κ-carrageenase and its preparation method and application
The κ-carrageenase Cgk-GDSX483 prepared through genetic engineering solves the problem of high cost of preparing carrageenan oligosaccharides by bio-enzymatic methods, realizes the industrial production of carrageenase and the efficient preparation of oligosaccharides, has clear enzymatic hydrolysis characteristics, and maintains high activity under suitable conditions.
Patent Information
- Application Number
- CN202411788452.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2044-12-06
AI Technical Summary
In the prior art, the sources of efficient enzymes for preparing carrageenan oligosaccharides by bioenzymatic methods are limited, resulting in high production costs and a complicated process, making industrial production difficult to achieve.
Through genetic engineering technology, a κ-carrageenanase (Cgk-GDSX483) was prepared with a clear amino acid sequence and coding gene. It was recombined and introduced into engineered bacteria for expression, and can specifically enzymatically hydrolyze κ-carrageenan to produce carrageenan oligosaccharides.
The industrial production of carrageenanase and the efficient preparation of carrageenan oligosaccharides have been achieved. The enzymatic hydrolysis characteristics are clear, the suitable temperature and pH range are wide, and metal ions promote the activity to generate carrageenan disaccharides, tetrasaccharides and hexasaccharides.
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Figure CN119331855B_ABST
Abstract
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 immunomodulation, anti-tumor, antiviral, antioxidant, and anticoagulant activities, and are widely used in biomedicine, agriculture, food and other fields.
[0003] Carrageenan oligosaccharides are primarily prepared through chemical hydrolysis, physical methods, and enzymatic methods. Physical and chemical degradation processes are difficult to control, with harsh reaction conditions, uncontrolled product formation, and varying degrees of structural damage. Enzymatic methods, however, offer advantages such as mild reaction conditions and high product specificity, and are therefore attracting widespread attention.
[0004] However, for the bioenzymatic preparation of carrageenan oligosaccharides, the sources of efficient bioenzymes are 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 discover efficient carrageenase to meet the production 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 their industrial production, the present invention provides a κ-carrageenanase 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 comprising the aforementioned κ-carrageenase encoding gene.
[0010] In a fourth aspect, the present invention further 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 further provides the use of the κ-carrageenanase 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-carrageenanase 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 uses genetic engineering technology to obtain a κ-carrageenanase (i.e., carrageenanase Cgk-GDSX483) that can specifically enzymatically degrade κ-carrageenan. The amino acid sequence of the κ-carrageenanase 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 an expression vector after enzyme digestion and introduced into an engineered bacterium to obtain a recombinant engineered bacterium. The engineered bacterium expresses the κ-carrageenanase, which can degrade κ-carrageenan and generate carrageenan disaccharides, carrageenan tetrasaccharides, and carrageenan hexasaccharides. The present invention has important contribution significance to the industrial production of carrageenanase and the preparation of carrageenan oligosaccharides.
[0019] The present invention clarifies the enzymatic hydrolysis characteristics of carrageenanase Cgk-GDSX483: its optimal reaction temperature is 40°C and its optimal reaction pH is 8.0. In addition, carrageenanase Cgk-GDSX483 can still retain more than 95% of its activity after incubation at 30°C for 7 hours, and maintains 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 colored pre-stained protein marker, and 1 represents carrageenase Cgk-GDSX483.
[0022] Figure 3 is the relative activity of carrageenase Cgk-GDSX483 at different temperatures.
[0023] Figure 4 The temperature stability of carrageenase Cgk-GDSX483.
[0024] Figure 5 is the relative activity of carrageenase Cgk-GDSX483 at different pH.
[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 table, 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] Figure 9 This is the liquid chromatography-mass spectrometry (Q-TOF-MS) analysis of carrageenase Cgk-GDSX483. Figure 9 In the figures, DP2, DP4 and DP6 represent carrageenan disaccharide, carrageenan tetrasaccharide and carrageenan hexasaccharide, respectively. DETAILED DESCRIPTION
[0029] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. It is obvious that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.
[0030] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.
[0031] Main test materials:
[0032] Escherichia coli expression vector pET-28a(+) was provided by Hangzhou Baosai Biotechnology Co., Ltd.
[0033] Escherichia coli competent cells BL21 (DE3) were purchased from 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] Primers for amplification of the gene encoding carrageenase Cgk-GDSX483 (amino acid sequence shown in SEQ ID NO: 1) (nucleotide sequence 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] The gene encoding the carrageenase Cgk-GDSX483 was amplified using the upstream and downstream primer sequences. The amplification system consisted of 25 μL of 2× pfu PCR mix, 2 μL of each upstream and downstream primer, 2 μL of template, and 19 μL of ddH2O, for a total of 50 μL. The amplification program was as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s, followed by a 10°C incubation period.
[0041] Figure 1 This 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 engineered 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). The recovered fragments were ligated to the E. coli expression vector pET-28a(+) digested at the same sites and transformed into competent E. coli BL21(DE3) cells. After transformation, the cells were plated on LB agar plates containing kanamycin and incubated overnight at 37°C. Monoclonal colonies that emerged were selected for positive verification and sequencing. Recombinant bacteria that were sequenced correctly were re-inoculated onto LB agar plates containing kanamycin to obtain the 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 volume. The culture was expanded at 37°C and 220 rpm. When the bacterial solution OD 600 =0.5, 0.1 mM IPTG was added and induced at 25°C for 12 h.
[0046] After induction, the culture medium was collected and centrifuged at 8000 rpm / min for 10 minutes. The cells were harvested and resuspended in phosphate buffer (pH 8.0). Ultrasonic disruption was performed on ice for 30 minutes. The disrupted liquid was centrifuged at 8000 rpm / min for 10 minutes. 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 analyzed by SDS-PAGE. Figure 2 This is the SDS-PAGE detection result of carrageenase Cgk-GDSX483. Figure 2 A purified single protein band was observed with a relative molecular weight of approximately 35 kDa, 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 the mixed system, the concentration of κ-carrageenan was 0.2%, and the concentration of carrageenase Cgk-GDSX483 was 2 U / mL. The mixture was incubated for 30 minutes, 500 μL of DNS reagent was added and mixed, and the mixture was boiled for 10 minutes for color development.
[0050] 1. Optimum reaction temperature
[0051] Temperature conditions of 4, 20, 30, 40, 50, 60, 70, and 100° C. were selected, 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. Figure 3 is the relative activity of carrageenase Cgk-GDSX483 at different temperatures. Figure 3 It was found that the optimal reaction temperature of carrageenase Cgk-GDSX483 was 40°C.
[0052] 2. Temperature stability
[0053] Carrageenase Cgk-GDSX483 was incubated at temperatures of 30, 40, 50, and 70°C for 7 hours. After the incubation, the enzyme solution was collected 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] At the optimal reaction temperature (40°C), the substrate was dissolved in buffers ranging from pH 3.0 to 11.0, and the relative activity of carrageenase Cgk-GDSX483 in buffers of varying pH values was determined. In these experiments, the buffers at pH values of 3.0, 4.0, 5.0, and 6.0 were citric acid buffer; the buffers at pH values of 6.0, 7.0, and 8.0 were phosphate buffer; the buffers at pH values of 8.0, 9.0, and 10.0 were Tris-HCl buffer; and the buffers at pH values of 9.0, 10.0, and 11.0 were sodium carbonate buffer. Figure 5 is the relative activity of carrageenase Cgk-GDSX483 at different pH values. Figure 5 It was found that the optimal reaction pH of carrageenase Cgk-GDSX483 was 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 was incubated with the substrate 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 4.0-11.0, and the relative activity of the carrageenase Cgk-GDSX483 is relatively high within this range.
[0058] 5. Influence of different metal ions
[0059] NiCl2, CuCl2, FeSO4, MnSO4, MgSO4, ZnSO4, CaCl2, KCl, BaCl2, and NaCl reagents were prepared with a final concentration of 5 mmol / L respectively. The enzyme solution and substrate were incubated at 40°C for 30 min, using no metal ions as a control, to determine the relative activity of carrageenase Cgk-GDSX483. 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 kappa-carrageenan oligosaccharides using carrageenase Cgk-GDSX483.
[0062] A 0.5% κ-carrageenan substrate solution was prepared with phosphate buffer (pH = 8.0), and 10% carrageenanase Cgk-GDSX483 enzyme solution was added according to the total reaction volume (the final concentration of the enzyme solution in the mixed system was 2 U / mL). The reaction was carried out at 40 ° C for 10 min, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 12 h, 24 h, 30 h, 36 h, 48 h, and 72 h, respectively. After the reaction, the reaction was terminated by heating and boiling for 10 min, cooled to room temperature, and the enzymatic hydrolysis products were analyzed.
[0063] 1. Thin Layer Chromatography Analysis
[0064] The silica gel thin layer plate was activated in a drying oven at 100°C for 1 h, 3 μL of the above reaction solution was spotted on the silica gel plate, and galactose, carrageenan disaccharide, carrageenan tetrasaccharide and carrageenan hexasaccharide were used as standard controls. The plate was developed in a developing agent of n-butanol: ethanol: water = 3:2:2 and then blown dry. The plate was then soaked in a color developer (2 g of diphenylamine, 2 mL of aniline, 10 mL of 85% phosphoric acid, 1 mL of concentrated hydrochloric acid, and 100 mL of acetone) and then blown dry. The plate was heated at 110°C for 10 min for color development.
[0065] Figure 8The following are the 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 tetraose, and carrageenan hexaose, 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 tetraose and carrageenan hexaose are mainly produced.
[0066] 2. Liquid chromatography-mass spectrometry (Q-TOF-MS) analysis
[0067] Reaction solutions at 10 min, 30 min, 1 h, 5 h, 12 h, and 24 h were diluted to 50 μg / mL, filtered through a 0.22 μm microporous filter, and analyzed on a TOF-MS system. Chromatographic conditions included an injection volume of 5 μL, a mobile phase of acetonitrile:1 mM formic acid in water (1:1), a flow rate of 0.2 mL / min, and a column temperature of 35°C. Mass spectrometry was performed using an electrospray ionization (ESI) source in negative ionization mode, with a mass scan range of m / z = 50 to 2000 Da. Figure 9 The results of liquid chromatography-mass spectrometry (Q-TOF-MS) analysis of carrageenase Cgk-GDSX483 are shown. Figure 9 In the formula, DP2, DP4 and DP6 represent carrageenan disaccharide, carrageenan tetrasaccharide and carrageenan hexasaccharide, respectively. Figure 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-carrageenanase (i.e., carrageenanase Cgk-GDSX483) that can specifically enzymatically hydrolyze κ-carrageenan by means of genetic engineering technology. The amino acid sequence of the κ-carrageenanase 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 cleavage and introduced into an engineering bacterium to obtain a recombinant engineering bacterium. The engineering bacterium expresses the κ-carrageenanase, and the κ-carrageenanase 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 carrageenanase and the preparation of carrageenan oligosaccharides. The present invention also clarifies the enzymatic hydrolysis characteristics of carrageenanase Cgk-GDSX483, which has guiding significance for the preparation or production of carrageenan oligosaccharides.
[0069] The embodiments described above are only some of the embodiments of the present invention, not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within 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: 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 according to claim 4 is transformed into an engineered bacterium to produce the κ-carrageenase.
6. An engineered bacterium, characterized in that: Comprising the expression vector according to claim 4.
7. The use of the κ-carrageenanase according to claim 1 in degrading κ-carrageenan, characterized in that: The κ-carrageenanase degrades κ-carrageenan to generate κ-carrageenan oligosaccharides; The kappa-carrageenan oligosaccharides are carrageenan disaccharide, carrageenan tetrasaccharide and carrageenan hexasaccharide.
8. 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 are carrageenan disaccharides, carrageenan tetrasaccharides and carrageenan hexasaccharides.