CN106884010A - A kind of method that nanoscale magnetic bead orients the controllable fixed multienzyme of ratio - Google Patents

Abstract

The invention relates to a method for immobilizing multi-enzymes with controllable directional ratio of nano magnetic spheres, which belongs to the technical field of enzyme immobilization. In this method, nano-magnetic spheres chelated with nickel ions are used as carriers, fluorescent proteins (GFP and DsRed) fused with anchor domains are used as models instead of enzymes fused with anchor domains, and the N-terminal His tag and DsRed are constructed by genetic recombination. The scaffold protein of cohesin (A and C) is the intermediary, and the fluorescent protein complex is immobilized on the carrier through the affinity adsorption of Ni ²⁺ and His tag and the specific binding between cohesin and anchor domain. The enzyme immobilization method of the invention has simple process, mild conditions, and can immobilize the enzyme in a directional and controllable manner, which is an innovation of the enzyme immobilization method.

Description

A method for immobilizing multi-enzyme with controllable orientation and ratio of nano-magnetic spheres

technical field

The invention belongs to the technical field of enzyme immobilization, and in particular relates to a multi-enzyme immobilization method, in particular to a multi-enzyme immobilization method which requires two different ratios of enzyme-catalyzed reactions in an enzyme cascade reaction.

Background technique

Enzymes are biocatalysts that lower the activation energy of a reaction and increase the rate of the reaction. Immobilized enzymes have remarkable catalytic performance and reusability, thus, they have great potential in green and sustainable applications, especially in industrial applications.

There have been many reports on efforts to develop efficient strategies for immobilizing enzymes, most of which mainly focus on the immobilization of single enzymes. However, in many cases, a single enzyme cannot fully catalyze the reaction, but multiple enzymes are required to act synergistically in a cascade reaction, so more and more people are interested in catalyzing biotransformation through multi-enzyme co-immobilization. Co-immobilizing multiple enzymes on the surface of the carrier, making full use of the catalytic properties of different enzymes, and increasing the concentration of intermediate products can not only expand the application range of enzyme catalysis, but also improve the overall reaction efficiency of the multi-enzyme system. To a certain extent, it overcomes the limitation of single-catalysis of single-enzyme reaction system.

Although different strategies for the co-immobilization of multiple enzymes have been explored, most of these methods are still based on the technology developed for the immobilization of single enzymes. In general, the main disadvantage of these methods is the difficulty in distinguishing one enzyme from another during co-immobilization, since many enzymes share common structural features. Therefore, the order and stoichiometric ratio of colocalized enzyme molecules are usually not well controlled, leading to a decrease in the overall catalytic efficiency of the cascade reaction. Compared with in vitro reaction systems, many cascade reactions in or on the surface of living cells are catalyzed by multienzyme complexes, which are self-assembled into highly ordered structures by different enzymes. Advantages of such multienzyme complexes include creating physical proximity effects for substrate access, maintaining high local concentrations of intermediates, and reducing substrate inhibition, thereby increasing the overall catalytic efficiency of the target pathway.

For the carrier of immobilized enzymes, most magnetic polymer microspheres are organic and inorganic hybrid materials that combine magnetic metals or oxides with organic polymers. They are inorganic materials such as magnetic metals or metal oxides and organic materials. Produced by polymer hybridization and compounding. The polymer part of the magnetic composite polymer microsphere can also easily introduce functional groups through copolymerization, covalent bonding with small molecules, adsorption, etc., so as to provide other organic or inorganic nanomaterials with magnetic polymer microspheres. Convenient to further implement new functions. Nanomagnetic balls are a new type of magnetic material with broad application prospects that have been gradually produced, developed, and expanded since the 1970s. Due to their good magnetic response, small particle size, large specific surface area, and good biocompatibility, etc. It is widely used in immobilized enzyme technology due to its advantages.

Contents of the invention

The present invention aims to provide a method for immobilizing multiple enzymes with controllable orientation and proportion of nano magnetic spheres. The catalytic efficiency of the enzyme cascade reaction; at the same time, the use of magnetic nanospheres as a carrier is beneficial to the separation and repeated utilization of enzymes, substrates, and products.

In order to achieve the above object, the specific technical solutions of the present invention are as follows:

1. A method for the fixed multi-enzyme of a nano-magnetic ball directional ratio controllable, it is characterized in that: utilize the Ni ²⁺ of chelating on the surface of the nano-magnetic ball and the affinity adsorption between the His tag of the scaffold protein, the scaffold protein is immobilized On the nano magnetic ball; according to the specific adsorption of cohesin and anchor domain on the scaffold protein, the enzyme fused with the anchor domain is immobilized on the scaffold protein; by controlling the type, quantity and amount of cohesin on the scaffold protein Sorting to achieve directional and ratio-controlled adsorption of enzymes.

2. The method for immobilizing multiple enzymes with controllable directional ratio of nano-magnetic spheres is used in cascade reactions that require multiple enzymes with different quantitative ratios.

3. Further, the method for controlling the directional ratio of the nano-magnetic spheres to immobilize multiple enzymes is characterized in that: it comprises the following steps:

(1) construct recombinant scaffold protein;

(2) constructing a fluorescent protein that fuses the anchor domain;

(3) Mixing the scaffold protein and magnetic spheres chelating Ni ²⁺ for culture, so that the scaffold protein is immobilized on the magnetic sphere;

( ₄ ) Separate the scaffold protein-treated nanomagnetic balls with a magnet, and then mix and adsorb with the fluorescent protein fused with the anchor domain. Combined to fix on the nano magnetic spheres.

4. Further, the scaffold protein described in step (1) comprises a His tag.

5. Further, the scaffold protein described in step (1) is a combination of multiple or multiple cohesin proteins.

6. Further, each fluorescent protein described in step (2) is combined with an anchor domain respectively, and these anchor domains are respectively combined with the cohesin of the scaffold protein described in step (1).

7. Further, the scaffold protein described in step (3) is a single scaffold protein, or multiple scaffold proteins are mixed and adsorbed in different molar ratios.

8. Further, the fluorescent protein complex described in step (4) is crude extract or pure protein solution.

A method for immobilizing multi-enzymes with controllable directional ratio of nano magnetic balls, characterized in that: magnetic nano microspheres chelated with Ni ²⁺ are used as carriers, and fluorescent proteins fused with anchor domains are used as model proteins instead of enzymes for immobilization research Effect, using the scaffold protein containing His tag and cohesin as an intermediate, through the affinity adsorption of Ni ²⁺ and His tag and the specific adsorption of cohesive domain-anchor domain, the fluorescence fused with the anchor domain Proteins are immobilized on magnetic nanospheres.

A method for immobilizing multi-enzymes with controllable directional ratio of nano magnetic spheres, characterized in that it comprises the following steps:

(1) Construction of scaffold proteins ACA and CAC by gene recombination construction. A and C were cloned into plasmid pYD1, and then the fused ACA and CAC were cloned into plasmid pETDuet-1 to obtain scaffold proteins pETDuet1-A-C-A and pETDuet1-C-A-C with N-terminal His tags. Among them, cohesin A comes from CbpA in C.cellulovorans, and cohesin C comes from the first cohesin of CipC in Clostridium cellulolyticum.

(2) The fluorescent proteins GFP-docA and DsRed-docC fused with the anchor domain were constructed by gene recombination. Insert GFP and docA into plasmid pYD1 to obtain GFP-docA (GA) with GS linker in the middle, then clone it into plasmid pET22b, and use the same method to obtain DsRed-docC (RC). Among them, the anchor domain docA is from EngY in C.cellulovorans, and the anchor domain docC is from celCCA in C.cellulolyticum.

(3) Cultivate the scaffold protein in a shaker at 37°C for about 2 hours until OD ₆₀₀ =0.6-0.8, induce with 1mM IPTG at 37°C for 3h-6h, and optimize it to 3h; put the fluorescent protein complex on a shaker at 37°C Cultivate in medium for about 2 hours until OD ₆₀₀ =0.6-0.8, and induce with 0.4 mM IPTG at 20°C for 12 hours. The obtained protein solution was centrifuged at 8000r for 5min, and washed once with water. The obtained bacteria were resuspended in 1×PBS buffer (pH≈7.4) to make OD ₆₀₀ =40. The cells were disrupted with an ultrasonic disruptor, and centrifuged at 12000 r for 10 min to obtain the supernatant, which is the crude extract of the target protein.

(4) Perform qualitative characterization by SDS-PAGE, measure protein content with Coomassie brilliant blue method, and use BandScan software to measure the percentage of target protein in crude protein extract, thereby calculating the concentration of target protein.

(5) Disperse magnetic nanospheres (MNPs) chelated with Ni ²⁺ into a 10mg/ml solution, take 200ul of MNPs and resuspend in 4ml of scaffold protein solution, and incubate on a shaking table at 15°C for 3-5h.

(6) The scaffold protein-treated nano-magnetic spheres were separated with a magnet and washed once with water, and then left at 4°C for 12 hours with 1.2ml fluorescent protein fused with the anchor domain, and CaCl ₂ was added to make the concentration 10mM.

(7) Make a standard curve according to the concentration of the fluorescent protein and the corresponding fluorescence intensity, and calculate the amount of fluorescent protein adsorbed by the magnetic sphere per unit according to the difference in fluorescence intensity of the fluorescent protein supernatant solution before and after the fluorescent protein is adsorbed by the magnetic spheres treated with the scaffold protein .

In step (5), the scaffold protein may be a single scaffold protein, or two scaffold proteins may be mixed in different proportions.

Compared with prior art, the beneficial effect of the present invention:

In the present invention, two recombinant scaffold proteins are used, both of which have three cohesive domains and an N-terminal His tag. GFP and DsRed genes are fused to the anchor domain at the C-terminus to ensure that they can bind to the scaffold protein through the specific adsorption of the cohesive domain-anchor domain. This protein-protein interaction has been reported to be species-specific, allowing us to control the amount and location of GFP and DsRed specific binding on scaffold proteins. The His tag of the scaffold protein fixes the fluorescent protein complex on the surface of the magnetic nanospheres through its affinity adsorption with nickel ions. The ratio of loaded GA and RC can be further tuned by adjusting the molar fixation ratio of the two scaffold proteins.

Description of drawings

Fig. 1 is the fluorescence scanning picture: (A) the fluorescence intensity of the protein supernatant after the nano-magnetic spheres adsorbed the fluorescent protein complex (GFP-docA) after being treated with BSA or scaffold protein; Fluorescence intensity of protein supernatant after adsorption of fluorescent protein complex (DsRed-docC) by magnetic nanospheres.

Figure 2 shows the amount of fluorescent protein complexes adsorbed by magnetic nanospheres.

Figure 3 shows the ratio of the two fluorescent protein complexes adsorbed by the magnetic nanospheres when the molar ratios of the two scaffold proteins are 0:5, 1:4, 2:3, 3:2, 4:1 and 5:0 .

Fig. 4 is a schematic flow chart of the method of the present invention.

detailed description

The present invention will be further described below in combination with examples. It should be noted that this example is only for explaining the present invention, rather than limiting the scope of the present invention.

Example 1: Qualitative characterization of fluorescent protein complexes (GA, RC) immobilized on magnetic nanospheres

(1) Disperse magnetic nanospheres (MNPs) chelated with Ni ²⁺ into a 10mg/ml solution, take 200ul MNPs dispersed in 4ml 0.02umol/ml scaffold protein (ACA, CAC) and the same concentration of control BSA The solution was cultured on a shaker at 15°C for 5h. Separate the magnetic balls with a magnet and wash them several times with water.

(2) Disperse scaffold protein or BSA-treated magnetic spheres in 1.2mL fluorescent protein complex GA (add CaCl ₂ to make the final concentration 10mM), and let it stand at 4°C for 12h, shaking it occasionally to keep it in the state of suspension.

(3) Separate the magnetic balls with a magnet and wash them several times with water. Disperse it in 1.2mL fluorescent protein complex RC (add CaCl ₂ to make the final concentration 10mM), and let it stand at 4°C for 12h, shaking it occasionally to make it in a suspended state.

(4) Scan the supernatant solution after separating the magnetic spheres with a fluorescence spectrophotometer to obtain a fluorescence spectrum (Fig. 1). Compared with the control BSA-treated magnetic balls, when the scaffold protein-treated magnetic balls were used for adsorption, the fluorescence intensity of the isolated fluorescent protein complexes decreased sharply, indicating that the scaffold protein-treated magnetic balls could well absorb fluorescence. protein complex.

Example 2: The amount of adsorption of fluorescent protein complexes (GA, RC) immobilized on magnetic nanospheres

(1) Make a standard curve according to the concentration of fluorescent protein and the corresponding fluorescence intensity.

(2) According to the same method as in Example 1, the magnetic balls were treated with scaffold proteins with ACA:CAC (molar ratio) of 0:5, 1:4, 2:3, 3:2, 4:1 and 5:0 respectively Finally, adsorb GA and RC in sequence, and measure the fluorescence intensity of the fluorescent protein complex before and after adsorption with a fluorescence spectrophotometer (GA excitation wavelength: 475cm ^-1 , emission wavelength: 499cm ^-1 ; RC excitation wavelength: 558cm ^-1 , emission wavelength: 583cm ^-1 ;)

(3) According to the standard curve in step (1), according to the fluorescence intensity difference of the fluorescent protein supernatant solution before and after the fluorescent protein complex is adsorbed by the magnetic ball treated by the scaffold protein, calculate the amount of the fluorescent protein complex adsorbed by the magnetic sphere per unit (figure 2). GA increases with the scaffold protein ACA:CAC, depending on the specific adsorption of the cohesin-anchor domain. The maximum adsorption capacity of magnetic balls can reach ~0.831μmol/g magnetic balls.

Example 3: Molar ratio of fluorescent protein complexes (GA, RC) immobilized on magnetic nanospheres

(1) Carry out adsorption and measurement according to the same method as in Example 2, and finally calculate the adsorption ratio of the two adsorbed fluorescent protein complexes (GA, RC) ( FIG. 3 ). The ratio of adsorbed GA and RC is mostly smaller than the theoretical value, which is due to the steric hindrance during adsorption. The ratio of the two fluorescent proteins adsorbed is controllable in the range of about 1:3 to 2:1.

Claims (8)

1. A method for the fixed multi-enzyme of a nano-magnetic ball directional ratio controllable, it is characterized in that: utilize the Ni ²⁺ of chelating on the surface of the nano-magnetic ball and the affinity adsorption between the His tag of the scaffold protein, the scaffold protein is immobilized On the nano magnetic ball; according to the specific adsorption of cohesin and anchor domain on the scaffold protein, the enzyme fused with the anchor domain is immobilized on the scaffold protein; by controlling the type, quantity and amount of cohesin on the scaffold protein Sorting to achieve directional and ratio-controlled adsorption of enzymes. 2 . The method for immobilizing multiple enzymes with controlled orientation and proportion of magnetic nanospheres according to claim 1 , characterized in that: it is used in cascade reactions requiring multiple enzymes with different quantitative ratios. 3 . 3. The method of nano-magnetic sphere directional ratio controllable immobilization of multiple enzymes according to claim 1, characterized in that: comprising the following steps: (1) construct recombinant scaffold protein; (2) constructing a fluorescent protein that fuses the anchor domain; (3) Mixing the scaffold protein and magnetic spheres chelating Ni ²⁺ for culture, so that the scaffold protein is immobilized on the magnetic sphere; ( ₄ ) Separate the scaffold protein-treated nanomagnetic balls with a magnet, and then mix and adsorb with the fluorescent protein fused with the anchor domain. Combined to fix on the nano magnetic spheres. 4 . The method for immobilizing multiple enzymes with controlled orientation and ratio of magnetic nanospheres according to claim 3 , wherein the scaffold protein described in step (1) comprises a His tag. 5. The method for immobilizing multiple enzymes with controllable orientation and ratio of magnetic nanospheres according to claim 3, characterized in that the scaffold protein described in step (1) is a combination of multiple or multiple cohesin proteins. 6. the method for the fixed multi-enzyme of nanometer magnetic ball directional ratio controllability according to claim 3, it is characterized in that, each kind of fluorescent protein described in step (2) is respectively combined with a kind of anchor domain, and these anchors The domains specifically bind to the cohesin of the scaffold protein described in step (1). 7. the method for the fixed multi-enzyme of nano magnetic ball directional ratio controllability according to claim 3, it is characterized in that, the scaffold protein described in step (3) is a single scaffold protein, or multiple scaffold proteins Mixed adsorption according to different molar ratios. 8. The method for immobilizing multi-enzymes with controllable orientation and ratio of magnetic nanospheres according to claim 3, characterized in that the fluorescent protein complex described in step (4) is a crude extract or a pure protein solution.