Gene encoding carrageenanase, encoding product therefor and use thereof

Abstract

Provided are a gene encoding a carrageenanase, an encoding product therefor and use thereof, belonging to the technical field of biological enzymes. The nucleotide sequence of the gene Cgk-GDSX477 encoding carrageenanase is as shown in SEQ ID NO: 1, and the amino acid sequence of the carrageenanase Cgk-GDSX477 encoded by the gene is as shown in SEQ ID NO: 2. The carrageenanase Cgk-GDSX477 has good relative activity at 4-70°C and is relatively stable in a buffer system having a pH of 3.0-11.0. The provided carrageenanase can degrade κ-carrageenan to produce carrageenan oligosaccharides. The carrageenan oligosaccharides comprise κ-neo-carrageenan tetraose and κ-neo-carrageenan hexaose. A carrageenanase having good heat resistance and stability is provided for the enzymolysis of carrageenan.

Description

A carrageenase-encoding gene, its encoding product and application TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological enzymes, and relates to a carrageenase-encoding gene, its encoding product and application. BACKGROUND

[0002] Carrageenan is a hydrophilic colloid, also known as Eucheuma gel, Gelidium gel, Furcellaria gel and Chondrus gel, which is a hydrophilic colloid extracted from Eucheuma, Gelidium, Furcellaria and other red seaweed. Carrageenan has a repeating alpha-1,4-D-galactopyranose-beta-1,3-D-galactopyranose disaccharide unit backbone structure. The main component of carrageenan is cellulose, which can swell and form a stable colloid when in contact with water. Carrageenan can stabilize food texture and improve taste, and is often used as a thickening agent, gelling agent, suspending agent, emulsifying agent and stabilizer. Carrageenan also has wide application in the medical field. Studies have found that carrageenan has biological activities such as antiviral, immunomodulatory, anticoagulant, antioxidant and anticancer activities.

[0003] Carrageenan oligosaccharide is a degradation product of carrageenan. Compared with carrageenan, carrageenan oligosaccharide has lower molecular weight, higher solubility, easier absorption, better stability and safety. At the same time, due to the full exposure of active groups on the molecular chain, the activity of carrageenan oligosaccharide is significantly higher than that of carrageenan, and even new activities are generated. Carrageenan oligosaccharide has immunomodulatory, antitumor, anti-radiation, antibacterial, antiviral, antioxidant and anticoagulant activities. At present, there are many methods for preparing carrageenan oligosaccharide by degrading carrageenan with microbial enzymes. Compared with the traditional acid hydrolysis method, the enzyme degradation method has the advantages of specific recognition site and cleavage site, uniform product, mild action mode, no damage to raw materials and products, and simple purification. However, the reported carrageenanases have poor stability, and the enzyme activity is greatly lost at a temperature higher than 40℃. Therefore, it is of great significance to provide a carrageenanase with good heat resistance and stability. SUMMARY

[0004] In order to study a carrageenanase with good heat resistance and stability, the present application provides a carrageenanase-encoding gene and its encoding product and application. The gene is Cgk-GDSX477, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the amino acid sequence of the gene encoding product carrageenanase Cgk-GDSX477 is shown in SEQ ID NO: 2. The carrageenanase Cgk-GDSX477 has good relative activity at 4-70℃, is relatively stable in a buffer system with pH 3.0-11.0, and can degrade kappa-carrageenan to obtain kappa-neo carrageenan tetrasaccharide and kappa-neo carrageenan hexasaccharide. The present application provides a carrageenanase with good heat resistance and stability for enzymatic degradation of carrageenan.

[0005] To achieve the technical purposes of the present application, in one aspect, the present application provides a gene encoding carrageenase, the gene is Cgk-GDSX477, the nucleotide sequence of which is shown as SEQ ID NO: 1.

[0006] In another aspect, the present application claims a recombinant expression vector comprising the gene encoding carrageenase, the gene encoding carrageenase is connected to the expression vector pET-28a(+) to obtain the recombinant expression vector.

[0007] Further, the present application double digests Cgk-GDSX477 and E. coli expression vector pET-28a(+) with restriction endonuclease NdeI and xhoI respectively, and connects the recovered fragment to the E. coli expression vector digested with the same site to obtain the recombinant expression vector.

[0008] In another aspect, the present application claims a recombinant engineering bacteria comprising the gene encoding carrageenase or the recombinant expression vector, and the host of the recombinant engineering bacteria is BL21(DE3).

[0009] Further, the present application transforms the BL21(DE3) competent cells with the recombinant expression vector, spreads on the LB agar plate with kanamycin resistance, incubates at 37℃ overnight, picks the single clone for positive verification and sequencing identification. The single clone with correct sequencing is inoculated to the LB agar plate with kanamycin resistance again to obtain the recombinant engineering bacteria.

[0010] In another aspect, the present application claims a carrageenase encoded by the gene producing carrageenase or produced by the recombinant engineering bacteria. The carrageenase is Cgk-GDSX477, and the amino acid sequence thereof is shown as SEQ ID NO: 2.

[0011] Further, the present application inoculates the recombinant engineering bacteria 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 30℃ for 12 h. After the fermentation is completed, the culture solution is collected by centrifugation at 8000 rpm / min for 10 min, the bacterial body is collected, resuspended with the phosphate buffer with pH 8.0, and ultrasonically broken in the ice bath for 30 min. The broken enzyme solution is centrifuged at 8000 rpm / min for 10 min, 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 thereof is about 52 kDa detected by SDS-PAGE.

[0012] Further, the application detects the enzyme activity of carrageenase under the condition of 4-70 DEG C and finds that the carrageenase Cgk-GDSX477 has good relative activity under 4-70 DEG C, and the optimum reaction temperature is 40 DEG C. After the carrageenase is dissolved in the buffer solution with pH 3.0-11.0, the enzyme activity is detected, and it is found that the carrageenase Cgk-GDSX477 has good relative activity at pH 8.0 or pH 9.0, the buffer solution is phosphate buffer solution at pH 8.0, and the buffer solution is sodium carbonate buffer solution at pH 9.0, and the optimum reaction pH is 8.0 or 9.0. After the carrageenase is mixed with the buffer solution with pH 3.0-11.0, it is found that the carrageenase Cgk-GDSX477 is relatively stable in the buffer solution system with pH 3.0-11.0 and can exert the enzymolysis effect.

[0013] Further, the application detects the enzyme activity of carrageenase under the condition of 4-70 DEG C and finds that the carrageenase Cgk-GDSX477 has good relative activity under 4-70 DEG C, and the optimum reaction temperature is 40 DEG C. After the carrageenase is dissolved in the buffer solution with pH 3.0-11.0, the enzyme activity is detected, and it is found that the carrageenase Cgk-GDSX477 has good relative activity at pH 8.0 or pH 9.0, the buffer solution is phosphate buffer solution at pH 8.0, and the buffer solution is sodium carbonate buffer solution at pH 9.0, and the optimum reaction pH is 8.0 or 9.0. After the carrageenase is mixed with the buffer solution with pH 3.0-11.0, it is found that the carrageenase Cgk-GDSX477 is relatively stable in the buffer solution system with pH 3.0-11.0 and can exert the enzymolysis effect.

[0014] Further, the application detects the enzyme activity of carrageenase under the condition of 4-70 DEG C and finds that the carrageenase Cgk-GDSX477 has good relative activity under 4-70 DEG C, and the optimum reaction temperature is 40 DEG C. After the carrageenase is dissolved in the buffer solution with pH 3.0-11.0, the enzyme activity is detected, and it is found that the carrageenase Cgk-GDSX477 has good relative activity at pH 8.0 or pH 9.0, the buffer solution is phosphate buffer solution at pH 8.0, and the buffer solution is sodium carbonate buffer solution at pH 9.0, and the optimum reaction pH is 8.0 or 9.0. After the carrageenase is mixed with the buffer solution with pH 3.0-11.0, it is found that the carrageenase Cgk-GDSX477 is relatively stable in the buffer solution system with pH 3.0-11.0 and can exert the enzymolysis effect.

[0015] Compared with the prior art, the technical scheme provided by the application has at least the following beneficial effects or advantages:

[0016] The application provides a carrageenase with good heat resistance and stability for preparing carrageenan oligosaccharide by degrading carrageenan. The carrageenase Cgk-GDSX477 provided by the application has good relative activity under 4-70 DEG C, and the optimum reaction temperature is 40 DEG C. The carrageenase Cgk-GDSX477 provided by the application has good relative activity at pH 8.0 or pH 9.0, the buffer solution is phosphate buffer solution at pH 8.0, and the buffer solution is sodium carbonate buffer solution at pH 9.0, and the optimum reaction pH is 8.0 or 9.0. After the carrageenase is mixed with the buffer solution with pH 3.0-11.0, it is found that the carrageenase Cgk-GDSX477 is relatively stable in the buffer solution system with pH 3.0-11.0 and can exert the enzymolysis effect.

[0017] The present application finds, through thin layer chromatography and liquid phase mass spectrometry analysis, that the enzymatic products of carrageenanase Cgk-GDSX477 after enzymatic hydrolysis of kappa-carrageenan include kappa-neocarratetraose and kappa-neocarratohexaose, the characteristic ion peak of kappa-neocarratetraose is a peak with mass-to-charge ratio m / z of 394.05, and the characteristic ion peak of kappa-neocarratohexaose is a peak with mass-to-charge ratio m / z of 391.05. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application.

[0019] Fig. 1 is an agarose gel electrophoresis diagram of the gene Cgk-GDSX477 encoding carrageenanase.

[0020] Fig. 2 is an SDS-PAGE diagram of carrageenanase Cgk-GDSX477.

[0021] Fig. 3 is the effect of different temperatures on the relative activity of carrageenanase Cgk-GDSX477.

[0022] Fig. 4 is the effect of different pHs on the relative activity of carrageenanase Cgk-GDSX477.

[0023] Fig. 5 is the effect of different pHs on the stability of carrageenanase Cgk-GDSX477.

[0024] Fig. 6 is a result diagram of thin layer chromatography analysis of the enzymatic products of carrageenanase Cgk-GDSX477 under different enzymatic hydrolysis times. Gal is galactose; two is standard kappa-neocarratetraose; four is standard kappa-neocarratetraose; six is standard kappa-neocarratohexaose; 1-15 are respectively enzymatic hydrolysis times 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, and 72 h.

[0025] Fig. 7 is a total ion chromatogram of mass spectrometry detection of the enzymatic products of carrageenanase Cgk-GDSX477 under different enzymatic hydrolysis times. DP4 is kappa-neocarratetraose; DP6 is kappa-neocarratohexaose.

[0026] Fig. 8 is a mass spectrometry detection result diagram of kappa-neocarratetraose.

[0027] Fig. 9 is a mass spectrometry detection result diagram of kappa-neocarratohexaose. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be described below 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.

[0029] LB agar plates and LB liquid media were purchased from Shenguo Bioengineering (Shanghai) Co., Ltd.

[0030] Kappa-carrageenan was purchased from Qingdao Haide Marine Oligosaccharide Technology Co., Ltd. Example 1

[0031] This example provides the preparation of carrageenase Cgk-GDSX477.

[0032] 1. Preparation of recombinant bacteria

[0033] The genome of Photobacterium rosenbergii GDSX-4 (preserved number GDMCC 63878) disclosed in the patent with application number CN202311542969.7 was used as a template, and the enzyme-producing gene (as shown in SEQ ID NO: 1) was subjected to PCR amplification. The upstream primer was designed with NdeI as the site 5'-GGAATTCCATATGAAATTACGTTATTTCTTGCT-3', and the downstream primer was designed with xhoI as the site 5'-CCGCTCGAGTGGCTCGATGCTAGTTATCTTTA-3'. The PCR amplification system was 50 μL (2×pfu PCR mix 25 μL, upstream primer and downstream primer each 2 μL, template 2 μL, ddH2O 19 μL), and the PCR amplification conditions were 94℃ 5min, 30 cycles (94℃ 30sec, 55℃ 30sec, 72℃ 40sec), 10℃ hold on. The PCR product was detected by agarose gel electrophoresis, and the results are shown in Figure 1. As shown in Figure 1, the enzyme-producing gene Cgk-GDSX477 was obtained by PCR amplification, and the size was 1353bp.

[0034] The PCR product and E. coli expression vector pET-28a(+) (purchased from Hangzhou Baosai Biological Technology Co., Ltd.) were subjected to double digestion with restriction enzymes (NdeI-xhoI), and the recovered fragment was connected to the same site digested E. coli expression vector, then the BL21 (DE3) competent cells (purchased from Hangzhou Baosai Biological Technology Co., Ltd.) were transformed, spread on LB agar plates (containing 30 μg / mL kanamycin), and incubated at 37℃ overnight. Single colonies were picked for positive verification and sequencing. The single colony with correct sequencing was inoculated again on LB agar plates (containing 30 μg / mL kanamycin) to obtain recombinant bacteria.

[0035] 2. Preparation of carrageenase Cgk-GDSX477

[0036] The recombinant bacteria were activated in 5 mL of LB sterile liquid medium (containing 50 μg / mL kanamycin) and inoculated into 100 mL of LB sterile liquid medium (containing 50 μg / mL kanamycin) at a volume ratio of 1% for expansion culture, at 37°C, 220 rpm, and when the OD 600 of the bacterial solution was 0.5, 0.1 mM of IPTG was added, and induction was carried out at 30°C for 12 h.

[0037] After fermentation, the culture solution was collected by centrifugation at 8000 rpm / min for 10 min, the bacterial bodies were collected, resuspended with a phosphate buffer at pH 8.0, and broken by ultrasonic treatment in an ice bath for 30 min. The broken enzyme solution 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 by Ni-NTA affinity chromatography. The amino acid sequence of the purified protein (carrageenase Cgk-GDSX477) is shown in SEQ ID NO: 2, and the result of SDS-PAGE detection is shown in FIG. 2, wherein a single band of electrophoretically pure protein was obtained, and the relative molecular weight was about 52 kDa. Example 2

[0038] This example provides the determination of the reaction conditions of carrageenase Cgk-GDSX477.

[0039] Carrageenase enzyme activity determination: 490 μL of substrate (0.2% κ-carrageenan) and 10 μL of carrageenase Cgk-GDSX477 were reacted at 40°C for 30 min, 500 μL of DNS reagent was added, mixed, boiled for 10 min, and colored.

[0040] 1. Determination of the reaction temperature of carrageenase Cgk-GDSX477

[0041] The reaction temperature in the enzyme activity determination was 4, 20, 30, 40, 50, 60, and 70°C, respectively, and each test was done in triplicate, and the relative activity of carrageenase Cgk-GDSX477 was calculated (FIG. 3).

[0042] The calculation formula of the relative activity is: relative activity = enzyme activity value / highest enzyme activity value in the same group x 100%.

[0043] As shown in FIG. 3, carrageenase Cgk-GDSX477 has good relative activity at 4-70°C, and the optimum reaction temperature is 40°C.

[0044] 2. Determination of the reaction pH of carrageenase Cgk-GDSX477

[0045] 1) The buffer solution (pH 3.0-6.0 is citric acid buffer, pH 6.0-8.0 is phosphate buffer, pH 8.0-10.0 is Tris-HCl buffer, and pH 9.0-11.0 is sodium carbonate buffer) with pH 3.0-11.0 was selected for dissolving the substrate in the enzyme activity assay. Each test was performed in triplicate, and the relative activity of carrageenase Cgk-GDSX477 was calculated (Figure 4).

[0046] As shown in Figure 4, carrageenase Cgk-GDSX477 has good relative activity at pH 8.0 or pH 9.0. The buffer solution is phosphate buffer at pH 8.0 and sodium carbonate buffer at pH 9.0, and the optimal reaction pH is 8.0 or 9.0.

[0047] 2) Carrageenase Cgk-GDSX477 was mixed with the above-mentioned buffer solution with pH 3.0-11.0, and placed for 24 h. The relative activity of carrageenase Cgk-GDSX477 in different pH buffer systems was determined, and each test was performed in triplicate. The determination results are shown in Figure 5.

[0048] As shown in Figure 5, carrageenase Cgk-GDSX477 is relatively stable in the buffer system with pH 3.0-11.0, and can exert enzymatic hydrolysis. Example 3

[0049] This example provides the application of carrageenase Cgk-GDSX477 in degrading carrageenan.

[0050] Preparation of enzymatic oligosaccharides: 0.5% κ-carrageenan substrate solution was prepared with phosphate buffer (pH 8.0), 10% enzyme solution was added according to the total reaction volume, and 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. The reaction was terminated by heating and boiling for 10 min, and then cooled to room temperature.

[0051] Thin layer chromatography analysis of kappa-carrageenan oligosaccharides: Take the silica gel thin layer plate in the 100℃ drying oven for 1h, take 3μL of the above sample on the silica gel plate, take galactose (Gal), standard kappa-neo carrageenan disaccharide (two), standard kappa-neo carrageenan tetrasaccharide (four), standard kappa-neo carrageenan hexose (six) as control, place in n-butanol: ethanol: water (3:2:2) developing agent and develop, then blow dry, immerse in color developing agent (diphenylamine 2g, aniline 2mL, 85% phosphoric acid 10mL, concentrated hydrochloric acid 1mL, acetone 100mL) and blow dry, color develop at 110℃ for 10min, the thin layer chromatography detection result is shown in Figure 6. As shown in Figure 6, the enzymatic products of carrageenanase Cgk-GDSX477 after enzymatic hydrolysis of kappa-carrageenan mainly consist of kappa-neo carrageenan tetrasaccharide and kappa-neo carrageenan hexose.

[0052] Liquid chromatography-mass spectrometry (Q-TOF-MS) analysis of degradation product structure: take the reaction solution in the above process and dilute to 50μg / mL, filter through 0.22μm microporous filter membrane, inject into TOF-MS system for analysis, the chromatographic conditions are as follows: injection amount 5μL; mobile phase: acetonitrile: 1mM formic acid (1:1); flow rate 0.2mL / min; column temperature 35℃. The mass spectrometry uses ESI point spray ion source, negative ionization mode; mass scan range: m / z 50~2000Da. The ESI-MS detection result is shown in Figure 7, the mass spectrum of kappa-neo carrageenan tetrasaccharide (DP4) is shown in Figure 8, and the mass spectrum of kappa-neo carrageenan hexose (DP6) is shown in Figure 9.

[0053] As shown in Figures 7-9, the enzymatic products of carrageenanase Cgk-GDSX477 after enzymatic hydrolysis of kappa-carrageenan are kappa-neo carrageenan tetrasaccharide and kappa-neo carrageenan hexose, the characteristic ion peak of kappa-neo carrageenan tetrasaccharide is the peak with mass-to-charge ratio m / z of 394.05, and the characteristic ion peak of kappa-neo carrageenan hexose is the peak with mass-to-charge ratio m / z of 391.05.

[0054] The above-described embodiments are part of the embodiments of the present application, rather than 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 only represents selected embodiments of the application. All other embodiments obtained by related deduction and replacement made by those of ordinary skill in the art under the condition of the concept of the present application, without making creative efforts, belong to the scope of protection of the present application.

Claims

1. A gene encoding a carrageenase, characterized by, The gene is Cgk-GDSX477, and the nucleotide sequence of the gene is shown as SEQ ID NO:

1.

2. A recombinant expression vector, characterized in that, The gene encoding carrageenase in claim 1 is connected to an expression vector pET-28a (+) to obtain the recombinant expression vector.

3. A recombinant engineered bacterium, characterized in that, The gene encoding carrageenase in claim 1 or the recombinant expression vector in claim 2.

4. The recombineering bacteria of claim 3, wherein, The host of the recombinant engineering bacteria is Escherichia coli; The Escherichia coli is BL21 (DE3).

5. A carrageenase enzyme, characterized in that, The carrageenase is encoded by the gene for producing carrageenase in claim 1 or produced by the recombinant engineering bacteria in claim 3. The carrageenase is Cgk-GDSX477, and the amino acid sequence of the carrageenase is shown as SEQ ID NO:

2.

6. Use of the carrageenase according to claim 5 for the enzymatic hydrolysis of carrageenan, characterized in that, The carrageen is kappa-carrageen, and the enzymatic product of the kappa-carrageen is kappa-carrageenan oligosaccharide, which includes kappa-neo carrageenan tetrasaccharide and kappa-neo carrageenan hexasaccharide.

7. Use according to claim 6, characterized in that, The enzymatic temperature of the carrageenase is 4-70 ℃. The carrageen is dissolved by using a buffer with pH 3.0-11.

0.

8. Use according to claim 6, characterized in that, The enzymatic temperature of the carrageenase is 40 ℃, and the carrageen is dissolved by using a buffer with pH 8.0 or a buffer with pH 9.

0.

9. Use according to claim 8, characterized in that, The buffer with pH 8.0 is a phosphate buffer, and the buffer with pH 9.0 is a sodium carbonate buffer.

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