PHA polymerase immobilized connecting peptide and PHA polymerase immobilization method

Abstract

The invention relates to the technical field of biology, in particular to a connecting peptide for PHA polymerase immobilization and a PHA polymerase immobilization method. The invention provides the connecting peptide used for PHA polymerase immobilization, a PHA polymerase immobilization connecting system is optimized, and experimental results show that under the connecting peptide and the PHA polymerase immobilization connecting system, correct expression of functional protein can be guaranteed, and meanwhile most catalytic activity of the functional protein can be maintained.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a connecting peptide for immobilizing PHA polymerase and a method for immobilizing PHA polymerase. Background Art

[0002] Polyhydroxyalkanoates (PHA) are a series of biopolyesters synthesized by microorganisms. When carbon sources are excessive and nitrogen and / or phosphorus sources are limited, they accumulate as intracellular particles to store carbon and energy. There are three main enzymes involved in its biosynthesis process, namely β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB) and PHA polymerase (PhaC).

[0003] PhaC can immobilize the enzyme by connecting it to form a fusion protein, thereby improving the biological stability of the enzyme, making it easy to separate from the reaction system, and easy to control. Fusion proteins, as artificially constructed multi-domain proteins, have various problems in expression compared to natural proteins, such as reduced expression, incorrect folding of the three-dimensional protein structure, and inhibition of biological activity. The emergence of this problem is mainly due to the mutual influence of the two adjacent domains, resulting in damage to the protein structure at both ends. Therefore, the design and selection of the connecting peptide as the buffer region between the two different domains is particularly important. At present, a connecting peptide that can be used in most fusion proteins has not been found. In actual application scenarios, it is impossible to optimize the design of the connecting peptide for each fusion protein. This is not only time-consuming and labor-intensive in operation, but also has limited effects. Therefore, it is of great research significance to try to dig out a connecting peptide that can be used in the immobilization of PHA polymerase, which is universal and can fully maintain the activity of the enzyme. Summary of the invention

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

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

[0006] The present invention obtains a connecting peptide suitable for PHA polymerase immobilization through screening. The connecting peptide is used in the immobilization of the PHA polymerase to achieve enzyme immobilization on the basis of maintaining enzyme activity; wherein, the connecting peptide with an amino acid sequence such as SEQ ID NO: 6 is Linker 2, and the connecting peptide with an amino acid sequence such as SEQ ID NO: 22 is Linker 10, wherein Linker 10 has the best effect of being used for PHA polymerase immobilization.

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

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

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

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

[0011] In the present invention, the functional protein comprises at least one of ATP-dependent carboxylic acid amine 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 carboxylic acid amine ligase-connector peptide with amino acid sequence as shown in SEQ ID NO:6-PHA polymerase; and / or

[0014] ATP-dependent carboxylic acid amine ligase-connector peptide with amino acid sequence as shown in SEQ ID NO:22-PHA polymerase; and / or

[0015] PHA polymerase-connector peptide having an amino acid sequence as shown in SEQ ID NO:6-ATP-dependent carboxylic acid amine ligase; and / or

[0016] PHA polymerase-connector peptide having an amino acid sequence as shown in SEQ ID NO: 22-ATP-dependent carboxylic acid amine ligase; and / or

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

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

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

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

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

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

[0023] The present invention develops a PHA polymerase immobilization system, which connects the PHA polymerase and the functional protein through the connecting peptide described in the present invention. The connection mode of the PHA polymerase immobilization system is optimized based on the screening of suitable connecting peptides. In a specific embodiment of the present invention, ATP-dependent carboxylic acid amine ligase (LAL134) is taken as an example to prove that in the PHA polymerase immobilization system, the functional protein expressed by connecting in the connection mode of functional protein-connecting peptide-PHA polymerase has the highest activity.

[0024] At the same time, the present invention expresses different enzymes by connecting the connecting peptide of the present invention to the PHA polymerase, proving that the connecting peptide of the present invention has universality and can ensure the correct expression of functional proteins while maintaining most of their catalytic activity.

[0025] The present invention provides a nucleic acid, which includes a nucleic acid encoding the connecting peptide of the present invention and / or the PHA polymerase immobilization system of the present invention.

[0026] The nucleic acid described in the present invention can be DNA, RNA, cDNA or PNA. In an embodiment of the present 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. 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). Nucleic acid can be linear or circular in topology. Nucleic acid can be a part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0027] In the present invention, the nucleic acid may be optimized or unoptimized, and the optimization includes but is not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites 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 comprises an expression element and the nucleic acid of the present invention; the expression element includes but is not limited to a promoter, a terminator, an enhancer and / or an expression tag, etc.

[0029] The present invention provides a recombinant vector, which comprises at least one of the nucleic acid of the present invention or the expression unit of the present invention and a vector backbone.

[0030] Furthermore, the sources of the recombinant vector of the present invention include plants, animals, bacteria, fungi, bacteriophages and / or viruses, which are not limited in the present invention. In a specific embodiment of the present invention, the recombinant vector is derived from bacteria, specifically Escherichia coli.

[0031] Recombinant vector of the present invention refers to the nucleic acid vector of recombinant, is a kind of recombinant DNA molecule, it comprises the desired coding sequence and the expression of the coding gene in the concrete host organism that is requisite suitable nucleotide sequence or element that can be operably connected.In this manual, "plasmid" and "vector" can be exchanged and general sometimes, because plasmid is the most commonly used vector form at present.But the present invention is intended to include such other forms of expression vector, it plays equivalent effect, it is known or will become known in the art, includes but not limited to: plasmid, phage particle, viral vector and / or is only potential genome insert.In a specific embodiment, the nucleic acid encoding the fusion protein provided by the present invention can be constructed in various prokaryotic expression vectors, specifically including pET28a.

[0032] The present invention provides a host cell transformed or transfected with the recombinant vector. The host cell is transformed or transfected with a vector constructed using recombinant DNA technology, so that the transformed host cell has the ability to replicate the protein-encoding vector or express the desired protein.

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

[0034] The host cell provided by the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages or viruses, which are not limited by the present invention. In a specific embodiment of the present invention, the host cell is a bacterium, specifically Escherichia coli, more specifically Escherichia coli DH5α and / or BL21 (DE3).

[0035] The present invention provides the use of at least one of the following I) to V) in enzyme catalysis reaction and / or biosynthesis:

[0036] 1), the connecting peptide of the present invention;

[0037] II), the PHA polymerase immobilization system of the present invention;

[0038] III), the nucleic acid of the present invention;

[0039] IV), the expression unit of the present invention;

[0040] V), the recombinant vector of the present invention;

[0041] VI), the host cell of the present invention.

[0042] The present invention provides a connecting peptide for PHA polymerase immobilization and optimizes the PHA polymerase immobilization connection system. Experimental results show that the connecting peptide and the PHA polymerase immobilization connection system of the present invention can ensure the correct expression of the functional protein while maintaining most of its catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram showing the production and purification method of polyhydroxyalkanoate (PHA) immobilized enzyme;

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

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

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

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

[0048] Figure 6 Table 4 shows the results of enzyme activity determination of strains;

[0049] Figure 7 Table 5 shows the enzyme activity determination of strains. DETAILED DESCRIPTION

[0050] The present invention provides a connecting peptide for immobilizing PHA polymerase and a method for immobilizing PHA polymerase. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0051] Polyhydroxyalkanoate (PHA) biosynthesis Figure 1 As shown;

[0052]

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

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

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

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

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

[0058] Connecting peptide 3 nucleotide sequence: gctgaagcagctgctgcgaaagaggcagccgcggcgaaagaggcagctgctgcgaaggaggcagccgcggcgaaggcactggaag cggaagcagcggcggcgaaagaagccgcggcggcgaaggaggccgcggctgccaaggaggccgctgctgcgaaagca (SEQ ID NO: 7);

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

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

[0061] Connector peptide 4 amino acid sequence: PAPAP (SEQ ID NO: 10);

[0062] Connector peptide 5 nucleotide sequence: ccggcgccggcgccggcgccggcgccggcgccg (SEQ ID NO: 11);

[0063] Connector peptide 5 amino acid sequence: PAPAPAPAPAP (SEQ ID NO: 12);

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

[0065] Connector peptide 6 amino acid sequence: VLAVIDKRGGGGG (SEQ ID NO: 14);

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

[0067] Connector peptide 7 amino acid sequence: VLAVIDKRGGGGGSGGGSGGGSGGGGS (SEQ ID NO: 16);

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

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

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

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

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

[0073] Connector peptide 10 amino acid sequence: AEAAAAKEAAAAKEAAAAKALEAEAAAAKEAAAAKEAAAAKA (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 the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0083] Example 1 Construction of Connecting Peptide in Fusion Protein

[0084] The main research objects of this example are polyhydroxy fatty acid synthase (PhaC) and ATP-dependent carboxylic acid amine ligase (ATP-grasp enzymes). ATP-dependent carboxylic acid amine ligase (ATP-grasp enzymes) can catalyze the formation of amide bonds by activating carboxylic acids as acyl phosphate intermediates accompanied by the hydrolysis of ATP, and are important participants in the synthesis of short oligopeptides. In this example, the dipeptide alanylphenylalanine (Ala-Phe) was catalyzed by the ATP-dependent carboxylic acid amine ligase LAL134, but in the actual catalytic synthesis of Ala-Phe dipeptide, it was found that the crude enzyme solution would cause the dipeptide product to decrease continuously with the extension of reaction time. In order to solve similar phenomena, a connecting peptide was designed;

[0085] In order to evaluate the effect of connection with different regions on the activity of the fusion protein, the functional proteins were connected to the N-terminus of PhaC through connecting peptides of different flexibility, rigidity and length (Linker1~Linker5, Linker8~Linker9), or the functional proteins were directly fused with PhaC to evaluate the effect of the binding between different types of connecting peptides and the N-terminus of PhaC on the activity of the fusion protein. When the functional protein was fused with the C-terminus of PhaC, in order to ensure the activity of the fusion protein and the exposure of the functional protein on the outside of PhaC, the design of the connecting peptide needed to have a certain hydrophobicity. Therefore, when designing the connecting peptide at the C-terminus of PhaC, only the effect of the hydrophobic connecting peptides of different lengths on the activity of the fusion protein was considered (specifically Linker6 and Linker7), and the fusion protein activity was detected after protein expression.

[0086] Example 2 Expression of fusion protein

[0087] Take the glycerol bacterial suspensions of strains PHA-S01 to PHA-S10 containing different connecting peptide plasmids constructed in Example 1 and streak them on LB plates (containing 100 mg / L Kan) and culture them at 37°C overnight; pick a single clone and transfer it to 50 mL LB and culture it at 37°C 220 rpm until OD 600 2.5~3.5, according to the actual OD of the secondary seed 600= 0.1. Transfer the bacterial solution from the primary shake flask to two 600 mL LB (2 L conical flask) secondary shake flasks and culture at 37 °C 220 rpm for 2-3 hours. 600 The value is 0.6 to 1.0. IPTG (final concentration 0.3 mM) and glucose (final concentration 1%) were added to each bottle and induced at 30°C overnight for 16 hours.

[0088] After the fermentation, 5 mL of bacterial solution was pipetted into 10 mL centrifuge tubes for measuring the OD of the harvested bacteria. 600 The remaining bacterial solution was centrifuged at 8000 rpm for 7 min, and the bacteria in each bottle of culture medium were collected in self-sealing bags, weighed, and stored in a -20°C refrigerator for later use.

[0089] SDS-PAGE gel electrophoresis: Take 5mL of bacterial solution from the previous step, take 150μL first, centrifuge at 12000rpm for 1min, remove the supernatant, add 40μL PBS (20mM, pH 7.0) buffer to resuspend the bacteria, add 10μL 5× protein loading buffer; after centrifuging the remaining bacterial solution at 12000rpm for 5min, add 2mL PBS (20mM, pH 7.0) buffer to resuspend and mix, then break in an ultrasonic disruptor for 20min, take 40μL, add 10μL 5× protein loading buffer, and heat all samples in a 100℃ metal bath for 10min. Take 10μL of all samples and load them on 12.5% ​​SDS-PAGE.

[0090] from Figure 2 to Figure 5 It can be clearly seen from the SDS-PAGE electrophoresis gel image that different connecting peptides and connection methods have a great influence on the expression of the fusion protein. The overall expression level of PHA-S08~S10 is slightly lower than that of PHA-S02~S07, which proves that connecting LAL134 to the N-terminus of the PhaC protein is more conducive to protein expression; in PHA-S02~S07, the connecting peptide L3 is more conducive to protein expression.

[0091] Example 3 Determination of the Enzyme Activity of ATP-Dependent Carboxylic Amine Ligase and Fusion Protein

[0092] Take 0.3 g of the centrifuged bacteria after induction, resuspend the bacteria in 2.7 g of 10 mM Tris buffer at 1:9 (m / m) to prepare a bacterial suspension, and ultrasonically disrupt the bacterial suspension at 40% and ultrasonicate it on ice for 30 minutes. Prepare the reaction buffer according to Table 1, and adjust the pH of the reaction buffer to 8.5.

[0093] A 25mL stoppered Erlenmeyer flask was used as the catalytic reactor, with a liquid volume of 10mL. First, 9.9mL of reaction buffer was preheated in a 38°C water bath incubator for 5 minutes, and then 100μL of the broken bacterial suspension was added to start the reaction. After that, samples were taken at 5min and 15min of reaction time, respectively, as the time interval for enzyme activity determination. 100μL was sampled each time, and 900μL of 60% methanol was immediately added for quenching. Each reaction was performed three times to take the average value and calculate the enzyme activity.

[0094] According to the results, the enzyme activity of the functional protein LAL134 alone is 967U / g. The enzyme activity of the fusion protein obtained by direct fusion is severely inhibited, especially when it is directly fused with the C-terminus of phaC. Direct fusion without a connecting peptide as a buffer will reduce the fusion resistance of the C-terminus and lead to decreased activity. The enzyme activity of the fusion proteins PHA-S09 and PHA-S10 fused with the C-terminus of phaC in the presence of a connecting peptide shows that appropriately extending the length of the connecting peptide can better ensure the activity of the fusion protein. This is because the shorter connecting peptide cannot fully separate the two protein domains and causes the subsequent translated peptide chain to fold incorrectly. However, the best fusion method is to fuse the functional protein with the N-terminus of phaC through a suitable connecting peptide. Among them, PHA-S05 has the best activity. Moreover, after statistical analysis, there is no significant difference in the data between PHA-S05 and the control PHA-S01 (P=0.1309024184>0.05), indicating that Linker3 will not inhibit the activity of the fusion protein ( Figure 6 ).

[0095] Table 3. Enzyme activity reaction solution configuration table

[0096]

[0097] Table 4. Results of strain enzyme activity determination

[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: The enzyme activity is the mean ± SE, and the number of repetitions n is 3;

[0100] Example 4 Optimization of the connecting peptide L3 sequence

[0101] In order to further optimize the best performing linker peptide Linker3 in Example 3, based on the PHA-P05 plasmid, the sequence length of the transition sequence (EAAAAK) in the linker peptide was optimized according to 1×, 2×, 3×, and 4× multiples, respectively. The constructed plasmids containing different linkers were respectively transferred into the host BL21 (DE3), and the enzyme activity was determined in the same way as in Example 3.

[0102] According to the results, compared with Linker3 with a longer transition peptide (PHA-S05 enzyme activity of 915.7U / g), appropriately reducing the length of the connecting peptide will be more conducive to maintaining the function of the fusion protein. However, too short a connecting peptide will cause the two protein domains to affect each other's domain functions. n AL(EAEAAAAK) n The length of the linker peptide of the A structure was optimized, and the optimal linker peptide A(EAAAAK)3AL(EAEAAAAK)3A(Linker10) was found. Compared with linkers of other lengths, its enzyme activity was the highest and most stable. Moreover, statistical analysis showed that there was a significant difference between PHA-S05 and the control PHA-S01 in the data (P=0.0009741591<0.001), indicating that the protease activity of PHA-S15 fusion protein was significantly higher than that of the single enzyme proteinase ( Figure 7 ).

[0103] Table 5. Results of enzyme activity assay of 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: The enzyme activity is the mean ± SE, and the number of repetitions n is 3;

[0106] Example 5 Application of Connector Peptides on Different Functional Proteins

[0107] In order 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 way, such as green fluorescent protein (EGFP) by Gibson Assembly technology to construct fusion protein genes with different functions into the pET28a vector. The results showed that both strains PHA-S11 and S12 were able to detect green fluorescence, proving that the fusion proteins connected 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 constructed into fusion protein genes with different functions by Gibson Assembly technology and inserted into pET28a vector to detect their enzyme activities.

[0109] Take 0.4 g of the centrifuged bacteria after induction, resuspend the bacteria in 4 mL of reaction solution to prepare a bacterial suspension, use ultrasonic disruption at 40%, ultrasonicate the bacterial suspension on ice for 30 min to obtain a crude enzyme solution, centrifuge after ultrasonication 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 50mL stoppered triangular flask was used as a catalytic reactor, and the liquid volume was 20mL. For the control reaction, 12mL of reaction buffer was preheated in a 30°C water bath shaking incubator for 5min, and then 4mL of SA01 and SA18 crude enzyme solutions were added to start the reaction. For the experimental reaction, 16mL of reaction buffer was preheated in a 30°C water bath shaking incubator for 5min, and then 4mg of solid enzyme and 4mL of crude enzyme solution were added to react (method as shown in Table 4). During the reaction, 1M NaOH was used to control pH 8.0, and then samples were taken at 5min as the time interval for enzyme activity determination. 20μL was sampled each time, and 1980μL of 60% acetonitrile was immediately added for quenching. Each reaction was performed three times to take the average value, and the enzyme activity was calculated.

[0111] The enzyme activities of SA01 and SA18 were tested, and the results showed that the fusion proteins of the two connecting peptides retained more than half of the catalytic activity of the single functional proteins, but the connecting peptide L3 performed better. Compared with the independent functional proteins, its catalytic activity of the connected fusion protein could reach more than 80%.

[0112] This indicates that the connecting peptide L3 can be applied to fusion proteins of different functional proteins, which can not only ensure the correct expression of functional proteins but also maintain most of their catalytic activity.

[0113] Table 6. Enzyme activity reaction solution configuration table

[0114]

[0115] Table 7. Enzyme activity reaction system

[0116] strain 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 strain enzyme activity determination

[0118] strain 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 only preferred embodiments 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A connecting peptide for use in immobilizing PHA polymerase, characterized in that: The amino acid sequence is shown in SEQ ID NO:6 and / or SEQ ID NO:

22.

2. PHA polymerase immobilization system, characterized in that: The method comprises PHA polymerase, at least one of the connecting peptides described in claim 1 and a functional protein.

3. The PHA polymerase immobilization system according to claim 2, characterized in that: The connection relationship between the PHA polymerase, at least one of the connecting peptides according to claim 1, and the functional protein comprises: PHA polymerase - at least one of the connecting peptides according to claim 1 - a functional protein; and / or Functional protein: at least one of the connecting peptides described in claim 1; PHA polymerase.

4. The PHA polymerase immobilization system according to claim 3, characterized in that: The functional protein comprises 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 carboxylic acid amine ligase-connector peptide with amino acid sequence as shown in SEQ ID NO:6-PHA polymerase; and / or ATP-dependent carboxylic acid amine ligase-connector peptide with amino acid sequence as shown in SEQ ID NO:22-PHA polymerase; and / or PHA polymerase-connector peptide having an amino acid sequence as shown in SEQ ID NO:6-ATP-dependent carboxylic acid amine ligase; and / or PHA polymerase-connector peptide having an amino acid sequence as shown in SEQ ID NO: 22-ATP-dependent carboxylic acid amine ligase; and / or PHA polymerase-amino acid sequence as shown in SEQ ID NO: 6 connecting peptide-fluorescent protein; and / or PHA polymerase-amino acid sequence as shown in SEQ ID NO: 22 connecting peptide-fluorescent protein; and / or N-acetylglucosamine-2-isomerase-connector peptide with amino acid sequence as shown in SEQ ID NO:6-PHA polymerase; and / or N-acetylglucosamine-2-isomerase-connector peptide with amino acid sequence as shown in SEQ ID NO:22-PHA polymerase; and / or N-acetylneuraminic acid lyase-connector peptide with amino acid sequence as shown in SEQ ID NO:6-PHA polymerase; and / or N-acetylneuraminic acid lyase-connector peptide with the amino acid sequence shown in SEQ ID NO: 22-PHA polymerase.

6. A nucleic acid, characterized in that It comprises a nucleic acid encoding the connecting peptide according to claim 1 and / or the PHA polymerase immobilization system according to any one of claims 2 to 5.

7. An expression unit, characterized in that Comprising an expression element and the nucleic acid according to claim 6.

8. A recombinant vector, characterized in that It comprises at least one of the nucleic acid according to claim 6 or the expression unit according to claim 7 and a vector backbone.

9. A host cell, characterized in that Transfect or transform the recombinant vector as described in claim 8.

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

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