A method for constructing immobilized enzyme to synthesize sulfonate donor PAPS based on self-assembly strategy

Abstract

The present invention discloses a method for constructing immobilized enzymes to synthesize sulfonate donor PAPS based on a self-assembly strategy, belonging to the field of biotechnology. The present invention provides a method for synthesizing bifunctional enzymes and ADP phosphorylase by using aggregating proteins to self-assemble PAPS, realizing the immobilization of enzymes for synthesizing PAPS. By further rationally designing and modifying the linker, the conversion efficiency is greatly improved. The present invention simultaneously realizes the co-immobilization of PAPS-synthesizing bifunctional enzymes and ADP phosphorylase through self-assembling peptides, directly breaks the cell wall and adds centrifugal precipitation to achieve one-step synthesis of PAPS. The immobilized PAPS-synthesizing bifunctional enzyme still has an ATP conversion rate of 45% after being recycled ten times, which is about 50% of that in the first use. This method simplifies the enzyme separation and purification steps, improves the utilization efficiency of enzymes, and significantly reduces the cost of industrial PAPS production.

Description

Technical Field

[0001] The present invention relates to a method for constructing an immobilized enzyme for synthesizing a sulfonate donor PAPS based on a self-assembly strategy, belonging to the field of biotechnology. Background Art

[0002] Enzymes are green and efficient biocatalysts, which are widely used in the production of high-value-added products in industry. At present, the main problems existing in the use of industrial enzyme preparations are problems in their production, separation, and application processes. Among them, the production process of enzyme preparations is too complex; the purification process is too cumbersome and costly; a series of problems such as the inability to reuse enzyme preparations have become the main limiting factors for the industrial production of high-value-added products by enzymatic methods. Since free enzymes are continuously consumed during the catalytic process, it is difficult to recover or impossible to recover them, resulting in great waste. In addition, in the actual industrial application process, there are still problems such as high purification cost, poor stability, and inability to reuse enzymes. Therefore, reducing the production and separation cost of enzymes, improving the stability of enzymes, and solving the problem of enzyme recovery are the keys to improving the industrial properties of enzymes and realizing their efficient industrial transformation. Applying enzymes through enzyme immobilization technology is a method to break through these limitations.

[0003] Enzyme immobilization technology is a kind of technology that binds enzymes in a certain space through physical, chemical, or biological means, retains most of their catalytic activities, and improves their stability and the number of times of reuse. The traditional preparation methods of immobilized enzymes are divided into two categories: physical methods and chemical methods. Physical methods include physical adsorption methods, embedding methods, etc. The adsorption method is based on physical adsorption on the solid surface to make the enzyme contact with the insoluble carrier to achieve the purpose of immobilization; the embedding method realizes the immobilization of the enzyme by encapsulating the enzyme in a polymer network or semi-permeable membrane. The advantages of immobilizing enzymes by physical methods are simple operation, large enzyme loading, and the enzyme does not participate in chemical reactions, and the structure and catalytic activity of the enzyme will not be affected. However, in the adsorption method, since no chemical bond is formed between the carrier and the enzyme, the binding between the enzyme and the carrier is not stable. In the embedding method, due to the spatial hindrance effect of the embedding material on the exchange of substrates and products, it is not applicable to some reactions. Chemical methods include binding methods and cross-linking methods. The binding method is to connect the enzyme to the carrier by forming a chemical bond; while the cross-linking method cross-links the groups on the enzyme surface through a cross-linking agent to form a macromolecular insoluble immobilized enzyme. Immobilized enzymes prepared by chemical methods have good thermal stability and storage properties. However, enzyme inactivation is easily caused during chemical modification.

[0004] Bio-immobilization is a new type of enzyme immobilization method, including whole-cell catalysis and active aggregation of enzymes. Active aggregation uses some short peptides (aggregation tags) and aggregation proteins or their combination to form macromolecular insoluble immobilized enzymes within cells. Inclusion bodies have long been regarded as misfolded and inactive protein aggregates. However, studies have shown that the binding of the target protein to some tags can promote the generation of active inclusion bodies, which consist of short peptide chains containing several amino acids and aggregation-induced tags. Active inclusion bodies have many advantages, such as high enzyme activity retention, simple purification, high stability, easy long-term storage, reusability, and being basically free of transgenes after separation from production cells. It is a carrier-free, bioproducible, and biodegradable immobilization technology.

[0005] 3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is a derivative of adenosine triphosphate (ATP), the activated form of sulfonic acid groups, and the only known active sulfonic acid group donor. It plays an important role in the biosynthesis of compounds such as chondroitin sulfate, heparin, and dermatan sulfate that become active after sulfonation modification. ADP phosphorylase refers to an enzyme capable of phosphorylating ADP to generate ATP, including polyphosphate kinase (ACS Catal. 2021, 11(16): 10405–10415) and polyphosphate exohydrolase (Enzyme Res. 2015; 2015: 404607.). ADP phosphorylase can simultaneously utilize the by-products ADP and pyrophosphate PPi during the synthesis of PAPS. When used in combination with the PAPS synthase dual-functional enzyme, it can achieve the equimolar synthesis of the product PAPS from the substrate ATP.

[0006] Due to the excessively high preparation cost of the enzymes for the current enzymatic synthesis of PAPS, it directly leads to the high price of PAPS, thus restricting its application. Summary of the Invention

[0007] Currently, for the in vitro enzymatic synthesis of PAPS, the enzymes in the process need to be purified and catalyzed using pure enzymes. There are disadvantages such as high enzyme separation and purification costs, enzyme instability during the catalytic process, and inability to be reused, which are not conducive to large-scale production. In the present invention, the artificial PAPS synthase and ADP phosphorylase in the PAPS synthesis process are immobilized by a bio-self-assembly method for the enzymatic synthesis of PAPS. Using ATP and sulfate as substrates, the immobilized enzymes catalyze the synthesis of PAPS, improving the enzyme utilization efficiency and reducing production costs.

[0008] Based on previous research, the present invention provides a method for synthesizing PAPS based on constructing immobilized enzymes, using different aggregation proteins to achieve the bifunctional enzyme for PAPS synthesis (ASAKS5 ) and the intracellular self-aggregation and assembly of ADP phosphorylase (PaPPX) to achieve enzyme immobilization. Among them, the PAPS synthetic bifunctional enzyme (ASAK S5 ) catalyzes Mg-ATP and SO4 2- to generate the product PAPS and by-products ADP and PPi. ADP phosphorylase catalyzes the by-products to generate Mg-ADP and PPi to generate ATP. Therefore, the two-enzyme cascade catalysis can achieve equimolar substrate ATP and product PAPS.

[0009] This method of immobilized enzyme-catalyzed synthesis of PAPS simplifies the steps of enzyme separation and purification in actual production, significantly improves the stability and reusability of the enzyme, and greatly reduces the production cost.

[0010] The present invention provides an immobilized enzyme, which is obtained by connecting the aggregation protein CipA or CipB derived from Photobacterium to the N-terminus or C-terminus of the enzyme protein through any one of the linker peptides shown in SEQ ID NOs. 11 to 15; the enzyme protein is the PAPS synthetic bifunctional enzyme with the amino acid sequence shown in SEQ ID NO. 16 and the ADP phosphorylase with the amino acid sequence shown in SEQ ID NO. 17, respectively, to obtain the PAPS synthetic bifunctional enzyme immobilized enzyme and the ADP phosphorylase immobilized enzyme.

[0011] In one embodiment of the present invention, the ADP phosphorylase is derived from Pseudomonas aeruginosa, and its Gene ID is 877947.

[0012] In one embodiment of the present invention, the Gene ID of the aggregation protein CipA derived from Photobacterium is 155404.

[0013] In one embodiment of the present invention, the Gene ID of the aggregation protein CipB derived from Photobacterium is 1890126.

[0014] In one embodiment of the present invention, it is obtained by connecting the aggregation protein CipA or CipB derived from Photobacterium to the N-terminus or C-terminus of the enzyme protein through any one of the linker peptides shown in SEQ ID NOs. 11 to 15.

[0015] In one embodiment of the present invention, the nucleotide sequence encoding the PAPS synthetic bifunctional enzyme is shown in SEQ ID NO. 18.

[0016] In one embodiment of the present invention, the nucleotide sequence encoding the ADP phosphorylase is shown in SEQ ID NO. 19.

[0017] The present invention also provides a gene encoding the above-mentioned immobilized enzyme.

[0018] The present invention also provides a recombinant vector carrying the above-mentioned gene.

[0019] In one embodiment of the present invention, the recombinant vector uses pET28a(+) and pCDF-Duet-1 as expression vectors.

[0020] The present invention also provides a recombinant cell expressing the above-mentioned immobilized enzyme or carrying the above-mentioned gene or carrying the above-mentioned recombinant vector.

[0021] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0022] The present invention also provides a recombinant Escherichia coli that expresses the above-mentioned immobilized enzyme.

[0023] In one embodiment of the present invention, the recombinant Escherichia coli uses E. coli BL21(DE3) as an expression host.

[0024] In one embodiment of the present invention, the recombinant Escherichia coli uses pET28a as an expression vector.

[0025] The present invention provides a method for synthesizing 3′-phosphoadenosine-5′-phosphosulfate. The method uses ATP and MgSO4 as substrates and uses the above-mentioned immobilized enzyme or the above-mentioned recombinant cell to catalytically convert and synthesize 3′-phosphoadenosine-5′-phosphosulfate.

[0026] The present invention provides an enzyme immobilization technology for synthesizing PAPS. The immobilization of the PAPS-synthesizing bifunctional enzyme and ADP phosphorylase is respectively achieved by different self-assembling peptides, and they are added separately to achieve one-pot dual-enzyme cascade catalysis for synthesizing PAPS. After each reaction, the enzyme is collected by low-temperature centrifugation and then used for the next catalysis.

[0027] In one embodiment of the present invention, the co-immobilization of the PAPS-synthesizing bifunctional enzyme and the PAPS-synthesizing bifunctional enzyme is simultaneously achieved by a self-assembling peptide. The cell wall is directly broken and the centrifugal precipitate is added to achieve one-step synthesis of PAPS. And it is collected by low-temperature centrifugation for the next catalysis.

[0028] In one embodiment of the present invention, the immobilized PAPS-synthesizing bifunctional enzyme and ADP phosphorylase are obtained by respectively connecting the aggregating protein with them through a fusion linker sequence.

[0029] In one embodiment of the present invention, the aggregating protein and the short peptide are fused to the N-terminus and C-terminus of the PAPS-synthesizing bifunctional enzyme and ADP phosphorylase.

[0030] In one embodiment of the present invention, the fusion linker sequence is as shown in SEQ ID NO.11-15.

[0031] In one embodiment of the present invention, the fusion linker sequence may be a repetitive sequence, and the number of repetitions is greater than or equal to 10 times.

[0032] In one embodiment of the present invention, the artificial PAPS-synthesizing bifunctional enzyme is from our laboratory. It is formed by fusing the ATP sulfurylase of Saccharomyces cerevisiae and the APS kinase of Escherichia coli through a flexible linker sequence;

[0033] In one embodiment of the present invention, the ADP phosphorylase refers to an enzyme that can phosphorylate ADP to generate ATP, including polyphosphate kinase PPK and polyphosphate exohydrolase PPX.

[0034] In one embodiment of the present invention, the polyphosphate exohydrolase is EC 3.6.1.11, and its sources include but are not limited to Pseudomonas aeruginosa, and its Gene ID is 877947.

[0035] In one embodiment of the present invention, the aggregatin is derived from natural microorganisms or is artificially designed.

[0036] The aggregatin CipA derived from Photorhabdus luminescens has a Gene ID of 155404;

[0037] The aggregatin CipB derived from Photorhabdus luminescens has a Gene ID of 1890126;

[0038] The aggregatin Aβ42 from amyloid-β precursor protein has a Gene ID of 112927;

[0039] The self-assembling proteins are 18A, L6KD, HVdoT, GFIL8, NSPdoT, DMdoT, ELK16, CipA, CipB, Aβ42, and preferably CipA or CipB.

[0040] The aggregating short peptide EAK16 derived from Saccharomyces cerevisiae comes from the Gene ID of 1806634842 of Saccharomyces cerevisiae zuotin.

[0041] The artificially designed aggregating short peptides 18A, L6KD, GFIL8, NSPdoT, HVdoT, DMdoT have sequences as shown in SEQ ID NO:1-6.

[0042] In one embodiment of the present invention, the aggregating short peptide EAK16 derived from Saccharomyces cerevisiae was mutated, and all A's were replaced with L's to enhance the hydrophobicity of the short peptide.

[0043] In one embodiment of the present invention, the number of cycles of the immobilized PAPS synthetic bifunctional enzyme is at least 10 times.

[0044] In one embodiment of the present invention, the buffer solution in the reaction system is 10 - 50 mM Tris-HCl, with a pH of 6.0 - 8.0, and 2 - 20 mM ATP, 4 - 40 mM magnesium sulfate, 2 - 200 mM sodium sulfate, 0.5 - 1.0 mg / mL of immobilized PAPS synthetic bifunctional enzyme, and ADP phosphorylase are added.

[0045] In one embodiment of the present invention, the catalytic reaction temperature is 20 - 40 °C, and the catalytic time is 2 - 10 h.

[0046] In one embodiment of the present invention, one or more of the protectants maltose, mannose, rhamnose, glucose, sucrose, sorbitol, glycerol, and BSA are added to the catalytic system. Among them, the addition amounts of maltose, mannose, rhamnose, glucose, and sucrose are 10 - 50 mg / mL in final concentration; the addition amount of glycerol is 10 - 50 mL / mL (v / v) in final concentration; and the addition amount of BSA is 20 - 100 mg / mL in final concentration.

[0047] The present invention also protects the application of the method for synthesizing 3'-phosphoadenosine-5'-phosphosulfate by the immobilized enzyme, or the method for increasing the synthesis amount of 3'-phosphoadenosine-5'-phosphosulfate in the synthesis of 3'-phosphoadenosine-5'-phosphosulfate.

[0048] The present invention also provides the application of the above-mentioned immobilized enzyme, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned method in the synthesis of 3'-phosphoadenosine-5'-phosphosulfate, or in related products produced using 3'-phosphoadenosine-5'-phosphosulfate.

[0049] Beneficial effects

[0050] 1. By screening different aggregation proteins and aggregation short peptides, the present invention realizes the intracellular self-assembly of PAPS synthetic bifunctional enzyme and ADP phosphorylase, and realizes the biological immobilization of PAPS synthetic bifunctional enzyme and ADP phosphorylase.

[0051] 2. By optimizing the binding positions of the aggregation protein and aggregation short peptide with the PAPS synthetic bifunctional enzyme and ADP phosphorylase, as well as the length and rigidity of the linker sequence, the catalytic activity of the immobilized PAPS synthetic bifunctional enzyme and ADP phosphorylase is significantly improved. Compared with the original catalytic system, it does not require complex purification, has higher stability, and the efficiency of producing PAPS is also higher.

[0052] 3. The present invention simultaneously achieves the co-immobilization of PAPS synthetic bifunctional enzyme and ADP phosphorylase through self-assembling peptides, directly breaks the cell wall and adds centrifugal precipitation to achieve one-step synthesis of PAPS.

[0053] 4. By adding stabilizers to the reaction system and optimizing the catalytic system, the present invention can keep the enzyme highly active for a long time. The combination of immobilized PAPS synthetic bifunctional enzyme and ADP phosphorylase can be reused ten times with high activity. Brief Description of the Drawings

[0054] Figure 1 It is a schematic diagram of agrin aggregation.

[0055] Figure 2 It is a fluorescence microscope image of agrin aggregated with green fluorescent protein.

[0056] Figure 3 It is an inclusion body protein gel image of PAPS bifunctional enzyme and ADP phosphorylase fused with different agrins.

[0057] Figure 4 It is a PAPS bifunctional enzyme gel image of agrin linked with different linker peptides.

[0058] Figure 5 It is a stability comparison diagram of immobilized PAPS bifunctional enzyme and ADP phosphorylase and their pure enzymes.

[0059] Figure 6 It is a diagram showing the influence of adding different protectants on the stability of immobilized PAPS bifunctional enzyme and ADP phosphorylase.

[0060] Figure 7 It is a diagram for optimizing the addition ratio of immobilized PAPS bifunctional enzyme and ADP phosphorylase in the reaction system.

[0061] Figure 8 It is a diagram of the conversion rate of 1 mol ATP to 1 mol PAPS when immobilized PAPS bifunctional enzyme and ADP phosphorylase are cycled different times.

[0062] Figure 9 It is a protein gel image of co-immobilized PAPS bifunctional enzyme and ADP phosphorylase.

[0063] Figure 10 It is a flow chart for synthesizing PAPS by immobilized co-immobilized PAPS bifunctional enzyme and ADP phosphorylase. Detailed Description of the Invention

[0064] Escherichia coli BL21(DE3), pET28a(+)(Takara, Japan), pCDF-Duet-1(Takara, Japan), pET28a-ASAK involved in the following examples S5 , pET28a-PaPPX are stored in the laboratory. The construction method of the said pET28a-ASAK S5 is disclosed in (ACS Catal. 2021, 11(16): 10405–10415); the said pET28a-PaPPX is generated by one-step self-assembly after linearizing pET28a(+) and PaPPX (amino acid sequence as shown in SEQ ID NO. 17) by digestion with BamHI / HindIII. PaPPX is codon-optimized according to Escherichia coli and synthesized by Shanghai Sangon. All plasmid construction reagents and sequencing verification involved are purchased and completed in Shanghai Sangon Biotech Co., Ltd. All kinds of analytical pure reagents involved are purchased from Sinopharm Group.

[0065] The media involved in the following examples are as follows:

[0066] LB medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract.

[0067] TB medium: 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol.

[0068] The detection methods involved in the following examples are as follows:

[0069] Protein quantification of inclusion bodies: Dissolve the inclusion bodies with 8 M urea and quantify using a Bradford protein concentration assay kit (detergent-compatible).

[0070] The enzyme activity definition of immobilized PAPS bifunctional enzyme is: the amount of enzyme required to synthesize 1 μM PAPS per hour starting from substrates ATP and magnesium sulfate at 35 °C.

[0071] The enzyme activity definition of immobilized PaPPX is: the amount of enzyme required to synthesize 1 μM ATP per hour using ADP as the substrate and pyrophosphate as the phosphate donor at 35 °C.

[0072] Detection method for the enzyme activity of ADP-phosphorylated PaPPX:

[0073] The ATP detection kit is purchased from Shanghai Sangon. The principle of action is that hexokinase catalyzes the formation of glucose 6-phosphate from glucose and ATP, and glucose 6-phosphate dehydrogenase further catalyzes glucose 6-phosphate and NADP +Dehydrogenation generates NADPH. NADPH has a characteristic absorption peak at 340 nm, and the amount of NADPH is proportional to the amount of ATP. All procedures were carried out according to the manual instructions.

[0074] Enzyme activity detection method for PAPS bifunctional enzyme:

[0075] Add 5 mM ATP and 5 mM MgSO4 to the reaction system, and make up the volume of the reaction system to 1.5 mL with 20 mM Tris-HCl buffer (pH 7.5). After incubating at 35 °C for 2 h, detect the yield of PAPS by high performance liquid chromatography.

[0076] PAPS detection method:

[0077] Use Agilent 1600 HPLC system, Polyamine II column (4.6×250 mm, 12 nm), mobile phase: 50 mM KH2PO4 and 0.1% triethylamine solution, flow rate: 0.6 mL·min -1 , injection volume: 5 μL, detection time 35 min, detector: UV254 nm.

[0078] PAPS synthesis conversion rate:

[0079] The molar ratio of the product PAPS to the substrate ATP.

[0080] Example 1: Immobilized expression and enzyme activity determination of PAPS synthesis bifunctional enzyme and ADP phosphorylase

[0081] The specific steps are as follows:

[0082] (1) Construction of pET28a-ASAK S5 , pET28a-PaPPX:

[0083] The construction method of the pET28a-ASAKS5 is disclosed in (ACS Catal. 2021, 11(16): 10405–10415); the pET28a-PaPPX is generated by one-step self-assembly after linearizing pET28a(+) and PaPPX (amino acid sequence as shown in SEQ ID NO. 17) by digestion with BamHI / HindIII, and PaPPX was synthesized by Shanghai Sangon.

[0084] (2) Selection of aggregating protein ( Figures 1 - 2 )

[0085] Select aggregation proteins CipA (Gene ID: 155404) from Photobacterium, aggregation protein CipB (Gene ID: 1890126) from Photobacterium, EAK16 (Gene ID: 1879200149) from Saccharomyces cerevisiae zuotin, and artificially designed aggregation short peptides 18A, L6KD, HVdoT, GFIL8, NSPdoT, DMdoT, Aβ42 (GeneID: 112927) for selection.

[0086] According to the codon preference rule of Escherichia coli, the amino acid sequences of aggregation proteins 18A, L6KD, GFIL8, NSPdoT, HVdoT, DMdoT, Aβ42, Saccharomyces cerevisiae zuotin (partial region), CipA, and CipB are shown in SEQ ID NO.1 - 10 respectively (the nucleotide sequences are shown in SEQ ID NO.20 - 29 respectively), and these sequences are used as aggregation proteins.

[0087] (3) Connect the above - optimized aggregation protein nucleotide sequences to the C - terminal between the Not I and Xho I restriction enzyme cleavage sites and the N - terminal between the Ncol I and Bamh I restriction enzyme cleavage sites of plasmid pET28a - ASAK through GGGGS linker to obtain different recombinant plasmids: pET28a - ASAK S5 - aggregation protein. S5 - aggregation protein.

[0088] (4) Connect the above - optimized nucleotide sequences to the C - terminal between the Not I and XhoI restriction enzyme cleavage sites and the N - terminal between the Ncol I and Bamh I restriction enzyme cleavage sites of plasmid pET28a - PaPPX to obtain different recombinant plasmids: pET28a - PaPPX - aggregation protein.

[0089] (5) Transfer the recombinant plasmids pET28a - ASAK S5 - aggregation protein and pET28a - PaPPX - aggregation protein into E.coli BL21(DE3). After verification, streak - culture on an LB plate containing kanamycin (50 μg / L). Pick a single colony and inoculate it into an LB seed medium. After culturing to the seed solution, transfer the seed solution to 50 mL of TB fermentation medium at a volume fraction of 1 mL / 50 mL, continue culturing for 1 - 2 h, then add 0.5 mM IPTG and induce culture at 30 °C for 8 - 12 h. After completion, collect the cells, weigh the wet weight of the cells, and ultrasonically disrupt them for purification and analysis. The obtained inclusion body gel diagram is as Figure 3 shown.

[0090] The results show that both ASAK and PaPPX are expressed in the form of intracellular precipitates.

[0091] (5) Different bifunctional enzyme ASAK S5 Enzyme activity detection of immobilized enzyme:

[0092] By sonicating the collected E. coli BL21(DE3) / pET28a-ASAK cells and centrifuging at high speed to collect the precipitate, the target protein was purified by washing three times with 20 mM Tris-HCl at pH 7.5; obtaining the bifunctional enzyme ASAK containing different aggregation proteins S5 of the immobilized enzyme; S5 In the catalytic system, add 50 - 100 mg of the immobilized bifunctional enzyme obtained by crushing the above different wet cells, ATP with a final concentration of 5 - 10 mM and MgSO4 with a final concentration of 10 - 20 mM, catalyze at 35 - 40 °C for 2 - 10 h, and use high-performance liquid chromatography to detect the generation of PAPS. The PAPS-synthesizing bifunctional enzyme ASAK

[0093] After fusing any aggregation protein at the N-terminus of the PAPS-synthesizing bifunctional enzyme ASAK S5 the inclusion bodies do not have catalytic activity, while the specific enzyme activity results of the inclusion bodies of the bifunctional enzyme with different aggregation proteins fused at the C-terminus are shown in Table 1.

[0094] Table 1: Comparison of specific enzyme activities of inclusion bodies of bifunctional enzymes synthesized by fusing different aggregation proteins at the C-terminus

[0095] Name Specific enzyme activity (U / g) <![CDATA[ASAK from Photobacterium mergence - derived CipA S5 > 270±12 <![CDATA[ASAK from Photobacterium fucatum-derived CipB S5 > 12±2 <![CDATA[ASAK derived from Saccharomyces cerevisiae EAK16 S5 > 76±10 <![CDATA[ASAK incorporating artificial self-assembling peptide 18A S5 > 218±13 <![CDATA[ASAK incorporating artificial self-assembling peptide L6KD S5 > 206±14 <![CDATA[ASAK incorporating artificial self-assembling peptide GFIL8 S5 > 0 <![CDATA[ASAK incorporating artificial self-assembling peptide HVdoT S5 > 155±10 <![CDATA[ASAK incorporating artificial self-assembling peptide NSPdoT S5 > 47±6 <![CDATA[ASAK incorporating artificial self-assembling peptide DMdoT S5 > 58±5 <![CDATA[ASAK incorporating artificial self-assembling peptide Aβ42 S5 > 142±8

[0096] The results show that after fusing any aggregation protein at the N-terminus of the PAPS-synthesizing bifunctional enzyme ASAK S5 the inclusion bodies do not have catalytic activity. The inclusion bodies of the bifunctional enzyme with the aggregation protein CipA from Photobacterium leiognathi fused at the C-terminus have the highest specific enzyme activity, with a specific enzyme activity of 270 U / g wet cells.

[0097] (6) Enzyme activity detection of immobilized enzymes of different ADP phosphorylases

[0098] The biomass specific activity yield, calculated by multiplying the specific activity of the enzyme aggregate (U / mg enzyme aggregate) by the yield (in mg enzyme aggregate / g wet cells), can be defined as "catalytic activity per gram of wet cells". This parameter allows for quantitative comparison of enzyme aggregates fused with different aggregation proteins, taking into account their specific activity and yield, as well as different expression levels and different degrees of stability.

[0099] The fermentation broth of E. coli BL21(DE3) / pET28a-PaPPX-aggregation protein obtained in step (4) is the immobilized enzyme of different ADP phosphorylases;

[0100] Add 50 - 100 mg of immobilized ADP phosphorylase obtained by breaking different wet bacterial cells into the catalytic system, with ADP at a final concentration of 5 - 10 mM, MgSO4 at 10 - 20 mM, and pyrophosphate at 5 - 10 mM, and catalyze for 2 h at 35 - 40 °C. Detect the generation of ATP using high-performance liquid chromatography. After fusing any aggregation protein at the N-terminus, it does not have catalytic activity. The specific enzyme activity results of ADP phosphorylase with different aggregation proteins fused at the C-terminus are shown in Table 2.

[0101] Table 2: Comparison of specific enzyme activities of ADP phosphorylase with different aggregation proteins fused at the C-terminus

[0102]

[0103]

[0104] The results show that after fusing any aggregation protein at the N-terminus, it does not have catalytic activity.

[0105] The PaPPX inclusion body with the aggregation protein CipA from Photobacterium fischeri fused at the C-terminus has the highest specific enzyme activity, with a specific enzyme activity of 950 U / g wet bacterial cells.

[0106] Example 2: Comparison of enzyme activities of PAPS synthase bifunctional enzyme and ADP phosphorylase with different linker sequences

[0107] Construction of the fused immobilized PAPS synthase bifunctional enzyme: Fuse the aggregation protein CipA from Photobacterium fischeri and the PAPS synthase bifunctional enzyme ASAK S5 into a fragment, and add different fusion linker sequences (linker) at the linker part respectively to fuse them into a fragment, so as to maintain a certain spatial position between the aggregation protein and the PAPS synthase bifunctional enzyme, avoid interference between proteins, and prevent the aggregation protein from forming a dense structure that affects the mass exchange of substrates.

[0108] Specifically, remove the stop codon of the previous gene, directly connect it to the linker, and then connect it to the start codon of the other gene; the sequence of the linker can be SEQ ID NO.11 - 15.

[0109] After designing the linker sequence on the primer, it is synthesized by Sangon Biotech (Shanghai). Add the linker through PCR amplification, and then use the blunting kination ligation (BKL) kit (Takara, Japan) to ligate the products. All constructs are verified by sequencing through Sangon Biotech (Shanghai) or Ascentage.

[0110] Similarly, construct the fused immobilized ADP phosphorylase and verify its activity.

[0111] E. coli BL21(DE3) / pET28a-ASAK containing different linkers was prepared separately S5 -linker-CipA and E. coli BL21(DE3) / pET28a-PaPPX-linker-CipA.

[0112] Expression conditions:

[0113] Pick a single colony and culture it overnight in LB medium. Inoculate the seeds into TB medium at a ratio of 1 mL / 50 mL and continue culturing until the OD 600 When it reaches 0.6 - 0.8, induce expression. Induction conditions: induce expression with 0.1 - 0.2 mM IPTG (30 °C, 220 rpm), and the expression time is 8 - 12 h. 50 mg / L kanamycin sulfate should be added during the cultivation of the recombinant strains to ensure the stability of the plasmid.

[0114] The results after expression are shown in Table 3. Among the linkers with sequences as shown in SEQ ID NO.11 - 15, the effect of SEQ ID NO.15 is better.

[0115] Table 3: Enzyme activity data after fusion with different linkers

[0116]

[0117] At the same time, taking the non-repeated linker as a comparison, the specific implementation method is the same as above. The difference is that the linkers are adjusted to (GGGGS)*1, (EAAAAK)*1, (GMALP)*1 respectively, and the results are shown in Table 4:

[0118] Table 4: Enzyme activity data after fusion with different linkers

[0119]

[0120] The results show that as the length of the linker increases, the specific enzyme activity of the obtained bifunctional enzyme also gradually increases. The specific enzyme activity of the bifunctional enzyme synthesized by PAPS linked with a rigid linker is higher than that of the flexible linker.

[0121] The final result shows that the specific enzyme activity of the bifunctional enzyme synthesized by PAPS fused with a rigid PT linker is the highest. Therefore, the bifunctional enzyme synthesized by PAPS fused with CipA using a rigid PT linker is simply referred to as the immobilized bifunctional enzyme for PAPS synthesis. Some protein gel diagrams are as Figure 4 shown.

[0122] Example 3: Comparison of the stability of immobilized enzyme and free enzyme

[0123] The specific steps are as follows:

[0124] (1) Preparation of immobilized enzyme

[0125] Prepare E.coli BL21(DE3) / pET28a-ASAK S5 -linker-CipA and E.coli BL21(DE3) / pET28a-PaPPX-linker-CipA according to the methods of Examples 1-2; where the linker used is: PT linker (PTPPTTPTPPTTPTPTP);

[0126] Prepare ASAK S5 -CipA immobilized enzyme and PaPPX-CipA immobilized enzyme respectively according to the methods of Examples 1-2.

[0127] (2) Preparation of free enzyme PAPS to synthesize bifunctional enzyme ASAK S5 and pure enzyme of ADP phosphorylase PaPPX

[0128] Respectively introduce pET28a-ASAK S5 and pET28a-PaPPX into E.coli BL21(DE3) to prepare E.coli BL21(DE3) / pET28a-ASAK S5 and E.coli BL21(DE3) / pET28a-PaPPX recombinant E.coli strains respectively;

[0129] Respectively culture the recombinant E.coli strains overnight in LB medium at 37°C and transfer them to TB medium. When OD600 reaches 0.6-0.8, add 0.5 mmol / L IPTG and continue to culture at 30°C for 8-12 h;

[0130] Then, the cells were collected by centrifugation at 8,000 - 10,000 g for 15 minutes at 4°C. The cells were disrupted by sonication in 50 mM Tris-HCl buffer pH 7.5. The cell debris was removed by centrifugation, and the crude enzyme solution was collected as the supernatant. Purification was carried out using an AKTA start 25 (GE Healthcare, USA) protein purifier, and the protein was purified from the crude enzyme solution by nickel affinity chromatography in a wash buffer (50 mM Tris-HCl, pH 7.5, 30 mM imidazole and 500 mM NaCl) and an elution buffer (20 mM Tris-HCl, pH 7.5, 500 mM imidazole and 500 mM NaCl). The purified protein solution was desalted using a HiTrap desalting column (GE Healthcare, USA) and a wash buffer (50 mM Tris-HCl, pH 7.5). The protein concentration was determined using a Bradford protein assay kit, and the free enzyme PAPS-synthesizing bifunctional enzyme ASAK S5 and the pure enzyme of ADP phosphorylase PaPPX were separately prepared;

[0131] (3) The obtained immobilized PAPS-synthesizing bifunctional enzyme ASAK S5 -CipA, immobilized ADP phosphorylase PaPPX-CipA, free enzyme PAPS-synthesizing bifunctional enzyme ASAK S5 and free enzyme ADP phosphorylase PaPPX pure enzyme were verified to be correct by SDS-PAGE. After dialysis to remove salt ions, the reaction was carried out;

[0132] (4) 0.5 g / L of free PAPS-synthesizing bifunctional enzyme ASAK was added to the catalytic system (containing 50 - 100 mM Tris-HCl buffer, pH 7.0 - 8.5);

[0133] At the same time, 0.5 g / L of immobilized PAPS-synthesizing bifunctional enzyme ASAK-CipA was added to another catalytic system (containing 50 - 100 mM Tris-HCl buffer, pH 7.0 - 8.5);

[0134] The above catalytic systems were incubated at 35°C for 2 - 24 h respectively. Samples were taken every two hours and 10 mM ATP and 20 mM MgSO4 were added, and the catalytic reaction was carried out at 35°C to detect the enzyme activity, and the production of PAPS was detected by high performance liquid chromatography.

[0135] (5) ADP phosphorylase stability system:

[0136] Add 0.5 g / L of free ADP phosphorylase PaPPX to the catalytic system (containing 50 - 100 mM Tris-HCl buffer, pH 7.0 - 8.5);

[0137] Meanwhile, add 0.5 g / L of immobilized ADP phosphorylase PaPPX-CipA to another catalytic system (containing 50 - 100 mM Tris-HCl buffer, pH 7.0 - 8.5);

[0138] Incubate the above catalytic systems at 35 °C for 2 - 24 h respectively. Sample every two hours and add 10 mM ADP and 10 mM sodium pyrophosphate, and conduct catalytic reactions at 35 °C to detect the enzyme activity, and use an ATP detection kit to detect the generation of ATP.

[0139] The results show that the stability of the immobilized bifunctional enzyme and ADP phosphorylase has been significantly improved compared with that of the free enzyme. The results are as Figure 5 shown. After incubating the free enzyme PaPPX at 35 °C for 24 hours, it still has 70% relative activity.

[0140] The relative activity of the immobilized enzyme PaPPX-CipA reaches 89%, which is 20% higher than that of the free enzyme PaPPX.

[0141] The relative activity of the free enzyme ASAK is only 44% of its initial activity, while the catalytic activity of the immobilized enzyme ASAK-CipA is 73% of the initial activity, which is 29% higher than that of the free enzyme.

[0142] Example 4: Effect of the addition of a protective agent on the stability of the immobilized PAPS bifunctional enzyme

[0143] (1) Preparation of the immobilized enzyme

[0144] Prepare E. coli BL21(DE3) / pET28a-ASAK S5 -linker-CipA according to the methods of Examples 1 - 2; where the linker used is: PT linker (PTPPTTPTPPTTPTPTP);

[0145] And prepare the ASAK S5 -CipA immobilized enzyme respectively according to the methods of Examples 1 - 2.

[0146] (2) Add to the bifunctional enzyme protein ASAK S5The addition amounts of different protectants maltose, mannose, rhamnose, glucose, sucrose, sorbitol, glycerol, and BSA in -CipA were 10 mg / mL at the final concentration. They were incubated at 37 °C for 24 h, and then the activity of the immobilized bifunctional enzyme was measured to characterize its stability.

[0147] The results are as Figure 6 shown. After adding sucrose, BSA, glycerol, and sorbitol, the stability of the enzyme was improved to varying degrees. Among them, the effects of adding BSA and glycerol were the most significant. After incubating for 24 h, the ASAK S5 -CipA still had 82% activity, which was 9% higher than that of the control.

[0148] Example 5: Optimizing the addition ratio of immobilized PAPS bifunctional enzyme and ADP phosphorylase to synthesize PAPS

[0149] (1) Preparation of immobilized enzyme

[0150] E. coli BL21(DE3) / pET28a-ASAK S5 -linker-CipA and E. coli BL21(DE3) / pET28a-PaPPX-linker-CipA were prepared according to the methods of Examples 1-2; among them, the linker used was: PT linker (PTPPTTPTPPTTPTPTP);

[0151] And the ASAK S5 -CipA immobilized enzyme and PaPPX-CipA immobilized enzyme were respectively prepared according to the methods of Examples 1-2.

[0152] (2) The addition ratio of immobilized PAPS synthetic bifunctional enzyme and ADP phosphorylase was studied.

[0153] The obtained immobilized PAPS synthetic bifunctional enzyme and ADP phosphorylase were added to the catalytic system in different ratios. The ratios of ASAK S5 -CipA immobilized enzyme and PaPPX-CipA immobilized enzyme were respectively: 1:1, 2:1, 3:1, 5:1, and 10:1; the total addition amount of the enzyme was: 1-2 g / L.

[0154] 5-10 mM ATP, 10-20 mM MgSO4, and 10 mg / mL glycerol were respectively added to the above catalytic systems, and the catalytic reaction was carried out at 35-40 °C for 2 h. After the reaction ended, the production of PAPS was detected by high performance liquid chromatography.

[0155] The results are as Figure 7As shown, when the ratio of immobilized PAPS synthase bifunctional enzyme to ADP phosphorylase is 5:1, 10 mM of ATP can be converted into 9 mM of PAPS, and the conversion rate of PAPS reaches 90%.

[0156] Example 6: Study on the recyclability of immobilized PAPS bifunctional enzyme and ADP phosphorylase

[0157] (1) Preparation of immobilized enzyme

[0158] E. coli BL21(DE3) / pET28a-ASAK S5 -linker-CipA and E. coli BL21(DE3) / pET28a-PaPPX-linker-CipA were prepared according to the methods of Examples 1-2; the linker used was: PT linker (PTPPTTPTPPTTPTPTP);

[0159] And ASAK S5 -CipA immobilized enzyme and PaPPX-CipA immobilized enzyme were respectively prepared according to the methods of Examples 1-2.

[0160] (2) The recyclability of immobilized PAPS synthase bifunctional enzyme and ADP phosphorylase was studied.

[0161] The obtained immobilized PAPS synthase bifunctional enzyme and ADP phosphorylase were added to the catalytic system, where ASAK S5 -CipA was 1 g / L and PaPPX-CipA was 0.2 g / L; then 10 mM ATP, 20 mM MgSO4, and 10 mg / mL of glycerol were added, and the catalytic reaction was carried out at 35-40 °C for 2 h. After the reaction, the formation of PAPS was detected by high performance liquid chromatography.

[0162] The results are as Figure 8 shown. The system after the reaction was centrifuged, and the supernatant was used to detect the formation of PAPS by high performance liquid chromatography.

[0163] (3) The precipitate obtained in step (2) was washed three times with pH = 7.5, 50-100 mM Tris-HCl and then re-added to a new catalytic system for the next round of catalysis. Specifically:

[0164] The obtained precipitate was added to the catalytic system, then 10 mM ATP, 20 mM MgSO4, and 10 mg / mL of glycerol were added, and the catalytic reaction was carried out at 35-40 °C for 2 h. After the reaction, the formation of PAPS was detected by high performance liquid chromatography.

[0165] According to the above method, continue the cycle eight times, for a total of ten cycles, and use high-performance liquid chromatography to detect the generation of PAPS. The results are as Figure 8 shown.

[0166] The results show that the immobilized PAPS-synthesizing bifunctional enzyme still has a 45% ATP conversion rate after being recycled ten times, which is about 50% of that in the first use.

[0167] Example 7: Co-immobilization of PAPS bifunctional enzyme and ADP phosphorylase to achieve the synthesis of PAPS

[0168] Select the immobilized PAPS bifunctional enzyme and ADP phosphorylase with the highest enzyme activity; among them, the linker used is: PTlinker (PTPPTTPTPPTTPTPTP).

[0169] Connect the immobilized ADP phosphorylase between the EcoRI and HindIII restriction enzyme cleavage sites of plasmid pCDFDuet to obtain the recombinant plasmid pCDFDuet-PaPPX-linker-CipA.

[0170] Transfer the recombinant plasmid pCDFDuet-PaPPX-linker-CipA and pET28a-CipAASAK S5 -linker-CipA (the preparation method is the same as in Examples 1-2) into E. coli BL21(DE3), verify and streak-culture on a plate containing kanamycin and spectinomycin (50 μg / L). Pick a single colony and inoculate it into the LB seed medium. After culturing to the seed liquid, transfer the seed liquid to 50 mL of TB fermentation medium at a volume fraction of 1 mL / 50 mL, continue culturing for 1-2 h, then add 0.5 mM IPTG and induce culture at 30 °C for 8-12 h.

[0171] After completion, collect the bacterial cells, ultrasonically disrupt them, and collect the precipitate by high-speed centrifugation. Use 20 mM Tris-HCl at pH 7.5 to wash three times to remove impurities from the target protein. The protein gel diagram is as Figure 9 shown.

[0172] Prepare a mixed enzyme containing ASAK S5 -CipA immobilized enzyme and PaPPX-CipA immobilized enzyme.

[0173] Add the obtained immobilized mixed enzyme to the catalytic system, then add 10 mM ATP and 120 mM MgSO4, and carry out the catalytic reaction at 35-40 °C for 2 h. After the reaction is completed, use high-performance liquid chromatography to detect the generation of PAPS ( Figure 10 ).

[0174] The results showed that 10 mM of ATP could synthesize 9 mM of PAPS, and the synthesis efficiency of PAPS reached 90%.

[0175] Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An immobilized enzyme, characterized in that, The immobilized enzyme is obtained by connecting the aggregation protein CipA from Photobacterium to the C-terminus of the enzyme protein through any one of the linker peptides of SEQ ID NOs. 11 to 15; the enzyme protein is the PAPS synthase bifunctional enzyme with the amino acid sequence shown in SEQ ID NO. 16 and the ADP phosphorylase with the amino acid sequence shown in SEQ ID NO. 17, and the immobilized PAPS synthase bifunctional enzyme and the immobilized ADP phosphorylase are obtained respectively.

2. A gene encoding the immobilized enzyme according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. A recombinant cell expressing the immobilized enzyme according to claim 1, or carrying the gene according to claim 2, or carrying the recombinant vector according to claim 3.

5. The recombinant cell according to claim 4, characterized in that, The recombinant cell uses bacteria or fungi as the expression host.

6. A method for synthesizing 3′-phosphoadenosine-5′-phosphosulfate, characterized in that, The method is to use the immobilized PAPS synthase bifunctional enzyme and the immobilized ADP phosphorylase according to claim 1, or the recombinant cell according to claim 4 or 5 expressing the immobilized PAPS synthase bifunctional enzyme and the immobilized ADP phosphorylase to catalytically convert and synthesize 3′-phosphoadenosine-5′-phosphosulfate with ATP and MgSO4 as substrates.

7. The method according to claim 6, wherein One or more of the protectants maltose, mannose, rhamnose, glucose, sucrose, sorbitol, glycerol, and BSA are added to the catalytic conversion system.

8. The method according to claim 7, wherein The catalytic conversion system is: the buffer solution is 10 - 50 mM Tris-HCl, with a pH of 6.0 - 8.0, adding 2 - 20 mM ATP, 4 - 40 mM magnesium sulfate, mixing 2 - 200 mM sodium sulfate and 0.1 - 5.0 mg / mL of the immobilized PAPS synthase bifunctional enzyme and ADP phosphorylase.

9. Use of the immobilized enzyme according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4 or 5, or the method according to any one of claims 6 - 8 in the synthesis of 3′-phosphoadenosine-5′-phosphosulfate.

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