt buffer filling 10mlpH=8, frequency be 40KHz, power be the ultrasound condition of 240w under ice-bath ultrasonic 30min, be uniformly dispersed;
(2) phosphate buffered saline buffer that 120mg lipase (in protein content) is placed in 120ml100mM is taken, room temperature lower magnetic force stirs 30min, make enzyme fully dissolve formation homogeneous phase solution, pour in dispersed magnetic granular system obtained in (1), shaken at room temperature 40min;
(3) in (2), dropwise add 150ml acetonitrile, then stir 30min at 4 DEG C;
(4) in (3), add the glutaraldehyde of 4ml25%, shaken at room temperature 8 hours, after Magneto separate, gets solid, fully washs with distilled water, and vacuum lyophilization 36 hours, obtains magnetic cross-linked lipase aggregate.
Enzyme work is according to document (Uyanik, A., Sen, N., Yilmaz, M., 2011.ImprovementofcatalyticactivityoflipasefromCandidaru gosaviasol-gelencapsulationinthepresenceofcalix (aza) crown.Bioresour.Technol.102,4313-4318) described mensuration, the enzyme yield alive of magnetic cross-linked lipase aggregate is 64%.Under the effect of externally-applied magnetic field, solid-liquid separation is carried out after every secondary response terminates, by magnetic cross-linked lipase aggregate with after distilled water repetitive scrubbing 5 times, rejoin the reaction that reactant carries out next round again, replication like this 10 times, the remnant enzyme activity (enzyme used relative to first time is lived) of magnetic cross-linked lipase aggregate is 90%.
Embodiment 4
(1) the functionalizing magnetic nanoparticle taking 20mg surface band dendroid amido is placed in the triangular flask of the 100mM phosphoric acid salt buffer filling 20mlpH=8, frequency be 40KHz, power be the ultrasound condition of 240w under ice-bath ultrasonic 30min, be uniformly dispersed;
(2) phosphate buffered saline buffer that 200mg lipase (in protein content) is placed in 200ml100mM is taken, room temperature lower magnetic force stirs 30min, make enzyme fully dissolve formation homogeneous phase solution, pour in dispersed magnetic granular system obtained in (1), shaken at room temperature 50min;
(3) in (2), dropwise add 180ml acetone, then stir 30min at 4 DEG C;
(4) in (3), add the glutaraldehyde of 7ml25%, shaken at room temperature 6 hours, after Magneto separate, gets solid, fully washs with distilled water, and vacuum lyophilization 36 hours, obtains magnetic cross-linked lipase aggregate.
Enzyme work is according to document (Uyanik, A., Sen, N., Yilmaz, M., 2011.ImprovementofcatalyticactivityoflipasefromCandidaru gosaviasol-gelencapsulationinthepresenceofcalix (aza) crown.Bioresour.Technol.102,4313-4318) described mensuration, the enzyme yield alive of magnetic cross-linked lipase aggregate is 85%.Under the effect of externally-applied magnetic field, solid-liquid separation is carried out after every secondary response terminates, by magnetic cross-linked lipase aggregate with after distilled water repetitive scrubbing 5 times, rejoin the reaction that reactant carries out next round again, replication like this 10 times, the remnant enzyme activity (enzyme used relative to first time is lived) of magnetic cross-linked lipase aggregate is 89%.
Embodiment 5
(1) the functionalizing magnetic nanoparticle taking 10mg surface band dendroid amido is placed in the triangular flask of the 100mM phosphoric acid salt buffer filling 10mlpH=8, frequency be 40KHz, power be the ultrasound condition of 240w under ice-bath ultrasonic 40min, be uniformly dispersed;
(2) phosphate buffered saline buffer that 100mg lipase (in protein content) is placed in 100ml100mM is taken, room temperature lower magnetic force stirs 30min, make enzyme fully dissolve formation homogeneous phase solution, pour in dispersed magnetic granular system obtained in (1), shaken at room temperature 50min;
(3) in (2), dropwise add the mixed solution (volume ratio is 1:1) of 90ml Virahol and acetone, then stir 30min at 4 DEG C;
(4) in (3), add the glutaraldehyde of 3ml25%, shaken at room temperature 10 hours, after Magneto separate, gets solid, fully washs with distilled water, and vacuum lyophilization 36 hours, obtains magnetic cross-linked lipase aggregate.
Enzyme work is according to document (Uyanik, A., Sen, N., Yilmaz, M., 2011.ImprovementofcatalyticactivityoflipasefromCandidaru gosaviasol-gelencapsulationinthepresenceofcalix (aza) crown.Bioresour.Technol.102,4313-4318) described mensuration, the enzyme yield alive of magnetic cross-linked lipase aggregate is 90%.Under the effect of externally-applied magnetic field, solid-liquid separation is carried out after every secondary response terminates, by magnetic cross-linked lipase aggregate with after distilled water repetitive scrubbing 5 times, rejoin the reaction that reactant carries out next round again, replication like this 10 times, the remnant enzyme activity (enzyme used relative to first time is lived) of magnetic cross-linked lipase aggregate is 90%.
The above embodiments are better embodiment of the present invention; but embodiments of the present invention are not limited to these; other is any deviate from connotation of the present invention and principle under do simplification, combination, substitute, the change of modification etc.; all be considered as the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (11)
1. a preparation method for magnetic cross-linked lipase aggregate, is characterized in that,
Said method comprising the steps of:
(1) the functionalizing magnetic nanoparticle taking appropriate surface band dendroid polyamines base is dissolved in phosphate buffered saline buffer, ice-bath ultrasonic makes it be uniformly dispersed, obtain the dispersion system of magnetic nanoparticle, in described dispersion system, the concentration of nano particle is 1 ~ 10mg/ml, the concentration of wherein said phosphate buffered saline buffer is 0.05 ~ 0.2mol/L, and pH value is 6.0 ~ 10.0;
(2) lipase solution being dissolved in phosphate buffered saline buffer poured in the described dispersion system of step (1), shaken at room temperature 30 ~ 50 minutes, the concentration of wherein said lipase solution is 0.5 ~ 20mg/ml;
(3) add the precipitation agent of described lipase in the solution obtained in step (2), low temperature stirs 20 ~ 50 minutes;
(4) linking agent is added in the solution obtained in step (3), crosslinked, shaken at room temperature 6 ~ 24 hours;
(5) by the solution solid-liquid separation that step (4) obtains, solid is got, after distilled water repetitive scrubbing, lyophilize.
2. preparation method according to claim 1, is characterized in that, the time of shaken at room temperature described in step (2) is 35 ~ 45 minutes.
3. preparation method according to claim 1, is characterized in that, the time that low temperature described in step (3) stirs is 30 ~ 40 minutes.
4. preparation method according to claim 1, is characterized in that, the time of step (4) described shaken at room temperature is 10 ~ 20 hours.
5. preparation method according to claim 1, is characterized in that,
The frequency of step (1) described ice-bath ultrasonic is 20 ~ 40KHz; Power is 240w and ultrasonic time is 15 ~ 30 minutes;
The temperature that the described low temperature of step (3) stirs is 0 ~ 4 degree Celsius;
Step (5) described solid-liquid separation is Magneto separate;
Step (5) described lyophilize is vacuum lyophilization and freeze-drying time is 36 ~ 72 hours.
6. preparation method according to claim 1, is characterized in that, described functionalizing magnetic nanoparticle adopts the Fe of homogeneous, the superparamagnetism of chemical precipitation method synthesis
3o
4for magnetic core, its surface be with primary amine base unit weight>=0.5mmol/g.
7. preparation method according to claim 1, is characterized in that, the particle diameter of described functionalizing magnetic nanoparticle is 100 ~ 400nm.
8. preparation method according to claim 1, is characterized in that, step (3) described precipitation agent is saturated solution or the organic solvent of ammonium sulfate;
Wherein,
The consumption of described ammonium sulfate saturated solution is 0.8 ~ 2.5 times of lipase solution by volume; Or
Described organic solvent is a kind of of ethanol, Virahol, acetone, acetonitrile or the trimethyl carbinol or two kinds and its consumption is 0.8 ~ 10 times of lipase solution by volume, and the feed postition of described organic solution is for slowly dropwise to add.
9. preparation method according to claim 1, is characterized in that, step (4) described linking agent is glutaraldehyde and its consumption is that final concentration is between 20 to 400mM.
10. adopt the magnetic cross-linked lipase aggregate that the preparation method of the magnetic cross-linked lipase aggregate according to any one of claim 1 ~ 9 obtains.
11. magnetic cross-linked lipase aggregates according to claim 10 are as the purposes of biological catalyst.
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| CN103937775A (en) * | 2014-03-07 | 2014-07-23 | 中国科学院过程工程研究所 | Magnetic cross-linked enzyme aggregate, preparation method and application thereof |
| CN104630322B (en) * | 2015-02-10 | 2018-04-13 | 江苏理工学院 | The method that immobilized enzyme method fractionation prepares optical voidness 1 (1 naphthyl) ethamine |
| CN107698773B (en) * | 2017-09-15 | 2021-04-13 | 江苏大学 | Magnetic dendritic polymer composite nanoparticle and preparation method and application thereof |
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| CN102392013A (en) * | 2011-11-09 | 2012-03-28 | 华南理工大学 | Magnetic immobilized cross-linking cellulase aggregates (CLEAs), preparation method and application thereof |
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2013
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392013A (en) * | 2011-11-09 | 2012-03-28 | 华南理工大学 | Magnetic immobilized cross-linking cellulase aggregates (CLEAs), preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| "Fabrication and Functionalization of Dendritic Poly(amidoamine)-Immobilized Magnetic Polymer Composite Microspheres";Hongbo Liu et al;《J. Phys. Chem. B》;20080219;第112卷;摘要，图1-2 * |
| "Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate";Bin Hu,et al;《Process Biochemistry》;20091231;第44卷;摘要、第1020页右栏第2段-1023页右栏第一段 * |
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