Self-assembled cross-linked enzyme aggregate based on protein scaffold and preparation method of self-assembled cross-linked enzyme aggregate

Abstract

The invention provides a self-assembled cross-linked enzyme aggregate based on a protein scaffold and a preparation method of the self-assembled cross-linked enzyme aggregate, and belongs to the technical field of biology. The preparation method comprises the following steps: connecting an enzyme connected with SpyTag to a SpyCatcher protein scaffold to form a self-assembled cross-linked enzyme aggregate; the amino acid sequence of the SpyCatcher protein is as shown in SEQ ID NO: 1. According to the self-assembled cross-linked enzyme aggregate prepared by the invention, the conformation and the enzyme activity of the enzyme are reserved to the maximum extent, meanwhile, the thermal stability and the reusability are improved, and a foundation is laid for subsequent industrial utilization.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a self-assembly cross-linked enzyme aggregate based on a protein scaffold and a preparation method thereof. Background Art

[0002] At present, the technologies for improving enzyme properties include directed evolution, site-directed mutagenesis, enzyme fusion, surface display and enzyme immobilization (LQeaCao, 2003; Madhavan et al., 2017). Immobilization technology can overcome the shortcomings of free enzymes, such as low catalytic efficiency, difficulty in reuse and lack of stability. Immobilized enzyme technology has promoted the development of the fermentation industry and other related industries. The carrier-based immobilization method is prone to changes in the enzyme structure due to the interaction between the carrier and the enzyme, resulting in enzyme inactivation. The fixed carrier usually has a certain volume and is not suitable for the catalytic reaction of macromolecular substrates due to diffusion limitations (Roessl et al., 2010). Moreover, since the enzyme and the carrier material usually have completely different properties, simply connecting them by covalent bonds may destroy the tertiary structure of the enzyme, making the enzyme passivated or inactivated.

[0003] The preparation of carrier-free immobilized enzymes does not require additional solid carriers, and the protease can be directly cross-linked. Cao et al. (2000) replaced the crystallization method with a simpler and cheaper precipitation method, first forming physical aggregation of enzyme molecules without denaturation, and then further cross-linking, and successfully constructed cross-linked enzyme aggregates (CLEAs) for the first time. Compared with traditional carrier-immobilized enzymes, carrier-free cross-linked enzyme biocatalytic systems have some obvious advantages, such as high catalyst specific surface area, high catalytic activity, good operating stability under extreme conditions and organic solvents, and significantly reduced costs. Although cross-linked enzyme aggregate technology has been successfully applied to the immobilization of a variety of enzymes, the commonly used cross-linking agent glutaraldehyde has low selectivity for protein surface reaction sites, resulting in deformation of the tertiary structure of the enzyme, which in turn affects the enzyme activity. In addition, due to the different number and position of surface lysine distribution of enzymes of different types and sources, the precipitation and cross-linking processes need to be optimized separately when preparing cross-linked enzyme aggregates, resulting in great limitations in the preparation technology of cross-linked enzyme aggregates. Therefore, the development of a cross-linked enzyme aggregate that greatly retains enzyme activity and does not require too many optimization steps and its preparation method is a technical problem that needs to be solved at this stage. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a self-assembled cross-linked enzyme aggregate based on a protein scaffold and a preparation method thereof. The self-assembled cross-linked enzyme aggregate based on a protein scaffold and a preparation method thereof retain the conformation and enzyme activity of the enzyme to the maximum extent, while improving thermal stability and reusability.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a self-assembling cross-linked enzyme aggregate based on a protein scaffold, wherein an enzyme connected to SpyTag is connected to a SpyCatcher protein scaffold to form a self-assembling cross-linked enzyme aggregate; the amino acid sequence of the SpyCatcher protein is shown in SEQ ID NO:1.

[0007] Preferably, the enzyme linked to the SpyTag comprises one or more of endoglucanase, cellobiohydrolase, β-glucosidase, endoxylanase, β-xylosidase, β-galactosidase, and α-amylase.

[0008] Preferably, the amino acid sequence of the endoglucanase connected to SpyTag is shown in SEQ ID NO:2, the amino acid sequence of the cellobiohydrolase connected to SpyTag is shown in SEQ ID NO:3, and the amino acid sequence of the β-glucosidase connected to SpyTag is shown in SEQ ID NO:4.

[0009] The present invention also provides a method for preparing the self-assembling cross-linked enzyme aggregate, comprising the following steps: mixing ammonium sulfate and SpyCatcher protein to obtain precipitated SpyCatcher protein; cross-linking the precipitated SpyCatcher protein with glutaraldehyde to obtain a SpyCatcher protein scaffold; and mixing an enzyme connected to SpyTag and the SpyCatcher protein scaffold to obtain a self-assembling cross-linked enzyme aggregate.

[0010] Preferably, the volume ratio of the SpyCatcher protein to ammonium sulfate is 1:1-1:5.

[0011] Preferably, the ammonium sulfate and SpyCatcher protein are mixed at a temperature of 2-6° C. and a time of 0.5-2 h.

[0012] Preferably, the mass ratio of glutaraldehyde to precipitated SpyCatcher protein is 50:1-1:1.

[0013] Preferably, the cross-linking temperature is 23-27° C. and the time is 1-5 h.

[0014] Preferably, the molar ratio of the enzyme connected to SpyTag to the SpyCatcher protein scaffold is 1:15-25.

[0015] Preferably, the enzyme connected to SpyTag and the SpyCatcher protein scaffold are mixed at a temperature of 23-27° C., a time of 1-3 h, and a speed of 50-150 rpm.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention first uses genetic engineering to synthesize K4-(GGGGS)2-SpyCatcher-His6 protein monomers, and then obtains a cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold (referred to as SpyCatcher protein scaffold) by ammonium sulfate precipitation and glutaraldehyde cross-linking. The binding ability of the constructed SpyCatcher protein scaffold can be maintained for at least 28 days under a 4°C environment, and has universal applicability. Through the specific binding effect between SpyCatcher and SpyTag, any enzyme molecule connected to SpyTag can be site-specifically connected to the cross-linked SpyCatcher protein scaffold to become a cross-linked enzyme aggregate, which solves the problem that the existing preparation of cross-linked enzyme aggregates requires separate optimization steps and enzyme inactivation.

[0018] Furthermore, the present invention utilizes the specific affinity between SpyCatcher-SpyTag, and combines the fusion protein of Egl01-ST, Cel5A-h38-ST and HOHC94-ST or Xynz-ST with the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold through self-assembly. The obtained self-assembled cross-linked enzyme aggregate retains the conformation and enzyme activity of the enzyme to the maximum extent, while improving the thermal stability and reusability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram for the construction of self-assembled cross-linked enzyme aggregates based on protein scaffolds.

[0020] Figure 2 This graph shows the effect of the amount of ammonium sulfate added on the precipitation rate of K4-(GGGGS)2-SpyCatcher-His6 protein monomer.

[0021] Figure 3 This graph shows the effect of the amount of anhydrous ethanol added on the precipitation rate of K4-(GGGGS)2-SpyCatcher-His6 protein monomer.

[0022] Figure 4 This is a graph showing the effect of the amount of PEG6000 added on the precipitation rate of K4-(GGGGS)2-SpyCatcher-His6 protein monomer.

[0023] Figure 5 This is a graph showing the effect of the amount of glutaraldehyde added on the cross-linking rate of K4-(GGGGS)2-SpyCatcher-His6 protein monomer.

[0024] Figure 6The figure shows the binding ratio of cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold and model protein EGFP-ST using ammonium sulfate as the precipitant. A is free EGFP-ST; S1 is the supernatant after EGFP-ST and cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold are combined at a molar ratio of 1:10; S2 is the supernatant after EGFP-ST and cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold are combined at a molar ratio of 1:20; S3 is the supernatant after EGFP-ST and cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold are combined at a molar ratio of 1:30; S4 is the supernatant after EGFP-ST and cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold are combined at a molar ratio of 1:50.

[0025] Figure 7 The figure shows the binding ratio of cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold and model protein EGFP-ST using ethanol as precipitant. A is free EGFP-ST; Y1 is the supernatant after EGFP-ST and cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold are combined at a molar ratio of 1:10; Y2 is the supernatant after EGFP-ST and cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold are combined at a molar ratio of 1:20; Y3 is the supernatant after EGFP-ST and cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold are combined at a molar ratio of 1:30; Y4 is the supernatant after EGFP-ST and cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold are combined at a molar ratio of 1:50.

[0026] Figure 8 Storage stability of the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold.

[0027] Fig. 9The results of the investigation of the binding of Egl01-ST, HOHC94-ST, Cel5A-h38-ST and the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold are shown in Figure 1. M is a marker, 1 is the supernatant after the binding of Egl01-ST, HOHC94-ST, Cel5A-h38-ST and the cross-linked K4-SpyCatcher protein scaffold, and 2 is the free cellulase after Egl01-ST, HOHC94-ST, Cel5A-h38-ST are mixed in a ratio of 92:6:2.

[0028] Fig.10 TEM characterization diagram, where A is free K4-SpyCatcher, B is the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold, and C is the cross-linked enzyme aggregate (C-CLEAs) bound to Egl01-ST / HOHC94-ST / Cel5A-h38-ST.

[0029] Fig.11 This is the result of investigating the optimum pH of the prepared self-assembled cross-linked enzyme aggregates.

[0030] Fig.12 This is the result of investigating the optimum temperature of the prepared self-assembled cross-linked enzyme aggregates.

[0031] Fig.13 This is a graph showing the results of investigating the repeated use stability of the prepared self-assembled cross-linked enzyme aggregates.

[0032] Fig.14 This is a graph showing the results of investigating the storage stability of the prepared self-assembled cross-linked enzyme aggregates.

[0033] Fig.15 This figure shows the comparison of the effects of the prepared self-assembled cross-linked enzyme aggregates and free enzymes on hydrolyzing corn straw.

[0034] Fig.16 The figure is a diagram of the binding of the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold and Xynz-ST. Among them, M is a marker, 1 is free endoxylanase Xynz-ST, and 2 is the supernatant after the endoxylanase Xynz-ST binds to the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold.

[0035] Fig.17 This figure shows the comparison of the hydrolysis effects of birch wood xylan by self-assembled cross-linked enzyme aggregates of endo-xylanase (X-CLEAs) and free endo-xylanase. DETAILED DESCRIPTION

[0036] The present invention provides a self-assembling cross-linked enzyme aggregate based on a protein scaffold, wherein the enzyme connected to SpyTag is connected to the SpyCatcher protein scaffold to form a self-assembling cross-linked enzyme aggregate; the amino acid sequence of the SpyCatcher protein is shown in SEQ ID NO: 1. The SpyCatcher protein scaffold described in the present invention is a K4-(GGGGS)2-SpyCatcher-His6 protein scaffold. The nucleotide sequence of the K4-(GGGGS)2-SpyCatcher-His6 protein described in the present invention is shown in SEQ ID NO: 5. The purpose of the four lysine groups at the N-terminus of the K4-(GGGGS)2-SpyCatcher-His6 gene of the present invention is to make glutaraldehyde react with the four lysines at the N-terminus as much as possible, avoid reaction with SpyCatcher, and ensure the normal structure of SpyCatcher, and (GGGGS)2 is a flexible linker.

[0037] In the present invention, the enzyme connected to SpyTag includes one or more of endoglucanase, cellobiohydrolase, β-glucosidase, endoxylanase, β-xylosidase, β-galactosidase, and α-amylase, preferably endoglucanase, cellobiohydrolase, β-glucosidase, and endoxylanase.

[0038] In the present invention, the amino acid sequence of the endoglucanase (Egl01-ST) connected to SpyTag is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 6; the amino acid sequence of the cellobiohydrolase (Cel5A-h38-ST) connected to SpyTag is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 7; the amino acid sequence of the β-glucosidase (HOHC94-ST) connected to SpyTag is shown in SEQ ID NO: 4, and the nucleotide sequence is shown in SEQ ID NO: 8; the amino acid sequence of the endoxylanase (Xynz-ST) connected to SpyTag is shown in SEQ ID NO: 12, and the nucleotide sequence is shown in SEQ ID NO: 13.

[0039] The present invention also provides a method for preparing the self-assembling cross-linked enzyme aggregate, comprising the following steps: mixing ammonium sulfate with SpyCatcher protein to obtain precipitated SpyCatcher protein; cross-linking the precipitated SpyCatcher protein with glutaraldehyde to obtain a SpyCatcher protein scaffold; mixing the enzyme connected to SpyTag with the SpyCatcher protein scaffold to obtain a self-assembling cross-linked enzyme aggregate. The method for preparing the SpyCatcher protein of the present invention comprises: selecting a K4-(GGGGS)2-SpyCatcher-His6 encoding gene (nucleotide sequence as shown in SEQ ID NO:5) to construct a plasmid, transferring the obtained plasmid into Escherichia coli BL21 (DE3) by transformation, inducing expression, and purifying it with a Ni-NTA column and a desalting column to obtain a K4-(GGGGS)2-SpyCatcher-His6 protein monomer (SpyCatcher protein).

[0040] In the present invention, the volume ratio of the SpyCatcher protein to ammonium sulfate is 1:1-1:5, preferably 1:1.5-1:4, and more preferably 1:2; the temperature for mixing the ammonium sulfate and the SpyCatcher protein is 2-6°C, preferably 3-5°C, and more preferably 4°C; the time for mixing the ammonium sulfate and the SpyCatcher protein is 0.5-2h, preferably 0.8-1.5h, and more preferably 1h. The ammonium sulfate of the present invention is a saturated ammonium sulfate solution. The purpose of mixing the ammonium sulfate and the SpyCatcher protein in the present invention is to obtain the precipitated SpyCatcher protein by salting out.

[0041] In the present invention, the mass ratio of glutaraldehyde to precipitated SpyCatcher protein is 50:1-1:1, preferably 40:1-10:1, and more preferably 30:1; the cross-linking temperature of glutaraldehyde and precipitated SpyCatcher protein is 23-27°C, preferably 24-26°C, and more preferably 25°C; the cross-linking time of glutaraldehyde and precipitated SpyCatcher protein is 1-5h, preferably 2-4h, and more preferably 3h.

[0042] In the present invention, the molar ratio of the SpyCatcher protein scaffold to the enzyme connected to SpyTag is 15-25:1, preferably 18-23:1, and further preferably 20:1; the temperature at which the enzyme connected to SpyTag and the SpyCatcher protein scaffold are mixed is 23-27°C, preferably 25°C; the time for mixing the enzyme connected to SpyTag and the SpyCatcher protein scaffold is 1-3h, preferably 2h; the speed at which the enzyme connected to SpyTag and the SpyCatcher protein scaffold are mixed is 50-150rpm, preferably 100rpm. The enzyme connected to SpyTag in the present invention is Egl01-ST, Cel5A-h38-ST and HOHC94-ST, and the mass ratio of Egl01-ST, Cel5A-h38-ST and HOHC94-ST is 90-94:4-8:1-3, preferably 92:6:2.

[0043] In the present invention, the preparation steps of Egl01-ST, Cel5A-h38-ST, HOHC94-ST or Xynz-ST include: selecting endoglucanase Egl01 gene or cellobiohydrolase Cel5A-h38 gene or β-glucosidase HOHC94 gene or endoxylanase Xynz gene and connecting them with SpyTag gene (nucleotide sequence as shown in SEQ ID NO: 9) to construct plasmids, respectively transferring them into Escherichia coli BL21 (DE3) by transformation, inducing expression, and purifying them with Ni-NTA column and desalting column to obtain Egl01-ST, Cel5A-h38-ST, HOHC94-ST and Xynz-ST.

[0044] In the present invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0045] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Example 1

[0047] A method for preparing self-assembled cross-linked enzyme aggregates based on protein scaffolds (flow chart as shown in Figure 1 The steps are as follows:

[0048] 1) The K4-(GGGGS)2-SpyCatcher-His6 coding gene (nucleotide sequence as shown in SEQ ID NO: 5) and the vector pET-22b were used to construct a recombinant plasmid, and the obtained recombinant plasmid was transformed into Escherichia coli BL21 (DE3) for induced expression, and purified by Ni-NTA column and desalting column to obtain K4-(GGGGS)2-SpyCatcher-His6 protein monomer (amino acid sequence as shown in SEQ ID NO: 1). The construction process of the recombinant plasmid in this step was synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0049] 2) The endoglucanase Egl01 gene (NCBI: AIY72753.1) or the cellobiohydrolase Cel5A-h38 gene (NCBI: MT764839.1) or the β-glucosidase HOHC94 gene (NCBI: EHJ97046.1) was selected and connected to the SpyTag gene (nucleotide sequence as shown in SEQ ID NO: 9), and then connected to the vector pET-22b or pET-28a or pET-21b in sequence to construct a recombinant plasmid, which was transformed into Escherichia coli BL21 (DE3) by transformation and induced expression. After purification by Ni-NTA column and desalting column, Egl01-ST protein (amino acid sequence as shown in SEQ ID NO: 2), Cel5A-h38-ST protein (amino acid sequence as shown in SEQ ID NO: 3) and HOHC94-ST protein (amino acid sequence as shown in SEQ ID NO: 4) were obtained, respectively. The construction process of the recombinant plasmid in this step was synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0050] 3) According to the volume ratio of K4-(GGGGS)2-SpyCatcher-His6 protein to saturated ammonium sulfate of 1:2, saturated ammonium sulfate was added to the obtained K4-(GGGGS)2-SpyCatcher-His6 and stirred evenly, and the reaction was salted out at a constant speed in a rotary mixer at a temperature of 4°C for 1 hour, and centrifuged at 5000 rpm for 10 minutes ， The precipitated K4-(GGGGS)2-SpyCatcher-His6 was obtained.

[0051] 4) According to the mass ratio of glutaraldehyde to K4-(GGGGS)2-SpyCatcher-His6 protein of 30:1, glutaraldehyde was added to the above precipitated K4-(GGGGS)2-SpyCatcher-His6 protein for cross-linking reaction. The cross-linking temperature was 25°C and the time was 3 h. After the reaction, the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold was obtained by centrifugation.

[0052] 5) The obtained Egl01-ST, Cel5A-h38-ST and HOHC94-ST were mixed in a mass ratio of 92:6:2, and the Egl01-ST / Cel5A-h38-ST / HOHC94-ST mixed protein was added to the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold at a molar ratio of 1:20 between the mixed protein and the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold, and stirred at 100 rpm at 25°C for 2 h to obtain self-assembled cross-linked enzyme aggregates (C-CLEAs) based on the protein scaffold.

[0053] Example 2

[0054] The difference from Example 1 is that in step 3), the mass ratio of K4-(GGGGS)2-SpyCatcher-His6 protein to saturated ammonium sulfate is 1:1, and the remaining steps are the same as in Example 1.

[0055] Example 3

[0056] The difference from Example 1 is that in step 3), the mass ratio of K4-(GGGGS)2-SpyCatcher-His6 protein to saturated ammonium sulfate is 1:3, and the remaining steps are the same as in Example 1.

[0057] Example 4

[0058] The difference from Example 1 is that in step 3), the mass ratio of K4-(GGGGS)2-SpyCatcher-His6 protein to saturated ammonium sulfate is 1:4, and the remaining steps are the same as in Example 1.

[0059] Example 5

[0060] The difference from Example 1 is that in step 3), the mass ratio of K4-(GGGGS)2-SpyCatcher-His6 protein to saturated ammonium sulfate is 1:5, and the remaining steps are the same as in Example 1.

[0061] Example 6

[0062] The difference from Example 1 is that in step 4), the mass ratio of cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold to glutaraldehyde is 1:50, and the remaining steps are the same as in Example 1.

[0063] Example 7

[0064] The difference from Example 1 is that endoglucanase, cellobiohydrolase and β-glucosidase are replaced by endoxylanase, the endoxylanase (gene NCBI: M22624) is obtained in the same way as in Example 1, the preparation and mixing steps of endoglucanase, cellobiohydrolase and β-glucosidase are discarded, and the remaining steps are the same as in Example 1. The amino acid sequence of Xynz-ST is shown in SEQ ID NO: 12, and the nucleotide sequence is shown in SEQ ID NO: 13.

[0065] Comparative Examples 1-5

[0066] The difference between Comparative Examples 1-5 and Examples 1-5 is that saturated ammonium sulfate is replaced with anhydrous ethanol, and the remaining steps are the same as Examples 1-5.

[0067] Comparative Examples 6-10

[0068] The difference between Comparative Examples 6-10 and Examples 1-5 is that saturated ammonium sulfate is replaced by PEG 6000, and the remaining steps are the same as Examples 1-5.

[0069] Comparative Example 11

[0070] The difference from Example 1 is that in step 4), the mass ratio of cross-linking K4-(GGGGS)2-SpyCatcher-His6 protein scaffold to glutaraldehyde is 1:1, and the remaining steps are the same as Example 1.

[0071] Comparative Example 12

[0072] The difference from Example 1 is that in step 4), the mass ratio of cross-linking K4-(GGGGS)2-SpyCatcher-His6 protein scaffold to glutaraldehyde is 1:2, and the remaining steps are the same as in Example 1.

[0073] Comparative Example 13

[0074] The difference from Example 1 is that in step 4), the mass ratio of cross-linking K4-(GGGGS)2-SpyCatcher-His6 protein scaffold to glutaraldehyde is 1:5, and the remaining steps are the same as Example 1.

[0075] Comparative Example 14

[0076] The difference from Example 1 is that in step 4), the mass ratio of cross-linking K4-(GGGGS)2-SpyCatcher-His6 protein scaffold to glutaraldehyde is 1:10, and the remaining steps are the same as Example 1.

[0077] Comparative Example 15

[0078] The difference from Example 1 is that in step 4), the mass ratio of cross-linking K4-(GGGGS)2-SpyCatcher-His6 protein scaffold to glutaraldehyde is 1:20, and the remaining steps are the same as in Example 1.

[0079] Test Example 1

[0080] The products after salting out of Example 1-5 and Comparative Example 1-10 were centrifuged, and the content of unprecipitated K4-(GGGGS)2-SpyCatcher-His6 in the supernatant was measured by BCA method, and then the precipitation rate was calculated. Figure 2-4 . Figure 2 It shows that when the volume ratio of K4-SpyCatcher protein to saturated ammonium sulfate addition is 1:2, the precipitation rate of K4-Spy Catcher reaches a maximum value of 86%. After that, the precipitation rate of K4-SpyCatcher does not change significantly when the amount of saturated ammonium sulfate addition is further increased. Therefore, the ratio of K4-SpyCatcher to saturated ammonium sulfate of 1:2 is subsequently selected as the optimal precipitant addition ratio. Figure 3 It shows that when the volume ratio of K4-SpyCatcher protein to anhydrous ethanol addition is 1:1, the precipitation rate of K4-SpyCatcher is 79%. As the ratio of anhydrous ethanol addition increases to 1:2, the precipitation rate of K4-SpyCatcher reaches a maximum value of 86%. Thereafter, when the amount of anhydrous ethanol added is further increased, the precipitation rate of K4-SpyCatcher does not change significantly. Therefore, the ratio of K4-SpyCatcher to anhydrous ethanol of 1:2 was subsequently selected as the optimal precipitant addition ratio. Figure 4 It shows that when the volume ratio of K4-SpyCatcher protein to PEG6000 addition is 1:1, the precipitation rate of K4-SpyCatcher is only 1.7%. As the ratio of PEG6000 addition increases to 1:5, the precipitation rate of K4-SpyCatcher reaches a maximum value of 16%. The precipitation effect of PEG6000 is worse than that of saturated ammonium sulfate and anhydrous ethanol. Therefore, the optimal precipitation ratio of the ammonium sulfate group and the ethanol group is 1:2 (v / v) for subsequent cross-linking.

[0081] Test Example 2

[0082] The cross-linked products of Example 1, Example 6 and Comparative Examples 11-15 were centrifuged, the supernatant was removed, washed twice with PBS, and then re-added with PBS to mix and cross-link the K4-(GGGGS)2-SpyCatcher-His6 protein. The amount of cross-linked protein was determined by the BCA method, and the cross-linking rate was calculated. Figure 5The results showed that the cross-linking rate of the precipitated K4-(GGGGS)2-SpyCatcher-His6 protein increased with the increase of the concentration of glutaraldehyde in the system. When the mass ratio of K4-(GGGGS)2-SpyCatcher-His6 protein to glutaraldehyde was 1:30, the cross-linking rate of K4-(GGGGS)2-SpyCatcher-His6 protein reached a maximum value of 97.7%. When the ratio was increased to 1:50, the cross-linking rate did not increase significantly. Therefore, 1:30 (w / w) was selected as the optimal cross-linking agent ratio in the subsequent step.

[0083] Test Example 3

[0084] EGFP-ST was used to detect the binding of K4-SpyCatcher protein scaffold after glutaraldehyde cross-linking.

[0085] 1. According to step 2) of Example 1, a model protein EGFP-ST was constructed. The amino acid sequence of the model protein is shown in SEQ ID NO: 10, and the nucleotide sequence is shown in SEQ ID NO: 11.

[0086] Referring to the steps of Example 1, the molar ratio of EGFP-ST to the cross-linked SpyCatcher protein scaffold was set to 1:10, 1:20, 1:30, and 1:50, respectively. The binding of EGFP-ST to the cross-linked SpyCatcher protein scaffold was determined by SDS-PAGE. The results are shown in FIG. Figure 6 As shown, the results showed that when ammonium sulfate was used as a precipitant, when the binding ratio of EGFP-ST to the cross-linked K4-SpyCatcher protein scaffold was 1:20, all EGF P-ST was bound to the protein scaffold.

[0087] 2. Referring to the steps of Example 1, ethanol was used as a precipitant, and the molar ratio of EGFP-ST to the cross-linked SpyCatcher protein scaffold was set to 1:10, 1:20, 1:30, and 1:50, respectively. The binding of EGFP-ST to the cross-linked SpyCatcher protein scaffold was determined by SDS-PAGE. The results are shown in FIG. Figure 7 As shown, when the binding ratio of EGFP-ST to the cross-linked K4-SpyCatcher protein scaffold reached a maximum value of 1:50, some EGFP-ST still did not bind to the protein scaffold, indicating that ethanol was used as a precipitant and glutaraldehyde was added for cross-linking, and the binding effect of EGFP-ST to the cross-linked SpyCatcher protein scaffold was poor.

[0088] Therefore, when C-CLEAs were subsequently prepared, saturated ammonium sulfate was used as the precipitant, and the volume ratio of K4-SpyCatcher to ammonium sulfate was 1:2 (v / v); glutaraldehyde was used as the cross-linking agent, and the mass ratio of K4-SpyCatcher protein to glutaraldehyde was 1:30 (w / w); the binding ratio of the enzyme connected to SpyTag to the cross-linked K4-SpyCatcher protein scaffold was a molar ratio of 1:20.

[0089] Test Example 4

[0090] In order to check the storage stability of the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold, the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold prepared in Example 1 was stored at 4°C for 28 days, and samples were taken every 7 days. Then, each sample was self-assembled with the EGF P-SpyTag prepared in Experimental Example 3 at a molar ratio of 20:1. Assembly process: in a constant temperature air bath shaker, 100rpm, 25°C and 2h. After the assembly is completed, the supernatant is centrifuged and analyzed by SDS-PAGE to see if there is any residual free EGFP-Sp yTag. The results are as follows Figure 8 As shown, the results showed that the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold still maintained a good binding rate with EGFP-SpyTag at 4°C for 28 days, indicating that the cross-linked K4-(GG GGS)2-SpyCatcher-His6 protein scaffold has good stability.

[0091] Test Example 5

[0092] 1. The free cellulase obtained by mixing Egl01-ST, Cel5A-h38-ST and HOHC94-ST in a mass ratio of 92:6:2 in step 5) of Example 1 and the supernatant obtained by combining the Egl01-ST / HOHC94-ST / Cel5A-h38-ST mixed protein obtained in step 5) of Example 1 with the cross-linked K4-SpyCatcher protein scaffold were subjected to SDS-PAGE analysis. The results are shown in Fig. 9 As shown, it indicates that the three Egl01-ST, Cel5A-h38-ST and HO HC94-ST exist separately in the mixed solution, and after mixed reaction with the cross-linked K4-SpyCatcher protein scaffold, they are completely bound to the cross-linked K4-SpyCatcher protein scaffold.

[0093] 2. The free K4-SpyCatcher obtained in step 3) of Example 1, the cross-linked K4-SpyCatcher protein scaffold obtained in step 4) of Example 1, and the self-assembled cross-linked enzyme aggregate obtained in step 5) of Example 1 were characterized by transmission electron microscopy (TEM). Fig.10 As shown, it was shown that after free K4-SpyCatcher was cross-linked with glutaraldehyde, a spherical cross-linked K4-SpyCatcher protein scaffold was obtained, and the protein scaffold effectively bound to Egl01-ST / HOHC94-ST / Cel5A-h38-ST.

[0094] Test Example 6

[0095] The self-assembled cross-linked enzyme aggregates (C-CLEAs) prepared in Example 1 and the mixed free cellulase (Free enzymes) obtained in step 5) of Example 1 were added to a buffer solution of pH 3-10 (pH 3-8 are phosphate buffers, pH 9-10 are glycine-NaOH buffers), and the system contained 2% microcrystalline cellulose (Avicel) as a substrate. The reaction was carried out in a constant temperature air bath shaker at 200 rpm and 50°C for 7 hours. The concentration of reducing sugars in the hydrolyzate was then determined by the DNS (3,5-dinitrosalicylic acid) method. A standard curve was prepared using glucose. The enzyme activity was set to produce 1 μmol of glucose per minute per unit enzyme solution, i.e. 1U. Each experimental condition was repeated three times. The results are as follows. Fig.11 As shown in the results, the mixed free cellulase and self-assembled cross-linked enzyme aggregates showed the best activity at pH 6. The activity of self-assembled cross-linked enzyme aggregates under extreme pH conditions (such as pH 3 and pH 10) was higher than that of the corresponding free enzymes. This shows that self-assembled cross-linked enzyme aggregates have greater application potential in lower pH or higher pH systems than free enzymes.

[0096] Test Example 7

[0097] The self-assembled cross-linked enzyme aggregates prepared in Example 1 and the mixed free cellulase obtained in step 5) of Example 1 were added to a phosphate buffer of pH 6, and the system contained 2% microcrystalline cellulose as a substrate. The reaction was carried out in a constant temperature air bath shaker or water bath at 200 rpm and a temperature range of 30-80°C for 7 hours. The concentration of reducing sugars in the hydrolyzate was then determined by the DNS (3,5-dinitrosalicylic acid) method. A standard curve was prepared using glucose. The enzyme activity was set to produce 1 μmol of glucose per minute per unit of enzyme solution, i.e. 1U. Each experimental condition was repeated three times. The results are as follows: Fig.12As shown in the results, the optimal hydrolysis temperature of the mixed free cellulase and the self-assembled cross-linked enzyme aggregates was 50°C; within the temperature range of 30-60°C, the self-assembled cross-linked enzyme aggregates could still maintain more than 70% activity, which was about 30% higher than the activity of the free enzyme. This may be because the immobilized multi-enzyme system not only provides a more suitable and stable environment for substrate capture and intermediate retention, but also increases the structural rigidity of the enzyme, making the enzymatic reaction more efficient than the free enzyme in solution.

[0098] Test Example 8

[0099] The hydrolysis system contains the self-assembled cross-linked enzyme aggregates prepared in Example 1 (2 mg / g substrate) and 2% corn stover powder pretreated with γ-butyrolactone or sodium carboxymethyl cellulose (CMC-Na). The hydrolysis conditions are 50°C, pH 6 phosphate buffer and 200 rpm, for 72 h (corn straw) or 30 min (CMC-Na). After the reaction, the supernatant is separated by centrifugation, and the concentration of reducing sugars therein is determined by the DNS method. The second and subsequent rounds of hydrolysis process are performed after the first round, after the hydrolysis supernatant is centrifuged and poured out, new buffer and substrate are added for further hydrolysis. The hydrolysis conditions are the same as those of the first round. The results are as follows. Fig.13 As shown in the results, the self-assembled cross-linked enzyme aggregates still maintained 85% hydrolysis efficiency after three hydrolysis cycles with CMC-Na. From the fifth cycle, the enzyme activity stabilized and the final residual activity was 54%. For the insoluble substrate corn stover, the activity exceeded 60% after three hydrolysis cycles.

[0100] Test Example 9

[0101] The self-assembled cross-linked enzyme aggregates prepared in Example 1 were stored at 4°C for 21 days, and samples were taken at specific time points. The samples were then added to a pH 6 phosphate buffer, and the system contained 2% Avicel as a substrate. The reaction was carried out in a constant temperature air bath shaker or water bath at 200 rpm and 50°C for 7 hours. The concentration of reducing sugars in the hydrolyzate was then determined by the DNS (3,5-dinitrosalicylic acid) method. A standard curve was prepared using glucose. The enzyme activity was set to produce 1 μmol of glucose per minute per unit enzyme solution, which is 1U. Each experimental condition was repeated three times. The results are as follows. Fig.14As shown, the results showed that both free enzyme and self-assembled cross-linked enzyme aggregates showed a slight decrease in activity after three days of storage, and the activity retention of self-assembled cross-linked enzyme aggregates was more significant after 12 days of storage. Finally, after 21 days of storage, the self-assembled cross-linked enzyme aggregates retained 76% of the initial activity, while the free enzyme only retained 66% of the initial activity. This may be due to the enhanced structural rigidity of the self-assembled cross-linked enzyme aggregates combined with the cross-linked K4-(GGGGS)2-SpyCatcher-His6 protein scaffold, which provided a protective support for the enzyme, thereby alleviating the impact of environmental changes during long-term storage.

[0102] Test Example 10

[0103] The hydrolysis system contains the self-assembled cross-linked enzyme aggregates prepared in Example 1 (2 mg / g substrate) or the mixed free cellulase (2 mg / g substrate) obtained in step 5) of Example 1 and 2% corn straw powder pretreated with γ-butyrolactone (pretreatment conditions: γ-butyrolactone / water = 4:1 (v / v), solid-liquid ratio of 1:10, sulfuric acid concentration of 150 mM, 120°C treatment for 1 h). The hydrolysis conditions are 50°C, pH 6 phosphate buffer and 200 rpm for 72 h. After the reaction, the supernatant is separated by centrifugation, and the concentration of reducing sugars therein is determined by the DNS method. The results are as follows Fig.15 As shown in the results, the hydrolysis performance of free enzyme and self-assembled cross-linked enzyme aggregates was comparable within 24 hours of the start of hydrolysis. However, after 48 and 72 hours, the reducing sugars produced by the hydrolysis of self-assembled cross-linked enzyme aggregates were 1.6 times that of free enzyme. This may be because free enzymes are easily inactivated when incubated for a long time or subjected to mechanical shear force. In contrast, cross-linked enzyme aggregates are more resistant to these adverse effects due to their enhanced structural rigidity and show stronger hydrolytic activity.

[0104] Test Example 11

[0105] The supernatant after cross-linking of Xynz-ST and K4-(GGGGS)2-SpyCatcher-His6 protein scaffold in Example 7 was verified by SDS-PAGE. Fig.16 As shown, there was almost no unbound Xynz-ST band in the supernatant, indicating that Xynz-ST had been maximally loaded and assembled onto the cross-linked SC scaffold.

[0106] The self-assembled cross-linked enzyme aggregates (X-CLEAs) and free endoxylanase prepared in Example 7 were added to a phosphate buffer at pH 6, and the system contained 2% birchwood xylan as a substrate. The reaction was carried out in a constant temperature air bath shaker or water bath at 200 rpm and a temperature range of 50°C for 30 minutes. The concentration of reducing sugars in the hydrolyzate was then determined by the DNS (3,5-dinitrosalicylic acid) method. A standard curve was prepared using xylose. The results are shown in FIG. Fig.17As shown in the results, the activity of X-CLEAs was retained by about 87% compared with the reducing sugars produced by the free enzyme (38.68 mg). This may be due to the lower mass transfer efficiency of X-CLEAs during the hydrolysis of insoluble substrates compared with the free enzyme.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A self-assembled cross-linked enzyme aggregate based on a protein scaffold, characterized in that: The enzyme linked to SpyTag is connected to the SpyCatcher protein scaffold to form a self-assembled cross-linked enzyme aggregate; The amino acid sequence of the SpyCatcher protein is shown in SEQ ID NO:

1.

2. The self-assembled cross-linked enzyme aggregate according to claim 1, characterized in that The enzyme connected to the SpyTag includes one or more of endoglucanase, cellobiohydrolase, β-glucosidase, endoxylanase, β-xylosidase, β-galactosidase, and α-amylase.

3. The self-assembling cross-linked enzyme aggregate according to claim 2, characterized in that The amino acid sequence of the endoglucanase linked to SpyTag is shown in SEQ ID NO:2, the amino acid sequence of the cellobiohydrolase linked to SpyTag is shown in SEQ ID NO:3, and the amino acid sequence of the β-glucosidase linked to SpyTag is shown in SEQ ID NO:

4.

4. The method for preparing the self-assembled cross-linked enzyme aggregate according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: mixing ammonium sulfate and SpyCatcher protein to obtain precipitated SpyCatcher protein; cross-linking the precipitated SpyCatcher protein with glutaraldehyde to obtain a SpyCatcher protein scaffold; and mixing an enzyme connected to SpyTag and the SpyCatcher protein scaffold to obtain a self-assembled cross-linked enzyme aggregate.

5. The preparation method according to claim 4, characterized in that: The volume ratio of the SpyCatcher protein to ammonium sulfate is 1:1-1:

5.

6. The preparation method according to claim 4, characterized in that: The ammonium sulfate and SpyCatcher protein are mixed at a temperature of 2-6° C. for a time of 0.5-2 h.

7. The preparation method according to claim 4, characterized in that: The mass ratio of the glutaraldehyde to the precipitated SpyCatcher protein is 50:1-1:

1.

8. The preparation method according to claim 4, characterized in that: The cross-linking temperature is 23-27° C. and the cross-linking time is 1-5 h.

9. The preparation method according to claim 4, characterized in that: The molar ratio of the enzyme connected to SpyTag to the SpyCatcher protein scaffold is 1:15-25.

10. The preparation method according to claim 4, characterized in that: The enzyme connected to SpyTag and the SpyCatcher protein scaffold are mixed at a temperature of 23-27° C., a time of 1-3 h, and a speed of 50-150 rpm.

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