Agarase Ag0139 and fusion enzyme and application thereof

Abstract

The invention provides agarase Ag0139 as well as a fusion enzyme and application thereof, and relates to the technical field of enzyme engineering. The agarase Ag0139 disclosed by the invention can be used as a catalytic degradation agarase, and plays a key role in a green biodegradation process of agarose; and the key technical problem of extremely low solubility of the GH118 family agarase in an escherichia coli expression system is successfully solved through fusion enzyme engineering. According to the constructed fusion enzyme MBP-Ag0139, the soluble high-efficiency expression is realized; according to the constructed fusion enzyme MBP-Ag0139-SpyTag, efficient and stable immobilization of agarase is achieved, the catalytic characteristic and thermal stability of agarase are improved, the number of times of recycling can be effectively increased, and the use cost is reduced. And a solid foundation is laid for green and low-cost enzymatic industrial production of high-polymerization-degree agaro-oligosaccharide.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically to an agarase Ag0139, its fusion enzyme, and its applications. Background Technology

[0002] Agar is a major component of the cell wall of red algae. Its degradation product, agaro-oligosaccharide (AOS), possesses many unique biological activities, such as promoting the growth of beneficial intestinal bacteria, antibacterial, antiviral, antitumor, enhancing immunity, antioxidant, moisturizing, and whitening effects. Therefore, it has significant potential applications in the pharmaceutical, food, and cosmetic industries. Currently, common methods for preparing agaro-oligosaccharide include chemical, physical, and enzymatic methods. Enzymatic hydrolysis, as a novel method for preparing agaro-oligosaccharide, not only offers mild and efficient reaction conditions and produces products with a single degree of polymerization, but is also environmentally friendly. Achieving industrial-scale enzymatic preparation of agaro-oligosaccharide has become a new research trend.

[0003] Agarases, as catalytic degradative enzymes, play a crucial role in the green biodegradation of agarose. Currently studied agarases, based on the type and sequence of glycosidic bonds they cleave in agarose, belong to five different glycosidic hydrolase families: GH16, GH50, GH86, GH117, and GH118. Among them, the main products of GH118 family agarases are hexasaccharides, octasaccharides, and decasaccharides, making them a primary tool for preparing high-polymerization-degree agar oligosaccharides. Current research on GH118 family agarases is limited, and no protein structure crystallization has been achieved to date. AlphaFold predicts that GH118 family agarases exhibit… β -helex structure, in the structure β - High proportion of folded plates. Overexpressed GH118 family agarases have extremely low solubility and poor stability, which is one of the key factors limiting their application. In addition, free agarases are difficult to separate from the decolorization reaction system and cannot be recovered and reused, increasing their application costs.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] This invention provides an agarase Ag0139, its fusion enzyme, and its applications. The agarase Ag0139 is obtained through isolation, and the fusion enzyme MBP-Ag0139 is obtained by modification based on the isolated agarase Ag0139. The fusion enzyme MBP-Ag0139-SpyTag is obtained by modification based on the fusion enzyme MBP-Ag0139. The fusion enzyme MBP-Ag0139 has higher solubility than the agarase Ag0139 itself, while the immobilized enzyme prepared from the fusion enzyme MBP-Ag0139-SpyTag has higher thermal stability than the fusion enzyme MBP-Ag0139. The immobilized enzyme shows almost no loss of activity after 10 consecutive cycles of recovery, and retains more than 50% of its activity after 13 cycles. This can be applied to the enzymatic industrial production of agar oligosaccharides, significantly reducing production costs.

[0006] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted: The present invention provides an agarase Ag0139, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] SEQ ID NO.1 ANYTASNASQLSARLHDAANNGTGVDVITIQGSIFTDQQIDIQTPVTIQGAAGFRVSRIIRTSDDGFQPLFNIQSSNVTIRNLLLIDEKGQNTNTQVASEAGNDHSNARL INIPYEDAYQQIANITIENNTFENTAVGVASSGLIPRNLSITNNDFIKVNRSVELLRDVGRVYNVWNVSANNVVLNGGTLNISNNRIRGNRVRLGISVDAGNDGVYVPPS FTNIPFFDAAAARAHFSDKPVVFANGSQVNSNTVEGANEFGIALATVANVTVAGNTVSTTEDDINSSDDIENNFTAGINVEHNSRDIVVDSNTITVGASGNFATGINVLAF QDHHAPLNHAQASSNITLVRNIFKGTGENTILAFGFSNLVVEDNNASQFTTRNPYQVTASFYNVPCGLSTSTARGTNNNIRYNQSSFNGTGNAPQYYDKNGNVVSGYTCQ Preferably, the nucleotide sequence of the agarase Ag0139 is as shown in SEQ ID NO.2.

[0008] SEQ ID NO.2 A fusion enzyme MBP-Ag0139, the amino acid sequence of the fusion enzyme MBP-Ag0139 is shown in SEQ ID NO.3.

[0009] SEQ ID NO.3 KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYGDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRVAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSGGSGENLYFQGANYTASNASQLSARLHDAANNGTGVDVITIQGSIFTDQQIDIQTPVTIQGAAGFRVSRIIRTSDDGFQPLFNIQSSNVTIRNLLLIDEKGQNTNTQVASEAGNDHSNARLINIPYEDAYQQIANITIENNTFENTAVGVASSGLIPRNLSITNNDFIKVNRSVELLRDVGRVYNVWNVSANNVVLNGGTLNISNNRIRGNRVRLGISVDAGNDGVYVPPSFTNIPFFDAAARAHFSDKPVVFANGSQVNSNTVEGANEFGIALATVANVTVAGNTVSTTEDDINSSDDIENNFTAGINVEHNSRDIVVDSNTITVGASGNFATGINVLAFQDHHAPLNHAQASSNITLVRNIFKGTGENTILAFGFSNLVVEDNNASQFTTRNPYQVTASFYNVPCGLSTSTARGTNNNIRYNQSSFNGTGNAPQYYDKNGNVVSGYTCQ Preferably, the nucleotide sequence of the fusion enzyme MBP-Ag0139 is shown in SEQ ID NO.4.

[0010] SEQ ID NO.4 A fusion enzyme MBP-Ag0139-SpyTag, which is obtained by modifying the above-mentioned fusion enzyme MBP-Ag0139, and the amino acid sequence of the fusion enzyme MBP-Ag0139-SpyTag is shown in SEQ ID NO.5.

[0011] SEQ ID NO.5 KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYGDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRVAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSGGSGENLYFQGANYTASNASQLSARLHDAANNGTGVDVITIQGSIFTDQQIDIQTPVTIQGAAGFRVSRIIRTSDDGFQPLFNIQSSNVTIRNLLLIDEKGQNTNTQVASEAGNDHSNARLINIPYEDAYQQIANITIENNTFENTAVGVASSGLIPRNLSITNNDFIKVNRSVELLRDVGRVYNVWNVSANNVVLNGGTLNISNNRIRGNRVRLGISVDAGNDGVYVPPSFTNIPFFDAAARAHFSDKPVVFANGSQVNSNTVEGANEFGIALATVANVTVAGNTVSTTEDDINSSDDIENNFTAGINVEHNSRDIVVDSNTITVGASGNFATGINVLAFQDHHAPLNHAQASSNITLVRNIFKGTGENTILAFGFSNLVVEDNNASQFTTRNPYQVTASFYNVPCGLSTSTARGTNNNIRYNQSSFNGTGNAPQYYDKNGNVVSGYTCQEFGGGGSGGGGSAHIVMVDAYKPTK Preferably, the nucleotides of the fusion enzyme MBP-Ag0139-SpyTag are as shown in SEQ ID NO.6.

[0012] SEQ ID NO.6 The present invention also provides the related biological materials described above with agarase Ag0139, or fusion enzyme MBP-Ag0139, or fusion enzyme MBP-Ag0139-SpyTag, wherein the biological materials comprise at least one of (a)-(e): (a) Encoding the agarase Ag0139 of claim 1, or the fusion enzyme MBP-Ag0139 of claim 3, or the polynucleotide encoding the fusion enzyme MBP-Ag0139-SpyTag of claim 4; (b) An expression cassette containing the polynucleotide described in (a); (c) A recombinant vector containing the polynucleotide described in (a); (d) Recombinant microorganisms containing the polynucleotides described in (a); (e) Recombinant microorganisms containing the recombinant vector described in (c).

[0013] A method for improving the soluble expression of agarase Ag0139 includes the following steps: fusing a nucleic acid molecule encoding the agarase Ag0139 with a nucleic acid molecule encoding a maltose-binding protein to construct a fusion gene, and expressing it in Escherichia coli.

[0014] Preferably, the nucleotide sequence of the fusion gene is shown in SEQ ID NO.4.

[0015] A method for preparing an immobilized enzyme includes the following steps: S1. SpyCatcher protein was mixed with sodium phosphate buffer and CaCl2 solution to form nanoflowers; S2. Mix the nanoflower with the fusion enzyme MBP-Ag0139-SpyTag solution, and fix the fusion enzyme MBP-Ag0139-SpyTag onto the nanoflower to obtain the final product.

[0016] Preferably, the molar ratio of SpyCatcher protein:sodium phosphate buffer:CaCl2 solution is (1-2):(1-2):(1-2).

[0017] Preferably, the molar ratio of SpyCatcher protein:sodium phosphate buffer:CaCl2 solution is 1:1:2.

[0018] Preferably, the amino acid sequence of the SpyCatcher protein is shown in SEQ ID NO.7.

[0019] SEQ ID NO.7 MAMVDTLSGLSSEQGQSGDMTIEEDDETHIKFSKRDEDDEELAGATMELRDSSGETISTWISDGEVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEEGEVTVDGKATKGDAHI The nucleotide sequence of the SpyCatcher protein is shown in SEQ ID NO.8.

[0020] SEQ ID NO.8 ATGGCGATGGTGGATACCCTGAGCGGCCTGAGCAGCGAACAAGGTCAGAGCGGCGATATGACCATTGAAGAAGATGATGAAACGCATATTAAATTTAGCAAACGCGATGAAGATGACGAGGAATTAGCGGGCGCGACGATGGAACTGCGCGATAGCAGCGGCGAAACCATTAGC ACCTGGATTAGCGATTGGCGAAGTGAAAGATTTTTATCTGTATCCGGGCAAATATACCTTTGTGGAAACCGCGGCGCCGGATGGCTATGAAGTGGCGACCGCGATTACCTTTACCGTGAACGAAGAAGGCGAAGTGACCGTGGATGGCAAAGCCACCAAGGGCGACGCGCACATC The immobilized enzyme prepared by the above-described method.

[0021] A method for degrading agarose or preparing agar oligosaccharides, using the above-mentioned agarase Ag0139, or the above-mentioned fusion enzyme MBP-Ag0139, or the above-mentioned fusion enzyme MBP-Ag0139-SpyTag, or the above-mentioned immobilized enzyme.

[0022] Application of agarase Ag0139, or fusion enzyme MBP-Ag0139, or fusion enzyme MBP-Ag0139-SpyTag, or immobilized enzyme in the degradation of agarose or the preparation of agar oligosaccharides.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention isolates and obtains agarase Ag0139, which can act as a catalytic degradative enzyme for agarose, playing a crucial role in the green biodegradation of agarose. Furthermore, the modification of agarase Ag0139 successfully solves the key technical challenge of extremely low solubility of GH118 family agarases in the *E. coli* expression system. The constructed fusion enzyme MBP-Ag0139 achieves highly efficient and soluble expression; while the constructed fusion enzyme MBP-Ag0139-SpyTag enables efficient and stable immobilization of agarase. The immobilized enzyme prepared from this enzyme not only improves its catalytic properties and thermal stability but also effectively increases the number of reusable enzymes and reduces operating costs. This lays a solid foundation for the green and low-cost enzymatic industrial production of high-polymerization agar oligosaccharides. Attached Figure Description

[0024] Figure 1 : ag0139 Gel electrophoresis image of PCR amplification, where M is the standard protein marker; 1 is... ag0139 Gene fragments; Figure 2 Seamless cloning ag0139 and mbp PCR amplification gel electrophoresis image of gene fragments, where M is the standard protein marker; 1 is... ag0139 Gene fragment; 2 is mbp Gene fragments; Figure 3 Gel electrophoresis images showing the induced expression results of Ag0139, MBP-Ag0139, MBP-Ag0139-SpyTag, and SpyCatcher in Escherichia coli. (a) is a gel electrophoresis image showing the induced expression results of Ag0139 in Escherichia coli, where M is the marker and 1 is the induction marker. E. coli pET-28a (+) bacterial culture protein; 2-3 are Ag0139 crude enzyme; 4-5 are MBP-Ag0139 crude enzyme; (b) is a gel electrophoresis image showing the induced expression results of MBP-Ag0139 in Escherichia coli, where M is the marker and 1 is the induction marker. E. coli pET-28a (+) bacterial culture protein; 2 is MBP-Ag0139-SpyTag crude enzyme; (c) is a gel electrophoresis image of the induced expression results of SpyCatcher in E. coli, where M is the marker; 1 is the purified SpyCatcher; 2 is the SpyCatcher complex with MBP-Ag0139-SpyTag; and 3 is the crude SpyCatcher protein. (d) is a gel electrophoresis image of the induced expression results of MBP-Ag0139 in Escherichia coli, M is the marker; 1 is the TEV digestion product of MBP-Ag0139; 2 is the purified MBP-Ag0139; 3 is the purified TEV digestion product of MBP-Ag0139; 4 is the TEV enzyme. Figure 4 The results show the comparison of catalytic properties of MBP-Ag0139 and Ag0139; (a) comparison of optimal reaction temperature; (b) comparison of specific enzyme activity; (c) comparison of thermal stability at 35 °C; and (d) comparison of thermal stability at 40 °C. Figure 5 Effect of the ratio of SpyCatcher, CaCl2 and PBS on the encapsulation efficiency of SpyCatcher in nanoflowers; Figure 6 Effect of reaction time on the binding rate of MBP-Ag0139-SpyTag and SpyCatcher-NFs (NFs: Nano Flowers); Figure 7 Enzyme activity of immobilized enzyme MBP-Ag0139-NFs after recycling; Figure 8 Comparison of catalytic properties of MBP-Ag0139 and MBP-Ag0139-NFs, including (a) specific enzyme activity; (b) optimal reaction temperature; (c) thermal stability at 40 °C; (d) optimal reaction pH; (e) pH stability of MBP-Ag0139; and (f) pH stability of MBP-Ag0139-NFs. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0026] Example 1: Construction of Ag0139 recombinant expression plasmid (1) Primers for amplifying the agarase Ag0139 gene FAg0139:5'-CATGCCATGGGCGCTAACTATACGGCCAGCAA-3' RAg0139:5'-CCGCTCGAGTTGGCAAGTATAACCTGATACAAC-3' (2) Construct primers for the fusion protein MBP-Ag0139 Fmbp:5'-TTTAAGAAGGAGATATACCATGGGCAAAATCGAAGAAGGTAAACTGG-3' Rmbp:5'-CTGGCCGTATAGTTAGCGCCCTGAAAATACAGGTTTT-3' Fmbp-2829:5'-ACCTGTATTTTCAGGGCGCTAACTATACGGCCAGC-3' Rmbp-2829:5'-TGGTGGTGGTGGTGGTGCTCGAGTTGGCAAGTATAACCTGATACAA -3' Using the genomic DNA of agar-degrading strains as a template, and by adding... Nco I and Xho I. Primers FAg0139 and RAg0139 for the restriction enzyme sites were used to cleave the gene according to the following reaction system and conditions. ag0139 The PCR amplification was performed, and the reaction system is shown in Table 1. The reaction process is shown in Table 2.

[0027] Table 1: PCR reaction system PCR reaction solution System (μL) Template (genomic DNA of agar-degrading strain) 1.0 Primer FAg0139 0.5 Primer RAg0139 0.5 2xTaq Mixture 25.0 <![CDATA[ddH2O]]> 23.0 Overall system 50.0 Table 2: PCR reaction process Reaction temperature (°C) reaction time 98 3 min 98 10 s 53 15 s 72 1.5 min 72 5 min 4 ∞ The target gene amplified by the above PCR ag0139 The fragment and the extracted pET-28a(+) plasmid were subjected to double enzyme digestion ( Nco I and Xho I), and the enzyme digestion products were recovered by gel extraction. According to the ligation system in Table 3, the double-digested linearized plasmid pET-28a(+) was ligated with... ag0139 The gene fragments were ligated to obtain the Ag0139 recombinant expression plasmid pET-28a(+)-Ag0139. The ligation system of the target gene and the vector is shown in Table 3.

[0028] Table 3: Target gene-vector ligation system Components System (μL) Purified target fragment 3.0 Linear vector pET28a(+) 2.0 Solution I 5.0 Overall system 10.0 The PCR amplification results of Ag0139 are as follows: Figure 1 As shown.

[0029] Example 2: Construction of the MBP-Ag0139 recombinant expression plasmid The MBP gene sequence was obtained from the NCBI database, and inserts at both ends of the sequence... Nco I and Xho To facilitate subsequent cleavage and separation of the MBP tag and Ag0139, GS flexible linker and TEV protease cleavage site sequences were introduced after the MBP gene sequence. These sequences were then synthesized by a gene company using a total chemical synthesis method. Finally, the recombinant expression plasmid pET-28a(+) was constructed by ligating it with the double-digested and linearized pET-28a(+) expression vector. -mbp Each was labeled with pET-28a(+). -ag0139 and pET-28a(+)- mbp Using primers Fmbp-2829 and Rmbp-2829, and Fmbp and Rmbp, gene sequencing was performed according to the following reaction system and conditions. ag0139 and mbp The PCR amplification reaction system is shown in Table 4, and the reaction process is shown in Table 5.

[0030] Table 4: PCR reaction system PCR reaction solution System (μL) 5×PrimeSTAR GXL Buffer 5.0 Template (pET-28a(+)- or pET-28a(+)-mbp) 1.0 Primer F 0.5 Primer R 0.5 dNTP Mixture 2.0 <![CDATA[ddH2O]]> 15.0 PrimeSTAR GXL DNA Polymerase 1.0 Overall system 25.0 Table 5: PCR reaction process Reaction temperature (°C) reaction time 95 3 min 95 30 s Annealing temperature 30 s 72 1 min 72 5 min 4 ∞ The PCR amplification obtained above ag0139 and mbp Fragments and double enzyme digestion ( Nco I and Xho I) The linearized plasmid pET-28a(+) was ligated according to the ligation system in Table 6 to obtain the MBP-Ag0139 recombinant expression plasmid pET-28a(+)- mbp-ag0139 The linking system between the target gene and the vector is shown in Table 6.

[0031] Table 6: Target gene-vector ligation system Components System (μL) Purified target fragment 2.0 Purified target fragment 2.0 Linear vector pET28a(+) 3.0 2×Seamless Cloning Mix 10.0 Nuclease free water 0 Overall system 20.0 ag0139 and mbp PCR amplification results of gene fragments are as follows Figure 2 As shown.

[0032] Example 3: Construction of MBP-Ag0139-SpyTag and SpyCatcher recombinant expression plasmids Will mbp , ag0139 and spyTag Three gene sequences are linked end to end to obtain mbp-ag0139-spyTag Gene sequence obtained from the NCBI database spycatcherThe gene sequence is shown in SEQ ID NO. 8, and the SpyCatcher amino acid sequence is shown in SEQ ID NO. 7.

[0033] exist mbp-ag0139-spyTag and spycatcher Add to both ends Nco I and Xho The enzyme was first digested at the I restriction site, then synthesized by a gene company using a total chemical synthesis method. This was then ligated into the double-digested linearized pET-28a(+) expression vector to construct the recombinant expression plasmid pET-28a(+) containing the agarase MBP-Ag0139-SpyTag. mbp-ag0139-spyTag Recombinant expression plasmid pET-28a(+)- with SpyCatcher spycatcher .

[0034] Example 4: Inducible expression of Ag0139, MBP-Ag0139, MBP-Ag0139-SpyTag and SpyCatcher in Escherichia coli Plasmid pET-28a(+)- ag0139 pET-28a(+)- mbp-ag0139 pET-28a(+)- mbp-ag0139- spyTag and pET-28a(+)- spycatcher Transfect into E. coli using the following method. E. coli Engineered strains of Ag0139, MBP-Ag0139, MBP-Ag0139-SpyTag, and SpyCatcher were obtained from competent cells of BL21(DE3)pLySs. E. coli BL21(DE3)pLySs / pET-28a(+)- ag0139 , E. coli BL21(DE3)pLySs / pET-28a(+)- mbp- ag0139 , E. coli BL21(DE3)pLySs / pET-28a(+)- mbp-ag0139-spyTag and E. coli BL21(DE3)pLySs / pET-28a(+)- spycatcher .

[0035] (1) Take 5 μL of recombinant plasmid sample and add it to 50-100 μL of competent cells. Mix slowly and place on ice for 30 min.

[0036] (2) Perform a 90-second heat shock (42°C water bath), then quickly return to the ice bath and wait for 3-5 minutes.

[0037] (3) Add 500 μL of LB liquid culture medium (without antibiotics), mix gently, and incubate with shaking for 1 h (37 °C).

[0038] (4) Centrifuge the bacterial culture (5000 rpm, 1 min) to precipitate the bacterial cells, and remove most of the supernatant (about 50-100 μL of bacterial cells are left to resuspend).

[0039] (5) Spread the bacterial culture evenly onto LB plates (containing Kana antibiotic) and incubate overnight in a 37 ℃ incubator.

[0040] (6) Pick a single colony from an LB plate and culture it. Take 50 μL of the bacterial solution and send it for testing to determine whether the recombinant strain is correct.

[0041] Successfully constructed genetically engineered strains E. coli BL21(DE3)pLySs / pET-28a(+)- ag0139 , E. coli BL21(DE3)pLySs / pET-28a(+)- mbp-ag0139 , E. coli BL21(DE3)pLySs / pET-28a(+)- mbp-ag0139-spyTag and E. coli BL21(DE3)pLySs / pET-28a(+)- spycatcher Inoculate each culture into 5 mL of LB medium and incubate on a shaker for 12 h (37 ℃, 220 rpm) to obtain the seed culture. Inoculate 200 μL of the seed culture into 200 mL of LB medium (containing Kana) and incubate with shaking for 2 h (37 ℃, 220 rpm). Then, add 200 μL of 0.5 M IPTG at a ratio of 1‰ and incubate with shaking for 20–24 h (16 ℃, 220 rpm) to induce the expression of the target protein. Collect the bacterial cells, centrifuge, and discard the supernatant.

[0042] Add 15 mL of lysis buffer (Tris 20 mM, NaCl 20 mM, DTT 1 mM, imidazole 20 mM, pH 7.0) to the bacterial cells and sonicate on an ice-water mixture (power 260-300 W, total lysis time 30 min, working time 3 s, interval time 3 s). After sonication, centrifuge again (4 ℃, 8000 rpm) for 40 min, and the resulting supernatant is the crude enzyme solution. The crude enzyme solution is purified using a His Trap™ HP-5 mL nickel column for recombinant enzymes Ag0139, MBP-Ag0139, MBP-Ag0139-SpyTag, and SpyCatcher. Add 5×SDS loading buffer to the enzyme solution, mix well, boil for 10 min, then centrifuge at 12000 rpm for 10 min. Perform SDS-PAGE protein electrophoresis on the supernatant using a 5% stacking gel and a 12% separating gel.

[0043] The results are as follows Figure 3 As shown, the same empty carrier bacteria E. coli Compared with the pET-28a(+) control group, the genetically engineered strain E. coli BL21(DE3)pLySs / pET-28a(+)- ag0139 , E. coli BL21(DE3)pLySs / pET-28a(+)- mbp- ag0139 , E. coli BL21(DE3)pLySs / pET-28a(+)- mbp-ag0139-spyTag and E. coli BL21(DE3)pLySs / pET-28a(+)- spycatcher After induction, overexpressed protein bands were observed in the supernatant after sonication. The protein size was consistent with expectations, indicating successful overexpression of Ag0139, MBP-Ag0139, MBP-Ag0139-SpyTag, and SpyCatcher in E. coli. The experimental results are as follows: Figure 3 As shown.

[0044] Example 5: Effect of MBP on the catalytic properties of agarase Ag0139 To investigate the effect of MBP on the catalytic properties of Ag0139, purified MBP-Ag0139 was digested with TEV protease. The digestion product was purified using a His Trap™ HP-5 mL nickel column to obtain electrophoretically pure Ag0139 without the MBP tag. The catalytic properties of the purified Ag00139 without the MBP tag were compared with those of MBP-Ag0139.

[0045] (1) Effect of MBP on the optimal reaction temperature of enzyme Add 270 μL of agarose solution (concentration 0.25%) to a 1.5 mL EP tube and incubate in a thermostatic water bath at temperatures of 25, 30, 30, 40, 45, 50 ℃ and 55 ℃ for 10 min. Then add 30 μL of enzyme and react for 15 min. Finally, add 300 μL of DNS and boil for 7 min to terminate the reaction. Measure the OD value of the reaction solution at 540 nm. According to the enzyme activity assay method, with the highest enzyme activity as 100%, obtain the relative enzyme activity at each temperature to determine the optimal temperature for the enzymatic reaction.

[0046] The optimal reaction temperature for both Ag0139 and MBP-Ag0139 is 35 ℃, but when the temperature exceeds 50 ℃, the enzyme activity of Ag0139 and MBP-Ag0139 is almost lost. Figure 4 a). This shows that the fusion of MBP does not affect the optimal reaction temperature of agarase Ag0139.

[0047] (2) Effect of MBP on enzyme activity The purified Ag0139 and MBP-Ag0139 were diluted to 0.006 mg / mL. Following the optimal reaction temperature as described above, the OD value at 540 nm was measured using the DNS method, and the amount of galactose was calculated. We define the amount of enzyme required to hydrolyze agarose to produce 1 mg of galactose per unit time (per minute) as one enzyme activity unit (U). Calculations showed that the specific enzyme activity of MBP-Ag0139 was 10% lower than that of Ag0139. Figure 4 (b) Therefore, the fusion of MBP has little effect on the specific enzyme activity of agarase Ag0139.

[0048] (3) Effect of MBP on enzyme thermostability The enzyme activities of Ag0139 and MBP-Ag0139 were measured after incubation at 35 ℃ and 40 ℃ for different times. The reaction was carried out for 15 min, followed by boiling for 7 min to terminate the reaction. The OD value of the reaction solution at 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The relative enzyme activity was calculated according to the enzyme activity assay method to determine the temperature stability of the enzyme-catalyzed reaction.

[0049] The temperature thermal stability of Ag0139 and MBP-Ag0139 at 35 ℃ and 40 ℃ is as follows: Figure 5As shown in c and d: When Ag0139 and MBP-Ag0139 were incubated at 35 ℃ for 20 min, their relative enzyme activities remained above 80%, and after 30 min, the residual enzyme activities were both above 50%. When Ag0139 and MBP-Ag0139 were incubated at 40 ℃ for 10 min, the enzyme activity of MBP-Ag0139 was approximately 70%, while that of Ag0139 was only about 30%. When incubated at 40 ℃ for 30 min, the residual enzyme activities of both were below 20%. Regardless of whether the incubation was at 35 ℃ or 40 ℃ for 10, 20, or 30 min, the relative enzyme activity of MBP-Ag0139 was higher than that of Ag0139, indicating that the fusion of MBP improved the thermal stability of Ag0139.

[0050] Example 6: Immobilization of MBP-Ag0139 (1) Preparation of nanoflowers SpyCatcher, 15 mM CaCl2 solution, and 10 mM sodium phosphate buffer (pH 7.0) were mixed in molar ratios of 1:1:1, 2:1:1, 1:2:1, and 2:1:2. After incubation at 16 °C for 6–36 h, the mixture was centrifuged at 8000 rpm for 10 min. The precipitate was washed three times with ultrapure water to obtain SpyCatcher-NFs nanoflowers. A SpyCatcher solution diluted with an appropriate amount of ddH2O was used as a control group. The protein concentration in the supernatant of the experimental group and the control group was determined by BCA method. To ensure the repeatability and reliability of the data, three parallel experiments were conducted. The binding rate of SpyCatcher during nanoflower formation was calculated, yielding the encapsulation efficiency.

[0051] By comparing the encapsulation effects of SpyCatcher under different molar ratios, the results showed that the encapsulation efficiency of SpyCatcher was the highest, reaching 91%, when the molar ratio of SpyCatcher, sodium phosphate buffer, and CaCl2 was 1:1:2. Figure 5 ).

[0052] (2) Immobilization of MBP-Ag0139 The nanoflowers were mixed with 10 mM sodium phosphate buffer (pH 7.0) containing MBP-Ag0139-SpyTag and incubated at 16 °C for 6 hours. After centrifugation at 8000 rpm for 10 min, the precipitate was washed three times with ultrapure water to obtain the immobilized enzyme MBP-Ag0139-NFs.

[0053] To assess the immobilization process and immobilization rate, samples were taken every 20 minutes (at time points of 0, 20, 40, 60, 80, 100, 120, 140, 160, 240, 360, and 480 min). The samples were centrifuged at 8000 rpm for 10 min, and the protein concentration in the supernatant was determined by the BCA method. The binding rate of SpyCatcher and MBP-Ag0139-SpyTag, i.e., the immobilization rate, was then calculated.

[0054] Comparison of protein concentration measurements in supernatants at different reaction times showed that the binding rate of MBP-Ag0139-SpyTag to SpyCatcher-NFs increased with increasing reaction time, reaching as high as 95% when the reaction time exceeded 140 min. Figure 6 This demonstrates that the SpyTag / Catcher system exhibits high immobilization efficiency for agarase MBP-Ag0139.

[0055] Example 7: Number of cycles of immobilized enzyme MBP-Ag0139-NFs Add 270 μL of 0.25% agarose solution and 0.18 μg of immobilized enzyme to a 2 mL EP tube and incubate at 40 °C for 15 min. Centrifuge at 8000 rpm for 10 min, and add the precipitate directly to the next identical reaction system. Repeat the same procedure to determine the reproducibility at this temperature. Measure the specific activity of the supernatant after each reaction, using the absorbance change after the first reaction as a control to determine the reproducibility.

[0056] Experimental results showed that, under a reaction temperature set at 40 ℃, the immobilized enzyme MBP-Ag0139-NFs exhibited good reusability. After 13 consecutive recovery cycles, its residual enzyme activity was still greater than 50%. Figure 8 ).

[0057] Example 8: Comparison of catalytic properties between immobilized enzyme MBP-Ag0139-NFs and free enzyme MBP-Ag0139 All reaction systems for catalytic characterization were performed according to Example 7. (1) Compared with enzyme activity The specific enzyme activity of the immobilized enzyme MBP-Ag0139-NFs was slightly lower than that of the free enzyme MBP-Ag0139, being 88.9% of that of MBP-Ag0139. Figure 8 a).

[0058] (2) Optimal reaction temperature and temperature stability The optimal reaction temperature for both MBP-Ag0139-NFs and MBP-Ag0139 is 35 °C. However, the immobilized enzyme exhibits significantly higher activity at higher temperatures than MBP-Ag0139. For example, at a reaction temperature of 50 °C, MBP-Ag0139-NFs retains 82.8% of its optimal activity, while the free enzyme retains only 7.82% of its optimal activity. Figure 8 b).

[0059] The immobilized enzyme MBP-Ag0139-NFs exhibited significantly higher thermostability at 40 °C than the free enzyme. At 40 °C and 45 °C, the half-lives of the immobilized enzyme were 44.29 min and 16.33 min, respectively, significantly longer than those of the free enzyme (9.30 min and 4.11 min, respectively). Figure 8 c).

[0060] (3) Optimal reaction pH and pH stability Both MBP-Ag0139-NFs and MBP-Ag0139 exhibited high enzyme activity under neutral and alkaline conditions, with optimal reaction pH of pH 8-9 for both. Within the pH range of 7-10, enzyme activity showed little fluctuation. Figure 8 d). Within the pH range of 5-6, the immobilized enzyme retains over 90% of its optimal pH enzyme activity, significantly higher than that of the free enzyme. This indicates that immobilization broadens the pH range of the enzyme-catalyzed reaction by MBP-Ag0139.

[0061] Immobilized enzymes also exhibit better pH stability than free enzymes. Immobilized enzymes, stored at 4°C for 25 days within a pH range of 5-9, retain over 70% of their enzyme activity. Figure 8 f). Free enzymes can only maintain this level within the pH range of 8-9; after 3 days of storage at pH 5-6, enzyme activity drops below 20%. At pH 7, after 20 days of storage, free enzyme activity drops to 50%. Figure 8 e).

[0062] This demonstrates that the pH stability range of immobilized enzymes is significantly better than that of free enzymes, especially in the slightly acidic pH range of 7. The improved acid resistance of agarases holds great promise for applications in the preparation of agar oligosaccharides based on acid hydrolysis-biocatalytic coupling.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gellulase AgO139, characterized by, The amino acid sequence of the agarase Ag0139 is shown as SEQ ID NO.

1.

2. A fusion enzyme MBP-AgO139, characterized by, The amino acid sequence of the fusion enzyme MBP-Ag0139 is shown as SEQ ID NO.

3.

3. A fusion enzyme MBP-AgO139-SpyTag, characterized in that, The fusion enzyme MBP-Ag0139-SpyTag is obtained by modification based on the fusion enzyme MBP-Ag0139 of claim 2, and the amino acid sequence of the fusion enzyme MBP-Ag0139-SpyTag is shown as SEQ ID NO.

5.

4. A biological material related to the agarase Ag0139 of claim 1, or the fusion enzyme MBP-Ag0139 of claim 2, or the fusion enzyme MBP-Ag0139-SpyTag of claim 3, the biological material comprising at least one of (a)-(e): (a) a polynucleotide encoding the agarase Ag0139 of claim 1, or the fusion enzyme MBP-Ag0139 of claim 2, or the fusion enzyme MBP-Ag0139-SpyTag of claim 3; (b) an expression cassette comprising the polynucleotide of (a); (c) a recombinant vector comprising the polynucleotide of (a); (d) a recombinant microorganism comprising the polynucleotide of (a); (e) a recombinant microorganism comprising the recombinant vector of (c).

5. A method for improving soluble expression of Agarase AgO139, characterized in that, The method comprises the following steps: fusing a nucleic acid molecule encoding the agarase Ag0139 with a nucleic acid molecule encoding maltose binding protein to construct a fusion gene, and expressing the fusion gene in Escherichia coli.

6. A method for the preparation of an immobilized enzyme, characterized by, The method comprises the following steps: S1, mixing the SpyCatcher protein with a sodium phosphate buffer and a CaCl2 solution to form nanoflowers; S2, mixing the nanoflowers with the fusion enzyme MBP-Ag0139-SpyTag solution, and fixing the fusion enzyme MBP-Ag0139-SpyTag on the nanoflowers.

7. The method for preparing an immobilized enzyme according to claim 6, characterized by, The molar ratio of the SpyCatcher protein, the sodium phosphate buffer, and the CaCl2 solution is (1-2):(1-2):(1-2).

8. An immobilized enzyme prepared by the method of any one of claims 6-7.

9. A method for degrading agar or preparing agarooligosaccharides, characterized by, The agarase Ag0139 of claim 1, or the fusion enzyme MBP-Ag0139 of claim 2, or the fusion enzyme MBP-Ag0139-SpyTag of claim 3, or the immobilized enzyme of claim 8.

10. Use of the agarase Ag0139 of claim 1, or the fusion enzyme MBP-Ag0139 of claim 2, or the fusion enzyme MBP-Ag0139-SpyTag of claim 3, or the immobilized enzyme of claim 8 in degrading agar or preparing agar oligosaccharides.

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