Linker peptide for immobilization of pha polymerase and method for immobilization of pha polymerase

Abstract

The present application relates to the field of biotechnology, in particular to a PHA polymerase immobilization connecting peptide and a PHA polymerase immobilization method. The present application provides a connecting peptide for PHA polymerase immobilization, and optimizes the PHA polymerase immobilization connecting system. Experimental results show that, under the connecting peptide and the PHA polymerase immobilization connecting system, the correct expression of the functional protein can be ensured, and most of the catalytic activity is maintained.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to PHA polymerase immobilized linker peptides and methods for PHA polymerase immobilization. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are a series of microbially synthesized biopolyesters that accumulate as intracellular particles to store carbon and energy when carbon sources are abundant and nitrogen and / or phosphorus sources are limited. Their biosynthesis mainly involves three enzymes: β-ketothiolase (PhaA), acetyl-CoA reductase (PhaB), and PHA polymerase (PhaC).

[0003] PhaC can immobilize enzymes by linking them to form fusion proteins, thereby improving enzyme biostability, making the enzyme easier to isolate from the reaction system, and facilitating control. As artificially constructed multi-domain proteins, fusion proteins present various challenges in expression compared to natural proteins, such as reduced expression levels, misfolding of the protein's three-dimensional structure, and inhibited biological activity. These problems mainly arise from the interaction between two adjacent domains, leading to structural damage at both ends of the protein. Therefore, the design and selection of linker peptides, serving as buffer regions between two different domains, is crucial. Currently, no linker peptide has been found that can be applied to most fusion proteins. In practical applications, it is impractical to optimize the design of linker peptides for every fusion protein; this is not only time-consuming and labor-intensive but also has limited effectiveness. Therefore, exploring a universal linker peptide that can be used for PHA polymerase immobilization while maintaining sufficient enzyme activity is of significant research importance. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a linker peptide immobilized with PHA polymerase and a method for immobilizing PHA polymerase.

[0005] The present invention provides a linker peptide for PHA polymerase immobilization, the amino acid sequence of which is shown in SEQ ID NO:6 and / or SEQ ID NO:22.

[0006] This invention obtains linker peptides suitable for PHA polymerase immobilization through screening. These linker peptides, when used in the PHA polymerase immobilization process, can achieve enzyme immobilization while maintaining enzyme activity. Among them, the linker peptide with the amino acid sequence SEQ ID NO:6 is Linker2, and the linker peptide with the amino acid sequence SEQ ID NO:22 is Linker10. Linker10 is the most effective for PHA polymerase immobilization.

[0007] The present invention provides a PHA polymerase immobilization system, which includes PHA polymerase, at least one of the linker peptides described in the present invention, and a functional protein.

[0008] Furthermore, in the PHA polymerase immobilization system of the present invention, the linkage relationship between the PHA polymerase, at least one of the linking peptides, and the functional protein includes:

[0009] PHA polymerase – at least one of the linked peptides – a functional protein; and / or

[0010] Functional protein - at least one of the linker peptides - PHA polymerase.

[0011] In this invention, the functional protein includes at least one of ATP-dependent carboxylamine ligase, fluorescent protein, N-acetylglucosamine-2-isomerase and / or N-acetylneuraminic acid lyase.

[0012] Furthermore, the PHA polymerase immobilization system of the present invention comprises:

[0013] ATP-dependent carboxylamine ligase-PHA polymerase with an amino acid sequence as shown in SEQ ID NO:6; and / or

[0014] ATP-dependent carboxylamine ligase – a ligase-PHA polymerase with an amino acid sequence as shown in SEQ ID NO:22; and / or

[0015] PHA polymerase – a linker peptide with an amino acid sequence as shown in SEQ ID NO:6 – an ATP-dependent carboxylamine ligase; and / or

[0016] PHA polymerase – a linker peptide with an amino acid sequence as shown in SEQ ID NO:22 – an ATP-dependent carboxylamine ligase; and / or

[0017] PHA polymerase – linker peptide with amino acid sequence as shown in SEQ ID NO:6 – fluorescent protein; and / or

[0018] PHA polymerase – linker peptide with amino acid sequence as shown in SEQ ID NO:22 – fluorescent protein; and / or

[0019] N-acetylglucosamine-2-isomerase-linked peptide-PHA polymerase with an amino acid sequence as shown in SEQ ID NO:6; and / or

[0020] N-acetylglucosamine-2-isomerase-linked peptide-PHA polymerase with an amino acid sequence as shown in SEQ ID NO:22; and / or

[0021] N-acetylneuraminic acid lyase – a linker peptide-PHA polymerase with an amino acid sequence as shown in SEQ ID NO:6; and / or

[0022] N-acetylneuraminic acid lyase - amino acid sequence as shown in SEQ ID NO:22 - linker peptide-PHA polymerase.

[0023] This invention develops a PHA polymerase immobilization system, which involves linking PHA polymerase and functional proteins using the linker peptides described in this invention. Based on the selection of suitable linker peptides, the linking method of the PHA polymerase immobilization system has been optimized. In a specific embodiment of this invention, ATP-dependent carboxylamine ligase (LAL134) is used as an example to demonstrate that the functional protein expressed by linking with the functional protein-linker peptide-PHA polymerase in the PHA polymerase immobilization system exhibits the highest activity.

[0024] Furthermore, this invention demonstrates, through the expression of different enzymes via the linker peptides described herein and PHA polymerase, that the linker peptides described herein have universality, ensuring the correct expression of functional proteins while maintaining most of their catalytic activity.

[0025] The present invention provides nucleic acids comprising nucleic acids encoding the linker peptides and / or the PHA polymerase immobilization system described herein.

[0026] The nucleic acid described in this invention can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be part of a vector (e.g., an expression or cloning vector) or a fragment thereof. The nucleic acid can be obtained directly from a natural source or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0027] In this invention, the nucleic acid may be optimized or unoptimized. The optimization includes, but is not limited to: codon usage bias, elimination of secondary structures that are unfavorable to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, mRNA secondary structure, hidden splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning.

[0028] The present invention provides an expression unit, which includes an expression element and the nucleic acid described in the present invention; the expression element includes, but is not limited to, a promoter, a terminator, an enhancer and / or an expression tag.

[0029] The present invention provides a recombinant vector comprising at least one of the nucleic acids or expression units described in the present invention and a vector backbone.

[0030] Furthermore, the recombinant vector of the present invention can be derived from plants, animals, bacteria, fungi, bacteriophages and / or viruses. The present invention does not limit the source of these sources. In a specific embodiment of the present invention, the recombinant vector is derived from bacteria, specifically Escherichia coli.

[0031] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts. In specific embodiments, the nucleic acid encoding the fusion protein provided by this invention can be constructed in various prokaryotic expression vectors, specifically including pET28a.

[0032] This invention provides host cells for transformation or transfection with the recombinant vector. By transforming or transfecting host cells with a vector constructed using recombinant DNA technology, the transformed host cells are capable of replicating the vector encoding a protein or expressing a desired protein.

[0033] Furthermore, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0034] The host cells provided by this invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention does not limit the sources. In a specific embodiment of this invention, the host cell is a bacterium, specifically *Escherichia coli*, and more specifically *Escherichia coli* DH5α and / or BL21(DE3).

[0035] This invention provides the application of at least one of the following (I) to (V) in enzyme-catalyzed reactions and / or biosynthesis:

[0036] I) The linker peptide described in this invention;

[0037] II) The PHA polymerase immobilization system described in this invention;

[0038] III) The nucleic acid described in this invention;

[0039] IV) The expression unit described in this invention;

[0040] V) The recombinant vector described in this invention;

[0041] VI) The host cell described in this invention.

[0042] This invention provides a linker peptide for PHA polymerase immobilization and optimizes the PHA polymerase immobilization linker system. Experimental results show that the linker peptide and PHA polymerase immobilization linker system described in this invention can ensure the correct expression of functional proteins while maintaining most of their catalytic activity. Attached Figure Description

[0043] Figure 1 A schematic diagram showing the production and purification method of polyhydroxyalkanoate (PHA) immobilized enzymes;

[0044] Figure 2 12.5% ​​SDS-PAGE gel electrophoresis of PHA-S01 and PHA-S02 strains;

[0045] Figure 3 12.5% ​​SDS-PAGE gel electrophoresis of strains PHA-S03, PHA-S04 and PHA-S05;

[0046] Figure 4 12.5% ​​SDS-PAGE gel electrophoresis of PHA-S05, PHA-S06 and PHA-S07 strains;

[0047] Figure 5 12.5% ​​SDS-PAGE gel electrophoresis of strains PHA-S08, PHA-S09, and PHA-S010;

[0048] Figure 6 Table 4 shows the enzyme activity assay results of the strains.

[0049] Figure 7 Table 5 shows the enzyme activity assay of the strain. Detailed Implementation

[0050] This invention provides a linker peptide immobilized with PHA polymerase and a method for PHA polymerase immobilization. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0051] Biosynthesis of polyhydroxy fatty acid esters (PHA) Figure 1 As shown;

[0052]

[0053] PhaC amino acid sequence: (SEQ ID NO:2);

[0054] Linker peptide 1 nucleotide sequence: ggggggggaggttcaggtggaggcggaagtggcggtggtggcagc (SEQ ID NO:3);

[0055] Linker peptide 1 amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO:4);

[0056] Linker peptide 2 nucleotide sequence: ggggggggaggaggcggaggtggt (SEQ ID NO:5);

[0057] Linker peptide 2 amino acid sequence: GGGGGGGG (SEQ ID NO:6);

[0058] Nucleotide sequence of connecting peptide 3: gctgaagcagctgctgcgaaagaggcagccgcggcgaaagaggcagctgctgcgaaggaggcagccgcggcgaaggcactggaag cggaagcagcggcggcgaaagaagccgcggcggcgaaggaggccgcggctgccaaggaggccgctgctgcgaaagca (SEQ ID NO: 7);

[0059] Linker peptide 3 amino acid sequence: AEAAAAAKEAAAAKEAAAAKEAAAAKALEAEAAAAKEAAAAKEAAAAKEAAAAKA (SEQ ID NO:8);

[0060] Linker peptide 4 nucleotide sequence: ccagcgccagctccg (SEQ ID NO:9);

[0061] The amino acid sequence of the linker peptide 4: PAPAP (SEQ ID NO:10);

[0062] Linker peptide 5 nucleotide sequence: ccggcgccggcgccggcgccggcgccggcgccggcgccg (SEQ ID NO:11);

[0063] The 5-amino acid sequence of the linker peptide is: PAPAPAPAPAP (SEQ ID NO:12);

[0064] Linker peptide 6 nucleotide sequence: gtactagctgttatagataaaaggggagggggcggtggt (SEQ ID NO:13);

[0065] The amino acid sequence of the linker peptide 6 is: VLAVIDKRGGGGG (SEQ ID NO:14);

[0066] Linker peptide 7 nucleotide sequence: gtactagctgttatagataaaagggggggaggcggtggctctggtggcggctccggcggtggtagcggtggcggtggtagc (SEQ ID NO:15);

[0067] The amino acid sequence of the linker peptide 7 is: VLAVIDKRGGGGGSGGGSGGGSGGGGS (SEQ ID NO:16);

[0068] Linker peptide 8 nucleotide sequence: gctgaggcagccgcggcgaaggcactggaagcggaagcagcggcggcgaaagca (SEQ ID NO:17);

[0069] Linker peptide 8 amino acid sequence: AEAAAAAKALEAAAAKA (SEQ ID NO:18);

[0070] Linker peptide 9 nucleotide sequence: gctgaggcagctgctgcgaaggaggcagccgcggcgaaggcactggaagcggaagcagcggcggcgaaagaagccgcggcggcg aaggca (SEQ ID NO:19);

[0071] Linker peptide 9 amino acid sequence: AEAAAAAKEAAAAKALEAAAAKEAAAAKA (SEQ ID NO:20);

[0072] Linker peptide 10 nucleotide sequence: gctgaggcagccgcggcgaaagaggcagctgctgcgaaggaggcagccgcggcgaaggcactggaagcggaagcagcggcggcg aaagaagccgcggcggcgaaggaggccgcggctgccaaggca (SEQ ID NO:21);

[0073] Linker peptide 10 amino acid sequence: AEAAAAAKEAAAAKEAAAAKALEAEAAAAKEAAAAKEAAAAKA (SEQ ID NO: 22);

[0074]

[0075] Amino acid sequence of LAL134: MGDSTVLLVYARGGPPPSDAIPKAAAATDRLHLLLLDPLPLPAVAPAARCCASVTDLTADRPEPGELIDRIVALARRTAADAVLTFSEFALLAVAEAAERLGLRGPGPGAARARSKRLMRRTWADAGLPVPRFRYVDSLTDLERAWAELHPPILLKSAWGAGSIGQTVLEQRAQLAGAWRHMLGALTAARARSMSELKAGEAGHELIAEELIPATTESWYDRSGYGDYLSVEGMVVDGVYHPVCITGRLPTVPDFTELGNMAPCVLPEPLQHTVSELSRQAVDALGLGTCGTHTEIKLMADRRLCLLETAARFGGLLLTQQIEAIHGVDLVGALTRAHLGQDPGLPDGLLTGPGRCAAGSVNVLATDSRGRPWTTRPLFLPEAVDWDRLLSPGSGIRVAPDFTVPSGTVMHRYRTGHGSLNLAGVLLLTAPTPPGSAARFLRGPQRSRRSLGGRRPRSWLTGPGHTSRRAVVPPVLRAPGSRRPGARRRPRPAP (SEQ ID NO:24);

[0076]

[0077] SA18 nucleotide sequence: atgaagaaattaacgggactgatcgccgcgccccatacaccgttcgactcgtcgtcgaacgtgaacttcgaagagattgataagattgccaagcacttaatcaacgatggtgtaaagggaatctacgtgtgtggtacgactggcgaaggtatccattgctcggtcgaggagcgtaaggcgattgccgaacgctgggtgtctgcgtgcaaccataagttggacatcattgtacataccggcgccttatctatcgtcgatactcttgaattaacgcgtcacgccgataccttagatattctggcaacttccgctattggcccctgcttctttaagccagggtctgtctccgacttagttgaatattgtgcaactatcgcagcagccgcaccttctaaggggttctattactatcacagtggtatgtcgggcgtaaatttgaatatggaggaatttctgatccaagcagacaagcgcattccaaacttatccggtttgaagttcaattcaggggacctttacgaatatcagcgttgtcttcgcgcttgtgacggtaagttcgatgtgccgtttggggtagacgagttccttcctggggcgcttgctgtaggagcaaagagtgcggttggatcaacgtataattacgctgctccacactttaacagtatcatcgaggcgttcaacaagggcgatcacgacgctgtatttaataaaatgactaacgttatcgagcttattcgtgttcttgtagaatttggaggtgtggccgccggcaagatcgctatggaattacatgacatcaacgcaggggatccccgtctgccgttgatgcccctttctgcggaacagaagctgaccgtggttgagaaaatgcgtgccgccaatttcctgaag (SEQ ID NO:26);

[0078] Plasmids and strains are shown in Table 1:

[0079] Table 1. Plasmids and strains

[0080]

[0081]

[0082] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0083] Example 1: Construction of linker peptides in fusion proteins

[0084] The main research focus of this example is polyhydroxy fatty acid synthase (PhaC) and ATP-dependent carboxylamine ligases (ATP-grasp enzymes). ATP-grasp enzymes catalyze the formation of amide bonds by activating carboxylic acids as acyl phosphate intermediates, accompanied by ATP hydrolysis, and are important participants in the synthesis of short oligopeptides. In this example, the dipeptide alanylphenylalanine (Ala-Phe) was catalyzed by the ATP-dependent carboxylamine ligase LAL134. However, in actual catalytic synthesis of the Ala-Phe dipeptide, it was found that the crude enzyme solution led to a continuous decrease in dipeptide product with increasing reaction time. To address this phenomenon, a linker peptide was designed.

[0085] To evaluate the impact of linking to different regions on the activity of the fusion protein, functional proteins were linked to the N-terminus of PhaC using linkers of varying flexibility, rigidity, and length (Linker1–Linker5, Linker8–Linker9), or the functional proteins were directly fused to PhaC. This approach evaluated the effect of different types of linker binding to the N-terminus of PhaC on the activity of the fusion protease. When fusing functional proteins to the C-terminus of PhaC, the linker peptides needed to be hydrophobic to ensure the activity of the fusion protein and the exposure of the functional protein on the outside of PhaC. Therefore, when designing the linker peptides for the C-terminus of PhaC, only the effect of hydrophobic linkers of different lengths on the activity of the fusion protease was considered (specifically, Linker6 and Linker7). The activity of the fusion protease was then measured after protein expression.

[0086] Example 2 Expression of fusion protein

[0087] Glycerol cultures of strains PHA-S01 to PHA-S10, each containing different linker peptide plasmids constructed in Example 1, were streaked onto LB agar plates (containing 100 mg / L Kan) and incubated overnight at 37°C. Single colonies were picked and transferred to 50 mL LB agar plates and incubated at 37°C and 220 rpm until OD500. 600 The value is 2.5–3.5, based on the actual OD of secondary seeds. 600=0.1 Transfer bacterial culture from the primary shake flask to two 600mL LB (2L Erlenmeyer flasks) secondary shake flasks, and incubate at 37℃ and 220rpm for 2–3 hours. OD 600 The value was 0.6–1.0. IPTG (final concentration 0.3 mM) and glucose (final concentration 1%) were added to each bottle, and the mixture was induced overnight at 30°C for 16 h.

[0088] After fermentation, 5 mL of the bacterial culture was transferred to 10 mL centrifuge tubes for determination of the OD of the harvested bacteria. 600 And SDS-PAGE. Centrifuge the remaining bacterial culture at 8000 rpm for 7 min, collect the bacterial cells from each bottle of culture medium into a resealable bag, weigh the bacterial cells, and store them in a -20℃ refrigerator for later use.

[0089] SDS-PAGE gel electrophoresis: Take 5 mL of bacterial culture from the previous step, centrifuge 150 μL at 12000 rpm for 1 min, discard the supernatant, and resuspend the bacteria in 40 μL of PBS (20 mM, pH 7.0) buffer. Add 10 μL of 5× protein loading buffer. Centrifuge the remaining bacterial culture at 12000 rpm for 5 min, resuspend in 2 mL of PBS (20 mM, pH 7.0) buffer, mix well, and sonicate for 20 min. Take 40 μL of the mixture and add 10 μL of 5× protein loading buffer. Heat all samples in a 100℃ metal bath for 10 min. Load 10 μL of each sample into a 12.5% ​​SDS-PAGE gel.

[0090] from Figures 2-5 The SDS-PAGE gel images clearly show that different linking peptides and linking methods have a significant impact on the expression of the fusion protein. The expression levels of PHA-S08 to S10 are generally slightly lower than those of PHA-S02 to S07, indicating that linking LAL134 to the N-terminus of the PhaC protein is more conducive to protein expression. Among PHA-S02 to S07, linking peptide L3 is more conducive to protein expression.

[0091] Example 3: Enzyme activity assay of ATP-dependent carboxylamine ligase and fusion protein

[0092] Take 0.3g of the centrifuged bacterial cells after induction, and resuspend the cells in 2.7g of 10mM Tris buffer at a ratio of 1:9 (m / m) to prepare a bacterial suspension. Disrupt the suspension by sonication to 40% and then sonicate on ice for 30 min. Prepare the reaction buffer according to Table 1, and adjust the pH of the reaction buffer to 8.5.

[0093] A 25 mL Erlenmeyer flask with a stopper was used as the catalytic reactor, with a liquid volume of 10 mL. First, 9.9 mL of reaction buffer was preheated in a 38°C water bath with shaking for 5 min, then 100 μL of the lysed bacterial suspension was added to initiate the reaction. Samples were taken at 5 min and 15 min of reaction time as the time intervals for enzyme activity assay. Each 100 μL sample was immediately quenched by adding 900 μL of 60% methanol. Each reaction was performed three times, and the average value was used to calculate the enzyme activity.

[0094] The results showed that the enzyme activity of the functional protein LAL134 alone was 967 U / g. The enzyme activity of the fusion protein obtained by direct fusion was severely inhibited, especially when fused directly to the C-terminus of phaC. Direct fusion without a linker peptide as a buffer reduces the fusion resistance of the C-terminus, leading to decreased activity. The enzyme activity of the fusion proteins PHA-S09 and PHA-S10, which have a linker peptide fused to the C-terminus of phaC, indicated that appropriately extending the length of the linker peptide better ensures the activity of the fusion protein. This is because a shorter linker peptide cannot adequately separate the two protein domains, leading to misfolding of the peptide chain during subsequent translation. However, the best fusion method is the fusion of the functional protein to the N-terminus of phaC via a suitable linker peptide. PHA-S05 showed the best activity, and statistical analysis showed no significant difference between PHA-S05 and the control PHA-S01 (P = 0.1309024184 > 0.05), indicating that Linker3 does not inhibit the activity of the fusion protein. Figure 6 ).

[0095] Table 3. Enzyme activation reaction solution preparation table

[0096]

[0097] Table 4. Results of enzyme activity assay for bacterial strains

[0098] sample Enzyme activity U / g PHA-S01 967.0±17.6 PHA-S02 290.7±17.4 PHA-S03 811.7±15.8 PHA-S04 853.9±14.7 PHA-S05 915.7±20.5 PHA-S06 637.8±15.4 PHA-S07 751.4±10.4 PHA-S08 127.4±14.9 PHA-S09 286.5±5.7 PHA-S10 517.2±15.5

[0099] Note: Enzyme activity is the mean ± SE, and the number of replicates n is 3.

[0100] Example 4: Optimization of the L3 linker sequence

[0101] To further optimize Linker3, the best-performing linker peptide in Example 3, the transition sequence (EAAAAK) in the linker peptide was optimized by multiples of 1×, 2×, 3×, and 4× based on the PHA-P05 plasmid. The constructed plasmids containing different linkers were then transformed into the host BL21(DE3), and the enzyme activity was measured in the same manner as in Example 3.

[0102] The results showed that, compared to Linker3 (PHA-S05 enzyme activity of 915.7 U / g), which has a longer linker peptide, appropriately reducing the length of the linker peptide is more conducive to maintaining the function of the fusion protein. However, an excessively short linker peptide can cause the two protein domains to interfere with each other's domain functions. This was achieved by adjusting the length of the linker peptide (A(EAAAAK)). n AL(EAEAAAAK) n The length of the linker peptide in structure A was optimized, and the optimal linker peptide A(EAAAAK)3AL(EAEAAAAK)3A(Linker10) was found. Compared with linkers of other lengths, it had the highest and most stable enzyme activity. Moreover, statistical analysis showed a significant difference between PHA-S05 and the control PHA-S01 (P = 0.0009741591 < 0.001), indicating that the activity of the PHA-S15 fusion protease was significantly higher than that of the individual functional enzyme protease. Figure 7 ).

[0103] Table 5. Results of enzyme activity assay for bacterial strains

[0104] sample Enzyme activity U / g PHA-S01 967.0±17.6 PHA-S05 915.7±20.5 PHA-S13 605.9±10.9 PHA-S14 788.0±10.9 PHA-S15 1167.6±15.0

[0105] Note: Enzyme activity is the mean ± SE, and the number of replicates n is 3.

[0106] Example 5: Application of linking peptides to different functional proteins

[0107] To verify the best-performing linker peptides Linker10 and Linker2 in Examples 3 and 4, we constructed them on different functional proteins in the same manner, such as green fluorescent protein (EGFP). We constructed fusion protein genes with different functions into the pET28a vector using Gibson Assembly technology. The results showed that green fluorescence could be detected in strains PHA-S11 and S12, proving that the fusion proteins linked by the two linker peptides did not affect the normal expression of green fluorescent protein.

[0108] In addition, N-acetylglucosamine-2-isomerase (SA01) and (SA18) genes were used to construct fusion protein genes with different functions into the pET28a vector using Gibson Assembly technology, and their enzyme activity was detected.

[0109] Take 0.4 g of the induced and centrifuged bacterial cells, resuspend the cells in 4 mL of reaction solution to prepare a bacterial suspension, and sonicate the suspension on ice for 30 min to obtain a crude enzyme solution. Centrifuge the solution after sonication and discard the supernatant to obtain a solid enzyme. Prepare the reaction buffer according to Table 3, and adjust the pH of the reaction buffer to 7.8.

[0110] A 50 mL Erlenmeyer flask with a stopper was used as the catalytic reactor, with a liquid volume of 20 mL. For the control reaction, 12 mL of reaction buffer was preheated in a 30°C water bath with shaking for 5 min, followed by the addition of 4 mL each of crude SA01 and SA18 enzyme solutions to initiate the reaction. For the experimental reaction, 16 mL of reaction buffer was preheated in a 30°C water bath with shaking for 5 min, followed by the addition of 4 mg of solid enzyme and 4 mL of crude enzyme solution (methods are shown in Table 4). During the reaction, the pH was maintained at 8.0 using 1 M NaOH. Samples were taken at 5-minute intervals as the time interval for enzyme activity determination. Each sample was 20 μL, and 1980 μL of 60% acetonitrile was immediately added for quenching. Each reaction was performed three times, and the average value was used to calculate the enzyme activity.

[0111] Enzyme activities of SA01 and SA18 were detected, and the results showed that the fusion proteins of the two linker peptides maintained more than half of the catalytic activity of the single functional proteins. However, the linker peptide L3 performed better, and its fusion protein achieved more than 80% of the catalytic activity compared with the independent functional protein.

[0112] This indicates that the linker peptide L3 can be applied to fusion proteins of different functional proteins, ensuring the correct expression of the functional proteins while maintaining most of their catalytic activity.

[0113] Table 6. Enzyme activation reaction solution preparation table

[0114]

[0115] Table 7. Enzyme Activation Reaction System

[0116] strains reaction buffer SA01 SA18 PHA-S13 12mL 4mL 4mL PHA-S14 16mL 4mg 4mL PHA-S15 16mL 4mg 4mL PHA-S16 16mL 4mL 4mg PHA-S17 16mL 4mL 4mg

[0117] Table 8. Results of enzyme activity assay for bacterial strains

[0118] strains Enzyme activity U / g PHA-S16 1154 PHA-S17 963.9 PHA-S18 1057.7 PHA-S19 916.4 PHA-S20 976.6

[0119] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A linker peptide used in PHA polymerase immobilization, characterized in that, The amino acid sequence is shown in SEQ ID NO:

22.

2. A PHA polymerase immobilization system, characterized in that, It includes PHA polymerase, the linker peptide as described in claim 1, and the functional protein.

3. The PHA polymerase immobilization system according to claim 2, characterized in that, The connection relationship between the PHA polymerase, the linker peptide of claim 1, and the functional protein includes: PHA polymerase – the linker peptide – functional protein as described in claim 1; and / or Functional protein - the linker peptide-PHA polymerase as described in claim 1.

4. The PHA polymerase immobilization system according to claim 3, characterized in that, The functional protein includes at least one of ATP-dependent carboxylamine ligase, fluorescent protein, N-acetylglucosamine-2-isomerase and / or N-acetylneuraminic acid lyase.

5. The PHA polymerase immobilization system according to claim 4, characterized in that, include: ATP-dependent carboxylamine ligase – a ligase-PHA polymerase with an amino acid sequence as shown in SEQ ID NO:22; and / or PHA polymerase – linker peptide with amino acid sequence as shown in SEQ ID NO:22 – fluorescent protein; and / or N-acetylglucosamine-2-isomerase-linked peptide-PHA polymerase with an amino acid sequence as shown in SEQ ID NO:22; and / or N-acetylneuraminic acid lyase - amino acid sequence as shown in SEQ ID NO:22 - linker peptide-PHA polymerase.

6. Nucleic acid, characterized in that, Includes nucleic acids encoding the linker peptide of claim 1 and / or the PHA polymerase immobilization system of any one of claims 2 to 5.

7. An expression unit, characterized in that, It includes the expression element and the nucleic acid as described in claim 6.

8. A recombinant vector, characterized in that, It includes the nucleic acid as described in claim 6 or the expression unit and vector backbone as described in claim 7.

9. A host cell, characterized in that, Transfection or transformation of the recombinant vector as described in claim 8.

10. The use of at least one of the following (I) to (VI) in enzyme-catalyzed reactions and / or biosynthesis: I) The linker peptide according to claim 1; II) The PHA polymerase immobilization system according to any one of claims 2 to 5; III) The nucleic acid as described in claim 6; IV) The expression unit as described in claim 7; V), the recombinant vector according to claim 8; VI) The host cell as described in claim 9.

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