κ-carrageenanase cgk-GDSX478 for directed preparation of κ-carrageenan disaccharide

Abstract

Provided is κ-carrageenanase Cgk-GDSX478 for directed preparation of κ-carrageenan disaccharide, belonging to the technical field of biological enzymes. The nucleotide sequence of the gene Cgk-GDSX478 encoding κ-carrageenanase is shown in SEQ ID NO: 1, and the encoded κ-carrageenanase Cgk-GDSX478 amino acid sequence is shown in SEQ ID NO: 2. Also provided is a method for preparing κ-carrageenan disaccharide: adding the κ-carrageenanase Cgk-GDSX478 to a κ-carrageenan solution, and performing enzymolysis to obtain κ-carrageenan disaccharide; the enzymolysis reaction conditions are: the temperature is 4-70°C, the pH is 3.0-11.0, and the duration is 30-1440 min. The κ-carrageenan disaccharide obtained by degrading κ-carrageenan using the κ-carrageenanase has a high degree of homogeneity, and is of great significance for the industrial production of κ-carrageenan disaccharide.

Description

Kappa-carrageenase Cgk-GDSX478 for directed preparation of kappa-carrageenan disaccharide TECHNICAL FIELD

[0001] The present application belongs to the field of biological enzymes, and relates to a kappa-carrageenase Cgk-GDSX478 for directed preparation of kappa-carrageenan disaccharide. BACKGROUND

[0002] As a sulfated polysaccharide, carrageenan has the effects of immunomodulation, anti-tumor, and anti-virus. Carrageenan oligosaccharide is a degradation product thereof, and has the advantages of lower molecular weight and higher solubility compared with carrageenan. At the same time, due to the full exposure of active groups, the activity is significantly improved, and it shows good application prospects in the fields of biological medicine, food, and agriculture. Due to the diverse structural types of carrageenan, there are various connection modes and branching forms. After certain biological technology treatment (such as physical degradation, oxidative degradation, acid degradation, enzymatic degradation, and molecular modification), the structure of the obtained carrageenan oligosaccharide is relatively complex, and the structures and activities of the oligosaccharides obtained by different preparation methods are different.

[0003] The prior art mostly uses chemical hydrolysis to prepare carrageenan oligosaccharide. Due to the fact that the chemical hydrolysis reaction process is often too violent, the structure of the product is damaged in the hydrolysis process, and therefore the application is greatly limited. The specific degradation method using carrageenanase is considered to be a promising preparation method due to the mild reaction conditions and good product specificity. At present, the products prepared by enzymatic hydrolysis of carrageenan to prepare carrageenan oligosaccharide are mostly disaccharides, tetrasaccharides, and hexasaccharides, and even a small amount of octasaccharides. Therefore, it is of important research value and broad development prospects to develop an enzymatic hydrolysis technology with single product prepared by carrageenanase, and it is also an important direction for the high-value development of the carrageenan industry.

[0004] In the present application, the genome of Photobacterium rosenbergii GDSX-4 disclosed in CN117247877A published on December 19, 2023 is used as a template, and the enzyme-producing gene thereof is subjected to PCR amplification. SUMMARY

[0005] In view of the above prior art, the present application provides a kappa-carrageenase Cgk-GDSX478 which can degrade kappa-carrageenan and produce kappa-carrageenan disaccharide. The enzyme can be used for directed preparation of kappa-carrageenan disaccharide, and has important significance for the industrial production of kappa-carrageenan disaccharide.

[0006] To achieve the technical purpose of the present application, in one aspect, the present application provides a gene encoding kappa-carrageenase, the gene is Cgk-GDSX478, the nucleotide sequence of the gene is shown as SEQ ID NO: 1.

[0007] Further, the gene Cgk-GDSX478 of the present application is derived from Photobacterium luciferium GDSX-4.

[0008] Further, the present application provides a recombinant expression vector, the present application uses restriction endonuclease (NdeI-xhoI) to double digest Cgk-GDSX478 and E. coli expression vector pET-28a (+) respectively, and connects the recovered fragment to the same site of the digested E. coli expression vector to obtain the recombinant expression vector.

[0009] On the other hand, the present application claims a recombinant engineering bacterium comprising the above-mentioned gene encoding kappa-carrageenase or the recombinant expression vector, the host of the recombinant engineering bacterium is E. coli, and the E. coli is BL21 (DE3).

[0010] Further, the present application transforms the BL21 (DE3) competent cell with the recombinant expression vector, spreads on the LB agar plate with kanamycin resistance, incubates at 37℃ overnight, picks the single colony for positive verification and identification sequencing. The single colony with correct sequencing is inoculated again on the LB agar plate with kanamycin resistance to obtain the recombinant engineering bacterium.

[0011] On the other hand, the present application claims a kappa-carrageenase encoded by the above-mentioned gene producing kappa-carrageenase or produced by the recombinant engineering bacterium. The carrageenase is Cgk-GDSX478, and the amino acid sequence is shown as SEQ ID NO: 2.

[0012] Further, the present application inoculates the recombinant engineering bacterium into 100 mL of LB sterile liquid medium containing 50 μg / mL of kanamycin after activation at a volume ratio of 1%, and incubates at 37℃, 220 rpm, and when the OD 600 of the bacterial liquid is 0.5, 0.1 mM of IPTG is added, and the induction is performed at 25℃ for 12 h. After the fermentation is completed, the culture solution is centrifuged at 8000 rpm / min for 10 min, the bacterial body is collected, the supernatant is filtered through a 0.45 μm filter membrane, and the crude enzyme solution is collected for protein purification by Ni-NTA affinity chromatography. The amino acid sequence of the purified protein is shown as SEQ ID NO: 2, and the relative molecular weight is about 35 kDa detected by SDS-PAGE.

[0013] The application also claims the use of the kappa-carrageenase in the targeted preparation of kappa-carrageenan disaccharide. The kappa-carrageenase has an enzymatic hydrolysis temperature of 4-60 DEG C, an enzymatic hydrolysis pH of 3.0-11.0, and an enzymatic hydrolysis time of 30-1440 min; preferably, the kappa-carrageenase has an enzymatic hydrolysis temperature of 40 DEG C, an enzymatic hydrolysis pH of 8.0, and an enzymatic hydrolysis time of 420 min.

[0014] Further, the application detects the enzyme activity of the kappa-carrageenase under the condition of 4-60 DEG C and finds that the kappa-carrageenase Cgk-GDSX478 has good relative activity under 4-60 DEG C, and the optimum reaction temperature is 40 DEG C. After the kappa-carrageenase Cgk-GDSX478 is used to detect the enzyme activity after carrageenan is dissolved in a buffer with a pH of 3.0-11.0, it is found that the kappa-carrageenase Cgk-GDSX478 is relatively stable in the buffer system with a pH of 3.0-11.0 and can exert enzymatic hydrolysis, and the optimum reaction pH is 8.0.

[0015] Further, the kappa-carrageenan oligosaccharide is prepared by using the kappa-carrageenase Cgk-GDSX478 to hydrolyze kappa-carrageenan, and it is found by thin layer chromatography and liquid chromatography-mass spectrometry analysis that the enzymatic hydrolysis product of the kappa-carrageenase Cgk-GDSX478 after hydrolyzing kappa-carrageenan is kappa-carrageenan disaccharide.

[0016] Compared with the prior art, the technical scheme provided by the application has at least the following beneficial effects or advantages:

[0017] The kappa-carrageenase Cgk-GDSX478 provided by the application has good relative activity under 4-60 DEG C, and the optimum reaction temperature is 40 DEG C. The kappa-carrageenase Cgk-GDSX478 is relatively stable in the buffer system with a pH of 3.0-11.0 and can exert enzymatic hydrolysis, and the optimum reaction pH is 8.0. It is found by thin layer chromatography and liquid chromatography-mass spectrometry analysis that the enzymatic hydrolysis product of the kappa-carrageenase Cgk-GDSX478 after hydrolyzing kappa-carrageenan is kappa-carrageenan disaccharide. The above results show that the kappa-carrageenase Cgk-GDSX478 has high degradation activity on kappa-carrageenan, and the product has high mononess. The application has important significance for the industrial production of kappa-carrageenan disaccharide. BRIEF DESCRIPTION OF DRAWINGS

[0018] Fig. 1 is an agarose gel electrophoresis diagram of the gene Cgk-GDSX478.

[0019] Fig. 2 is an SDS-PAGE diagram of the kappa-carrageenase Cgk-GDSX478.

[0020] Figure 3 is the effect of different temperatures on the enzymatic reaction of the kappa-carrageenase Cgk-GDSX478.

[0021] Figure 4 is the effect of different temperatures on the incubation of the kappa-carrageenase Cgk-GDSX478.

[0022] Figure 5 is the effect of different pH on the enzymatic reaction of the kappa-carrageenase Cgk-GDSX478.

[0023] Figure 6 is the effect of different pH on the incubation of the kappa-carrageenase Cgk-GDSX478.

[0024] Figure 7 is the effect of different metal ions on the enzymatic reaction of the Cgk-GDSX478 enzyme.

[0025] Figure 8 is a result diagram of the thin layer chromatography analysis of the kappa-carrageenase Cgk-GDSX478 enzymatic product at different times. Wherein, Gal is galactose; DP2 is a kappa-carrageenan disaccharide standard; DP4 is a kappa-carrageenan tetrasaccharide standard; DP6 is a kappa-carrageenan hexose standard; 1-15 respectively represent the enzymatic reaction time of 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, 72 h.

[0026] Figure 9 is an ESI-MS detection structure diagram of the kappa-carrageenase Cgk-GDSX478 enzymatic product. Wherein, DP2 is a kappa-carrageenan disaccharide standard.

[0027] Figure 10 is a mass spectrum of DP2. Wherein, DP2 is a kappa-carrageenan disaccharide monomer. DETAILED DESCRIPTION

[0028] In the following, the technical solutions of the present application are described in conjunction with examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. Example 1

[0029] This embodiment provides a preparation method of the recombinant enzyme Cgk-GDSX478.

[0030] 1. Acquisition of the gene Cgk-GDSX478

[0031] The experiment takes the genome of Photobacterium rosenbergii GDSX-4 disclosed in the patent with application number CN117247877A as a template, and the enzyme-producing gene thereof is amplified by PCR. The upstream primer is designed with NdeI as the site 5'-GGAATTCCATATGACTTTAAACTTGTCTAATAA-3', and the downstream primer is designed with xhoI as the site 5'-CCGCTCGAGCTCTCTGCTCCATACACGAACGTAC-3'. The PCR amplification system is 50μL (2×pfu PCR mix 25μL, 2μL of primer, 2μL of template, and 19μL of ddH2O), and the amplification condition is 94℃ 5min, 30Cycle (94℃ 30sec, 55℃ 30sec, 72℃ 40sec) 10℃ hold on, to obtain the PCR product. The PCR product is detected by agarose gel electrophoresis, and the result is shown in Figure 1. As shown in Figure 1, the enzyme-producing gene Cgk-GDSX478 is obtained by PCR amplification, and the size is 912bp.

[0032] The Photobacterium rosenbergii in the present embodiment has the following preservation information:

[0033] Preservation time: October 13, 2023;

[0034] Preservation unit: Guangdong Microbial Culture Collection Center;

[0035] Address of the preservation unit: 5th floor, Experimental Building, No.100, Martyrs' Road, Yuexiu District, Guangzhou, Guangdong Province;

[0036] Preservation number: GDMCC No.63878.

[0037] 2. Obtaining of recombinant bacteria Cgk-GDSX478

[0038] The PCR product and the E. coli expression vector pET-28a(+) (purchased from Hangzhou Baosai Biological Technology Co., Ltd.) are double-digested with restriction enzymes (NdeI-xhoI), respectively, the recovered fragment is connected to the expression vector digested with the same site, and then the BL21(DE3) competent cells (purchased from Hangzhou Baosai Biological Technology Co., Ltd.) are transformed, spread on LB agar plates (kanamycin resistance), incubated at 37℃ overnight, and then single colonies are picked for positive verification and sequencing. The correct sequencing clone is inoculated again on LB agar plates (kanamycin resistance) to obtain the recombinant bacteria Cgk-GDSX478.

[0039] 3. Obtaining of Cgk-GDSX478 enzyme

[0040] Recombinant bacteria Cgk-GDSX478 was activated in 5 mL LB sterile liquid medium (containing 50 μg / mL kanamycin), and was inoculated into 100 mL LB sterile liquid medium (containing 50 μg / mL kanamycin) at a 1% inoculation amount for expansion culture, 37°C, 220 rpm, and when the bacterial liquid OD 600 was 0.5, 0.1 mM IPTG was added, and induction was carried out at 25°C for 12 h.

[0041] After fermentation, the culture solution was collected by centrifugation at 8000 rpm / min for 10 min, the supernatant was passed through a 0.45 μm filter membrane, and the Cgk-GDSX478 crude enzyme solution was collected for protein purification by Ni-NTA affinity chromatography. The purified protein was detected by SDS-PAGE, and the results are shown in Figure 2. It is a single band electrophoretic pure protein, indicating that Cgk-GDSX478 (kappa-carrageenase) is obtained, and its relative molecular weight is about 35 kDa. Example 2

[0042] This example provides the optimum enzyme hydrolysis reaction conditions of kappa-carrageenase Cgk-GDSX478 and the optimum incubation conditions of Cgk-GDSX478 enzyme.

[0043] The enzyme activity determination method of kappa-carrageenase Cgk-GDSX478 in this example is: 490 μL substrate (0.2% kappa-carrageenan) and 10 μL kappa-carrageenase Cgk-GDSX478, 40°C reaction for 30 min. After the reaction is completed, 500 μL of DNS reagent is added and mixed, boiled for 10 min, and then colored.

[0044] Kappa-carrageenase Cgk-GDSX478 enzyme reaction temperature: kappa-carrageenase Cgk-GDSX478 was subjected to enzyme hydrolysis reaction with kappa-carrageenan at temperatures of 4, 20, 30, 40, 50, 60, and 70°C, and the results are shown in Figure 3. Kappa-carrageenase Cgk-GDSX478 can well exert enzyme activity at 4-60°C, and realizes the degradation of kappa-carrageenan.

[0045] Optimum temperature of kappa-carrageenase Cgk-GDSX478 enzyme reaction: kappa-carrageenase Cgk-GDSX478 was incubated at temperatures of 30, 40, 50, and 70°C for 7 h, and the temperature stability of kappa-carrageenase Cgk-GDSX478 was determined, respectively. The results are shown in Figure 4. After incubation at 30°C and 40°C for 7 h, kappa-carrageenase Cgk-GDSX478 can still retain more than 95% activity.

[0046] Kappa-carrageenase Cgk-GDSX478 enzymatic reaction pH: At 40℃, kappa-carrageenan was dissolved in buffers with pH values of 3.0-11.0 (pH values of 3.0, 4.0, 5.0, and 6.0 were citric acid buffers, pH values of 6.0, 7.0, and 8.0 were phosphate buffers, pH values of 8.0, 9.0, and 10.0 were Tris-HCl buffers, and pH values of 9.0, 10.0, and 11.0 were sodium carbonate buffers) to test the optimal pH of kappa-carrageenanase Cgk-GDSX478 enzymatic reaction. The results are shown in Figure 5, and the optimal reaction pH of kappa-carrageenanase Cgk-GDSX478 was 8.0.

[0047] Kappa-carrageenanase Cgk-GDSX478 enzyme solution was mixed with the above different pH buffers and placed for 24 h to determine the stability of kappa-carrageenanase Cgk-GDSX478 enzyme solution in different pH buffers. The results are shown in Figure 6, and kappa-carrageenanase Cgk-GDSX478 enzyme solution was relatively stable in buffer systems with pH values of 3.0-11.0 and could exert enzymatic effects. Example 3

[0048] This example provides the effects of different metal ions on the activity of kappa-carrageenanase Cgk-GDSX478.

[0049] Test group: Metal ion reagents with a final concentration of 5 mmol / L of Ni 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Mg 2+ , Zn 2+ , Ca 2+ , K + , Ba 2+ , and Na + were prepared, 490 μL of substrate (0.2% kappa-carrageenan) and 10 μL of kappa-carrageenanase Cgk-GDSX478 were added, and the reaction was carried out at 40℃ for 30 min. After the reaction, 500 μL of DNS reagent was added and mixed, boiled for 10 min, and then colored.

[0050] Control group: Without adding metal ions, 490 μL of substrate (0.2% kappa-carrageenan) and 10 μL of kappa-carrageenanase Cgk-GDSX478 were reacted at 40℃ for 30 min.

[0051] The results are shown in Figure 7, and Fe 2+ , Na + , Ni 2+ , Ba 2+ , and Mn 2+ can promote the activity of kappa-carrageenanase Cgk-GDSX478. Example 4

[0052] The present example provides a method for preparing kappa-carrageenan oligosaccharides by enzymatic hydrolysis of kappa-carrageenan using kappa-carrageenase Cgk-GDSX478.

[0053] (1) Preparation of kappa-carrageenan oligosaccharides

[0054] Prepare a 0.5% kappa-carrageenan substrate solution with a phosphate buffer (pH 8.0), and add 10% kappa-carrageenase Cgk-GDSX478 enzyme solution to the total reaction volume. React 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 is completed, terminate heating by boiling the mixture for 10 min, and cool to room temperature to obtain a kappa-carrageenan oligosaccharide solution.

[0055] (2) Analysis of kappa-carrageenan oligosaccharides by thin layer chromatography

[0056] Activate a silica gel thin layer plate in a 100°C drying oven for 1 h. Spot 3 μL of the above kappa-carrageenan oligosaccharide solution on the silica gel plate, use galactose, standard disaccharide, tetrasaccharide, and hexasaccharide as controls, and develop in a n-butanol: ethanol: water (3:2:2) developing agent. After development, dry the silica gel thin layer plate. After soaking in a color developing agent (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), dry the silica gel thin layer plate. Color develop by heating the silica gel thin layer plate at 110°C for 10 min.

[0057] The results, as shown in Figure 8, indicate that the product of kappa-carrageenase Cgk-GDSX478 enzymatic hydrolysis of kappa-carrageenan is mainly carrageenan disaccharide.

[0058] (3) Analysis of the structure of the degradation product by liquid chromatography mass spectrometry (Q-TOF-MS)

[0059] Dilute the kappa-carrageenan oligosaccharide solution to 50 μg / mL, filter through a 0.22 μm microporous filter, and inject into a TOF-MS system for analysis. The chromatographic conditions are as follows: injection volume is 5 μL; mobile phase is acetonitrile: 1 mM formic acid water = 1:1; flow rate is 0.2 mL / min; and column temperature is 35°C. The mass spectrometry uses an ESI point spray ion source, negative ionization mode; and mass scan range: m / z 50~2000 Da. The ESI-MS detection results, as shown in Figure 9, indicate that the product of kappa-carrageenase Cgk-GDSX478 enzymatic hydrolysis of kappa-carrageenan is only carrageenan disaccharide.

[0060] The above-described embodiments are merely some of the embodiments of the present application, but not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but merely represents selected embodiments of the application. All other embodiments obtained by persons of ordinary skill in the art based on the concept of the present application, without making creative labor, are within the scope of the present application.

Claims

1. A gene encoding a kappa-carrageenase, characterized in that, The gene is Cgk-GDSX478, and the nucleotide sequence of the gene is shown as SEQ ID NO:

1.

2. The gene of claim 1, wherein The Cgk-GDSX478 is derived from Photobacterium leiognathi GDSX-4.

3. A recombinant expression vector, characterized in that, The gene encoding the kappa-carrageenase is connected to an expression vector to obtain a recombinant expression vector.

4. A recombinant engineered bacterium, characterized in that, The gene encoding the kappa-carrageenase or the recombinant expression vector.

5. The recombineering bacteria of claim 4, wherein, The host of the engineered bacteria is Escherichia coli; The Escherichia coli is BL21.

6. A kappa-carrageenase characterized in that, The kappa-carrageenase is Cgk-GDSX478, and the amino acid sequence of the kappa-carrageenase is shown as SEQ ID NO:

2.

7. The kappa-carrageenase in claim 6 is used in the preparation of kappa-carrageenan disaccharide. The enzymatic temperature of the kappa-carrageenase is 4-60℃, the enzymatic pH is 3.0-11.0, and the enzymatic time is 30-1440 min.

8. Use according to claim 6, characterized in that, The enzymatic temperature of the kappa-carrageenase is 40℃, the enzymatic pH is 8.0, and the enzymatic time is 420 min.

9. Use according to claim 6, characterized in that, ​

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