Kappa-carrageenase as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses kappa-carrageenase as well as a preparation method and application thereof. The amino acid sequence of the kappa-carrageenase is as shown in SEQ ID NO: 1, and the nucleotide sequence of the coding gene of the kappa-carrageenase is as shown in SEQ ID NO: 2. The coding gene and an expression vector subjected to enzyme digestion are recombined and introduced into engineering bacteria, the recombinant engineering bacteria are obtained, the engineering bacteria express kappa-carrageenase (namely carrageenase Cgk-GDSX483), and the kappa-carrageenase can degrade kappa-carrageenan and generate carrageenan disaccharide, carrageenan tetraose and carrageenan hexaose. The method disclosed by the invention has important contribution significance for realizing industrial production of carrageenase and preparation of carrageenan oligosaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and discloses a kappa-carrageenase and a preparation method and application thereof. Background Art

[0002] Carrageenan oligosaccharides produced by the degradation of carrageenan have a degree of polymerization of 2 to 10, a small molecular weight, and good water solubility. They have multiple biological activities such as immunomodulatory, anti-tumor, antiviral, antioxidant, and anticoagulant activities, and are widely used in biomedicine, agriculture, food and other fields.

[0003] The main methods for preparing carrageenan oligosaccharides include violent chemical hydrolysis, physical method and bio-enzyme method. The degradation process of physical and chemical methods is difficult to control, the reaction conditions are violent, the products are uncontrolled, and their structures are damaged to varying degrees. The specific degradation method of bio-enzyme method has attracted widespread attention due to its advantages such as mild reaction conditions and good product specificity.

[0004] However, for the preparation of carrageenan oligosaccharides by bioenzymatic method, the source of efficient bioenzymes is very limited, and the enzyme preparation process is cumbersome and the production cycle is long, resulting in high production costs. Therefore, it is necessary to explore efficient carrageenase to meet the preparation needs of carrageenan oligosaccharides. Summary of the invention

[0005] In order to meet the preparation requirements of carrageenan oligosaccharides, reduce the preparation cost of carrageenan oligosaccharides, and help realize the industrial production thereof, the present invention provides a κ-carrageenase and a preparation method and application thereof.

[0006] First, the present invention provides a κ-carrageenase, the amino acid sequence of the κ-carrageenase is shown in SEQ ID NO:1.

[0007] In a second aspect, the present invention further provides a κ-carrageenase encoding gene, wherein the κ-carrageenase encoding gene comprises a nucleotide sequence encoding the above-mentioned κ-carrageenase.

[0008] Furthermore, the nucleotide sequence is shown in SEQ ID NO:2.

[0009] In a third aspect, the present invention further provides an expression vector, wherein the expression vector contains the above-mentioned κ-carrageenase encoding gene.

[0010] In a fourth aspect, the present invention also provides a method for preparing the κ-carrageenase, comprising: transferring the expression vector into an engineered bacterium to obtain the κ-carrageenase.

[0011] In a fifth aspect, the present invention also provides an engineered bacterium comprising the above-mentioned expression vector.

[0012] In a sixth aspect, the present invention also provides the use of the κ-carrageenase in degrading κ-carrageenan, wherein the κ-carrageenanase degrades κ-carrageenan to generate κ-carrageenan oligosaccharides.

[0013] Furthermore, in the above application, the κ-carrageenan oligosaccharide includes: carrageenan disaccharide, carrageenan tetrasaccharide and carrageenan hexasaccharide.

[0014] In a seventh aspect, the present invention further provides a method for preparing κ-carrageenan oligosaccharides, comprising: using the above-mentioned κ-carrageenase to degrade κ-carrageenan to generate κ-carrageenan oligosaccharides, wherein the κ-carrageenan oligosaccharides include: carrageenan disaccharides, carrageenan tetrasaccharides and carrageenan hexasaccharides.

[0015] Finally, the present invention also claims to protect the use of the above-mentioned κ-carrageenase in preparing algae protoplasts.

[0016] Compared with the prior art, the present invention "a κ-carrageenase and its preparation method and application" has the following advantages:

[0017] Beneficial effects:

[0018] The present invention obtains a κ-carrageenase (i.e., carrageenase Cgk-GDSX483) capable of specifically enzymolyzing κ-carrageenan by means of genetic engineering technology, wherein the amino acid sequence of the κ-carrageenase is shown in SEQ ID NO: 1, and the nucleotide sequence of the encoding gene thereof is shown in SEQ ID NO: 2. The encoding gene is recombined with the expression vector after enzyme cutting and introduced into an engineering bacterium to obtain a recombinant engineering bacterium, wherein the engineering bacterium expresses the κ-carrageenase, and the κ-carrageenase can degrade κ-carrageenan and generate carrageenan disaccharides, carrageenan tetrasaccharides, and carrageenan hexasaccharides. The present invention has an important contribution to the realization of the industrial production of carrageenase and the preparation of carrageenan oligosaccharides.

[0019] The present invention clarifies the enzymatic hydrolysis characteristics of carrageenase Cgk-GDSX483: its optimal reaction temperature is 40°C, and its optimal reaction pH is 8.0. In addition, carrageenase Cgk-GDSX483 can still retain more than 95% of its activity after incubation at 30°C for 7 hours, and maintains a relatively stable activity in a buffer system with a pH value of 4.0 to 11.0. The present invention also clarifies Fe 2+ 、Na + , Ba 2+ It can promote the enhancement of the activity of carrageenase Cgk-GDSX483. The above characteristics of carrageenase Cgk-GDSX483 have guiding significance for the preparation or production of carrageenan oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the PCR amplification product of the gene encoding carrageenase Cgk-GDSX483. Figure 1 In the figure, M represents DNA Marker and 1 represents PCR amplification product.

[0021] Figure 2 This is a diagram showing the results of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection of carrageenase Cgk-GDSX483. Figure 2 In the figure, M represents the colored pre-stained protein marker, and 1 represents the carrageenase Cgk-GDSX483.

[0022] Figure 3 Relative activities of carrageenase Cgk-GDSX483 at different temperatures.

[0023] Figure 4 The temperature stability of carrageenase Cgk-GDSX483.

[0024] Figure 5 The relative activities of carrageenase Cgk-GDSX483 at different pH values.

[0025] Figure 6 pH stability of carrageenase Cgk-GDSX483.

[0026] Figure 7 This is the effect of metal ions on the relative activity of carrageenase Cgk-GDSX483.

[0027] Figure 8 These are the thin layer chromatography results of the enzymatic hydrolysis products of carrageenase Cgk-GDSX483 at different reaction times. Figure 8 In the figure, Gal represents galactose, di, tetra, and hexa represent carrageenan disaccharide, carrageenan tetrasaccharide, and carrageenan hexasaccharide, respectively, and 1 to 15 represent different reaction times.

[0028] Fig. 9 This is the liquid chromatography-mass spectrometry (Q-TOF-MS) analysis of carrageenase Cgk-GDSX483. Fig. 9 In the figure, DP2, DP4 and DP6 represent carrageenan disaccharide, carrageenan tetrasaccharide and carrageenan hexasaccharide, respectively. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0031] Main test materials:

[0032] Escherichia coli expression vector pET-28a(+), Hangzhou Baosai Biotechnology Co., Ltd.;

[0033] Escherichia coli competent cells BL21 (DE3), Hangzhou Baosai Biotechnology Co., Ltd.

[0034] Example 1

[0035] This example provides a method for preparing carrageenase Cgk-GDSX483.

[0036] 1. Amplification of the gene encoding carrageenase Cgk-GDSX483

[0037] The primers for amplification of the gene encoding carrageenase Cgk-GDSX483 (amino acid sequence as shown in SEQ ID NO: 1) (nucleotide sequence as shown in SEQ ID NO: 2) were designed, and the upstream and downstream primers were:

[0038] F: 5′-GGAATTCCATATGACAAATATAAAAAGCGTGAG-3′ (SEQ ID NO: 3);

[0039] R: 5′-CCGCTCGAGTTTATCCGCGAGTTTCCAAGCT-3′ (SEQ ID NO: 4).

[0040] According to the upstream and downstream primer sequences, the coding gene of carrageenase Cgk-GDSX483 was amplified. The amplification system was: 2×pfu PCR mix 25μL, upstream and downstream primers 2μL each, template 2μL, ddH2O 19μL, total 50μL. The amplification program was: 94℃5min; 94℃30s, 55℃30s, 72℃40s, 30 cycles; 10℃ insulation.

[0041] Figure 1 It is the PCR amplification product of the gene encoding carrageenase Cgk-GDSX483. Figure 1 It can be seen that the gene encoding carrageenase Cgk-GDSX483 was successfully amplified.

[0042] 2. Obtaining recombinant engineering bacteria producing carrageenase Cgk-GDSX483

[0043] The PCR product and the E. coli expression vector pET-28a(+) were double-digested with restriction endonucleases (NdeI-xhoI), and the recovered fragments were connected to the E. coli expression vector pET-28a(+) digested at the same site, and then transformed into E. coli BL21(DE3) competent cells. After the transformation, they were spread on LB agar plates containing kanamycin, cultured at 37°C overnight, and the grown monoclonal colonies were picked for positive verification and sequencing identification. The recombinant bacteria with correct sequencing were re-inoculated on LB agar plates containing kanamycin to obtain recombinant bacteria.

[0044] 3. Preparation of carrageenase Cgk-GDSX483 using recombinant bacteria

[0045] The recombinant bacteria were inoculated into 5 mL of LB sterile liquid medium (containing 50 μg / mL kanamycin) for activation, and then inoculated into 100 mL of LB sterile liquid medium (containing 50 μg / mL kanamycin) at a 1% inoculum, and cultured at 37°C and 220 rpm. When the bacterial solution OD 600 =0.5, add 0.1 mM IPTG and induce at 25°C for 12 h.

[0046] After induction, the culture fluid was collected, centrifuged at 8000 rpm / min for 10 min, the bacteria were collected, resuspended in a phosphate buffer at pH = 8.0, and ultrasonically disrupted in an ice bath for 30 min. The disrupted liquid was centrifuged at 8000 rpm / min for 10 min, the supernatant was filtered through a 0.45 μm filter membrane, and the crude enzyme solution was collected for protein purification using Ni-NTA affinity chromatography. The purified protein was detected by SDS-PAGE. Figure 2 This is the SDS-PAGE detection result of carrageenase Cgk-GDSX483. Figure 2 A purified single band protein with a relative molecular weight of about 35 kDa was observed, indicating that the carrageenase Cgk-GDSX483 was successfully prepared.

[0047] Example 2

[0048] This example describes the enzymatic hydrolysis properties of carrageenase Cgk-GDSX483.

[0049] In this example, the relative activity of carrageenase Cgk-GDSX483 was determined according to the following method: the carrageenase Cgk-GDSX483 was mixed with 490 μL of substrate, in which the concentration of κ-carrageenan was 0.2% and the concentration of carrageenase Cgk-GDSX483 was 2 U / mL, and they were incubated together for 30 min, 500 μL of DNS reagent was added and mixed, and the mixture was boiled for 10 min for color development.

[0050] 1. Optimum reaction temperature

[0051] The temperature conditions of 4, 20, 30, 40, 50, 60, 70 and 100° C. were selected respectively, and the carrageenase Cgk-GDSX483 prepared in Example 1 was incubated with the substrate for 30 min under these temperature conditions, and the relative activity of the carrageenase Cgk-GDSX483 was measured respectively. Figure 3 is the relative activity of carrageenase Cgk-GDSX483 at different temperatures. Figure 3 It can be seen that the optimal reaction temperature of carrageenase Cgk-GDSX483 is 40°C.

[0052] 2. Temperature stability

[0053] Temperatures of 30, 40, 50, and 70°C were selected respectively, and the carrageenase Cgk-GDSX483 was incubated at the above temperatures for 7 hours. After the incubation, the enzyme solution was taken and incubated with the substrate at 40°C for 30 minutes according to the above method to determine the enzyme activity, which was used to characterize the temperature stability of the enzyme. Figure 4 The temperature stability of carrageenase Cgk-GDSX483. Figure 4 It can be seen that carrageenase Cgk-GDSX483 can still retain more than 95% of its activity after incubation at 30°C for 7 hours.

[0054] 3. Optimal reaction pH

[0055] Under the optimum reaction temperature (40°C), buffers with pH values ​​of 3.0 to 11.0 were selected to dissolve the substrate, and the relative activity of carrageenase Cgk-GDSX483 in buffers with different pH values ​​was determined. In the above test, the buffers with pH values ​​of 3.0, 4.0, 5.0, and 6.0 were citric acid buffers; the buffers with pH values ​​of 6.0, 7.0, and 8.0 were phosphate buffers; the buffers with pH values ​​of 8.0, 9.0, and 10.0 were Tris-HCl buffers; and the buffers with pH values ​​of 9.0, 10.0, and 11.0 were sodium carbonate buffers. Figure 5 is the relative activity of carrageenase Cgk-GDSX483 at different pH values. Figure 5 It can be seen that the optimal reaction pH of carrageenase Cgk-GDSX483 is 8.0.

[0056] 4. pH stability

[0057] The enzyme solution of carrageenase Cgk-GDSX483 was mixed with the above-mentioned buffer solutions of different pH values ​​and allowed to stand for 24 hours. After the standing time, the enzyme solution and substrate were incubated at 40°C for 30 minutes to determine the relative activity, thereby characterizing the pH stability of carrageenase Cgk-GDSX483. Figure 6 is the pH stability of carrageenase Cgk-GDSX483. Figure 6It can be seen that the Cgk-GDSX483 enzyme is relatively stable in a buffer system with a pH value of 4.0 to 11.0, and the relative activity of the carrageenase Cgk-GDSX483 is relatively high within this range.

[0058] 5. Effects of different metal ions

[0059] NiCl2, CuCl2, FeSO4, MnSO4, MgSO4, ZnSO4, CaCl2, KCl, BaCl2, and NaCl reagents with a final concentration of 5 mmol / L were prepared respectively. The enzyme solution and substrate were incubated at 40°C for 30 min, using no metal ions as a control, and the relative activity of carrageenase Cgk-GDSX483 was determined. Figure 7 The effect of metal ions on the relative activity of carrageenase Cgk-GDSX483. Figure 7 It can be seen that Fe 2+ 、Na + , Ba 2+ Both can promote the increase of carrageenase Cgk-GDSX483 activity.

[0060] Example 3

[0061] This example describes the preparation of κ-carrageenan oligosaccharides using carrageenase Cgk-GDSX483.

[0062] Use phosphate buffer (pH=8.0) to prepare 0.5% κ-carrageenan substrate solution, add 10% carrageenanase Cgk-GDSX483 enzyme solution according to the total reaction volume (the final concentration of the enzyme solution in the mixed system is 2U / mL), and react at 40℃ for 10min, 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 8h, 12h, 24h, 30h, 36h, 48h, and 72h, respectively. After the reaction, heat and boil for 10min to terminate the reaction, cool to room temperature, and analyze the enzymatic products.

[0063] 1. Thin Layer Chromatography Analysis

[0064] Activate the silica gel thin layer plate in a drying oven at 100°C for 1h, take 3μL of the above reaction solution and spot it on the silica gel plate, use galactose, carrageenan disaccharide, carrageenan tetrasaccharide and carrageenan hexasaccharide as standard controls, develop it in a developing agent of n-butanol: ethanol: water = 3:2:2, blow dry it, soak it in a color developer (2g of diphenylamine, 2mL of aniline, 10mL of 85% phosphoric acid, 1mL of concentrated hydrochloric acid, 100mL of acetone), blow dry it, and heat it at 110°C for 10min for color development.

[0065] Figure 8The results of thin layer chromatography of the hydrolysis products of carrageenase Cgk-GDSX483 at different reaction times. Gal represents galactose, di, tetra, and hexa represent carrageenan disaccharide, carrageenan tetrasaccharide, and carrageenan hexasaccharide, respectively, and 1 to 15 represent different reaction times. Figure 8 It can be seen that after the enzymatic hydrolysis of κ-carrageenan by carrageenase Cgk-GDSX483, carrageenan disaccharide, carrageenan tetrasaccharide and carrageenan hexasaccharide are mainly produced.

[0066] 2. Liquid chromatography mass spectrometry (Q-TOF-MS) analysis

[0067] The reaction solution of 10min, 30min, 1h, 5h, 12h, and 24h was selected, diluted to 50μg / mL, filtered through a 0.22μm microporous filter membrane, and injected into the TOF-MS system for analysis. The chromatographic conditions were as follows: injection volume of 5μL; mobile phase of acetonitrile: 1mM formic acid water = 1:1; flow rate of 0.2mL / min; column temperature of 35°C. The mass spectrometer used an electrospray ionization (ESI) ion source, negative ionization mode; mass scanning range: m / z = 50-2000Da. Fig. 9 This is the result of liquid chromatography-mass spectrometry (Q-TOF-MS) analysis of carrageenase Cgk-GDSX483. Fig. 9 In the above, DP2, DP4, and DP6 represent carrageenan disaccharide, carrageenan tetrasaccharide, and carrageenan hexasaccharide, respectively. Fig. 9 It can be seen that after κ-carrageenan is hydrolyzed by carrageenase Cgk-GDSX483, the hydrolysis products are composed of carrageenan disaccharide, carrageenan tetrasaccharide and carrageenan hexasaccharide.

[0068] In summary, the present invention obtains a κ-carrageenase (i.e., carrageenase Cgk-GDSX483) that can specifically enzymatically hydrolyze κ-carrageenan by means of genetic engineering technology. The amino acid sequence of the κ-carrageenase is shown in SEQ ID NO: 1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 2. The encoding gene is recombined with the expression vector after enzyme cutting and introduced into the engineering bacteria to obtain a recombinant engineering bacteria, and the engineering bacteria express the κ-carrageenase, and the κ-carrageenase can degrade κ-carrageenan and generate carrageenan disaccharides, carrageenan tetrasaccharides and carrageenan hexasaccharides. The present invention has an important contribution to the realization of industrial production of carrageenase and the preparation of carrageenan oligosaccharides. The present invention also clarifies the enzymatic hydrolysis characteristics of carrageenase Cgk-GDSX483, which has guiding significance for the preparation or production of carrageenan oligosaccharides.

[0069] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A κ-carrageenase, characterized in that The amino acid sequence of the κ-carrageenase is shown in SEQ ID NO:

1.

2. A κ-carrageenase encoding gene, characterized in that: The κ-carrageenase encoding gene comprises: a nucleotide sequence encoding the κ-carrageenase according to claim 1.

3. The κ-carrageenase encoding gene according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO:

2.

4. An expression vector, characterized in that: The expression vector contains the κ-carrageenase encoding gene according to claim 2 or 3.

5. The method for preparing κ-carrageenase according to claim 1, characterized in that: include: The expression vector of claim 4 is transferred into an engineered bacterium to obtain the κ-carrageenase.

6. An engineered bacterium, characterized in that: Comprising the expression vector according to claim 4.

7. Use of the κ-carrageenase according to claim 1 in degrading κ-carrageenan, characterized in that: The κ-carrageenase degrades κ-carrageenan to generate κ-carrageenan oligosaccharides.

8. The use according to claim 7, characterized in that: The κ-carrageenan oligosaccharides include carrageenan disaccharides, carrageenan tetrasaccharides and carrageenan hexasaccharides.

9. A method for preparing kappa-carrageenan oligosaccharides, characterized in that: include: The κ-carrageenan is degraded by the κ-carrageenanase according to claim 1 to generate κ-carrageenan oligosaccharides, wherein the κ-carrageenan oligosaccharides include carrageenan disaccharides, carrageenan tetrasaccharides and carrageenan hexasaccharides.

10. Use of the κ-carrageenase according to claim 1 in preparing algae protoplasts.

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