CN110894497B - Method for fixing protein by using biofilm - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
- C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
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- C07K—PEPTIDES
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- C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
Abstract
The invention discloses a method for fixing protein by utilizing a biofilm, which takes the biofilm of microorganisms as an initial material to obtain the biofilm material for fixing a target object by chemical modification of polysaccharide, wherein the target object comprises protein containing free amino and/or nano-particles containing amino. The biofilm material disclosed by the invention can be used for immobilizing proteins with catalytic activity or without catalytic activity and nanoparticles containing free amino groups, can obviously improve the stability of immobilized proteins, has universality, can be expanded to the use of general biofilms, and greatly improves the utilization range of the biofilm material.
Description
Technical Field
The invention relates to the field of protein immobilization, in particular to an immobilized enzyme based on a biofilm, an immobilized carrier and a preparation method thereof.
Background
Biofilm is a natural microbial community formed by microorganisms adhering to the surface of materials and secreting Extracellular Polymeric Substances (EPS), the main component of which comprises water, proteins, polysaccharides, lipids, ions, etc. Reports indicate that more than 99% of microorganisms in nature can form biofilms to resist the harsh environment of the outside world.
In the field of biocatalysis, researchers have demonstrated that biofilms are good natural materials that can be used for enzyme immobilization and improve enzyme stability. The current method for immobilizing enzymes on biofilms mainly utilizes amyloid in biofilms to realize specific immobilization of enzymes, such as based on curli system in escherichia coli and TasA system in bacillus subtilis. Since the mechanism of secretion and assembly of amyloid in other microorganisms is unclear, the method for immobilizing biofilm based on amyloid cannot be well expanded to the utilization of other biofilms. Studies have also shown that the composition and amount of different biofilms varies, giving each biofilm unique properties. Therefore, the current enzyme immobilization method based on amyloid cannot meet the requirement of fully utilizing biofilm materials, and the development of a general method capable of utilizing various biofilms for enzyme immobilization is urgently needed.
Disclosure of Invention
A first object of the present invention is to provide a method for fixing an object using a biofilm.
The second purpose of the invention is to provide the application of the biofilm in target object immobilization.
It is a third object of the present invention to provide a biofilm catalyst.
The fourth purpose of the invention is to provide the application of the biofilm catalyst in preparing acetate perfume.
In order to realize the first purpose of the invention, the invention discloses the following technical scheme: a method for fixing a target object by using a biofilm of a microorganism is used as a starting material, and the target object comprises a protein containing a free amino group and/or a nanoparticle containing the amino group through chemical modification of polysaccharide.
As a preferred embodiment, the chemical modification of the polysaccharide means that the polysaccharide is oxidized by TEMPO and modified by EDC/NHS.
Preferably, the microorganism comprises one of clostridium acetobutylicum, bacillus subtilis, escherichia coli or staphylococcus aureus.
As a preferred embodiment, the protein containing free amino groups comprises one or more of lipase, protease, amylase, transaminase, bovine serum albumin.
In order to realize the second purpose of the invention, the invention discloses the following technical scheme: use of a biofilm for the immobilisation of a target, said biofilm being chemically modified by a polysaccharide, said target comprising a protein comprising free amino groups and/or a nanoparticle comprising amino groups.
Preferably, the chemical modification of the polysaccharide is the oxidation of the polysaccharide by TEMPO and the modification of EDC/NHS.
As a preferred embodiment, the microorganism comprises one of clostridium acetobutylicum, bacillus subtilis, escherichia coli or staphylococcus aureus.
As a preferred embodiment, the protein containing free amino group comprises one or more of lipase, protease, amylase, transaminase, bovine serum albumin.
In order to achieve the third purpose of the invention, the invention discloses the following technical scheme: a biofilm catalyst, the catalyst comprises a biofilm chemically modified by polysaccharide and catalytic protein, wherein the polysaccharide is chemically modified by TEMPO oxidation and EDC/NHS modification.
As a preferred embodiment, the catalyst further comprises aminated magnetic nanoparticles.
Preferably, an organic solvent-tolerant biofilm is used in the organic phase catalyst and an aqueous solvent-tolerant biofilm is used in the aqueous phase reaction catalyst.
In order to achieve the fourth object of the present invention, the present invention discloses the following technical solutions: the application of biofilm catalyst in preparing acetate perfume.
The invention constructs a magnetic biofilm catalyst applied to the preparation of acetate spices, which comprises the following steps:
a. performing biofilm culture on clostridium acetobutylicum, and dynamically detecting the change of polysaccharide in the growth process of the biofilm by using FITC-labeled lectin;
b. modifying polysaccharide in the clostridium acetobutylicum biomembrane;
c. preparation of aminated silica-coated Fe3O4Magnetic nanoparticles;
d. co-immobilizing to prepare a magnetic catalyst;
e. the magnetic catalyst is applied to the preparation of acetate spices.
Wherein the lectin in step a comprises PNA and UEA I for labeling D (+) -galactose and L (-) -trehalose respectively.
Wherein, the modification in the step b is divided into two steps: firstly, TEMPO is used for mediating the oxidation of polysaccharide in the biofilm, and EDC/NHS is used for modifying the oxidized polysaccharide.
Wherein, in the step c, the coprecipitation method is firstly used for preparing Fe3O4Nanoparticles, and preparation of SiO by sol-gel method2Encapsulated Fe3O4And finally, modifying the nanoparticles by using APTES to prepare the aminated magnetic nanoparticles.
And c, in the step d, co-incubating the lipase, the aminated magnetic nanoparticles and the modified biofilm material to realize co-immobilization of the lipase and the nanomaterial on the biofilm material.
In the step e, the catalytic system adopts: 10mM alcohol, 30mM vinyl acetate, 20mg/mL catalyst and normal hexane are used as solvents, and the reaction temperature is 35 ℃.
Further, the alcohols include cinnamyl alcohol, citronellol, geraniol, 2-phenylethyl alcohol, linalool and alpha-terpineol.
The invention has the advantages that: the biofilm material disclosed by the invention can be used for immobilizing proteins with catalytic activity or without catalytic activity and nanoparticles containing free amino groups, can obviously improve the stability of immobilized proteins, has universality, can be expanded to the use of general biofilms, and greatly improves the utilization range of the biofilm material.
Drawings
FIG. 1: the CLSM results of the present invention for staining biofilm represent PI staining and lectin staining, respectively.
FIG. 2 is a schematic diagram: the preparation process of the magnetic biocatalyst of the present invention; panel a is biofilm after incubation, panel b is biofilm material after TEMPO oxidation and EDC/NHS modification, and panel c is magnetic catalyst after immobilization.
FIG. 3: FE-SEM image of magnetic nanoparticle preparation; FIG. a shows Fe3O4Nanoparticles, panel b is Fe3O4-SiO2And (3) nanoparticles.
FIG. 4 is a schematic view of: FT-IR results plot for magnetic nanoparticles of the invention; curve 1 is Fe3O4-SiO2Nanoparticles, curve 2 Fe3O4-SiO2-NH2And (3) nanoparticles.
FIG. 5 is a schematic view of: the influence of the concentration of the lipase solution on the immobilization activity and the enzyme loading amount.
FIG. 6: structural analysis of the lipase of the present invention.
FIG. 7 is a schematic view of: the stability of the immobilized enzyme is researched; wherein, panel a is thermal stability, panel b is storage stability, panel c is pH stability, and panel d is organic solvent tolerance.
FIG. 8: the immobilized enzyme batch preparation method of the invention is a result chart of cinnamyl acetate preparation.
FIG. 9: relative activity of the immobilized enzyme of the invention for preparing other acetate spices.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1: culture of clostridium acetobutylicum biofilm
The clostridium acetobutylicum preserved at-80 ℃ is inoculated into a P2 culture medium, the culture is carried out under anaerobic condition at 37 ℃ to prepare a seed solution, then the seed solution is inoculated into an anaerobic culture bottle (P2 culture medium plus cotton towel), and the static culture and the fermentation are carried out at 37 ℃. Biofilms grown on cotton towels were sampled at 3, 5, and 7 days. The obtained biofilm is washed by PBS and used for polysaccharide staining and the preparation of subsequent immobilized materials.
Biofilm polysaccharide was stained with FITC labeled PNA and UEA I, and dead cells were stained with PI, where PNA specifically recognizes D (+) -galactose and UEA I specifically recognizes L (-) -trehalose. The results of CLSM are shown in fig. 1, where green indicates lectin-labeled polysaccharide, red indicates PI-labeled dead cells, and yellow is the color of the combination of the two. The results show that the content of polysaccharide in the biofilm increases gradually with the increase of the culture time, and the polysaccharide is an important component of the biofilm. The biofilm structure was analyzed by FE-SEM and the results are shown in FIG. 2a, from which it can be seen that Clostridium acetobutylicum cells are encapsulated by extracellular EPS, further illustrating the formation of biofilms.
Example 2: modification of biofilm Material
The biofilm is firstly oxidized by TEMPO, and the method comprises the following steps: 10g of biofilm was added to a solution containing 1mM TEMPO and 10mM NaBr, and then a NaClO solution was added to the mixture to start the reaction. And in the reaction process, continuously adding NaOH solution into the system to control the pH of the system to be 10, adding a small amount of ethanol to stop the reaction when the pH is not changed any more, and adjusting the pH to be 7.0 by using HCl solution. And (4) carrying out high-speed centrifugation on the oxidized biofilm material, collecting the precipitate, and washing 3 times by using deionized water.
The oxidized biofilm material is further modified by EDC/NHS by the following method: the oxidized biofilm material was immersed in MES solution (100mM, pH 6.0) containing 434mM EDC and 53.2mM NHS and incubated with shaking at room temperature for 3 h. And after centrifuging and collecting the precipitate, washing the precipitate for three times by using deionized water to obtain the final modified biofilm material.
The modified biofilm material is shown in fig. 2b, and the results show that the number of microbial cells in the biofilm is reduced after oxidation and modification, but the overall biofilm structure is maintained, and therefore, the change is acceptable.
Example 3: preparation of magnetic nanoparticles
Preparation of Fe using chemical coprecipitation method3O4Magnetic nanoparticles, the method comprising: to 100mL of deionized water was added 1.25g of FeCl2·4H2O and 3.40g FeCl3·6H2O, making Fe3+And Fe2+The ratio of (A) to (B) was controlled at 2:1 and the mixture was pre-stirred at 60 ℃ under nitrogen. Then, 6mL of aqueous ammonia was added thereto, and stirring was continued for 30-40 minutes. The black precipitate was collected by centrifugation and washed thoroughly with deionized water and ethanol.
Preparation of SiO by sol-gel method2A covered magnetic nanoparticle, the method comprising: taking 0.145g of Fe3O4Magnetic nanoparticles, dispersed in ethanol, and 6mL of deionized water and 3mL of ammonia water were added thereto. The reaction was started by adding 0.4mL of TEOS thereto and the mixture was incubated at room temperature with stirring. Collecting solid Fe3O4-SiO2And washed several times with deionized water and ethanol.
Prepared Fe3O4And Fe3O4-SiO2Magnetic nanoparticles, characterized using FE-SEM, are shown in figure 3. As can be seen from the results, synthesized Fe3O4The magnetic nanoparticles have a diameter of about 10nm, when SiO is used2After being wrapped, the diameter of the coated iron core becomes about 35nm, which indicates that Fe is successfully prepared3O4-SiO2Magnetic nanoparticles.
Fe3O4-SiO2The nano particles are further subjected to amination modification by the following method: firstly Fe3O4-SiO2The nanoparticles were well dispersed in ethanol, and APTES was then added to the system and incubated at room temperature for 2h and then at 50 ℃ for 5 h. Collecting the modified nanoparticlesThe pellet was washed thoroughly with ethanol and acetonitrile and dried under vacuum. The amination results of the nanoparticles were characterized using FT-IR and are shown in fig. 4. Curve 1 is Fe without amino modification3O4-SiO2Nanoparticles, curve 2 Fe after amination modification3O4-SiO2Nanoparticles, comparing curves 1 and 2, curve 2 was found to be 1677cm-1Has a distinct absorption peak, which is in accordance with NH2Bending vibration coincidence of (2), also accounting for Fe3O4-SiO2The nanoparticles were successfully aminated.
Example 4: immobilization of Lipase and study of enzyme Loading
Lipase Lip181 purified from Bacillus subtilis was provided by the laboratory, and the specific immobilization method was as follows: 10mg of the modified biofilm material was dispersed in a solution containing Lipase Lip181 and incubated at 20 ℃ for 2h with shaking (100 rpm). Then, 1mg of dispersed aminated nanoparticles (Fe) was added thereto3O4-SiO2-NH2) The incubation was performed at 20 ℃ with shaking again (100rpm) for 1 h. Unbound lipase was removed by washing well with PBS. The morphology after immobilization was characterized using FE-SEM, and the results are shown in fig. 2 c. The results show that after co-immobilizing magnetic nanoparticles and lipase, the surface of the material becomes more complex with particles around 35nm in diameter, which is Fe, appearing as compared to fig. 2b3O4-SiO2-NH2The phenomenon that the nanoparticles are immobilized on the surface of the material cannot be seen by the immobilized lipase Lip181 because of its small size.
The immobilization was performed using lipase Lip181 solutions (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0mg/mL) at different concentrations to investigate the changes in the enzyme loading and the immobilization activity of the biofilm material, and the results are shown in fig. 5. With the increase of the concentration of the solution, the enzyme loading amount of the biofilm material is gradually increased, but the activity of the immobilized lipase shows a tendency of increasing firstly and then decreasing, which is caused by that the active pocket of Lip181 is blocked by excessive immobilized protein to influence the nano-channel of the substrate and the product. When the concentration of the solution is 0.6mg/mL, the enzyme-carrying enzyme activity is the highest, and the enzyme-carrying amount is 3.856mg per g of wet biological membrane material.
Example 5: lipase immobilization site analysis
The specific activity of the free lipase is 6.39 +/-0.43U/mg, and the specific activity of the immobilized lipase is 6.78 +/-0.50U/mg by detecting the change of the specific activity of the lipase before and after immobilization. Although immobilized enzymes by chemical methods often cause reduction of specific activity of the enzymes, the specific activity of the lipases in the present study does not decrease, but slightly increases. In order to study the reason that the specific activity of lipase is not reduced after immobilization, the three-dimensional structure of lipase Lip181 is optimized by using a molecular dynamics method, and amino groups on the surface of protein and the positions of catalytic triads of Lip181 are analyzed by using Chimera software, and the result is shown in FIG. 6. Red indicates the position of the free amino group and blue indicates the catalytic triad position of the enzyme. From the results, it can be seen that the free amino group on the surface of lipase Lip181 is far from its catalytic triad position. After immobilization, the matrix material does not block the catalytic pocket for substrate to enter and exit Lip181, and does not adversely affect the specific activity of lipase.
Example 6: study of immobilized Lipase stability
The immobilized lipase is used for researching the stability change of the immobilized lipase, and in the research of thermal stability, the thermal stability of the immobilized lipase is determined by placing the immobilized lipase at 35 ℃ for incubation for 24h and sampling at regular time to detect the residual activity. As shown in FIG. 7a, the immobilized lipase has significantly improved stability at 35 ℃, and after 24h incubation, the activity of the free lipase is substantially lost, while the residual activity of the immobilized lipase can still reach 57.82%. Storage stability was investigated by incubating the immobilized lipase at 4 ℃ for several days and periodically sampling to determine the residual activity. As shown in FIG. 7b, the immobilized lipase has greatly improved stability when stored at 4 ℃, the residual activity of the free lipase after 10 days of storage is only 3.56%, while the activity of the immobilized lipase can still reach 82.58% at 10 days, and the residual activity of the immobilized lipase is still 74.23% even after 14 days of incubation. The pH stability was investigated by incubating the immobilized lipase in a buffer of pH 4 to 11 at 25 ℃ for 4h and determining its storage stability by measuring its residual activity. The results are shown in fig. 7c, the activity of the free lipase is reduced obviously after incubation in buffers with different pH values, at pH 10, the stability of the free lipase is highest, and the residual activity is 96.10%, while at other pH values, the residual activity of the free lipase is far lower than that of the immobilized lipase. The residual activity of the immobilized lipase can reach more than 90% in the environment of pH 5 to 11, which shows that the pH change resistance of the immobilized lipase is greatly improved. Organic solvent tolerance was determined by freeze-drying the immobilized lipase, incubating in n-hexane at 25 ℃ for several hours, and measuring the residual activity by sampling at regular intervals. In the stability study, free lipase was used as a control, and the effect of immobilization on the stability of lipase Lip181 was determined by comparing changes in stability. As shown in FIG. 7d, the immobilized lipase also has improved stability in n-hexane, and after 12h incubation, the residual activity of the free lipase is 23.52%, while the residual activity of the immobilized lipase is 42.34%. By analyzing the changes of the lipase in the aspects of thermal stability, storage stability, pH stability and organic solvent tolerance before and after immobilization, we finally conclude that the stability of the lipase Lip181 can be remarkably improved by using the biofilm scaffold material of the research.
Example 7: preparation of acetate perfume by immobilized lipase
The immobilized lipase is firstly frozen and dried, and then is used for preparing acetate perfume. The reaction system is as follows: 10mM alcohol, 30mM vinyl acetate, 20mg/mL catalyst, n-hexane as solvent. The mixture was incubated with shaking (150rpm) at 35 ℃ and the substrate and product were detected by gas chromatography. Using Shimadzu GC-2014 gas chromatography, the stationary phase was Rtx-5 capillary column (30m 0.25mm 0.25 μm) and the mobile phase was N2. The gradient elution procedure is shown in the table below.
The immobilized lipase after freeze-drying was used for studying the application of the immobilized lipase in batch preparation of cinnamyl acetate, and the result is shown in fig. 8, the yield of cinnamyl acetate in 3h in the first batch reaction is 85.09%, and the yield of cinnamyl acetate in 3h in the third batch reaction is 62.56%, which shows the application of the catalytic system in batch reaction. By immobilizing other lipases with superior properties on the biofilm material of this study, higher yields and more batch reactions were obtained.
The immobilized lipase is used for researching the application of the immobilized lipase in the preparation of other acetate spices, citronellol, geraniol, 2-phenethyl alcohol, linalool and alpha-terpineol are used as substrates in the process, and the difference of conversion efficiency is researched. The results show that the immobilized enzyme has no catalytic reaction on linalool and alpha-terpineol, the corresponding acetate perfume cannot be prepared by transesterification, and in the substrate with catalytic activity (figure 9), the lipase has the highest conversion efficiency on cinnamyl acetate, the second highest conversion efficiency on 2-phenethyl alcohol and the lowest conversion efficiency on geraniol.
Example 8: biofilm material immobilization of other proteins
On the basis of the study in example 4, it was investigated whether this biofilm material could be used for the immobilization of other proteins. The process uses protease, amylase, transaminase and bovine serum albumin as target proteins to carry out enzyme immobilization research, and the result shows that the researched proteins can be immobilized on the surface of our materials in a covalent connection mode. Further, the biofilm material prepared in the present study can be used for immobilizing proteins with catalytic activity or without catalytic activity and nanoparticles containing free amino groups.
Example 9: other biological envelope immobilized lipase
On the basis of examples 1 and 2, it was investigated whether this biofilm polysaccharide display strategy could be applied to the preparation of other biofilm materials. According to the process, bacillus subtilis, escherichia coli, staphylococcus aureus and the like are selected as research objects, the microorganisms are grown and gathered to form biofilms through a specific means, and polysaccharides in the biofilms are modified by means of TEMPO oxidation and EDC/NHS modification. The modified biofilm materials are used for immobilizing lipase, and the result shows that the immobilized biofilm materials all show corresponding lipase activity and the enzyme stability is improved. The results further illustrate that the biofilm polysaccharide display strategy of the research has universality, can be easily expanded to the use of other biofilms, and can greatly improve the utilization range of biofilm materials.
Polysaccharides are widely present in biofilms and are an important component of biofilms. Without understanding the mechanism of polysaccharide formation, the polysaccharides in biofilms can be modified by simple chemical reactions for rapid and simple immobilization of enzymes, which we call "biofilm polysaccharide display technology". Clostridium acetobutylicum is a gram-positive bacterium that has been demonstrated to be industrially fermented to produce butanol in the form of biofilms. The presence of EPS obviously improves the stability of clostridium acetobutylicum in an organic solvent, thereby improving the yield of butanol. Since the EPS of Clostridium acetobutylicum has a resistance to the toxicity of organic solvents, it may be a good choice for immobilizing enzymes and for applications in organic phase catalysis. Therefore, the acetone butanol clostridium biofilm is used for developing a universal enzyme immobilization method, which is beneficial to widening the available range of the biofilm which is a natural material.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (12)
1. A method for fixing a target object by using a biofilm is characterized in that the biofilm material for fixing the target object is obtained by taking the biofilm of microorganisms as a starting material and chemically modifying polysaccharide, wherein the target object comprises protein containing free amino groups, and the chemically modifying polysaccharide refers to the oxidation of polysaccharide by TEMPO and the modification of EDC/NHS.
2. The method of claim 1, wherein the target further comprises magnetic nanoparticles comprising amino groups.
3. The method of claim 1, wherein the microorganism comprises one of clostridium acetobutylicum, bacillus subtilis, escherichia coli, or staphylococcus aureus.
4. The method of claim 1, wherein the protein containing free amino groups comprises one or more of lipase, protease, amylase, transaminase, and bovine serum albumin.
5. Use of a biofilm for immobilizing a target, wherein the biofilm is chemically modified by a polysaccharide, the target comprises a protein comprising free amino groups, and the chemical modification of the polysaccharide is carried out by TEMPO oxidation and EDC/NHS modification.
6. The biofilm use in target immobilization according to claim 5, wherein said target further comprises magnetic nanoparticles comprising amino groups.
7. The use of a biofilm according to claim 5 for the immobilisation of a target, wherein said microorganism comprises one of Clostridium acetobutylicum, Bacillus subtilis, Escherichia coli or Staphylococcus aureus.
8. The use of biofilm to immobilize a target, wherein said protein containing free amino groups comprises one or more of lipase, protease, amylase, transaminase and bovine serum albumin.
9. A biofilm catalyst is characterized by comprising a biofilm chemically modified by polysaccharide and catalytic protein, wherein the polysaccharide is chemically modified by TEMPO oxidation and EDC/NHS, and the catalytic protein is protein containing free amino groups.
10. A biofilm catalyst according to claim 9, further comprising aminated magnetic nanoparticles.
11. A biofilm catalyst according to claim 9 or 10, wherein said organic phase catalyst comprises an organic phase solvent-tolerant biofilm and said aqueous phase reaction catalyst comprises an aqueous phase solvent-tolerant biofilm.
12. Use of a biofilm catalyst according to claim 9 or 10 in the preparation of acetate based fragrances, wherein the protein comprising free amino groups in said biofilm catalyst is a lipase.
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| Biofilm Polysaccharide Display Platform: A Natural, Renewable, and Biocompatible Material for Improved Lipase Performance;Hao Dong et al.;《J. Agric. Food Chem.》;20200113;第20卷;第1373-1381页 * |
| Programmable and printable Bacillus subtilis biofilms as engineered living materials;Jiaofang Huang et al.;《NATURE CHEMICAL BIOLOGY》;20190131;第15卷;第34-41页 * |
| Programmable biofilm-based materials from engineered curli nanofibres;Peter Q. Nguyen et al.;《NATURE COMMUNICATIONS》;20140917;第5卷;第1-10页 * |
| 丙酮丁醇梭菌生物膜结构与组成分析;杨 静等;《高校化学工程学报》;20161228;第31卷(第1期);第111-118页 * |
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