Method for improving enzyme catalytic activity by constructing enzyme polymer

Abstract

The invention relates to a method for constructing an enzyme polymer to improve enzyme catalytic activity. The method comprises the following steps: modifying a phage sponge protein Acb2 at a C terminal or an N terminal of glucosidase, glucuronidase, sucrose phosphorylase and the like through a genetic engineering means; the enzyme polymer with better catalytic activity is obtained by screening and optimizing the types of connecting peptides between the sponge protein and the natural enzyme and the number of amino acids. The specific enzyme activity of the sponge protein driven clustered enzyme aggregate enzyme designed and constructed in the invention is improved by 3.43 times compared with that of a wild enzyme, and kcat is improved by 32.3 times. The sponge protease polymer is high in catalytic activity, simple in preparation process and wide in application range, and has the potential of being applied to large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of catalytic technology, and in particular to a method for constructing enzyme multimers to enhance enzyme catalytic activity. Background Art

[0002] Enzymes are a class of natural biocatalysts that are widely used in fields such as food processing, drug synthesis, chemical and biological substance analysis, textiles, and the pulp industry. Since enzymes can catalyze biochemical conversions far more efficiently than artificial catalysts, applying natural enzymes to industrial processes is an extremely attractive strategy. Many important proteins are assembled in the form of functional domains (clusters) to organize and coordinate internal activities in living cells, such as channel proteins, ribosomes, RNA polymerases, etc. Cellular activators that form higher-order polymers - protein clusters help cells better complete biological processes by enhancing the intensity of gene expression. Inspired by natural protein clusters in cells, various protein assembly strategies have been artificially designed in recent years to mimic natural protein clusters and improve catalytic performance in vivo and in vitro.

[0003] However, artificial vesicles and DNA / RNA scaffolds are difficult to synthesize intracellularly, which hinders further applications. In in vitro self-assembly processes such as bacterial microcompartments and virus-like particles, complex operation steps are usually required to control the connection between the outer shell protein and the enzyme. In addition, macromolecular shells assembled in vivo impose a huge burden on metabolism. Insufficient stoichiometric control of encapsulated enzymes, lack of specificity in enzyme encapsulation, and low enzyme loading are also problems that urgently need to be solved. Inspired by nature, by utilizing the symmetry of natural polymeric proteins, macromolecular protein clusters have been constructed based on proteins with different oligomerization characteristics and developed as an ideal platform for nanotechnology. However, synthetic protein scaffolds may have an adverse effect on the folding of target proteins, resulting in a decrease or even complete loss of enzyme activity. In addition, the formation of fibrous connections in the scaffold also affects cell division. Therefore, it is of great significance to develop a biomimetic macromolecular protein cluster construction strategy with a simple preparation process. Summary of the Invention

[0004] The purpose of the present invention is to provide a sponge protein-driven cluster construction technology with extremely high catalytic activity enhancement for improving the problems existing in the preparation process of existing protein multimers, such as complex processes, uncontrolled loading, and enzyme activity loss.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions, including the following steps:

[0006] 1) Using homologous recombination means, sequence 2 is respectively modified at the N-terminus of sequence 1. The expression vector plasmid pET28a / pETduet1, with restriction enzyme sites BamHI and XhoI, is used for protein expression through Escherichia coli BL21(DE3), and the crude enzyme solution is obtained by high-pressure homogenization and crushing.

[0007] 2) The protein gel used in SDS-PAGE is composed of 8-15% acrylamide for denaturing electrophoresis. The chromatographic columns required for molecular sieving are SD75 / SD200 / SD600. The elution mobile phase is composed of 10 mM - 50 mM Tris-HCl, 3 mM - 10 mM dithiothreitol, 200 mM - 500 mM NaCl solution, with a pH of 6.5 - 8.5, and the elution flow rate is 0.5 - 2 mL / min.

[0008] 3) Weigh p-nitrophenyl β-D-glucopyranoside and dissolve it in water at a concentration of 0.5 - 3.5 mg / mL. Add the crude enzyme solution, with the final enzyme concentration being 0.005 - 0.05 mg / mL. At 20 - 50 °C and pH 7 - 10, after catalyzing the reaction for 1 - 3 min, add 1M Na 2 CO 3 to terminate the reaction and measure the absorbance.

[0009] Change the types of linkers and the number of amino acids. The sequences of linker1 - 10 are

[0010] linker 1: GSG; linker 2: GSGGSG; linker 3: GSGGSGGSG

[0011] linker 4: GSGGSGGSGGSG; linker 5: GSGGSGGSGGSGGSG;

[0012] linker 6: APA; linker 7: APAAPA; linker 8: APAAPAAPA;

[0013] linker 9: APAAPAAPAAPA; linker 10: APAAPAAPAAPAAPAAPAAPA

[0014] Use the enzyme multimers constructed with different linkers to carry out the catalytic reaction described in step 2, detect the absorbance and calculate the catalytic activity.

[0015] 4) At the junctions of enzyme protein subunits and the binding sites of spongin proteins, mutate pairs of amino acids into cysteine through genetic engineering. The mutation sites can be any pair of amino acids adjacent to the subunit boundary. Express and measure their stability. The specific steps are as follows: Place the crude enzyme solution at 20 - 50 °C for one hour. Weigh p-nitrophenyl β-D-glucopyranoside and dissolve it in water at a concentration of 0.25 - 2.5 mg / mL. Add the incubated crude enzyme solution, with the final enzyme concentration being 0.005 - 0.05 mg / mL. At 30 °C and pH 7, react for 1 min, add 1M Na 2 CO 3 to terminate the reaction and measure the absorbance.

[0016] 5) According to the methods described in Steps 1 and 2, select the optimal linker peptide obtained in Step 3 to construct the enzyme clusters GlMH@Acb2, BLSP@Acb2, and AbGUS@Acb2 of glucosidase GlMH, sucrose phosphorylase BLSP, and glucuronidase AbGUS. Express the enzyme clusters and the wild enzymes GlMH, BLSP, and AbGUS and obtain crude enzyme solutions. Verify the expression of the enzyme clusters by SDS-PAGE and determine the molecular weight level of the clusters.

[0017] The beneficial effects of the present invention include the following points:

[0018] 1) The method of the present invention provides a new general means for constructing enzyme multimers. Without in vitro assembly, it successfully constructs single-subunit and multimer wild enzymes into macromolecular cluster enzymes. Among them, the specific enzyme activity and catalytic efficiency of the cluster glycosidase are increased by 3.43 times and 32.3 times compared with the wild single-subunit enzyme.

[0019] 2) The present invention optimizes the types and amino acid numbers of the linker peptides of ligase and spongin, and discovers a linker that can greatly improve the catalytic activity.

[0020] 3) The multimeric enzyme clusters constructed by the present invention are used for the preparation of high-value compounds ginsenoside F1, CK, glyceroglucoside, and glycyrrhetinic acid, and the yield is greatly increased compared with the wild-type enzyme. Description of the Drawings

[0021] Figure 1 It is a schematic diagram for the construction of enzyme clusters.

[0022] Figure 2 SDS-PAGE and molecular sieve diagrams of wild enzymes, spongin, and enzyme clusters;

[0023] Figure 3 Schematic diagram of Linker, enzyme activity determination diagrams of wild enzymes and enzyme clusters with different linkers;

[0024] Figure 4 Molecular sieve diagrams of glucosidase GlMH, sucrose phosphorylase BLSP, glucuronidase AbGUS, and corresponding enzyme clusters

[0025] Figure 5 Yield diagrams of high-value compounds prepared by glucosidases GlSK, GlMH, sucrose phosphorylase BLSP, glucuronidase AbGUS, and corresponding enzyme clusters Detailed Embodiments

[0026] The present invention provides a method for constructing enzyme protein clusters to improve enzyme catalytic activity. The following examples are specific examples using this method:

[0027] Example 1

[0028] This example constructs an enzyme cluster based on the wild glucosidase GlSK SEQ ID NO.1 and the spongin Acb2 SEQ ID NO.5. SEQ ID NO.1 and SEQ ID NO.5 are constructed onto the expression vector pETduet1 using the linker GSG by homologous recombination enzymes, with the restriction enzyme sites being BamHI and XhoI. The constructed plasmid GlSK-9R@Acb2 is introduced into Escherichia coli BL21(DE3) by chemical transformation to obtain the bacteria that can express enzyme cluster 1. Inoculate the bacteria of enzyme cluster 1 and the wild enzyme GlSK bacteria, culture them overnight at 37°C, and scale up the system to induce protein expression at 16°C for 22 h. After the expression is completed, collect the bacterial cells and resuspend them in a pH 8.0 phosphate buffer (20 mM), and use high-pressure homogenization to break them to obtain a crude enzyme solution. Weigh 5 g of pNPG and dissolve it in 5 mL of deionized water. Take 0.3 mL of the solution, add GlSK-9R@Acb2 and the crude enzyme solution of GlSK, which contains 0.005 mg of glucosidase, react at 30°C for 1 min, and add 0.2 mL of 1M Na 2 CO 3 , measure the absorbance at 405 nM and calculate to obtain the final specific enzyme activity of 198.98 U / mg and 57.984 U / mg. Referring to the above method, using the same restriction enzyme sites, SEQ ID NO.1 and SEQ ID NO.5 are ligated with different linkers (linker1 to linker10) to obtain enzyme clusters 2-10 (GlSK-6F@Acb2, GlSK-9F@Acb2, GlSK-12F@Acb2, GlSK-15F@Acb2, GlSK-3R@Acb2, GlSK-6R@Acb2, GlSK-9R@Acb2, GlSK-12R@Acb2, GlSK-15R@Acb2). Next, use the above method to induce the expression of the enzyme clusters, and use the same system to measure the catalytic activity with pNPG. After calculation, the enzyme activities of enzyme clusters 2-10 are 67.987 U / mg, 86.831 U / mg, 71.375 U / mg, 61.737 U / mg, 147.48 U / mg, 168.08 U / mg, 198.98 U / mg, 141.68 U / mg, and 119.55 U / mg respectively.

[0029] Next, ginsenoside F1 was prepared using glucosidase GLSK and the enzyme cluster 8 with the highest activity. Ginsenoside Rg1 was weighed and dissolved in water to prepare a substrate solution with a concentration of 5 mg / ml. The above enzyme was added according to Rg1 / enzyme (w / w = 2:1). The reaction was carried out at 30 °C for 6 h, and the product ginsenoside F1 was detected by liquid phase. The conversion rates of ginsenoside Rg1 hydrolyzed by glucosidase GLSK and enzyme cluster 8 were 51% and 94% respectively, and the yields of ginsenoside F1 were 2.03 g / L and 3.75 g / L respectively.

[0030] Example 2

[0031] In this example, according to the experimental method described in 1, an enzyme cluster was constructed based on glucosidase GlMH SEQ ID NO.2, spongin Acb2 SEQ ID NO.5, and linker8 APAAPAAPA to obtain enzyme cluster 11GlMH@Acb2, and the crude enzymes GlMH and GlMH@Acb2 were expressed. Ginsenoside Rb1 was weighed and dissolved in water to prepare a substrate solution with a concentration of 5 mg / ml. The crude enzymes GlMH and GlMH@Acb2 were added according to Rb1 / enzyme (w / w = 2:1). The reaction was carried out at 30 °C for 6 h, and the product ginsenoside CK was detected by liquid phase. The conversion rates of ginsenoside Rb1 hydrolyzed by GlMH and enzyme cluster 11 were 89% and 96% respectively, and the yields of ginsenoside CK were 0.499 g / L and 1.89 g / L respectively.

[0032] Example 3

[0033] In this example, according to the experimental method described in 1, an enzyme cluster was constructed based on sucrose phosphorylase BLSP SEQ ID NO.3, spongin Acb2 SEQ ID NO.5, and linker8 APAAPAAPA to obtain enzyme cluster 12BLSP@Acb2, and the crude enzymes BLSP and BLSP@Acb2 were expressed. Sucrose-glycerol (w / w 1.2:1) was weighed and dissolved in water to prepare a substrate solution with a concentration of 5 mg / ml. The crude enzymes BLSP and enzyme cluster 12 were added according to sucrose / enzyme (w / w = 2:1). The reaction was carried out at 30 °C for 6 h, and the product glycerol glucoside was detected by liquid phase. The conversion rates of sucrose hydrolyzed by BLSP and enzyme cluster 12 were 61% and 93% respectively, and the yields of glycerol glucoside were 2.26 g / L and 3.45 g / L respectively.

[0034] Example 4

[0035] In this example, according to the experimental method described in 1, based on glucuronidase AbGUS SEQ ID NO.4, sponge protein Acb2 SEQ ID NO.5, and linker8 APAAPAAPA, an enzyme cluster was constructed to obtain the enzyme cluster 13AbGUS@Acb2, and the crude enzymes of AbGUS and AbGUS@Acb2 were expressed. Glycyrrhizic acid was weighed and dissolved in water to prepare a substrate solution with a concentration of 5 mg / ml. AbGUS and the crude enzyme of the enzyme cluster 13 were added according to glycyrrhizic acid / enzyme (w / w = 2:1), and the reaction was carried out at 45 °C for 2 h. The product glycyrrhetinic acid was detected by liquid phase. The conversion rates of GlMH and the enzyme cluster 11 for hydrolyzing glycyrrhizic acid were 57% and 97%, respectively, and the yields of glycyrrhetinic acid were 1.63 g / L and 2.77 g / L, respectively.

[0036] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

[0037] Wild glucosidase GlSK SEQ ID NO.1:

[0038] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSI

[0039] Wild glucosidase GlMH SEQ ID NO.2

[0040] MGSSHHHHHHSQDPTPTYLTAPDGTRFRDLNGNGVMDPYEDPRLSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADAGTLESGKETAAAKFERQHMDSSTSAA

[0041] Wild glucuronidase BLSP SEQ ID NO.3

[0042] MGSSHHHHHHSQDPEIQNKAMLITYADSLGKNLKDVHQVLKEDIGDAIGGVHLLPFFPSTGDRGFAPADYTRVDAAFGDWKDVEALGEEYYLMFDFMINHISRESVMYQDFKKNHDDSKYKDFFIRWEKFWAKAGENRPTQADVDLIYKRKDKAPTQEITFDDGTTENLWNTFGEEQIDIDVNSAIAKEFIKTTLEDMVKHGANLIRLDAFAYAVKKVDTNDFFVEPEIWDTLNEVREILTPLKAEILPEIHEHYSIPKKINDHGYFTYDFALPMTTLYTLYSGKTNQLAKWLKMSPMKQFTTLDTHDGIGVVDARDILTDDEIDYASEQLYKVGANVKKTYSSASYNNLDIYQINSTYYSALGNDDAAYLLSRVFQVFAPGIPQIYYVGLLAGENDIALLESTKEGRNINRHYYTREEVKSEVKRPVVANLLKLLSWRNESPAFDLAGSITVDTPTDTTIVVTRQDENGQNKAVLTADAANKTFEIVENDQTVMSSDNLTQ

[0043] Wild glucuronidase AbGUS SEQ ID NO.4

[0044] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSIPTDAQTHRLREDLAREDYSKVMLKPQQTVTRELVSLDGLWKFALVSDSNNNTQPWTGPLDTPLECPVPASYNDIFVDREIHDHVGWVYYQRNVFVPKGWSEDRYLVRCESATHHGRIYVNDHLVADHVGGYTPFEADITDLVAAGEQFRLTIGVNNELTYQTIPPGKVETLELTGKKVQTYQHDFYNYAGLARSVWLYSVPPQHIQDITVQTDVQGTTGVIDYNVVSNMIKGTMQITVLDEDGTTVATMTGSNGTIQIPSANLWHPGAAYLYQFHASIIDSSSNKTIDTYNLAVGIRTVKVQGTQFLINDKPFYFTGFGKHEDTAVRGKGHDRAFMVHDFQLLHWIGANSFRTSHYPYAEEVMEYADRQGLVVIDETPAVGLSFSLGVGVQVSNPPSTFGPDAINNKTREAHAQAIRELIHRDKNHPSVVMWSIANEPASSEDGAREYFAPLPKLARHLDPTRPVTFANVASATYKVDRISDLFDVLCLNRYYGWYTQTGELAEAEAALTEELNGWTEKFKKPIVMTEYGADTVAGLHSVMETPWSEEFQVGMLDMYHRVFDQVEAMAGEHVWNFADFQTAVGIIRVDGNKKGVFTRDRQPKAAAFLLKKRWTELHNGTAEEGQACQVDKLAAAL

[0045] Sponge protein Acb2 SEQ ID NO.5

[0046] HMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRG QYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0047] Enzyme cluster 1SEQ ID NO.6

[0048] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIGSGHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF Enzyme Cluster 2 SEQ ID NO.7

[0049] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIGSGGSGHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0050] Enzyme Cluster 3 SEQ ID NO.8

[0051] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIGSGGSGGSGHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0052] Enzyme Cluster 4 SEQ ID NO.9

[0053] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIGSGGSGGSGGSGHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0054] Enzyme Cluster 5 SEQ ID NO.10

[0055] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIGSGGSGGSGGSGGSGGSGHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0056] Enzyme cluster 6 SEQ ID NO.11

[0057] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIAPAHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF Enzyme Cluster 7 SEQ ID NO.12

[0058] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIAPAAPAHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0059] Enzyme Cluster 8 SEQ ID NO. 13

[0060] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIAPAAPAAPAHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0061] Enzyme Cluster 9 SEQ ID NO.14

[0062] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIAPAAPAAPAAPAHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0063] Enzyme Cluster 10 SEQ ID NO.15

[0064] MGSSHHHHHHSQDPPTPLTTLTAPDGTVFRDLDKDGVMAPFEDPRESVETRVEDLLGRMNLEEKAGLMFQTVIETSPDGTLVEQTGAISKSPTTVVVQEKLLNHFNVHVLPEGRLAARWHNNLQAVAEQTRLGIPVTVSTDPRHAFHENTGASFAAGHFSQWPDSLGLAAIGDTELVRQFADAARQEYLSVGIRAALHPCVDLATEPRWARQLNTFGETSQLVSDFTAAYLDGLQGPGGALSAESVACTTKHFPGGGPQKDGEDAHFPYGREQVYTGGTFEEHLAPFKVALEHKTAAMMPYYGMPVDLEIDGEKIEEVGFGYNKQIVTGLLREQMGFDGVVVTDWELVNDNKVATGQVLPARAWGVEHLDAPGRMEKIIHAGCDQFGGEECPDLLVQLVREGRVTEDRIDASVRRLLRVKFELGLFDDPYVDEDAADEIVGRADLVAAGLAAQSRSVTVLKNGDVDGSPVLPLTGSQRVYVVGMSDEDAARLGTVVTDPADADVAVVRLPAPWEHRDSMFLEAWFHQGSLDFSAETVAQVTELAAQVPVVLDVMLDRPAILTPLVDVATAIVGTYGTSDPALVAALTGEVKPEGRLPFQLPRSMEAVAASRPDVASDTTDPVFPVGHGLSIAPAAPAAPAAPAAPAHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0065] Enzyme Cluster 11 SEQ ID NO.16

[0066] MGSSHHHHHHSQDPTPTYLTAPDGTRFRDLNGNGVMDPYEDPRLSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADAGTLESGKETAAAKFERQHMDSSTSAAAPAAPAAPAHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0067] Enzyme Cluster 12 SEQ ID NO.17

[0068] MGSSHHHHHHSQDPEIQNKAMLITYADSLGKNLKDVHQVLKEDIGDAIGGVHLLPFFPSTGDRGFAPADYTRVDAAFGDWKDVEALGEEYYLMFDFMINHISRESVMYQDFKKNHDDSKYKDFFIRWEKFWAKAGENRPTQADVDLIYKRKDKAPTQEITFDDGTTENLWNTFGEEQIDIDVNSAIAKEFIKTTLEDMVKHGANLIRLDAFAYAVKKVDTNDFFVEPEIWDTLNEVREILTPLKAEILPEIHEHYSIPKKINDHGYFTYDFALPMTTLYTLYSGKTNQLAKWLKMSPMKQFTTLDTHDGIGVVDARDILTDDEIDYASEQLYKVGANVKKTYSSASYNNLDIYQINSTYYSALGNDDAAYLLSRVFQVFAPGIPQIYYVGLLAGENDIALLESTKEGRNINRHYYTREEVKSEVKRPVVANLLKLLSWRNESPAFDLAGSITVDTPTDTTIVVTRQDENGQNKAVLTADAANKTFEIVENDQTVMSSDNLTQAPAAPAAPAHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF

[0069] Enzyme Cluster 13 SEQ ID NO.18

[0070] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSIPTDAQTHRLREDLAREDYSKVMLKPQQTVTRELVSLDGLWKFALVSDSNNNTQPWTGPLDTPLECPVPASYNDIFVDREIHDHVGWVYYQRNVFVPKGWSEDRYLVRCESATHHGRIYVNDHLVADHVGGYTPFEADITDLVAAGEQFRLTIGVNNELTYQTIPPGKVETLELTGKKVQTYQHDFYNYAGLARSVWLYSVPPQHIQDITVQTDVQGTTGVIDYNVVSNMIKGTMQITVLDEDGTTVATMTGSNGTIQIPSANLWHPGAAYLYQFHASIIDSSSNKTIDTYNLAVGIRTVKVQGTQFLINDKPFYFTGFGKHEDTAVRGKGHDRAFMVHDFQLLHWIGANSFRTSHYPYAEEVMEYADRQGLVVIDETPAVGLSFSLGVGVQVSNPPSTFGPDAINNKTREAHAQAIRELIHRDKNHPSVVMWSIANEPASSEDGAREYFAPLPKLARHLDPTRPVTFANVASATYKVDRISDLFDVLCLNRYYGWYTQTGELAEAEAALTEELNGWTEKFKKPIVMTEYGADTVAGLHSVMETPWSEEFQVGMLDMYHRVFDQVEAMAGEHVWNFADFQTAVGIIRVDGNKKGVFTRDRQPKAAAFLLKKRWTELHNGTAEEGQACQVDKLAAALAPAAPAAPAHMDNQHKKIKGYRDLSQEEIDMMNRVKELGSQFEKLIQDVSDHLRGQYNASLHNRDEITRIANAEPGRWLAIGKTDIQTGMMAIIRAIAQPDSF。

Claims

1. An enzyme polymer (enzyme cluster), characterized in that: The enzyme polymer is a phage sponge protein Acb2 modified by a linker at the N-terminus or C-terminus of the catalytic enzyme; Wherein, the catalytic enzyme is any one of glucosidase GlSK, glucosidase GlMH, sucrose phosphorylase BLSP, and glucuronidase AbGUS; the amino acid sequences of the glucosidase GlSK, glucosidase GlMH, sucrose phosphorylase BLSP, and glucuronidase AbGUS are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively; The amino acid sequence of the bacteriophage sponge protein Acb2 is shown in SEQ ID NO.

5.

2. The enzyme polymer according to claim 1, wherein the linker peptide is selected from one of the following: linker 1: GSG; linker 2: GSGGSG; linker 3: GSGGSGGSG; linker4:GSGGSGGSGGSG; linker 5:GSGGSGGSGGSGGSG; linker 6:APA; linker 7:APAAPA; linker 8:APAAPAAPA; linker 9:APAAPAAPAAPA; linker 10:APAAPAAPAAPAAPAAPAAPA.

3. A nucleotide sequence, characterized in that An amino acid sequence encoding the enzyme multimer according to any one of claims 1 to 2.

4. A recombinant vector, characterized in that: Comprising the nucleotide sequence of claim 3.

5. A host cell, characterized in that Comprising the recombinant vector according to claim 4.

6. A method for constructing an enzyme polymer as claimed in any one of claims 1 or 2, characterized in that: The steps include: 1) Modify the bacteriophage sponge protein Acb2 at the N-terminus or C-terminus of glucosidase GlSK / glucosidase GlMH / sucrose phosphorylase BLSP / glucuronidase AbGUS as an assembly scaffold; 2) constructing the fusion protein GlSK@Acb2 / GlMH@Acb2 / BLSP@Acb2 / AbGUS@Acb2 in the pETduet1 or pET28a plasmid, introducing the constructed fusion protein sequence into Escherichia coli by genetic engineering means and completing protein expression, and breaking the Escherichia coli cells after protein expression by high-pressure homogenization to obtain a crude enzyme solution; 3) The protein expression and molecular weight distribution were analyzed by SDS-PAGE and molecular sieve to verify the polymer construction result of the enzyme polymer.

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