A kind of kelp mannuronate C5 isomerase and its application

Abstract

The present invention aims to provide a kelp mannuronic acid C5 isomerase gene and its application in the synthesis of alginate. The amino acid sequence of the kelp mannuronic acid C5 isomerase provided is SEQ ID NO: 1. The kelp MC5E protein of the present invention can be soluble expressed in a prokaryotic expression system, and can convert β-D-mannuronic acid (M) into α-L-guluronic acid (G) at the level of polymer polyM. The conversion efficiency is improved, and the content of G residues in the reaction product is increased, so as to synthesize alginate with higher hardness.

Description

Technical Field

[0001] The invention belongs to the technical field of enzymology, and in particular relates to a laminaria mannuronate C5 isomerase gene and application thereof. Background Art

[0002] Algin, also known as alginic acid polysaccharide, is a general term for a class of polysaccharides produced by brown algae and bacteria. Algin is a linear polymer composed of two monomers, β-D-mannuronic acid (M) and its C5 reverse isomer α-L-guluronic acid (G), and is the main carbon metabolite of kelp. Mannuronate C5 isomerase (referred to as MC5E) is an important modification enzyme in the synthesis pathway of alginate. It can convert β-D-mannuronic acid into α-L-guluronic acid (G) on the basis of polymer polyM. The lower the G content in alginate, that is, the higher the M / G ratio, the lower the gel strength and higher the flexibility. The higher the G content in alginate, that is, the lower the M / G ratio, the higher the gel strength and higher rigidity, as well as better mechanical properties and higher antibacterial activity. Summary of the invention

[0003] The present invention aims to provide a kelp mannuronate C5 isomerase gene and its application in synthesizing alginate.

[0004] The present invention first provides a kelp mannuronate C5 isomerase, comprising:

[0005] 1) a protease having an amino acid sequence of SEQ ID NO: 1,

[0006] 2) Proteases derived from 1) by replacing, deleting or adding one or more amino groups to 1);

[0007] MRPVLEAAAILSVFVSRLGAFARDTSSPNSAISKPSCNKGIDVSVRYSGRSDRLYIESANGRTRGGCITLKQIWNIRDGEGPLYAVDKNGDISDKVTGRWLLTDDLYVKDGITLKVHGKKGGGDA DELRLLSTKKKYINLRGHGGNLDFVHTKVWAWDTDRNYYDKDESDGRSFISVISEVITNSRESCSGKAKDDMGEARMDIVDSEMAYLGFQDSESYGLTWKVRGYCSDKRNPDVFDKVNVYGNIYDS EIHHNNFGVYTYGHKRGDWRRNRVHDNSGYGFDPHDDSDYLTIHDNKVFDNEWHGIIASERCNGVSIQGNEVYGGSSSNSAGIFLHRSSNNAIVKDNYVHDNGDAGIALLEVFDAEVSHNTFKDNKY GMRLSVGCGDNFIYNNDFIDSSKHNLLTYIGADKPEISKNARSKDNVFKENKFSGKGESVKLKEADDTQFLYNRFYSADKIRFYDCEGTIMKGNTGLSSVDVKVTHSSCFDHKCDDGFEPMC (SEQ ID NO:1).

[0008] The present invention also provides a gene for encoding the above-mentioned Laminaria japonica mannuronate C5 isomerase, a nucleotide sequence of which is SEQ ID NO: 2;

[0009]

[0010] Another aspect of the present invention provides a recombinant plasmid, into which the above-mentioned gene is inserted.

[0011] The present invention also provides a recombinant engineering strain, wherein the recombinant engineering strain carries the recombinant plasmid;

[0012] The recombinant engineering strain is a prokaryotic recombinant engineering strain.

[0013] The present invention also provides a use of the laminaria kelp mannuronic acid C5 isomerase, which is used in catalyzing the conversion of β-D-mannuronic acid (M) into α-L-guluronic acid (G);

[0014] The kelp mannuronate C5 isomerase provided by the present invention is also used for synthesizing alginate.

[0015] The kelp MC5E protein of the present invention can be soluble expressed in a prokaryotic expression system, and can convert β-D-mannuronic acid (M) into α-L-guluronic acid (G) at the level of polymer polyM, thereby improving the conversion efficiency and the content of G residues in the reaction product, thereby synthesizing alginate with higher hardness. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The electrophoresis diagram and western blot result diagram of the MC5E gene cloning of the present invention are shown; Figure a is a diagram of MC5E gene amplification; and Figure b is a diagram of western blot detection results.

[0017] Figure 2 This is a graph showing the results of nuclear magnetic resonance hydrogen spectrum analysis of MC5E gene recombinant protease activity detection provided by the present invention as an example. DETAILED DESCRIPTION

[0018] Mannuronate C5 isomerase (MC5E) is an important modifying enzyme in the alginate synthesis pathway. It can catalyze the isomerization of β-D-mannuronate (M) to α-L-guluronate (G) at the level of polymer polyM, further increasing the G residue content in alginate and improving the mechanical properties.

[0019] Based on the results of transcriptome sequencing of Laminaria japonica, the present invention cloned a cDNA sequence of a mannuronic acid C5 isomerase (MC5E) gene from Laminaria japonica, whose open reading frame (ORF) is 1500bp and encodes a protein sequence containing 499 amino acids. Soluble recombinant protein expression was successfully obtained by using an in vitro prokaryotic expression system. Nuclear magnetic resonance analysis showed that it can convert mannuronic acid (M) into guluronic acid (G) at the level of polymer polyM, and has a higher conversion efficiency, so that the prepared alginate has a higher hardness.

[0020] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0021] Example 1: Cloning of genes

[0022] The cloning method of the Laminaria japonica MC5E gene mainly includes the following steps:

[0023] 1. Extraction of total RNA from kelp:

[0024] Take an appropriate amount of kelp sample (≤100 mg), grind it in a mortar precooled with liquid nitrogen, repeat 3 to 5 times, and extract RNA from the ground powder. Extract total RNA from kelp according to the steps of SparkJade's Polysaccharide Polyphenol / Complex Plant RNA Rapid Extraction Kit (centrifugal column type). Use SparkJade's SPARKscript Ⅱ RT Plus Kit (With gDNAEraser) synthesis kit to reverse transcribe and synthesize cDNA for gene amplification.

[0025] 2. Synthesis of MC5E gene-specific primers and sequencing of PCR amplification products: Based on the Laminaria japonica transcription data, the specific primer sequences MC5E-F and MC5E-R (Table 1) for amplifying the MC5E gene ORF were designed using Primer Premier 5 software. The amplification system was: 4µl cDNA, 1µl MC5E-F, 1µl MC5E-R, 10µl Vazyme 2xPhanta Master Mix, 4µld dH2O. The reaction procedure was: 95℃ pre-denaturation for 3min; then 35 cycles: 95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 100s; finally 72℃ extension for 5 min. The PCR product was detected by DNA electrophoresis, gel recovery, connection and transformation, and then sequenced for verification. The results showed that the nucleotide sequence of the Laminaria japonica mannuronate C5 isomerase MC5E screened by the present invention was SEQ ID NO: 2, and the amino acid sequence of the encoded protein was SEQ ID NO: 1.

[0026] 3. Analysis of MC5E protein structure: The ORF finder tool was used to detect the open reading frame of the MC5E gene sequence with the nucleotide sequence of SEQ ID NO: 2, and the nucleic acid sequence was translated into an amino acid sequence as shown in SEQ ID NO: 1. The molecular weight (MW) and isoelectric point (pI) of the protein were measured using the ProtParam online tool, and the signal peptide sequence of the protein was predicted using SignalP 4.1, and whether the protein contained a transmembrane structure was predicted using TMHMM Server v. 2.0. The full length of the MC5E gene ORF was 1500 bp, encoding a 499 amino acid sequence, the molecular weight of the encoded protein was 55.92 kDa, and the theoretical isoelectric point was 5.91.

[0027] Table 1: Primers and their nucleotide sequence information

[0028] Primer name Primer sequence (5'-3') MC5E-F CCGGAATTCATGAGGCCGGTTCTT MC5E-R TTGCGGCCGCCTAACACATGGGCTC

[0029] Example 2: Expression and purification of Laminaria japonica MC5E recombinant protein

[0030] 1. Construction and transformation of MC5E recombinant plasmid: In order to achieve soluble expression of the target protein, the cold shock protein expression vector pET-32(a) stored in the laboratory was selected for the expression of the target protein. The protein expressed by this vector has a His tag at the N-terminus, which can be used for subsequent fusion protein purification. EcoR I and Not I was used as a double restriction site, and amplification primers with restriction sites and protective bases, namely MC5E-F and MC5E-R (Table 1), were designed. The cDNA described in Example 1 was used as a template to amplify the MC5E gene. The PCR amplification conditions were the same as those of the ORF, and the target band was amplified. The electrophoresis pattern is shown in Figure 1 a. The expression vector was constructed according to the instructions of Ligation Protocol with T4 DNA Ligase to obtain the recombinant plasmid, which was transformed into Trans1-T1 competent cells. The strains with positive sequencing results were inoculated into 100 mL LB medium, shaken overnight at 37°C, the plasmid was extracted, and then transformed into BL21(DE3)pLysS competent cells for sequencing verification.

[0031] 2. Expression of MC5E recombinant protein: Take an appropriate amount of the above positive bacterial solution and shake it overnight at 37°C; inoculate it into 4LLB medium (containing ampicillin). When OD600 is about 0.6, precool the culture flask in a 15°C ice bath for 30 min, add 0.1 mM IPTG, and shake it at 15°C and 120 rpm for 24 h.

[0032] 3. Detection of MC5E recombinant protein solubility: The bacterial solution was centrifuged at 4500 rpm and 4°C for 30 min, the bacterial precipitate was collected, resuspended in sodium phosphate buffer (20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 30 mM imidazole), and ultrasonically disrupted in an ice bath for 5 s, with an interval of 10 s, until the bacterial solution was clarified; the disrupted solution was centrifuged at 4°C and 13000 rpm for 45 min, and the supernatant obtained by centrifugation was filtered with a 0.45µm filter membrane. 15µl of the protein supernatant was mixed with 5µL of 4X LDS Sample Buffer (GenScript) in a centrifuge tube, denatured at 95°C for 10 min in a PCR instrument, and reacted at 120V for 55 min by SDS-PAGE. According to the instructions of Anti His-Tag Mouse Monoclonal Antibody and Goat Anti-Mouse IgG, HRPConjugated, the primary antibody and secondary antibody were diluted at a ratio of 1:500 and 1:2000 (v / v) respectively. The protein transfer and incubation were completed using the iBlot transfer instrument and iBind protein blotting instrument. After color development, the membrane was observed using the BIO-RAD gel imaging system. Obvious bands appeared at the predicted position, indicating that the recombinant protein obtained was a soluble protein. The results are shown in Figure 1 As shown in b.

[0033] 4. Detection of MC5E recombinant protease activity: First, prepare the following enzyme activity reaction detection system: 300µl MOPS (100mM); 300µl NaCl (1M); 12µl CaCl2 (1M); 300µl polyM (2.5%); 60µl MC5E; 1.92ml ddH2O. At the same time, use the MC5E protease inactivated by the same system as the control group. After preparation, react at 30℃ for 40h. Then heat at 95℃ for 10min, centrifuge at 12000rpm for 15min, transfer the supernatant to a 50ml centrifuge tube, add 4 times the volume of anhydrous ethanol, vortex to mix, centrifuge at 12000rpm for 15min, and discard the supernatant. According to the above steps, wash the precipitate with anhydrous ethanol 3 times. After the obtained precipitate was dried, 1 ml of D2O (deuterated water) was added, and it was quickly frozen with liquid nitrogen, and then repeatedly freeze-dried 3 times with a freeze dryer, and finally dissolved in 1 ml of D2O and measured on a nuclear magnetic resonance hydrogen spectrometer (Bruker Avance Ⅲ 600MHz ) and the detection temperature was 25°C. Figure 2 As shown, the G1 peak value increased significantly, indicating that the G content in alginate increased.

[0034] In summary, the Laminaria japonica MC5E protein obtained by the present invention can be soluble expressed in a prokaryotic expression system, and the sequence is found to contain an isomerase domain. According to nuclear magnetic resonance hydrogen spectrum analysis, the Laminaria japonica MC5E protein obtained by the present invention can convert β-D-mannuronic acid (M) into α-L-guluronic acid (G) at the level of polymer polyM. The conversion efficiency is improved, the content of G residues in the reaction product is increased, and the M / G ratio in the product is reduced, thereby synthesizing alginate with higher hardness.

Claims

1. A mannuronate C5 isomerase, characterized in that The amino acid sequence of the mannuronate C5 isomerase is SEQ ID NO:

1.

2. A gene, characterized in that The gene encodes the mannuronate C5 isomerase according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

2.

4. A recombinant plasmid, characterized in that: The gene according to claim 2 is inserted into the recombinant plasmid.

5. A recombinant engineering strain, characterized in that: The recombinant engineering strain carries the recombinant plasmid described in claim 4.

6. The recombinant engineered strain according to claim 5, characterized in that The recombinant engineering strain is a prokaryotic recombinant engineering strain.

7. Use of the mannuronate C5 isomerase according to claim 1 in catalyzing the conversion of β-D-mannuronate into α-L-guluronate.

8. A method for catalyzing the conversion of β-D-mannuronic acid into α-L-guluronic acid, characterized in that: The method is catalyzed by using the mannuronate C5 isomerase according to claim 1.

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