CN101914520B - Preparation of nano gel immobilized multienzyme system and application thereof in synthesizing 1,3-propylene glycol - Google Patents
Preparation of nano gel immobilized multienzyme system and application thereof in synthesizing 1,3-propylene glycol Download PDFInfo
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- CN101914520B CN101914520B CN2010102223716A CN201010222371A CN101914520B CN 101914520 B CN101914520 B CN 101914520B CN 2010102223716 A CN2010102223716 A CN 2010102223716A CN 201010222371 A CN201010222371 A CN 201010222371A CN 101914520 B CN101914520 B CN 101914520B
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Abstract
The invention relates to preparation of a nano gel immobilized multienzyme system. The preparation method comprises the following steps: constructing a nano gel-multienzyme assembling system by using a nano gel dispersion-adsorption immobilized enzyme technology, carrying out self-coupling, and catalyzing by multiple enzymes to synthesize the 1,3-propylene glycol. The nano gel suspension, which can be evenly dispersed in the water solution, is prepared to directly absorb the immobilized multienzyme system. The method can effectively inhibit the aggregation and enhance the dispersibility; the invention fully displays the excellent properties of nano gel carrier, such as high specific surface and the like, provides a location for simultaneously coupling multiple biological enzymes (multienzyme immobilization), and reduces the influence of the carrier on the dispersion of a substrate and products; and compared with the microbe fermentation method, the invention has the advantages of low cost, simple reaction, no cell pollution and the like, and the nano gel immobilized multienzyme system can not be easily degraded by the microbes. The invention provides a new effective catalytic method for synthesizing nano gel immobilized enzymes.
Description
Technical field
The present invention is a kind of preparation of nano gel immobilized multienzyme system and 1; (1, the application in 3-PD) synthetic is through being prepared in homodisperse nanogel suspension-s in the aqueous solution for ammediol; Use dispersion-adsorption of immobilization technology; Make up nanogel-multienzyme assembly system, multienzyme coupling catalysis synthesizes 1,3-PD.This method can effectively suppress to reunite; Improve dispersiveness, form evenly and the stabilized nano gel suspension, excellent properties such as performance nano-carrier high-ratio surface; Both, reduced carrier extending influence again to substrate and product for the multiple enzyme of coupling simultaneously is that multienzyme fixedly provides the place.Use nano gel immobilized multienzyme system catalysis synthetic 1,3-PD, it is low to have a cost than microbe fermentation method etc., and reaction is simple, acellular pollution, advantage such as be difficult for being degraded by microorganisms.Belong to 1, the nano gel immobilized enzyme catalysis synthesis technical field of 3-PD.
Background technology
Biopolyol 1; Ammediol (is called for short 1; 3-PD) be a kind of important hardware and software platform compound; With 1,3-PD is that the novel shape memory fiber material of monomer synthetic
is the focus of present synthon new varieties.
Microbial fermentation glycerine produces 1, and 3-PD mainly utilizes oxidation and the coupling of reduction approach to carry out catalyzed conversion.Intracellular oxidative pathway and reduction approach are through NADH
2/ NAD
+And energy ATP/ADP is coupled at together.In oxidative pathway, (GDH, EC 1.1.1.6) is oxidized to otan (DHA) to glycerine through glycerol dehydrogenase, generates reducing equivalent NADH simultaneously
2In the reduction approach, glycerine is with vitamins B
12Change into 3-hydroxy propanal (3-HPA) under the effect for the glycerol dehydratase (GDHt, EC 4.2.1.30) of coenzyme, and 3-HPA is reduced into 1 under the effect of oxydo-reductase (PDOR, EC 1.1.1.202), 3-PD, NADH
2As 1, the electron donor of 3-PD oxydo-reductase, reduction 3-HPA synthesizes 1,3-PD.Catalytic process is accompanied by self link-coupled regeneration of nicotinamide adenine dinucleotide.
With key enzyme immobilizations such as GDH, GDHt, PDOR, coupling Production by Enzymes 1,3-PD utilizes two parallel redox reaction enzyme systems exactly, and the reductase enzyme catalyzing glycerol changes into 1, and 3-PD, oxydase be catalysis coenzyme NAD H then
2/ NAD
+Cyclic regeneration.
Use immobilized multienzyme system catalysis synthetic 1,3-PD, it is simple to have a reaction than microbe fermentation method etc., and acellular pollution helps advantages such as operate continuously and later separation.And use the immobilized multienzyme catalystsystem, can make large fermentation tank be substituted by small-sized immobilized enzyme reactor, make integrated mill's miniaturized, therefore become biological process preparation 1, the research focus of 3-PD.
In enzyme immobilization technology, except process for fixation, seek novel carrier, have great importance with the various performances that improve immobilized enzyme.Nano carrier material is compared with corresponding block materials, has high-specific surface area, high reactivity, and strong adsorptive power, excellent specific properties such as high catalytic efficiency (can effectively improve the charge capacity and stability (the Wang et al.2006 of enzyme molecule; Liu et al.2009).Particularly the inorganic oxide nano carrier material has good thermal stability, chemicalstability and biocompatibility, is widely used in many fields such as miniature organism device, biological device array and electricity, enzyme catalysis, photochemical catalysis, delivery of drug.
Inorganic oxide nano-powder (being mostly coacervate) is a kind of nontoxic, pollution-free, ceramic that cost is low.Like nano-TiO
2Being difficult for being degraded by microorganisms during as enzyme immobilization carrier, acid and alkali-resistance, much more more fixedly sites can being provided, is a kind of the enzyme molecule to be had the highly non aqueous carrier of adsorptive power.Nano carrier material has unique surface and surface reaction performance, and the particle diameter of nano carrier material is more little, and its specific surface area is big more, and adsorptive power is strong more, and enzyme molecule charge capacity is big, and the catalytic activity of immobilized enzyme is just high more.
But the nano carrier material particle diameter is little, specific surface area is big, in water medium, has higher surface energy, reunites easily.Can form when in water, disperseing and be difficult to the dispersive coacervate, produce deposition and caking, this reduces the adsorptive power of nano carrier material to enzyme greatly.How effectively to suppress to reunite, improve dispersiveness, form evenly and the stabilized nano gel suspension, excellent properties such as performance nano-carrier high-ratio surface; Efficient adsorptive enzyme molecule reduces carrier to the extending influence of substrate and product, and just need carry out surface-treated to coacervate; Method commonly used is to add interface modifier (Cui Aili, 2001), i.e. dispersion agent; Suppress to reunite, obtain homodisperse nanogel suspension-s in the aqueous solution, give full play to excellent properties such as nano-carrier high-ratio surface.The huge specific surface area that homodisperse forms for the multiple enzyme of while coupling is that multienzyme fixedly provides the place, more helps the carrying out of multienzyme Kettenreaktion.
The present invention intends and is prepared in homodisperse nanogel suspension-s in the aqueous solution; Utilization dispersion-adsorption of immobilization technology makes up nanogel-multienzyme assembly system, develops to multi-enzyme system from single enzyme system; Utilize parallel redox reaction enzyme system, self cyclic regeneration NADH
2/ NAD
+, multienzyme coupling production 1,3-PD.
Compare with collosol and gel (sol-gel) nano immobilized enzyme, dispersion-absorption method has been eliminated the strong interaction between carrier and the enzyme, can make enzymic activity be able to keep (Kim et al.2006).The general catalyzer that adopts acid, alkali or salt as the sol-gel process in the sol-gel nano immobilized enzyme process, enzyme at higher, low pH or than the condition of high ionic strength under inactivation very easily; In the sol-gel process, a large amount of OH, COOH and the gel network formation initial stage that the enzyme molecular surface exists contains M-O (H)-M and the M-OH polymer segments exists intensive to interact (Xu Songwei, 2004), causes the change of enzyme texture image, causes that enzymic activity reduces.The ethanol that produces of the sol-gel process presoma hydrolysis microenvironment, the gel that destroy suitable enzyme existence is limited in some with enzyme and is not suitable in the microenvironment that enzymic activity keeps in addition, and the conformational change of enzyme and molecular motion are restricted with in solution, comparing.
Utilize the enzyme molecule in the nano-carrier dispersion-adsorbent solution, make up nanogel-multienzyme assembly system, the carrier particle diameter is little, and specific surface is big, and it is active to keep the enzyme Journal of Molecular Catalysis effectively, is more suitable in the multienzyme linked reaction than water-soluble enzyme.The enzyme charge capacity is big, and the reaction diffusional limitation is little, and cost is low, and acellular pollution is difficult for being degraded by microorganisms, and is a kind of effective nano gel immobilized enzyme catalysis synthetic novel method.
[1]Wang?P，Nanoscale?biocatalyst?systems.Current?Opinion?in?Biotechnology，2006，17(6)：574-579.
[2]Liu?W，Zhang?S，Wang?P，Nanoparticle-supported?multi-enzyme?biocatalysis?with?insitu?cofactor?regeneration.Journal?of?Biotechnology，2009，139(1)：102-107.
[3] Cui Aili, Wang Tingjie, He Hong, gold gushes, the dispersion of superfine titanic oxide powder in the aqueous solution, process engineering journal, 2001,1 (1): 99-101.
[4]Kim?MI，Ham?HO，Oh?SD，et?al.Immobilization?of?Mucor?javanicus?lipase?oneffectively?functionalized?silica?nanoparticles.J?Mol?Catal?B-Enzymatic，2006，39(1/4)：62-68.
[5] Xu Songwei, Jiang Zhongyi, Wu Hong, Huang Shufang, the preparation and the characteristic of the biological capsulation thing of sol-gel.Chemical progress, 2004,16 (3): 443-449.
Summary of the invention
Technical problem: the present invention is a kind of preparation of nano gel immobilized multienzyme system and 1, and the carrier of inorganic oxide nano-powder as immobilized enzyme adopted in the application during ammediol is synthetic; Utilize dispersion agent to suppress to reunite between nano particle, obtain dispersive nanogel suspension-s in the water, through dispersion-adsorption of immobilization method; Preparation nanogel-multienzyme assembly system; Self coupling, multienzyme catalytic production 1, ammediol.
Use nano gel immobilized multienzyme system catalysis to synthesize 1, ammediol is given full play to excellent properties such as nano-carrier high-ratio surface, both for the multiple enzyme of coupling simultaneously is that multienzyme fixedly provides the place, reduces carrier extending influence to substrate and product again.It is low to have a cost than microbe fermentation method etc., and reaction is simple, acellular pollution, and advantage such as be difficult for being degraded by microorganisms.
Technical scheme: the purpose of this invention is to provide homodisperse nanogel suspension-s in the aqueous solution; Utilization contains the hydroxyl alcohol groups of hydrophilic group; Disperse the inorganic oxide nano-powder; Prevent the nanoparticle reunion, give full play to excellent properties such as nano-carrier high-ratio surface, active adsorption is the method for conjugate enzyme system fixedly.Monobasic or polyvalent alcohol dispersion agent have the certain protection effect to enzyme molecule disulfide linkage and three-dimensional structure, and alcohol dispersion-adsorption of immobilization zymotechnic can fetter the conformation of enzyme and keep microenvironment stable effectively, is one of active important method of stabilized enzyme.Its characteristic may further comprise the steps: 1) cell-free extract preparation; 2) dispersion of nano-carrier; 3) nanogel absorption load immobilized multienzyme system.
The practical implementation process is:
1) cell-free extract prepares: after klebsiella spp is carried out multiplication culture, and centrifugal collection thalline; Washing is also weighed; The saline water that adds 2～10 times of quality, behind the mixing, ultrasonication under the ice bath environment; Centrifugal back obtains crude enzyme liquid; Again through the ammonium sulfate precipitation of saturation ratio 30%～70%, dialysis desalting, using the Tris-HCl buffer preparation of pH=6.5～10.0 to become concentration is the multi-enzyme system solution of 5.0～100g/L;
2) dispersion of nano-carrier: dispersion agent alcohol with the dissolving of Tris-HCl buffered soln, is pressed
Quality proportioning and TiO
2Nano-carrier mixes, and ultra-sonic dispersion 30～60min obtains dispersive nano-TiO in the water
2Gel suspension;
3) nanogel absorption load immobilized multienzyme system: with multi-enzyme system solution and nano-TiO
2The gel suspension mixing, volume ratio 1: 1～10, vibration absorption 0.5～6h in the shaking table in the time of 37-50 ℃; Centrifugal mixed system keeps supernatant and measures enzyme content, and lower sediment is washed with buffered soln not to be had Protein Detection to supernatant and go out; Obtain nano gel immobilized multienzyme system, cryopreservation;
Described dispersion agent alcohol is monobasic or polyvalent alcohol dispersion agent, is in polyoxyethylene glycol, terepthaloyl moietie, glycerine, Hydrocerol A, WR 34678 or the beta-mercaptoethanol one or more.
The prepared nano gel immobilized multienzyme system that obtains comprises: glycerol dehydrogenase, glycerol dehydratase and 1, ammediol oxydo-reductase.
Described nano gel immobilized multienzyme system is 1, and the application during ammediol is synthetic is characterized in that: multienzyme coupling catalysis 1, the ammediol compound method is: reaction solution consist of glycerine 100～650mmol/L, NAD
+0.5～5mmol/L, VB
125～30 μ mol/L, (NH
4)
2 SO
420～50mmol/L, (NH
4)
2Fe (SO
4)
20.5～2 μ mol/L, Mg
2+0.5～2mmol/L, Hydrocerol A 0.2～2mmol/L, pH=6.5～10.0 buffered soln; Add nano gel immobilized multienzyme system 10～100g/L, the shaking table vibration was reacted 2～12 hours under 30～45 ℃ of conditions, and the centrifuging and taking supernatant measures 1, ammediol content.
Beneficial effect: the invention provides a kind of effective nano gel immobilized enzyme catalysis synthetic novel method.Use nanogel dispersion-adsorption of immobilization technology, make up nanogel-multienzyme assembly system, multienzyme coupling catalysis synthesizes 1, ammediol, and dispersive nanogel carrier has big specific surface area, can effectively improve the charge capacity of enzyme; The carrier particle diameter is little, has little diffusional limitation; Preparation technology is easy, mild condition, and it is active to keep the enzyme Journal of Molecular Catalysis effectively, is suitable for the multienzyme linked reaction.Compare with microbe fermentation method, it is simple to have a reaction, and acellular pollution is difficult for being degraded by microorganisms, low cost and other advantages.
Description of drawings
Fig. 1. the thermostability of resolvase and nano gel immobilized multienzyme system is relatively.
Embodiment
1) cell-free extract prepares: after klebsiella spp is carried out multiplication culture, and centrifugal collection thalline; Washing is also weighed; The saline water that adds 2～10 times of quality, behind the mixing, ultrasonication under the ice bath environment; Centrifugal back obtains crude enzyme liquid; Again through the ammonium sulfate precipitation of saturation ratio 30%～70%, dialysis desalting, using the Tris-HCl buffer preparation of pH=6.5～10.0 to become concentration is the multi-enzyme system solution of 5.0～100g/L;
2) dispersion of nano-carrier: dispersion agent alcohol with the dissolving of Tris-HCl buffered soln, is pressed
Quality proportioning and TiO
2Nano-carrier mixes, and ultra-sonic dispersion 30～60min obtains dispersive nano-TiO in the water
2Gel suspension;
3) nanogel absorption load immobilized multienzyme system: with multi-enzyme system solution and nano-TiO
2The gel suspension mixing, volume ratio 1: 1～10, vibration absorption 0.5～6h in the shaking table in the time of 37-50 ℃; Centrifugal mixed system keeps supernatant and measures enzyme content, and lower sediment is washed with buffered soln not to be had Protein Detection to supernatant and go out; Obtain nano gel immobilized multienzyme system, cryopreservation;
Embodiment one different alcohols dispersion agent is to TiO
2The load factor influence
Bacterial classification: Cray uncle pneumobacillus (K.pneumoniae)
Get the centrifugal collection thalline of 1000mL fermented liquid; Washing is also weighed, and adds 10 times of quality 0.9% saline water, behind the mixing; Ultrasonication under the ice bath environment, centrifugal back obtain crude enzyme liquid; With the ammonium sulfate precipitation of saturation ratio 50%, behind the dialysis desalting, use the multi-enzyme system solution of Tris-HCl buffer preparation concentration as 20.0g/L.
Take by weighing 50mg TiO respectively
2(particle diameter 16nm, specific surface 280m
2/ g) nano-carrier is in the 1-7 test tube, and No. 1 test tube does not have dispersion agent, makes blank, ultra-sonic dispersion 30min with 5mLTris-HCl buffered soln.Add 5mL different alcohols dispersant solution in the 2-7 test tube, ultra-sonic dispersion 30min.Add 5mL multi-enzyme system solution respectively toward the 1-7 test tube again; The about 1h of vibration absorption in the shaking table in the time of 37 ℃; Taking-up is centrifugal 10min under 8000rpm; Lower sediment with the Tris-HCl buffered soln of pH=7.0 wash do not have albumen to supernatant after, merge the analysis that washings and supernatant are used for enzyme content and load factor.
The zymoprotein assay adopts the Bradford method to measure, and is standard protein with the bovine serum albumin.According to the protein content C in the solution before and after the absorption
0, C
t, calculate nano-TiO
2Particle is to the load factor ε of zymoprotein:
C in the formula
0Be initial enzyme concn (gL
-1), C
tBe absorption t moment residual enzyme concentration (gL
-1), ε is a load factor.
Table 1 different alcohols dispersion agent is to TiO
2The load factor influence
Embodiment one has studied the different alcohols dispersion agent to nano-carrier TiO
2The influence of load factor.Table 1 is the result show: add pure dispersion agent and effectively improve nano-TiO
2Dispersiveness can be given full play to excellent properties such as nano-carrier high-ratio surface, has effectively improved TiO
2Load factor to zymoprotein.
Wherein, the dispersal mechanism of polyoxyethylene glycol dispersion agent belongs to sterically hindered stabiliser.Polyoxyethylene glycol is as non-ionics, and is main through forming adsorption layer at the nanogel particle surface, produces and the reinforcement steric effect, makes to produce strong steric hindrance repulsive force between micelle, suppresses nanoparticle and reunites.The different molecular weight polyoxyethylene glycol is to TiO
2The load factor influence is different.
The glycerine specific inductivity is high, can dissolve mineral compound, to TiO
2Certain improvement effect is also played in the dispersion of nano-carrier.Glycerine is to TiO in the solution
2The dissemination of gel particle is the result of solvation.The hydroxyl and the TiO of alcohol molecule
2The hydroxyl oxygen on surface forms the ol bridge structure through hydrogen bond, thereby at TiO
2The surface forms solvated layer, suppresses nano-TiO
2Reunite between particle.The dispersive nano-carrier has big specific surface area, effectively improves the load factor of enzyme.
In addition, glycerine is again zymolyte, but the inducible enzyme sterie configuration, and the conformation that fetters enzyme effectively is with to keep microenvironment stable, to activity of the immobilized enzyme is stable certain booster action arranged.
Embodiment two different pH load process are to the influence of the carrier loaded glycerol dehydrogenase load factor of nanogel
The multi-enzyme system formulations prepared from solutions is with embodiment one.
With glycerine is dispersion agent, takes by weighing equivalent TiO
2(particle diameter 16nm, specific surface 280m
2/ g) 12 parts of powders; Add different pH Tris-HCl buffered soln (pH=5.0～10.0) respectively; Ultra-sonic dispersion 30min adds a certain amount of enzyme liquid vibration absorption 1h in the shaking table in the time of 37 ℃, takes out centrifugal 10min under 8000rpm; Lower sediment with Tris-HCl buffered soln wash do not have albumen to supernatant after, merge the load factor that washings and supernatant are used to analyze enzyme under the condition of different pH.Two parallel appearance are averaged experimental result such as table 2.
The different pH load process of table 2 are to the influence of the carrier loaded glycerol dehydrogenase load factor of nanogel
Embodiment two is a dispersion agent with glycerine, has contrasted condition of different pH to TiO
2The influence of gel load enzyme molecule load factor.The result shows that the pH condition is bigger to the load factor influence of immobilized enzyme, and the pH8-9 load factor is higher.Possibly be TiO
2All have functional groups such as hydroxyl, amino with the enzyme molecular surface, both sexes can take place dissociate under condition of different pH, so TiO
2Gel particle load enzyme divides the period of the day from 11 p.m. to 1 a.m, and condition of different pH can influence the electrostatic interaction between charged corpuscle, influences carrier absorption property etc.
The thermostability of embodiment three nano gel immobilized multienzyme systems
Bacterial classification: Cray uncle pneumobacillus (K.pneumoniae)
Glycerol dehydrogenase GDH enzyme activity determination: 5mL GDH reaction solution (contains 30mmol/L (NH
4)
2SO
4, 0.2mol/L glycerine, 2mmol/L NAD
+, 1 μ mol/L (NH
4)
2Fe (SO
4)
2, with the salt of wormwood buffer preparation of pH=11) under 37 ℃ of conditions, add experiment and start reaction with enzyme, isothermal reaction is 40 minutes in shaking table, transfers in the centrifuge tube in 8000r/min centrifugal 10 minutes, and supernatant is measured absorbance A down in 340nm.An enzyme activity unit (U) is in the needed enzyme amount of this condition next minute internal consumption 1mol substrate.
The multi-enzyme system formulations prepared from solutions is with embodiment one.
Take by weighing a certain amount of TiO
2Powder is a dispersion agent with glycerine, with Tris-HCl buffered soln ultra-sonic dispersion 30min, gets nanogel suspension-s, the about 1h of vibration absorption in the shaking table when wherein adding a certain amount of enzyme liquid in 37 ℃, and centrifuge washing gets nano gel immobilized multienzyme system.Get each 6 groups of resolvase liquid and the nano gel immobilized multienzymes of equivalent, be placed on 4,20,37,50,60 and 70 ℃ of insulation 2h down respectively, survey its glycerol dehydrogenase GDH enzyme activity, result such as Fig. 1.
Embodiment three has compared resolvase liquid and nano gel immobilized multienzyme relative enzyme situation alive under condition of different temperatures.The result shows that zymoprotein has improved thermostability after fixing.Resolvase is at 70 ℃ of insulation 2h; Enzyme activity only remains 11.7%; Zymoprotein this moment sex change inactivation basically all, and nano gel immobilized multienzyme has kept 77.5% enzyme activity at 70 ℃ of insulation 2h, and nano gel immobilized multienzyme specific ionization enzyme has had wideer suitable reaction condition scope.This maybe be because dispersion agent alcoholic extract hydroxyl group and enzyme molecule have formed hydrogen bond; Improve the thermal denaturation temperature of enzyme, or pure molecule is wrapped in around the enzyme molecule, stablized the space structure of enzyme; Three-dimensional structure to enzyme molecule disulfide linkage has the certain protection effect, thereby has improved the thermostability of immobilized enzyme.
Embodiment four nanogels-multienzyme assembly system coupling catalysis 1, ammediol is synthetic
The multi-enzyme system formulations prepared from solutions is with embodiment one.
With glycerine is dispersion agent, takes by weighing an amount of TiO
2(particle diameter 16nm, specific surface 280m
2/ g), adding Tris-HCl buffered soln (pH=5.0～10.0), ultra-sonic dispersion 30min adds a certain amount of enzyme liquid vibration absorption 1h in the shaking table in the time of 37 ℃, and centrifuge washing gets nano gel immobilized multienzyme system.
Get the nano gel immobilized multienzyme system 1.5g for preparing and mix with the 50mL reaction solution, reaction solution consist of glycerine 0.50mol/L, NAD
+2mmol/L, VB
1215 μ mol/L, (NH
4)
2SO
430mmol/L, (NH
4)
2Fe (SO
4)
21 μ mol/L, Mg
2+1.7mmol/L, Hydrocerol A 1.1mmol/L, Tris-HCl buffered soln (pH=7.0).Multienzyme coupling catalyzed reaction is carried out in shaking table, and through shaking table reaction 2～12 hours, the centrifuging and taking supernatant was with gas chromatography determination 1, ammediol concentration, result such as table 3.
Table 3 nano gel immobilized multienzyme system coupling catalysis produces 1, the ammediol situation
Above result shows, adopts dispersion-adsorption of immobilization technology, makes up nanogel-multienzyme assembly system, and this nano gel immobilized multienzyme system can be realized NADH
2/ NAD
+Self cyclic regeneration, coupling catalysis is synthetic 1, ammediol.
Use nanogel dispersion-adsorption of immobilization multi-enzyme system, catalyzing glycerol synthesizes 1, ammediol; Give full play to excellent properties such as nanogel high-ratio surface, both reacted simple, acellular pollution; For the multiple enzyme of coupling simultaneously is that multienzyme fixedly provides the place, method is easy, cost is low again.
Claims (4)
1. the preparation method of a nano gel immobilized multienzyme system is characterized in that with the many alcohol groups that contain hydrophilic group, dispersing nanometer carrier; Suppressing nanoparticle reunites; Obtain dispersive nanogel suspension-s in the water, give full play to excellent properties such as nano-carrier high-ratio surface, with dispersion-adsorption of immobilization method; The preparation nano gel immobilized multienzyme system is specially:
1) cell-free extract prepares: after klebsiella spp is carried out multiplication culture, and centrifugal collection thalline; Washing is also weighed; The saline water that adds 2～10 times of quality, behind the mixing, ultrasonication under the ice bath environment; Centrifugal back obtains crude enzyme liquid; Again through the ammonium sulfate precipitation of saturation ratio 30%～70%, dialysis desalting, using the Tris-HCl buffer preparation of pH=6.5～10.0 to become concentration is the multi-enzyme system solution of 5.0～100g/L;
2) dispersion of nano-carrier: dispersion agent alcohol with the dissolving of Tris-HCl buffered soln, is pressed
Quality proportioning and TiO
2Nano-carrier mixes, and ultra-sonic dispersion 30～60min obtains dispersive nano-TiO in the water
2Gel suspension;
3) nanogel absorption load immobilized multienzyme system: with multi-enzyme system solution and nano-TiO
2The gel suspension mixing, volume ratio 1: 1～10, vibration absorption 0.5～6h in the shaking table in the time of 37-50 ℃; Centrifugal mixed system keeps supernatant and measures enzyme content, and lower sediment is washed with buffered soln not to be had Protein Detection to supernatant and go out; Obtain nano gel immobilized multienzyme system, cryopreservation.
2. the preparation method of nano gel immobilized multienzyme system according to claim 1 is characterized in that in the dispersion of nano-carrier, and described dispersion agent alcohol is one or more in polyoxyethylene glycol, terepthaloyl moietie, glycerine or the WR 34678.
3. the preparation method of nano gel immobilized multienzyme system according to claim 1 is characterized in that the prepared nano gel immobilized multienzyme system that obtains comprises: glycerol dehydrogenase, glycerol dehydratase and 1, ammediol oxydo-reductase.
4. a nano gel immobilized multienzyme system as claimed in claim 1 is 1, and the application during ammediol is synthetic is characterized in that: multienzyme coupling catalysis 1, the ammediol compound method is: reaction solution consist of glycerine 100～650mmol/L, NAD
+0.5～5mmol/L, VB
125～30 μ mol/L, (NH
4)
2SO
420～50mmol/L, (NH
4)
2Fe (SO
4)
20.5～2 μ mol/L, Mg
2+0.5～2mmol/L, Hydrocerol A 0.2～2mmol/L, pH=6.5～10.0 buffered soln; Add nano gel immobilized enzyme 10～100g/L, the shaking table vibration was reacted 2～12 hours under 30～45 ℃ of conditions, and the centrifuging and taking supernatant measures 1, ammediol content.
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