CN103343115A - Magnetic immobilized cell, and preparation method and application thereof - Google Patents
Magnetic immobilized cell, and preparation method and application thereof Download PDFInfo
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- CN103343115A CN103343115A CN2013102957395A CN201310295739A CN103343115A CN 103343115 A CN103343115 A CN 103343115A CN 2013102957395 A CN2013102957395 A CN 2013102957395A CN 201310295739 A CN201310295739 A CN 201310295739A CN 103343115 A CN103343115 A CN 103343115A
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Abstract
The invention provides a magnetic immobilized cell, and a preparation method and the application thereof. The preparation method of the magnetic immobilized cell comprises the following steps of: (1) providing a catechol chitosan solution; (2) providing a bacterium suspension of a cell, adding magnetic nano ferroferric oxide into the bacterium suspension, and uniformly stirring; (3) adding the catechol chitosan solution into a solution prepared in the step (2), regulating the pH value to be neutral, and performing uniform stirring and magnetic separation to obtain the magnetic immobilized cell. Magnetic immobilized nitrilase recombinant escherichia coli provided by the invention can be used for continuously catalyzing mandelonitrile to be converted into R-(-)-mandelic acid for 15 batches in an aqueous phase, the yield reaches 100 percent, an ee value is 97 percent, and the mandelonitrile can be efficiently catalyzed to be converted into the R-(-)-mandelic acid in an ethyl-acetate-phase/aqueous-phase system, and the cell can be effectively separated by an external magnetic field. Therefore, a magnetic immobilized method has a wide application prospect and a practical value.
Description
Technical field
The present invention relates to full cell fixation field, more specifically, relate to a kind of magnetic immobilized cell and its preparation method and application.
Background technology
So-called immobilized cell technology, the biomass cells that will have certain physiological function exactly, for example microorganism cells, vegetable cell or zooblast etc. are fixed with certain method, a special kind of skill that is used as the solid biologic catalyzer.
Be compared to free cell, immobilized cell has significant advantage.For example immobilization can improve the stability of thalline, and immobilized cell is easy to separate and then simplify the separation and purification of product, and the immobilized cell of different shape can be applied in the dissimilar reactors in addition.Make a general survey of the development of immobilized cell technology in recent years, the most general immobilized cell method is entrapping method and absorption method.Entrapping method is by some high polymers thalline to be fixed in the carrier firmly, though this method immobilization efficiency is higher, because thalline is wrapped up fully, influences the transmission of substrate and converted product, causes the catalytic efficiency of immobilized bacterium lower.The method of traditional chitosan imbedded immobilized cell adopts the glutaraldehyde with toxicity as linking agent usually, and cell activity is impacted.The absorption rule is to utilize effects such as static between fixation support and the thalline surface, surface tension with the surface of thalline immobilization carrier, though this method is to the activity influence minimum of thalline, but immobilization efficiency is not high, and the weak force between thalline and the carrier causes when reacting influencing the repeating utilization factor of immobilized bacterium owing to stir or the effect of solvent etc. causes thalline to come off.
Along with the development of immobilized cell technology, more and more researchers has concentrated on sight in the colibacillary immobilization research.Escherichia expression system is as one of at present the most frequently used exogenous protein expression system, have that genetic background is clear, expression level is high, processing ease and low cost and other advantages, it is synthetic to be widely used in medicine intermediate at present, the fields such as production of energy development and bulk chemical.
The kind name of oxidizing glucose acidfast bacilli derives from Latin oxydans, and the meaning is to produce very sour material, and this kind belongs to gluconobacter suboxydans and belong to.The oxidizing glucose acidfast bacilli also is one of maximum microorganism of industrial application, and under lower pH situation, the oxidizing glucose acidfast bacilli can be carbohydrate and pure incomplete oxidation generate corresponding aldehyde, ketone or sour and famous widely.Most importantly its zone and stereoselectivity oxidation characteristic in conjunction with chemical technology, can be applied to complicated chemical synthesis process, and this makes the oxidizing glucose acidfast bacilli be widely used in industrial production.For example, the oxidizing glucose acidfast bacilli can be used for the production of ascorbic production, gluconic acid and glucose ketone acid and the production of otan.
Yet the existing fixed cell technology all cuts both ways, and therefore develops the focus that a kind of new immobilized cell technique becomes those skilled in the art's current research.
The adhesive capacity of pyrocatechol group be recent years the investigator find that by the analysis research that ocean mussel class bio-adhesive protein structure is formed it can form strong adhesion with organism and inorganics surface under the environment of the aqueous solution.Utilize this characteristic, the derivative that pyrocatechol is relevant can be used for biomacromolecule for example enzyme, DNA, antibody immobilization.But, up to this point, also do not occur catechol derivatives is applied to correlative study in the cell fixation.
Summary of the invention
In order to solve the problems of the technologies described above, the present invention aims to provide a kind of magnetic immobilized cell and its preparation method and application, this preparation method can improve the transmission between substrate and the converted product, improve catalytic efficiency, simultaneously can improve immobilization efficiency and repeating utilization factor again, and avoid the use of poisonous linking agent.
The invention provides a kind of preparation method of magnetic immobilized cell, described preparation method may further comprise the steps: (1) provides the pyrocatechol chitosan solution; (2) provide the bacteria suspension of cell, in described bacteria suspension, add the magnetic Nano Z 250, stir; (3) add described pyrocatechol chitosan solution in the solution for preparing in the step (2), regulate pH to neutral, stir, adopt magnetic resolution to obtain magnetic immobilized cell.
The structural formula of described pyrocatechol chitosan is:
Described pyrocatechol chitosan solution is to make by the pyrocatechol chitosan is dissolved in the 2wt% acetic acid solution.
The concentration of bacteria suspension is 1-10mg/mL described in the step (2), adding described magnetic Nano Z 250 to the concentration that accounts for described bacteria suspension is 0.04-1mg/mL, and the described pyrocatechol chitosan solution of adding to the ultimate density of pyrocatechol chitosan is 0.01-0.2mg/mL in the step (3).
Described magnetic Nano Z 250 adopts following method preparation: with FeCl
36H
2O and FeCl
24H
2O is dissolved in the ultrapure water under nitrogen protection, drips NaOH solution and regulates pH to 9.0, and stirring also, magnetic resolution obtains described magnetic Nano Z 250.
Described cell is to produce nitrilase recombination bacillus coli or oxidizing glucose acidfast bacilli.
The present invention also provides a kind of magnetic immobilized cell that makes according to above-mentioned preparation method, and described magnetic immobilized cell is that nitrilase recombination bacillus coli or magnetic immobilization oxidizing glucose acidfast bacilli are produced in the magnetic immobilization.
The present invention also provides a kind of application of aforesaid magnetic immobilized cell, and described magnetic immobilized cell is that the nitrilase recombination bacillus coli is produced in the magnetic immobilization, is used to the catalysis mandelonitrile and is converted into R-(-)-amygdalic acid.
The nitrilase recombination bacillus coli is produced in described magnetic immobilization can be converted into R-(-)-amygdalic acid by continuous 15 batches of catalysis mandelonitriles at aqueous phase, and productive rate reaches 100%.
Described magnetic immobilization is produced nitrilase recombination bacillus coli efficient catalytic mandelonitrile in ethyl acetate/water two-phase system and is converted into R-(-)-amygdalic acid.
The present invention proposes a kind of method that under the condition of gentleness, prepares magnetic immobilized cell.Main principle be adhesive capacity by the pyrocatechol chitosan with cell and magnetic-particle realization immobilization crosslinked together, the biocompatibility that the adding of chitosan stent has improved carrier has improved the physical strength of immobilized cell simultaneously.The pyrocatechol chitosan that the present invention adopts has excellent biological compatibility, and crosslinked method can be improved mass transfer, has avoided normally used glutaraldehyde with toxicity in the prior art.
The immobilization rate that the nitrilase recombination bacillus coli is produced in magnetic immobilization provided by the invention reaches 95.8%, activity recovery reaches 83.5%, can be converted into R-(-)-amygdalic acid at the continuous 15 batches of catalysis mandelonitriles of aqueous phase, and productive rate can both reach 100%, the ee value is 97%, phase specific ionization bacterium catalytic efficiency improves greatly, and mandelonitrile that also can the efficient catalytic high density in ethyl acetate/water two-phase system is converted into R-(-)-amygdalic acid.
It is on the low side that magnetic process for fixation provided by the invention had both overcome in the prior art catalytic efficiency of using embedding immobilization, and it is not high to have overcome the immobilized immobilization efficiency of absorption method again simultaneously, the defective that repeating utilization factor is low.In addition, this magnetic immobilized cell can obtain simple and effective separation by the magnetic field that adds, and simplify the operation course, so this process for fixation is with a wide range of applications and practical value.
Description of drawings
Fig. 1 is that the nitrilase recombination bacillus coli generates R-(-)-amygdalic acid at the continuous 16 batches of catalysis mandelonitriles of aqueous phase result schematic diagram is produced in the magnetic immobilization that preparation in accordance with the present invention is prepared;
Fig. 2 is that the reaction process curve that nitrilase recombination bacillus coli catalysis mandelonitrile in ethyl acetate/water two-phase system generates R-(-)-amygdalic acid is produced in the magnetic immobilization that preparation in accordance with the present invention is prepared.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.Should be appreciated that following examples are only for explanation the present invention but not for limiting the scope of the invention.
Synthetic and the sign of embodiment 1 pyrocatechol chitosan
Take by weighing the 0.1g chitosan and be dissolved in the 20ml1%(massfraction) acetic acid solution in, with 0.3g3, the 4-Dihydroxy benzaldehyde joins in the chitosan solution after being dissolved in the 5ml dimethyl formamide, and the stirring at room reaction added the 80ml ethanol sedimentation and obtains intermediate product after 6 hours.Above-mentioned intermediate product is suspended in the 20ml deionized water, adds the 0.3g sodium cyanoborohydride, stirring at room reaction 6 hours adds the 80ml ethanol sedimentation and obtains the pyrocatechol chitosan, and product cleans the final vacuum drying with deionized water and ethanol and obtains pale yellow powder.
Product is dissolved in the heavy water that contains 1% deuterochloroform is used for
1The analysis of H-NMR, the substitution value that goes out the pyrocatechol group by the cubage of measuring phenyl ring hydrogen in the product is 52.2%.
The preparation of embodiment 2 magnetic Nano Z 250s
Take by weighing 2.7g FeCl
36H
2O and 0.99g FeCl
24H
2O is dissolved under nitrogen protection in the 250ml ultrapure water, regulates about pH to 9 by adding 5M NaOH, and 40 ℃ were stirred 2 hours, separated obtaining nano ferriferrous oxide granule by magnet.Then it is dissolved in the magnetic Nano Z 250 solution that is configured to 1mg/ml in the ultrapure water.
The immobilization that embodiment 3 produces the nitrilase recombination bacillus coli
The selected product nitrilase recombination bacillus coli of present embodiment makes up (about this bacterial strain for this laboratory, can be referring to Wang, H., Sun, H., Wei, D., (2013) Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315by phylogeny-based enzymatic substrate specificity prediction.BMC biotechnology13,14.).After the cultivation, get the 0.1g(weight in wet base) intestinal bacteria bacterium mud is suspended in it in 20ml physiological saline, adds the magnetic Nano Z 250 solution (1mg/ml) of configuration among the 5ml embodiment 2, and magnetic agitation is even.Preparation 10ml2%(massfraction) acetic acid solution, and to the 10mg pyrocatechol chitosan that wherein slowly adds preparation among the embodiment 2, stirring and dissolving makes pyrocatechol chitosan solution (1mg/ml).Get in the bacteria suspension that 1ml pyrocatechol chitosan solution slowly joins the above-mentioned nano ferriferrous oxide of mictomagnetism solution thalline and magnetic Nano ferriferrous oxide particles crosslinked precipitation gradually under the effect of pyrocatechol chitosan.Regulate about the pH to 7 of above-mentioned solution with 1M NaOH solution, after the stirring at room 30 minutes, carry out magnetic resolution with magnet, by near beaker, placing a strong magnets, after leaving standstill for some time, have the immobilized cell of magnetic by attraction, carefully topple over the removal supernatant, the employing deionized water obtains magnetic immobilization recombination bacillus coli after cleaning for several times to lower floor's immobilized cell.Calculate by following formula, the immobilization rate of this magnetic immobilization recombination bacillus coli is 95.8%.Wherein, before the OD600(immobilization) refer to before the immobilization OD value to the 600nm place of measured in solution, after the OD600(immobilization) refer to that employing magnet after separating is to the OD value at the 600nm place of measured in solution after immobilization.
The activity of immobilization recombination bacillus coli detects by the enzyme work of measuring its nitrilase, and concrete reaction system is as follows: (pH8.0 100mM) contains 20mM mandelonitrile and an amount of recombination bacillus coli or corresponding immobilized bacterium to the 1ml phosphoric acid buffer.Add 0.1ml1M HCl termination reaction after 10 minutes 30 ℃ of reactions.The concentration of product R-(-)-amygdalic acid detects by HPLC.The result shows that the activity recovery of this immobilized bacterium is 83.5%.
The immobilization of embodiment 4 oxidizing glucose acidfast bacillis
Get the 0.1g(weight in wet base) the oxidizing glucose acidfast bacilli is (about this bacterial strain, can be referring to Guodong Wei, Xuepeng Yang, Tula Gan, Wenyu Zhou, Jinping Lin, Dongzhi Wei.High cell density fermentation of Gluconobacter oxydans DSM2003for glycolic acid production, J Ind Microbiol Biotechnol (2009) 36:1029 – 1034) bacterium mud is suspended in the 20ml physiological saline, add the magnetic Nano Z 250 solution (1mg/ml) of configuration among the 5ml embodiment 2, magnetic agitation is even.Obtain the pyrocatechol chitosan solution according to embodiment 3 described method configurations.Get in the bacteria suspension that 1ml pyrocatechol chitosan solution slowly joins the above-mentioned nano ferriferrous oxide of mictomagnetism solution thalline and magnetic Nano ferriferrous oxide particles crosslinked precipitation gradually under the effect of pyrocatechol chitosan.Regulate about the pH to 7 of above-mentioned solution with 1M NaOH solution, after the stirring at room 30 minutes, carry out magnetic resolution with magnet, by near beaker, placing a strong magnets, after leaving standstill for some time, have the immobilized cell of magnetic by attraction, carefully topple over the removal supernatant, the employing deionized water obtains magnetic immobilization oxidizing glucose acidfast bacilli after cleaning for several times to lower floor's immobilized cell.The immobilization rate of calculating the oxidizing glucose acidfast bacilli by the formula among the embodiment 3 is 63.7%.
By utilizing oxidizing glucose acidfast bacilli glycerine converting to generate the activity that the 2-pyruvic alcohol detects immobilized cell.Get in the phosphate buffered saline buffer that 0.5g glycerine is dissolved in 10ml pH=6.5, add said fixing cell or corresponding free oxidation grape acidfast bacilli reaction two hours.The output of product 2-pyruvic alcohol detects by HPLC.The result shows that the activity recovery of this immobilized bacterium is 76%.
Should be appreciated that above-described embodiment only is for the preparation method of a kind of magnetic immobilized cell according to a preferred embodiment of the invention is shown, this preparation method is not limited in above-described embodiment.
The reusability of nitrilase recombination bacillus coli is produced in embodiment 5 magnetic immobilizations
Utilize the immobilization of preparation among the embodiment 3 to produce continuous multiple batches of catalysis mandelonitrile generation R-(-)-amygdalic acid of nitrilase recombination bacillus coli, the system of reaction is 100ml, wherein the concentration of substrate mandelonitrile is 100mM, the temperature of reaction is 30 ℃, keeps reaction process pH about 8.0 by adding 1M NaOH solution automatically.The amount of adding biological catalyst in the reaction system is the free bacterium of 10g or corresponding immobilized bacterium.Every batch of reaction times is 1 hour, and reaction finishes the free bacterium in back by centrifugation, immobilized bacterium separate by magnet and with after the washed with de-ionized water three times for after a collection of reaction.
Reaction has been carried out 16 batches altogether, the result as shown in Figure 1, for free bacterium, immobilized bacterium has better stability, it is 97% that the productive rate of preceding 15 batches of reaction R-(-)-amygdalic acids of its catalysis can both reach 100%, ee value.Free bacterium then can only reach 100% yield by preceding 10 batch reactions of catalysis.
The organic phase solvent that two phase reaction adopts is ethyl acetate, and its volume ratio shared in two-phase is 30%.That react totally is 100ml, and wherein substrate mandelonitrile concentration is 1M, will be used for catalyzed reaction behind the free bacteria immobilization of 10g.PH by adding 1M NaOH maintenance system in the reaction process is about 8.0, and temperature of reaction is 30 ℃.In reaction process, by constantly sampling, utilize HPLC to detect the process of following response.
The result as shown in Figure 2, the reaction 4 hours after, it is 95% that the productive rate of product R-(-)-amygdalic acid has reached 99.2%, ee value.
Above-described, be preferred embodiment of the present invention only, be not in order to limiting scope of the present invention, the above embodiment of the present invention can also make a variety of changes.Be that simple, the equivalence that every claims according to the present patent application and description are done changes and modification, all fall into the claim protection domain of patent of the present invention.The present invention not detailed description be the routine techniques content.
Claims (10)
1. the preparation method of a magnetic immobilized cell is characterized in that, described preparation method may further comprise the steps:
(1) provides the pyrocatechol chitosan solution;
(2) provide a kind of bacteria suspension of cell, in described bacteria suspension, add the magnetic Nano Z 250, stir;
(3) add described pyrocatechol chitosan solution in the solution for preparing in the step (2), regulate pH to neutral, stir, magnetic resolution obtains magnetic immobilized cell.
2. preparation method according to claim 1 is characterized in that, the structural formula of described pyrocatechol chitosan is:
3. preparation method according to claim 1 is characterized in that, described pyrocatechol chitosan solution is to make by the pyrocatechol chitosan is dissolved in the 2wt% acetic acid solution.
4. preparation method according to claim 1, it is characterized in that, the concentration of the bacteria suspension of cell is 1-10mg/mL described in the step (2), adding described magnetic Nano Z 250 to the concentration that accounts for described bacteria suspension is 0.04-1mg/mL, and the described pyrocatechol chitosan solution of adding to the final concentration of pyrocatechol chitosan is 0.01-0.2mg/mL in the step (3).
5. preparation method according to claim 1 is characterized in that, described magnetic Nano Z 250 adopts following method preparation:
With FeCl
36H
2O and FeCl
24H
2O is dissolved in the ultrapure water under nitrogen protection, drips NaOH solution and regulates pH to 9.0, and stirring also, magnetic resolution obtains described magnetic Nano Z 250.
6. preparation method according to claim 1 is characterized in that, described cell is to produce nitrilase recombination bacillus coli or oxidizing glucose acidfast bacilli.
7. magnetic immobilized cell that preparation method according to claim 6 makes, described magnetic immobilized cell are that nitrilase recombination bacillus coli or magnetic immobilization oxidizing glucose acidfast bacilli are produced in the magnetic immobilization.
8. the application of a magnetic immobilized cell according to claim 7, described magnetic immobilized cell are that the nitrilase recombination bacillus coli is produced in the magnetic immobilization, are used to the catalysis mandelonitrile and are converted into R-(-)-amygdalic acid.
9. application according to claim 8 is characterized in that, the nitrilase recombination bacillus coli is produced in described magnetic immobilization can be converted into R-(-)-amygdalic acid by continuous 15 batches of catalysis mandelonitriles at aqueous phase, and productive rate reaches 100%.
10. application according to claim 8 is characterized in that, described magnetic immobilization is produced nitrilase recombination bacillus coli efficient catalytic mandelonitrile in ethyl acetate/water two-phase system and is converted into R-(-)-amygdalic acid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107059395A (en) * | 2017-05-16 | 2017-08-18 | 无锡协新毛纺织股份有限公司 | A kind of Laccase Catalyzed wool fabric is grafted the method for sorting of phenolic hydroxy group chitosan |
| CN108949738A (en) * | 2018-07-25 | 2018-12-07 | 嘉兴学院 | A kind of preparation method and applications of the microsphere immobilized cell of magnetic loading ionic liquid |
Citations (1)
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|---|---|---|---|---|
| CN102373193A (en) * | 2011-09-27 | 2012-03-14 | 华东理工大学 | Microbial cell immobilizing carrier and related application |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102373193A (en) * | 2011-09-27 | 2012-03-14 | 华东理工大学 | Microbial cell immobilizing carrier and related application |
Non-Patent Citations (2)
| Title |
|---|
| KEFENG NI ET AL.: "Magnetic catechol-chitosan with bioinspired adhesive surface：preparation and immobilization of ω-transaminase", 《PLOS ONE》 * |
| 赵旭东 等: "固定化酵母细胞生物合成谷胱甘肽的研究", 《华东理工大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107059395A (en) * | 2017-05-16 | 2017-08-18 | 无锡协新毛纺织股份有限公司 | A kind of Laccase Catalyzed wool fabric is grafted the method for sorting of phenolic hydroxy group chitosan |
| CN107059395B (en) * | 2017-05-16 | 2019-07-23 | 无锡协新毛纺织股份有限公司 | A kind of method for sorting of Laccase Catalyzed wool fabric grafting phenolic hydroxy group chitosan |
| CN108949738A (en) * | 2018-07-25 | 2018-12-07 | 嘉兴学院 | A kind of preparation method and applications of the microsphere immobilized cell of magnetic loading ionic liquid |
| CN108949738B (en) * | 2018-07-25 | 2020-10-23 | 嘉兴学院 | Preparation method and application of magnetic-loaded ionic liquid microsphere immobilized cell |
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Application publication date: 20131009 |