Brogia fusco-purpurea polysaccharide degrading enzyme gene, degrading enzyme as well as preparation method and application of degrading enzyme
By cloning the Gal43B gene of the erythropolysaccharide degradation enzyme and recombinantly expressing it, the problem of insufficient adaptability of the erythropolysaccharide degradation enzyme in the prior art is solved, and efficient hydrolysis of the erythropolysaccharide in a wide temperature and pH range is achieved. Provides the foundation and stability of industrial applications.
Patent Information
- Application Number
- CN202411264636.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-10
- Publication Date
- 2024-12-10
AI Technical Summary
The existing Aphrodisiac polysaccharide degradation enzymes are suitable within the range of acidic and neutral, which limits the application of Aphrodisiac polysaccharide in industrial production, especially in extreme acidic and alkaline conditions, which are difficult to effectively degrade.
The Gal43B gene of the erythrocyte polysaccharide degradation enzyme was obtained by cloning and transforming it into the host cell through recombinant expression vectors. A stable erythrocyte polysaccharide degradation enzyme in a wide temperature and pH range was prepared, which can efficiently hydrolyze the erythrocyte polysaccharide. , the products are mainly monosaccharides and small amounts of disaccharides and trisaccharides.
The efficient hydrolysis of polysaccharides of erythromycetes has been achieved, providing the basis for industrial application, and the enzyme has an activating effect on calcium ions, is highly resistant, is sensitive to certain inhibitors, but it shows good stability and active.
Smart Images
Figure CN119101694A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of gene engineering and enzyme engineering, and specifically relates to a red hair algae polysaccharide degrading enzyme gene, a degrading enzyme, and a preparation method and application thereof. Background Art
[0002] Bangia fusco-purpurea is also known as red hair algae and red hair algae. It contains essential amino acids, vitamins, polysaccharides, unsaturated fatty acids, phycobiliproteins and other biologically active substances. B. fusco-purpurea polysaccharide (BFP) is a sulfated galactan with 1,3-β-D-Galp as the main glycosidic bond configuration, combined with 1,3-β-D-Galp6S, 1,4-α-D-Galp, 1,4-α-L-AnGalp and other glycosidic bond configurations alternately linked. It is widely used in medical care, cosmetics, food industry and agriculture.
[0003] However, the adaptability of existing red algae polysaccharide degrading enzymes to pH values is in the acidic and neutral range, which limits the development of red algae polysaccharide bioresources. Therefore, the discovery of new red algae polysaccharide degrading enzymes that are resistant to extreme acidity and alkalinity is of great significance to industrial production. Summary of the invention
[0004] The purpose of the present invention is to provide a red hair algae polysaccharide degrading enzyme gene, a degrading enzyme, and a preparation method and application thereof. The red hair algae polysaccharide degrading enzyme can be used for degrading red hair algae polysaccharides to produce monosaccharides and a small amount of disaccharides and trisaccharides.
[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0006] A red hair algae polysaccharide degrading enzyme Gal43B gene, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0007] Preferably, the gene size of the red hair algae polysaccharide degrading enzyme Gal43B is 849 bp.
[0008] Another aspect of the present invention provides a red hair algae polysaccharide degrading enzyme Gal43B, whose amino acid sequence is shown in SEQ ID NO.2.
[0009] Preferably, the red hair algae polysaccharide degrading enzyme Gal43B encodes 282 amino acid residues.
[0010] The present invention also provides a recombinant expression vector, comprising the red hair algae polysaccharide degradation enzyme Gal43B gene.
[0011] The present invention also provides a recombinant expression strain, comprising the recombinant expression vector.
[0012] The present invention also provides a method for preparing red hair algae polysaccharide degrading enzyme Gal43B, comprising the following steps:
[0013] Provide red hair algae polysaccharide degradation enzyme Gal43B gene, expression vector and host cells;
[0014] Amplifying the Gal43B gene of the red hair algae polysaccharide degrading enzyme, and connecting the obtained amplification product with the expression vector to obtain a recombinant expression vector;
[0015] Transforming the recombinant expression vector into a host cell to obtain a recombinant expression strain;
[0016] Cultivating the recombinant expression strain to obtain the red hair algae polysaccharide degrading enzyme Gal43B;
[0017] The nucleotide sequence of the red algae polysaccharide degrading enzyme Gal43B gene is shown in SEQ ID NO:1.
[0018] Preferably, the host cell is one of Escherichia coli or a eukaryotic host.
[0019] Further preferably, the host cell is Escherichia coli BL21 (DE3).
[0020] Further preferably, the host cell is a eukaryotic host, including yeast or mammalian cells.
[0021] Preferably, the recombinant expression vector is transformed into Escherichia coli and induced by IPTG to obtain the red hair algae polysaccharide degrading enzyme Gal43B.
[0022] Preferably, the recombinant expression vector is transformed into Escherichia coli BL21 (DE3), and induced by IPTG to obtain the red hair algae polysaccharide degrading enzyme.
[0023] Preferably, the final concentration of IPTG is 0.5 mM and the induction temperature is 16°C.
[0024] The present invention also provides the method for preparing the red hair algae polysaccharide degrading enzyme Gal43B to prepare the obtained red hair algae polysaccharide degrading enzyme Gal43B.
[0025] The present invention also provides the red hair algae polysaccharide degrading enzyme Gal43B prepared by the preparation method or the use of the red hair algae polysaccharide degrading enzyme Gal43B in hydrolyzing red hair algae polysaccharide.
[0026] Preferably, in the reaction of hydrolyzing red hair algae polysaccharide, the reaction temperature of the red hair algae polysaccharide degrading enzyme Gal43B is 35-65° C.; and / or the reaction pH of the red hair algae polysaccharide degrading enzyme Gal43B is 5.0-8.0.
[0027] Preferably, in the reaction of hydrolyzing red hair algae polysaccharide, the optimal reaction temperature of the red hair algae polysaccharide degrading enzyme Gal43B is 55° C.; and / or the optimal reaction pH of the red hair algae polysaccharide degrading enzyme Gal43B is 6.0.
[0028] Compared with the prior art, the present invention has the following advantages and technical effects:
[0029] The present invention cloned a new red algae polysaccharide degrading enzyme Gal43B gene from Priestia megaterium JMU13, and found that the enzyme protein encoded by the gene has application potential in the fields of food, medicine, industry, etc. At the same time, the invention also cloned a large number of expressed engineering bacteria, which can realize the large-scale production of the red algae polysaccharide degrading enzyme, providing a good foundation for subsequent industrial applications.
[0030] In the preparation method of the red hair algae polysaccharide degrading enzyme Gal43B provided by the present invention, the red hair algae polysaccharide degrading enzyme Gal43B expression vector is transformed into a host cell, and the transformed host cell is then cultured to obtain the recombinant red hair algae polysaccharide degrading enzyme Gal43B from the culture. The method has simple steps, the process is easy to control, and the red hair algae polysaccharide degrading enzyme Gal43B can be stably and efficiently produced.
[0031] The red algae polysaccharide degrading enzyme Gal43B of the present invention or the red algae polysaccharide degrading enzyme Gal43B prepared by the preparation method of the red algae polysaccharide degrading enzyme Gal43B can be used in the field of hydrolyzing red algae polysaccharides. Since the red algae polysaccharide degrading enzyme Gal43B shows good stability in a wide temperature and pH range, when used to hydrolyze red algae polysaccharides, the hydrolysis reaction can be carried out efficiently and stably, which is conducive to increasing the yield of the hydrolyzate. The red algae polysaccharide degrading enzyme Gal43B provided by the present invention hydrolyzes red algae polysaccharides, and the obtained hydrolyzate is mainly monosaccharides and a small amount of disaccharides and trisaccharides. The product has good application prospects in multiple industries.
[0032] Calcium ions have an activating effect on the activity of Gal43B, a polysaccharide-degrading enzyme of red hair algae. 2+ The results showed that Ca 2+It has an activating effect on the red hair algae polysaccharide degrading enzyme Gal43B, and the activation effect of 10mmol / L is the strongest. The inhibitors EDTA, β-ME and DTT have an inhibitory effect on the activity of the red hair algae polysaccharide degrading enzyme Gal43B. The red hair algae polysaccharide degrading enzyme Gal43B has good resistance to Triton X-100. On the contrary, EDTA and CTAB greatly inhibit the activity of the enzyme. Under the conditions of 1% CTAB and 1mmol / L EDTA, the enzyme almost completely loses its activity.
[0033] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 The figure is an SDS-PAGE diagram of the induced expression and purification detection of the red hair algae polysaccharide degrading enzyme Gal43B; M is a band of a molecular weight standard protein, 1 is a band of pET-30a-gal43b-positive bacteria that are not induced in Comparative Example 1, 2 is a band of pET-30a-gal43b-positive bacteria (15°C, 16h) induced by IPTG in Comparative Example 2, 3 is a band of pET-30a-gal43b-positive bacteria (37°C, 16h) induced by IPTG in Comparative Example 3, 4 is a band of the supernatant after the whole bacteria of the red hair algae polysaccharide degrading enzyme Gal43B are broken in Example 1, and 5 is a band of the precipitate after the whole bacteria of the red hair algae polysaccharide degrading enzyme Gal43B are broken in Example 1;
[0035] Figure 2 The figure is a graph showing the effect of temperature on the activity and stability of the red algae polysaccharide degrading enzyme Gal43B provided in Example 1, wherein: Figure 2 a in the figure is the effect of temperature on the activity of red hair algae polysaccharide degradation enzyme Gal43B. Figure 2 b is a curve diagram showing the effect of temperature on the stability of red hair algae polysaccharide degradation enzyme Gal43B;
[0036] Figure 3 The graph is a graph showing the effect of pH on the activity and stability of the red algae polysaccharide degrading enzyme Gal43B provided in Example 1, wherein: Figure 3 a in the figure is the effect of pH on the activity of Gal43B, a polysaccharide-degrading enzyme of red hair algae. Figure 3 b is the curve diagram of the effect of pH on the stability of red hair algae polysaccharide degradation enzyme Gal43B;
[0037] Figure 4 This is a curve diagram showing the effect of metal ions on the activity of the red hair algae polysaccharide degrading enzyme Gal43B provided in Example 1;
[0038] Figure 5This is an analysis diagram of the red hair algae polysaccharide degrading enzyme Gal43B provided in Example 1 acting on red hair algae polysaccharide;
[0039] Figure 6 This is a graph showing the molecular weight changes of the enzymatic hydrolysis products of red hair algae polysaccharide degrading enzyme Gal43B provided in Example 1 acting on red hair algae polysaccharide;
[0040] Figure 7 This is an analysis diagram of the enzymatic hydrolysis products of red hair algae polysaccharide acting on red hair algae polysaccharide provided in Example 1. DETAILED DESCRIPTION
[0041] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.
[0042] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the common meanings understood by people with ordinary skills in the field to which the present invention belongs. If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer; if the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.
[0043] In the description of the present invention, the term "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.
[0044] The molecular biology experimental methods not specifically described in the examples of the present invention are all carried out with reference to the specific methods listed in the Molecular Cloning Experiment Guide, or according to the kits and product instructions.
[0045] The experimental materials and reagents involved in the following examples are:
[0046] Strains and vectors: Bacillus megaterium JMU13 was screened and preserved in the laboratory, and deposited in the China Center for Type Culture Collection on June 20, 2023, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20231073; Escherichia coli BL21 (DE3) was purchased from Shanghai Weidi Biotechnology Co., Ltd.; the expression vector pET-30a (+) was purchased from Youbao Biotechnology;
[0047] The bacterial genome extraction kit was purchased from Shanghai Huiling Biotechnology Co., Ltd.; the BCA protein assay kit was purchased from Beijing Solebow Technology Co., Ltd.
[0048] Example 1
[0049] Nucleotide sequence of the polysaccharide-degrading enzyme Gal43B gene from Bangia fusco-purpurea (SEQ ID NO.1):
[0050] Atgaaaacggatttaaacatcgcctttattggagctggatcaatggctgaagccatgatctcagggctgctgtcagataaaaagcttcgccccaatcaaatcaccgtaacaaatcatagcaacgataaaagacttcacgaacttcgaaacgcctatggaattaacattacgcgtaatcatgccttgttaatagaaaaaagcgatatcgtcgtgcttgctattaaaccaaaagacgtagccgaaagcatcaatacgattaagtcctatattcgtccccatcacctcgttctctctgttttagcgggtgtatcgactgccacaattgtaaacttgtttgaccaagaagtagcggttgtaagagctatgccaaatacatctgcttctattggattatcggctacagccattgcacccgggaagtttgctacagcggagcatatgaacgttactactgctttatttgaaacgattggtacggttacaactgtagaggaagagcagcttcacgctgtaacaggactatctggaagcggcccagcgtatgtctattatctagtagaagcaatggaaaaagcagctgaaaaacaaggcttggatacgtcagtgtcaaatgaactaattcttcaaacgcttatcggtgctgcagaaatgctgaaacagtcacctaaaacaccggccgtacttcgaaaagaagttatgagtcctggcggtacaacagaagcaggaatcgaacagcttatttctcataacttccaagaagcgatgatgcagtgtattgaacaagctaccattcgctctaaatatttaggagatacgattgacgaaaaaatctcgagtaaacgttcgtaa。
[0051] This example provides a polysaccharide-degrading enzyme Gal43B from Bangia fusco-purpurea
[0052] Construction of the recombinant expression vector and recombinant expression strain of the red hair algae polysaccharide degrading enzyme Gal43B gene: The genome of Priestia megaterium JMU13 was extracted using a bacterial genome extraction kit, and the red hair algae polysaccharide degrading enzyme Gal43B gene was amplified using the enzyme gene-specific primers.
[0053] The primers used to amplify the Gal43B gene of the red hair algae polysaccharide degrading enzyme include an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown in SEQ ID NO:3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:4.
[0054] Upstream primer (SEQ ID NO.3):
[0055] 5'-CGCCATATGATGAAAACGGATTTAAAC-3';
[0056] Downstream primer (SEQ ID NO.4):
[0057] 5'-CCCAAGCTTTTACGAACGTTTACT-3'.
[0058] The PCR reaction conditions were: 98°C for 1 min; 98°C for 10 sec, 55°C for 5 sec, 72°C for 5 sec; 72°C for 4 min. The amplified product was detected by agarose gel electrophoresis, and the target gene was recovered by gel cutting. The product was connected to the pET-30a(+) vector after being digested by NdeI and HindIII. The target gene was inserted into the pET-30a(+) vector to obtain a recombinant expression vector named pET-30a-gal43b. The gene sequence was confirmed by sequencing, and the recombinant expression vector was transformed into Escherichia coli BL21(DE3) to obtain a recombinant expression strain.
[0059] Cultivation of recombinant expression strains: After overnight culture of the recombinant expression strain, centrifuge at 8000r for 15min at 4°C, transfer to 50mL LB liquid medium (containing 50μg / mL kanamycin) at a volume ratio of 1:100, and culture at 37°C and 180rpm until OD 600When the pH value reaches 0.8, IPTG is added to a final concentration of 0.5 mM, and cultured at 16°C and 180 rpm for 20 h. The cells are collected by centrifugation at 4°C and 8000 rpm for 15 min. The cells are resuspended in binding buffer (15 mmol / mL imidazole, 300 mmol / mL NaCl and 50 mmol / mL NaH2PO4, pH 8.0) and ultrasonically disrupted on ice. Centrifuge at 8000 rpm for 15 min at 4°C, collect the supernatant, and then perform Ni-NTA affinity chromatography. After washing with washing buffer (30 mmol / mL imidazole, 300 mmol / mL NaCl and 50 mmol / mL NaH2PO4, pH 8.0), the supernatant is eluted with elution buffer (250 mmol / mL imidazole, 300 mmol / mL NaCl and 50 mmol / mL NaH2PO4, pH 8.0), and the eluate is collected to obtain the red hair algae polysaccharide degrading enzyme Gal43B.
[0060] Correspondingly, the amino acid sequence of the red hair algae polysaccharide degrading enzyme Gal43B is shown in SEQ ID NO.2:
[0061] MKTDLNIAFIGAGSMAEAMISGLLSDKKLRPNQITTVNHSNDKRLHELRNAYGINITRNHALLIEKSDIVVLAIKPKDVAESINTIKSYIRPHHLVLSVLAGVSTATIVNLFDQEVAVVRAMPNTSASIGLSATAIAPGKF ATAEHMNVTTALFETIGTVTTVEEEQLHAVTGLSGSGPAYVYYLVEAMEKAAEKQGLDTSVSNELILQTLIGAAEMLKQSPKTPAVLRKEVMSPGGTTEAGIEQLISHNFQEAMMQCIEQATIRSKYLGDTIDEKISSKRS.
[0062] The preparation method of Comparative Example 1 is the same as that of Example 1, except that IPTG induction is not performed during the cultivation of the recombinant expression strain.
[0063] The preparation method of Comparative Example 2 is the same as that of Example 1, except that during the cultivation of the recombinant expression strain, IPTG was added to a final concentration of 0.5 mM, and the culture was carried out at 15° C. and 180 rpm for 16 h.
[0064] The preparation method of Comparative Example 3 is the same as that of Example 1, except that during the cultivation of the recombinant expression strain, IPTG is added to a final concentration of 0.5 mM, and the culture is carried out at 37° C. and 180 rpm for 16 h.
[0065] The effect of the red algae polysaccharide degrading enzyme Gal43B provided in Example 1 and Comparative Examples 1-3 was verified by the following test:
[0066] 1. The red algae polysaccharide degrading enzyme Gal43B provided in Example 1 and Comparative Examples 1-3 was resuspended in PBS buffer, and after the protein loading buffer was denatured, it was separated and detected by SDS-PAGE, and the protein concentration was determined using a BCA protein assay kit. The results are as follows: Figure 1 .
[0067] Depend on Figure 1 It can be seen that M is a band of molecular weight standard protein, 1 is a band of pET-30a-gal43b-positive bacteria not induced in Comparative Example 1, 2 is a band of pET-30a-gal43b-positive bacteria (15°C, 16h) induced by IPTG in Comparative Example 2, 3 is a band of pET-30a-gal43b-positive bacteria (37°C, 16h) induced by IPTG in Comparative Example 3, 4 is a band of the supernatant after the whole bacteria of red hair algae polysaccharide degrading enzyme Gal43B are broken in Example 1, 5 is a band of the precipitate after the whole bacteria of red hair algae polysaccharide degrading enzyme Gal43B are broken in Example 1, and the molecular weight of red hair algae polysaccharide degrading enzyme Gal43B is 30.39KDa.
[0068] 2. Activity detection of red hair algae polysaccharide degradation enzyme Gal43B
[0069] The activity of polysaccharide-degrading enzyme Gal43B was detected by measuring the amount of reducing sugar released using the 3,5-dinitrosalicylic acid (DNS) method.
[0070] The specific operation is as follows: add 50 μL enzyme solution (enzyme solution diluted with pH 8.0 dissolution buffer) to 450 μL containing 1% red hair algae polysaccharide substrate solution (50mM, pH 6.0 NaH2PO4-Na2HPO4 buffer), react at 55°C for 15 minutes, add 500 μL DNS reagent, and heat in a boiling water bath for 10 minutes to terminate the reaction. After cooling to room temperature, measure the absorbance at 520nm, and calculate the amount of reducing sugar generated using the D-galactose standard curve. The definition of the enzyme activity unit is: under certain conditions, the amount of enzyme required to generate 1 μmol of reducing sugar per minute in 1mL enzyme solution is defined as one activity unit.
[0071] 3. Effects of test temperature and pH on the red algae polysaccharide degrading enzyme Gal43B provided in Example 1
[0072] (1) The optimal reaction temperature of the polysaccharide degrading enzyme of red hair algae was determined in the range of 35℃-65℃. The system used in the previous article was adopted, and the reaction was carried out at 35, 40, 45, 50, 55, 60 and 65℃ for 15 minutes, and the absorbance at 520nm was measured. The results are shown in Figure 2 .
[0073] The thermal stability analysis of the red hair algae polysaccharide degrading enzyme was carried out at 35, 45, 55 and 65°C. The purified enzyme solution was treated at different temperatures for 0-5h. After the heat treatment, the sample was immediately cooled on ice. The residual activity of the enzyme was measured by the standard method described above, and the untreated enzyme activity was defined as 100%. The results are shown in Figure 2 .
[0074] Depend on Figure 2 As shown in a, the optimal reaction temperature of the red hair algae polysaccharide degrading enzyme Gal43B is 55°C, and it exhibits a maximum activity of more than 60% in the temperature range of 50-65°C, and the enzyme activity decreases relatively at 65°C.
[0075] Depend on Figure 2 As shown in b, the enzyme still retains more than 90% activity at 35, 45, and 55°C. After being treated at 65°C for 1-5h, the relative enzyme activity gradually decreased, and the relative enzyme activity remained at 77.9% at 5h. These results indicate that the red hair algae polysaccharide degrading enzyme Gal43B has good temperature stability at 35-65°C.
[0076] (2) The optimal reaction pH of the red hair algae polysaccharide degrading enzyme was determined in the range of 3.0-9.0. The buffers used in the determination were: 50mmol / L citric acid-Na2HPO4 buffer (pH 3.0-6.0), 50mmol / L NaH2PO4-Na2HPO4 buffer (pH6.0-7.0), and 50mmol / LTris-HCl buffer (pH 7.0-9.0). The results are shown in Figure 3 .
[0077] The pH stability of the polysaccharide-degrading enzyme of the red hair algae was determined in the range of 5.5-7.0. The enzyme was placed in a buffer solution with different pH values and incubated at 55°C for 1 hour. The remaining activity of the enzyme was determined, and the untreated enzyme activity was defined as 100%. Figure 3 ) showed that the stability of the recombinant enzyme remained above 60% of the initial activity in the pH range of 5.5-8.0, indicating that the recombinant enzyme had good pH stability. Figure 3 .
[0078] Depend on Figure 3 As shown in a, the optimal reaction pH of the red hair algae polysaccharide degrading enzyme Gal43B is 6.0, and the enzyme shows a maximum activity of more than 60% in the pH range of 5.0-8.0.
[0079] Depend on Figure 3 As shown in b, the stability of the red hair algae polysaccharide degrading enzyme Gal43B is maintained at more than 60% of the initial activity in the pH range of 5.5-8.0, indicating that the red hair algae polysaccharide degrading enzyme Gal43B has good pH stability.
[0080] 4. Effects of testing additives on the red algae polysaccharide degrading enzyme Gal43B provided in Example 1
[0081] (1) Different metal ions with a final concentration of 1 mmol / L or 10 mmol / L were added to the polysaccharide degrading enzyme of red hair algae, including Na + , K + , Ca 2+ Mg 2+ , Ba 2+ , Mn 2+ 、Ni 2+ , Cu 2+ 、Co 2+ 、Zn 2+ , AL 3+ , Fe 3+ and Fe 2+ After being placed at 4°C for 1 hour, the remaining activity of the enzyme was measured, with the enzyme activity without the addition of metal ions being 100%. Figure 4 .
[0082] Depend on Figure 4 It can be seen that K + , Ca 2+ Mg 2+ , Ba 2+ , AL 3+ , Fe 3+ Can significantly promote enzyme activity. Among them, 10mmol / LCa 2+ After treatment, the relative activity increased to 174%; while the concentration of Cu at 10mmol / L 2+ and Fe 2+ After treatment, the enzyme activity was significantly inhibited. + , K + , Mn 2+ 、Ni 2+ , Cu 2+ 、Co 2+ , Fe 3+ and Fe 2+ The activity of recombinase Gal43B was significantly inhibited under treatment.
[0083] (2) The effect of adding inhibitors, detergents and denaturants on the activity of the polysaccharide degrading enzyme of red hair algae was determined. Inhibitors (EDTA, β-ME, DTT and PMSF) with a final concentration of 1mmol / L or 10mmol / L and surfactants (SDS, Tween 20, Tween 80 and TritonX-100, CTAB) of 0.1% and 1%, and denaturants (urea and guanidine hydrochloride) of 1mol / L and 5mol / L were added to the polysaccharide degrading enzyme of red hair algae, respectively. After being placed at 4℃ for 1h, 1% polysaccharide substrate solution of red hair algae was added, and the reaction was carried out at 55℃ for 15min. The residual activity of the enzyme was determined, and the enzyme activity without adding inhibitors, surfactants and denaturants was 100%. The results of the determination are shown in Table 1.
[0084] Table 1 Effects of inhibitors, detergents and denaturants on the activity of polysaccharide-degrading enzymes of R.
[0085]
[0086]
[0087] Note: “#” in the table means P<0.001; “*” in the table means P<0.05; “**” in the table means P<0.01.
[0088] As shown in Table 1, both β-ME and DTT showed inhibitory effects on enzyme activity at the two concentrations tested. The red hair algae polysaccharide degrading enzyme has good resistance to TritonX-100. In contrast, EDTA and CTAB greatly inhibited the activity of the enzyme. Under the conditions of 1% CTAB and 1mmol / L EDTA, the enzyme almost completely lost its activity.
[0089] 5. The red algae polysaccharide degrading enzyme Gal43B provided in Example 1 was used to act on the red algae polysaccharide, and 50 mL of a 2% red algae polysaccharide substrate solution (50 mM NaH2PO4-Na2HPO4 buffer, pH 6.0) was prepared. 1 U of red algae polysaccharide degrading enzyme was added, and the enzymatic reaction was carried out at 55°C, and samples were taken every 2 hours. After sampling, the changes in the reducing sugar content were detected. When the reducing sugar content tended to be stable, it was inactivated in a boiling water bath for 10 minutes, and the sample was centrifuged at 4°C, 8000 rpm for 10 minutes. The amount of reducing sugar released in the supernatant was determined by the DNS method. The substrate that was not treated with the recombinant enzyme was used as a negative control. The results are as follows. Figure 5 .
[0090] Depend on Figure 5It can be seen that as the treatment time increases, the reducing sugar concentration increases. After the red hair algae polysaccharide degrading enzyme Gal43B treats the substrate for 12 hours, the released reducing sugar is stable, so 12 hours is selected to extract the oligosaccharide sample. These results show that the red hair algae polysaccharide degrading enzyme Gal43B can act on red hair algae polysaccharides to produce reducing sugars.
[0091] 6. The red hair algae polysaccharide degrading enzyme Gal43B provided in Example 1 was used to act on the red hair algae polysaccharide to analyze the molecular weight change of the enzymatic hydrolysis product. The enzymatic hydrolyzate of different recombinant enzyme hydrolysis times was collected and inactivated and centrifuged. HPLC-ELSD was used to detect the change in substrate molecular weight during the reaction and the distribution of oligosaccharide products. The TSKgel SuperAW4000 column was used to detect the change in substrate molecular weight. The mobile phase was ultrapure water at a flow rate of 0.8 mL / min. The results are shown in Figure 6 .
[0092] Depend on Figure 6 It can be seen that at 0h of reaction, the sample retention time was 18.229min. As the reaction time increased, the sample retention time increased to 19.467min (6h), 19.73min (12h), and 20.23min (24h). From this analysis, as the enzymatic hydrolysis time increased, the molecular weight of the polysaccharide also decreased. At the same time, an oligosaccharide peak was observed at the retention time of 20min-30min, and the oligosaccharide content continued to accumulate as the reaction time increased.
[0093] 7. Analysis of the enzymatic hydrolysis products of the red hair algae polysaccharide degrading enzyme Gal43B provided in Example 1, take 1U of the red hair algae polysaccharide degrading enzyme and add it to 50mL of a 2% red hair algae polysaccharide substrate solution, and react at 55°C. Samples were taken every 2h, and the amount of reducing sugar released was determined by the DNS method. When the reducing sugar tended to be stable, it was inactivated in a boiling water bath for 10min, centrifuged at 4°C and 8000rpm for 10min, and 3 times the volume of anhydrous ethanol was added to the supernatant for alcohol precipitation, overnight at 4°C, and the supernatant was centrifuged to remove the anhydrous ethanol by rotary evaporation, and the oligosaccharide sample was obtained by freeze-drying. The enzymatic hydrolysis products were identified using Waters I-Class and Waters XEVO-TQS micro equipped with an ACQUITY UPLCr BEH C18 column (1.7μm). The two mobile phases were solution A: ultrapure water, solution B: acetonitrile (95%: 5%), and the gradient flow rate was 0.2mL / min. Mass spectra were obtained using a Waters I-Class, Waters XEVO-TQS micro mass detector. Figure 7 .
[0094] Depend on Figure 7It can be seen that the fragmentation peak m / z 177 represents unsaturated monosaccharides, the fragmentation peaks m / z 357 and m / z 397 represent unsaturated disaccharides, and the fragmentation peaks m / z 547 and m / z 578 represent unsaturated trisaccharides. This indicates that the red hair algae polysaccharide degrading enzyme Gal43B degrades the red hair algae polysaccharide to generate monosaccharides and a small amount of disaccharides and trisaccharides.
[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.
Claims
1. A red algae polysaccharide degrading enzyme Gal43B gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.
1.
2. A red algae polysaccharide degrading enzyme Gal43B, characterized in that: Its amino acid sequence is shown in SEQ ID NO.
2.
3. A recombinant expression vector, characterized in that: It comprises the red hair algae polysaccharide degrading enzyme Gal43B gene as described in claim 1.
4. A recombinant expression strain, characterized in that: Comprising the recombinant expression vector according to claim 3.
5. A method for preparing a red algae polysaccharide degrading enzyme Gal43B, characterized in that: The steps include: Provide red hair algae polysaccharide degradation enzyme Gal43B gene, expression vector and host cells; Amplifying the Gal43B gene of the red hair algae polysaccharide degrading enzyme, and connecting the obtained amplification product with the expression vector to obtain a recombinant expression vector; Transforming the recombinant expression vector into a host cell to obtain a recombinant expression strain; Cultivating the recombinant expression strain to obtain the red hair algae polysaccharide degrading enzyme Gal43B; Wherein, the nucleotide sequence of the red hair algae polysaccharide degrading enzyme Gal43B gene is shown in SEQ ID NO: 1; The host cell is one of Escherichia coli or a eukaryotic host.
6. The preparation method according to claim 5, characterized in that: The recombinant expression vector is transformed into Escherichia coli and induced by IPTG to obtain the red hair algae polysaccharide degrading enzyme Gal43B.
7. The preparation method according to any one of claims 5 to 6, characterized in that: The final concentration of IPTG was 0.5 mM, and the induction temperature was 16°C.
8. The red hair algae polysaccharide degrading enzyme Gal43B prepared by the method for preparing the red hair algae polysaccharide degrading enzyme Gal43B according to any one of claims 5 to 7.
9. Use of the red hair algae polysaccharide degrading enzyme Gal43B prepared by the preparation method according to any one of claims 5 to 7 or the red hair algae polysaccharide degrading enzyme Gal43B according to claim 8 in hydrolyzing red hair algae polysaccharides.
10. The use according to claim 9, characterized in that: In the reaction of hydrolyzing red hair algae polysaccharide, the reaction temperature of the red hair algae polysaccharide degrading enzyme Gal43B is 35-65° C.; and / or the reaction pH of the red hair algae polysaccharide degrading enzyme Gal43B is 5.0-8.0.