Random type difunctional carrageenase and application thereof

Abstract

The invention relates to the technical field of biology, in particular to random type difunctional carrageenase and application. The invention provides a random type difunctional carrageenase, the amino acid sequence of which is SEQ ID NO.1. The random type difunctional carrageenase can degrade iota-carrageenan and kappa-carrageenan in a random type internally tangent action mode to generate oligosaccharides with different polymerization degrees, is beneficial to research on the structure-function relationship of the carrageenan and the oligosaccharides thereof, and can be applied to the fields of food industry, chemical analysis and the like. The random type difunctional carrageenase can be applied to preparation of iota-carrageenan with different molecular weights and a series of oligosaccharides.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a random bifunctional carrageenase and its application. Background Art

[0002] Carrageenan is a natural anionic sulfated linear polysaccharide extracted from red algae, which is composed of alternating β-D-galactopyranose and α-D-galactopyranose residues or 3,6-endo-α-D-galactopyranose residues. There are three common types of carrageenan on the market: κ-carrageenan, ι-carrageenan and λ-carrageenan. Carrageenan has excellent physical functional properties, such as thickening, gelling, elasticity and stabilization ability, so carrageenan is widely used as a food additive for dairy products, beverages, jellies, etc. In addition, carrageenan also has a variety of biological activities, such as antioxidant, antiviral, anti-tumor and gastrointestinal protection.

[0003] Polysaccharides have the disadvantages of large molecular weight, high viscosity, poor solubility and low bioavailability, so they need to be degraded to obtain low molecular weight polysaccharides or oligosaccharides for use. Low molecular weight carrageenan and oligosaccharides obtained by degradation of carrageenan have attracted more and more attention due to their excellent solubility, high bioavailability and multiple physiological activities, so they have considerable application potential in the fields of food, medicine, cosmetics and agriculture. At present, the most common carrageenan degradation methods are acid hydrolysis and enzymatic degradation. Compared with acid hydrolysis, enzymatic degradation to obtain low molecular weight carrageenan and oligosaccharides has mild reaction conditions and clear product structure, which is in line with the current development trend of green and clean production.

[0004] ι-carrageenase can specifically degrade the β-1,4-glycosidic bonds of ι-carrageenan and produce low molecular weight ι-carrageenan and its oligosaccharides. Random ι-carrageenase is an ideal tool for preparing ι-carrageenan and a series of oligosaccharides with different molecular weights. However, the ι-carrageenase reported in current studies is a continuous ι-carrageenase, which can only continuously cut ι-carrageenan to produce fixed oligosaccharide fragments, and the products are mainly concentrated in oligosaccharides with a certain degree of polymerization, such as ι-carrageenan tetrasaccharide. The discovery of random ι-carrageenase can provide an important tool for the degradation of ι-carrageenan and provide a prerequisite for the preparation of ι-carrageenan and a series of oligosaccharides with different molecular weights. Summary of the invention

[0005] The technical problem to be solved by the present invention is that no random ι-carrageenase has been reported, and there is no enzyme available for preparing low molecular weight carrageenan and oligosaccharides with different polymerization degrees.

[0006] To solve the above problems, the present invention has found a protein sequence containing an N-terminal signal peptide, an iota-carrageenase catalytic domain and a C-terminal secretory domain, wherein the catalytic domain consists of 425 amino acids, and the amino acid sequence of the catalytic domain is shown in SEQ ID NO.1, and further illustrates that the enzyme can act on iota-carrageenan in a random endo-cutting manner and can act on κ-carrageenan at the same time. On this basis, the random bifunctional carrageenase is used to realize the preparation of iota-carrageenan with different molecular weights and iota-carrageenan oligosaccharides with different polymerization degrees.

[0007] To achieve the above-mentioned object, the present invention is implemented through the following technical scheme: a random bifunctional carrageenase and its application, the amino acid sequence of the enzyme is SEQ ID NO.1 and an enzyme derived from SEQ ID NO.1 which has been substituted, deleted or added with one or more amino acids and still has random bifunctional carrageenase activity, and other sequences such as SEQ ID NO.2 and SEQ ID NO.3 having a homology with SEQ ID NO.1 greater than 70%.

[0008] SEQ ID NO.1:

[0009] QPDAPSMFPVDRVESDFFTDDSASFPPAQRITLVNNVGDDSTTLQTAIDYLTGLGGGVITISA

[0010] GTWEFNEIRLRSNVHLMFDALAVVKPYETADVNSLPNLSVFRIGYTSVKSSNVSIRALSGQF

[0011] KIDLTHLAKETRITPFNVKEAENFMVADCYIDDNGTIHAALNCSASKRNDIWTGPVKGLIK

[0012] NLTSINAHGGYGVVQVRTGERLLFKDLTSLRGGVTLRLESDDVSASGGAIPLSVAKMSEISG

[0013] YNIKCTEGNAAVMLQPWDATNGWVDVQKIQATGCMAAVRIDKAYLSTIGTFDPDSRITDV

[0014] TTSYGTDAQVKQGVHPWVPCVLRMNPAILRPNPIPDMETYHLGPSIAPLLYRASTTGGATG

[0015] NGYYSVNVPTSTELMNKATGFNWSVSLVISRDDNQVNTNNCPPLSVDSYDSVNNLK

[0016] SEQ ID NO.2:

[0017] QPDAPSMFPVDRVESDFFTDDSASFPPAQRITLVNNVGDDSTTLQTAINTLTGLGGGVVTIS

[0018] AGNWEFNEIQLKSNVHLMFDALAVVKPYETADVNSLPNLSVFRIGYTSVKSSNVSIRALSG

[0019] QFKIDLTHLAKETRITPFNVKETENFMVADCYIDDNGTIHAALNCSASKRNDIWTGPVKGLI

[0020] KNLTSINAHGGYGVVQVRTGERLLFKDLTSLRGGVTLRLESDDVSASGGAIPLSVAKMSEIS

[0021] GYNIKCTEGNAAVMLQPWDATNGWVDVQKIEATGCMAAVRIDRAYLSTIGTFDPDSRITD

[0022] VTSSYGTNAQVKQGVFPWVPCVLRTDPNILRPNPIPDMETYHLGPSIAPLLYRASTTGGATG

[0023] NGYYSINVPTSTELMNKATGFNWSVSLVISRDNNQVNSNNCPALSVNSFDIESHVK

[0024] SEQ ID NO.3:

[0025] SPLPEFRNEADFFTDSSGKYPAAPSSQRITLVYNPATGDDSPALQTAINTLTNLTVMVNGENL

[0026] KGGVITIPAGTWEFNEIQLKSDVHLVFDPLAIVKPYDAVSPTNPQIPNMSIFRIGYTGVLSEN

[0027] VSIRASSGQFKVDLTHLDKETRITPFNVKEAKNFMVADCFVDDNGTIHAALNCSASKRNNI

[0028] WAGPVKGLVKNLTIINGHGGYGVVQVRTGEKLLFKNLTSLRGGVTLRLESDDVSASGGVIP

[0029] QEVAKMSEIFGYNIKCTDGNAAVMLQPWSAVNGWIDIQKIEATGCMAAVRIDKAFDKVSPF

[0030] VGVGFFDSSSRITDITSAYGINAHVKQGIFPWVPCKLRTNPEILRPNPIPNMETFYLGPSVAPL

[0031] LYRASTTGGAVGDGYYSINVPTENELKTNASGFDWSISLVISRDDNQVNTSNCPPALAVNTF

[0032] EIDNNLK

[0033] Polygalacturonase can hydrolyze the α-1,4 glycosidic bonds between galacturonic acid in polygalacturonic acid and belongs to the 28-glycoside hydrolase family. Carrageenase can act on the β-1,4 glycosidic chains in carrageenan molecules to produce a series of carrageenan oligosaccharides with even degrees of polymerization. Therefore, the mechanism of action of random bifunctional carrageenase is different from that of polygalacturonase.

[0034] Furthermore, when used, the reaction temperature for ι-carrageenan is 45-60°C, and the optimum reaction temperature is 55°C; the reaction temperature for κ-carrageenan is 40-55°C, and the optimum reaction temperature is 50°C.

[0035] Furthermore, when used, the optimum reaction pH for both ι-carrageenan and κ-carrageenan is 7.0.

[0036] Based on the random bifunctional carrageenase, a molecular weight controllable enzymatic hydrolysis model of iota-carrageenan was established; and iota-carrageenan with different molecular weights was obtained by controlling the enzymatic hydrolysis time and the amount of enzyme added.

[0037] Furthermore, the fitting equation of the model is Log 10 (Y)=3.190863-0.0082655X1-0.1347936X2+0.0054209X2 2 ; Among them, the molecular weight of the degradation product is Y, the amount of enzyme added is X1, and the reaction time is X2.

[0038] The nucleotide sequence corresponding to the gene SEQ ID NO.1 encoding the random bifunctional carrageenase is shown in SEQ ID NO.4 and all genes of SEQ ID NO.1 can be translated.

[0039] SEQ ID NO.4:

[0040] CAACCTGATGCACCTTCAATGTTTCCAGTCGATAGAGTAGAAAGTGATTTTTTTACTGAT

[0041] GATTCGGCTAGTTTTCCTCCAGCACAAAGAATAACCTTGGTTAATAATGTGGGAGACGAT

[0042] TCTACTACATTACAAACTGCTATAGATTACCTAACTGGTCTTGGAGGTGGTGTTATTACGA

[0043] TTTCTGCTGGAACTTGGGAATTTAATGAAATCCGATTGCGCTCTAATGTGCATTTAATGTT

[0044] TGATGCTTTAGCAGTAGTTAAACCATATGAAACGGCGGACGTTAATAGTCTTCCTAATTTA

[0045] TCTGTTTTTCGTATTGGATATACCTCGGTAAAATCAAGTAACGTGAGCATTAGAGCCTTA

[0046] AGCGGTCAATTTAAAATAGATTTAACCCATTTAGCGAAAGAAACCAGGATTACACCCTTC

[0047] AACGTAAAAGAAGCTGAAAATTTTATGGTGGCCGATTGTTATATAGATGATAACGGCACC

[0048] ATTCATGCGGCTCTAAATTGTTCTGCTAGTAAGAGAAACGACATTTGGACAGGACCAGT

[0049] TAAAGGTTTAATAAAAAATTTAACCAGTATAAATGCCCATGGCGGTTATGGTGTTGTTCA

[0050] GGTACGAACCGGAGAACGGTTGCTGTTTAAAAGATTTAACATCCTTACGTGGTGGTGTAA

[0051] CACTTCGTCTTGAAAGCGATGACGTTAGTGCCTCTGGAGGAGCTATTCCACTAAGTGTT

[0052] GCAAAAATGTCTGAGATTTCAGGATATAATATTAAATGTACCGAAGGAAATGCAGCAGTG

[0053] ATGCTGCAACCGTGGGATGCCACAAATGGATGGGGTTGATGTACAAAAAAATTCAAGCGAC

[0054] TGGTTGTATGGCGGCGGTACGTATTGATAAGGCTTATTTAAGCACCATTGGAACCTTCGA

[0055] TCCAGATTCAAGAATAACAGATGTTACCACCAGTTATGGTACAGATGCGCAGGTAAAAAC

[0056] AAGGAGTACATCCATGGGTGCCATGTGTATTAAGAATGAATCCAGCTATTTTAAGACCTA

[0057] ATCCTATTCCCGATATGGAAACATATCATCTTGGACCCTCAATTGCCCCATACTTTTATCGT

[0058] GCAAGTACCACTGGAGGTGCTACAGGTAACGGATATTATAGCGTAAATGTCCCAACTTC

[0059] AACTGAGCTTATGAATAAAGCAACAGGTTTTAATTGGTCTGTTCATTAGTTATATCAAG

[0060] AGATGATAATCAGGTAAAACAAATAATTGTCCACCATTATCAGTTGATTCTTATGATAGT

[0061] GTTAACAATTTGAAATAA

[0062] The protein characterized by the above gene can be used to degrade iota-carrageenan and kappa-carrageenan to obtain carrageenan oligosaccharides with various polymerization degrees.

[0063] The present invention provides a method for preparing the above-mentioned random bifunctional carrageenase, wherein the enzyme is heterologously expressed in systems such as Escherichia coli, Bacillus subtilis, and Pichia pastoris, and the random bifunctional carrageenase can be prepared in large quantities by inducing enzyme production. The above-mentioned random bifunctional carrageenase is successfully heterologously expressed in systems such as Escherichia coli, Bacillus subtilis, and Pichia pastoris, and can be used for mass production and preparation of target enzymes, and has the highest expression activity in the Escherichia coli expression system, and can be effectively applied to chemical analysis, food industry, and other fields.

[0064] The beneficial effects of the present invention are:

[0065] (1) The random bifunctional carrageenase gene of the present invention can be efficiently prepared by cloning and expressing the random bifunctional carrageenase;

[0066] (2) The random bifunctional carrageenase of the present invention can degrade ι-carrageenan and κ-carrageenan in a random endo-cutting manner;

[0067] (3) The random bifunctional carrageenase of the present invention can realize the preparation of carrageenan oligosaccharides with different polymerization degrees;

[0068] (4) The random bifunctional carrageenase of the present invention can achieve the precise preparation of carrageenans with different molecular weights. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 : SDS-PAGE result diagram of the random bifunctional carrageenase of the present invention;

[0070] Figure 2 : Schematic diagram of the optimal reaction conditions of the random bifunctional carrageenase of the present invention. 2A: Optimum reaction temperature; 2B: Optimum reaction pH;

[0071] Figure 3 : Schematic diagram of the action mode analysis of the random bifunctional carrageenase of the present invention. 3A: Acts on ι-carrageenan; 3B: Acts on κ-carrageenan;

[0072] Figure 4 : Extracted ion current chromatograms of the random bifunctional carrageenanase of the present invention degrading iota-carrageenan and κ-carrageenan. 4A: Products of degradation of iota-carrageenan; 4B: Products of degradation of κ-carrageenan;

[0073] Figure 5 : Schematic diagram of the random bifunctional carrageenase of the present invention producing κ-carrageenan of different molecular weights when the amount of enzyme added is controlled;

[0074] Figure 6 : Schematic diagram of the random bifunctional carrageenase of the present invention producing κ-carrageenan of different molecular weights under the condition of controlling the reaction time. DETAILED DESCRIPTION

[0075] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention. The various raw materials used in the following embodiments, unless otherwise specified, are purchased from the market.

[0076] Example 1: Heterologous expression of random bifunctional carrageenase in Escherichia coli

[0077] Escherichia coli carrying the random bifunctional carrageenase target gene (as shown in SEQ ID NO.4) was subcultured in LB liquid medium containing kanamycin and cultured at 37°C and 170 rpm until OD 600 About 0.4, and isopropylthiogalactoside was added to induce expression, the induction temperature was 17°C, and the induction time was 16h. The cells were collected by centrifugation, and a certain amount of 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate (PBS) buffer was added to resuspend, and then ultrasonically disrupted in an ice water bath, and the supernatant was collected by centrifugation at 4°C to obtain a crude enzyme solution containing random bifunctional carrageenase.

[0078] Example 2: Heterologous expression of random bifunctional carrageenase in Bacillus subtilis

[0079] Design upstream and downstream primers (5′-CGCGCGGCAGCCATATGCAACCTGATGCACCTTCAA-3′; 5′-GGTGGTGGTGCTCGAGTTATTTCAAATTGTTAACACTATCATAAGAATCAAC-3′), retrieve the target gene by PCR, and the PCR reaction conditions are 95℃3min, 95℃20s, 42℃30s, 72℃60s, 24 cycles, and finally 72℃ for 5min to obtain the target gene fragment. Connect the target gene to the pMA5 vector by seamless cloning, and transform the constructed expression vector into the Bacillus subtilis BS WB600 expression host. Screen and obtain positive clones and inoculate them in LB medium, and use isopropylthiogalactoside to induce expression, the induction temperature is 37℃, and the induction time is 16h. Collect the bacteria by centrifugation, add a certain amount of 20mM PBS buffer to resuspend, and then perform ultrasonic disruption in an ice water bath. Centrifuge and collect the supernatant at 4℃ to obtain the crude enzyme solution containing random bifunctional carrageenase.

[0080] Example 3: Heterologous expression of random bifunctional carrageenase in Pichia pastoris

[0081] Design upstream and downstream primers (5′-CGCGCGGCAGCCATATGCAACCTGATGCACCTTCAA-3′; 5′-GGTGGTGGTGCTCGAGTTATTTCAAATTGTTAACACTATCATAAGAATCAAC-3′), and retrieve the target gene by PCR. The PCR process is the same as in Example 2. Connect the target gene to the pPIC9K vector by seamless cloning, and electrotransform the constructed expression vector into GS115 competent cells. Screen and obtain positive clones and inoculate them in YPD medium, and culture them at 30°C and 220rpm for 16h. Then inoculate them in pH 6.0 BMGY medium and culture them at 30°C and 220rpm until OD 600 Centrifuge at about 5.0, then add pH 6.0 BMGY medium for culture. Add 0.3% methanol to induce expression, the induction temperature is 29°C, the induction time is 72h, and centrifuge at 4°C to collect the supernatant to obtain a crude enzyme solution containing random bifunctional carrageenase.

[0082] Example 4: Comparison of the activity of random bifunctional carrageenase in various expression systems

[0083] 375 μL of appropriately diluted enzyme solution in Example 1-Example 3 was mixed with 375 μL of 2 mg / mL ι-carrageenan solution and reacted at 55°C for 5 minutes, and then inactivated at 100°C for 10 minutes. 375 μL of appropriately diluted inactivated enzyme solution was also used to mix with ι-carrageenan solution and react under the same conditions as a control. The reducing sugar in the experimental group and the control group system was detected by the reducing sugar increment method pHBH method, and the enzyme activity of the bifunctional carrageenase on ι-carrageenan was calculated. Similarly, 75 μL of appropriately diluted enzyme solution was mixed with 75 μL of 2 mg / mL κ-carrageenan solution and reacted at 50°C for 5 minutes, and then inactivated at 100°C for 10 minutes. 75 μL of appropriately diluted inactivated enzyme solution was also used to mix with κ-carrageenan solution and reacted under the same conditions as a control. The reducing sugar in the experimental group and the control group system was detected by the reducing sugar increment method pHBH method, and the enzyme activity of the bifunctional carrageenase on κ-carrageenan was calculated. 1U activity is defined as the activity of generating 1 μmol of reducing sugar within 1 minute. The activity of 1 mL of fermentation broth under different expression systems was detected by pHBH method as shown in Table 1:

[0084] Table 1: Enzyme activities of bifunctional carrageenase prepared by different expression systems against ι-carrageenan and κ-carrageenan

[0085]

[0086] From the above results, it can be seen that the bifunctional carrageenase in the present invention can be successfully expressed in heterologous systems such as Escherichia coli, Bacillus subtilis, and Pichia pastoris, and the expression activity in Escherichia coli is the highest, and it can be used for the production of chemical reagents. The Pichia pastoris expression system can realize the extracellular expression of the recombinant enzyme, and the exogenous protein content is low, which simplifies the subsequent separation and purification of the recombinant enzyme, and is conducive to its application in the development and production of health products, food and medicine.

[0087] Example 5: Optimal reaction conditions for random bifunctional carrageenase

[0088] In order to obtain the optimal reaction conditions of random bifunctional carrageenase, temperature and pH were investigated respectively.

[0089] 1) Optimum reaction temperature

[0090] 375 μL of the appropriately diluted enzyme solution in Example 1 was mixed with 375 μL of pH 7.02 mg / mL ι-carrageenan solution, and 75 μL of the appropriately diluted enzyme solution was mixed with 75 μL of pH 7.02 mg / mL κ-carrageenan solution. After reacting at 20, 25, 30, 35, 40, 45, 50, 55, 60, and 70°C for 5 min, the mixture was inactivated at 100°C for 10 min. The enzyme activity was determined using the pHBH method. The results are shown in FIG. Figure 2As shown in A. It can be concluded that the optimum reaction temperature for ι-carrageenan is 55°C, and the optimum reaction temperature for κ-carrageenan is 50°C.

[0091] 2) Optimal reaction pH

[0092] Mix 375 μL of the appropriately diluted enzyme solution in Example 1 with 375 μL of 2 mg / mL ι-carrageenan solution at different pH values, and mix 75 μL of the appropriately diluted enzyme solution with 75 μL of 2 mg / mL κ-carrageenan solution at different pH values, so that the reaction is carried out under different pH conditions for 5 minutes and inactivated at 100°C for 10 minutes. The enzyme activity is determined using the pHBH method. The pH buffer systems used are: pH 4.0-7.0: 20 mM citric acid-phosphate buffer; pH 6.5-9.0: 20 mM PBS buffer; pH 9.0-11.0: 20 mM sodium carbonate-sodium bicarbonate buffer. The results are as follows: Figure 2 B. It can be concluded that the optimal reaction pH for ι-carrageenan and κ-carrageenan is 7.0.

[0093] Example 6: HPLC analysis of random bifunctional carrageenase reaction progress

[0094] 2U of the enzyme solution obtained in Example 1 was added to 100mg 2mg / mL ι-carrageenan and κ-carrageenan solution, and pH 7.0 PBS buffer was added to make up the reaction system to 100mL, reacted at 30°C, sampled at different times (10min, 30min, 1h, 2h, 4h, 12h, 24h), and inactivated at 100°C for 10min to terminate the reaction. The enzymatic hydrolysis product was analyzed by HPLC. HPLC analysis conditions: chromatographic column: Superdex 30increase 10 / 300GL; mobile phase: 50mM ammonium formate; flow rate 0.5mL / min. The results are as follows Figure 3 As shown in the figure, at the beginning of the reaction, the exclusion peak at 15 min shifted backward and decreased, accompanied by the generation of oligosaccharides with different polymerization degrees. As the enzymatic hydrolysis time increased, large molecular weight oligosaccharides were first generated and then degraded, while small molecular weight oligosaccharides continued to accumulate. This indicates that the enzyme is a random endonuclease.

[0095] Example 7: LC-MS analysis of the reaction products of random bifunctional carrageenase

[0096] 2U of the enzyme solution obtained in Example 1 was added to 100mg of 2mg / mL iota-carrageenan and κ-carrageenan solution, and pH 7.0 PBS buffer was added to make up the reaction system to 100mL, reacted at 30°C for 2h, and inactivated at 100°C for 10min to terminate the reaction. LC-MS analysis of the enzymatic hydrolysis product was performed using a monopole mass spectrometer. Figure 4As shown, mass spectrometry analysis shows that the degradation products are mainly composed of disaccharides, tetrasaccharides, hexasaccharides and octasaccharides. In combination with Example 6, it can be seen that the final products of the random bifunctional carrageenanase degrading iota-carrageenan and kappa-carrageenan are both tetrasaccharides and disaccharides.

[0097] According to the different modes of action, ι-carrageenase can be divided into random ι-carrageenase and continuous ι-carrageenase. Continuous ι-carrageenase continuously cuts ι-carrageenan to produce oligosaccharide fragments with a fixed degree of polymerization, i.e., tetrasaccharides. In the present invention, the random bifunctional carrageenase degrades ι-carrageenan, randomly cuts ι-carrageenan, produces oligosaccharide fragments (disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, etc.) with different degrees of polymerization, and shows polydispersity of enzymatic oligosaccharides at the initial stage of the reaction, so the enzyme exhibits the characteristics of a typical random ι-carrageenase, i.e., as described in Example 6. In addition, according to existing reports, ι-carrageenase specifically degrades ι-carrageenan, and the enzyme can degrade κ-carrageenan and ι-carrageenan, so the enzyme is a bifunctional carrageenase, i.e., Example 4. In summary, the enzyme is a random bifunctional carrageenase.

[0098] According to existing reports, ι-carrageenase is continuous type, and the oligosaccharides produced in the whole reaction process are mainly tetrasaccharides. The present invention provides a random bifunctional carrageenase. Although the final products of the random bifunctional carrageenase in this patent are also tetrasaccharides and disaccharides, small molecular oligosaccharides (disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, etc.) with various polymerization degrees can be produced by controlling the enzymolysis time.

[0099] Example 8: Preparation of iota-carrageenan oligosaccharides with different degrees of polymerization by random bifunctional carrageenase

[0100] 2U of the enzyme solution obtained in Example 1 was added to 20mL of 5mg / mL ι-carrageenan solution at pH 7.0 and reacted at 50°C for 4h. After the reaction was completed, the obtained enzymatic hydrolysate was lyophilized and concentrated, and the enzymatic hydrolysate was purified using GE's HiLoadTM 26 / 600SuperdexTM30pg chromatographic column, with a mobile phase of 5mm ammonium formate and a flow rate of 2.6mL / min. The fractionation tubes of ι-carrageenan tetrasaccharide and ι-carrageenan disaccharide were collected separately, dissolved and lyophilized to volatilize the ammonium formate for desalting. Finally, ι-carrageenan tetrasaccharide and ι-carrageenan disaccharide (purity higher than 99%) were obtained.

[0101] 0.2U of the enzyme solution obtained in Example 1 was added to 20mL of 5mg / mL ι-carrageenan solution at pH 7.0, and reacted at 50°C for 4h. The purification conditions were the same as above. The fractionation tubes of ι-carrageenan decasaccharide, ι-carrageenan octasaccharide and ι-carrageenan hexasaccharide were collected separately, dissolved and freeze-dried to volatilize the ammonium formate for desalination. Finally, ι-carrageenan decasaccharide, ι-carrageenan octasaccharide and ι-carrageenan hexasaccharide (purity higher than 99%) were obtained.

[0102] Example 9: Preparation of κ-carrageenan oligosaccharides with different polymerization degrees by random bifunctional carrageenase

[0103] 2U of the enzyme solution obtained in Example 1 was added to 20mL of 5mg / mL κ-carrageenan solution at pH 7.0, and reacted at 55°C for 4h. The purification conditions were the same as those in Example 8. The fractionation tubes of κ-carrageenan tetrasaccharide and κ-carrageenan disaccharide were collected separately, dissolved and freeze-dried to volatilize the ammonium formate to desalt. Finally, κ-carrageenan tetrasaccharide and κ-carrageenan disaccharide (purity higher than 99%) were obtained.

[0104] 0.3U of the enzyme solution obtained in Example 1 was added to 20mL of 5mg / mL κ-carrageenan solution at pH 7.0, and reacted at 55°C for 4h. The purification conditions were the same as those in Example 8. The fractionation tubes of κ-carrageenan decasaccharide, κ-carrageenan octasaccharide and κ-carrageenan hexasaccharide were collected separately, dissolved and freeze-dried to volatilize the ammonium formate for desalting. Finally, κ-carrageenan decasaccharide, κ-carrageenan octasaccharide and κ-carrageenan hexasaccharide (purity higher than 99%) were obtained.

[0105] Example 10: Establishment of a molecular weight controllable enzymatic hydrolysis model of ι-carrageenan based on random bifunctional carrageenanase

[0106] The concentration of ι-carrageenan was controlled to be 1%, and the amount of enzyme added and the reaction time were used as variables to prepare the reaction product. The molecular weight of the obtained product was determined by high performance gel exclusion chromatography combined with multi-angle laser light scattering and differential detector (HPSEC-MALLS-RI). Chromatographic column: Shodex OHpak LB-806M (8.0×300mm); mobile phase: pH 7.420mM PBS solution (containing 0.15MNaCl); flow rate: 0.5mL / min; column temperature: 40℃; injection volume: 80μL. The molecular weight test results are shown in Table 2.

[0107] Table 2: Molecular weight of iota-carrageenan hydrolyzed by random bifunctional carrageenanase

[0108]

[0109]

[0110]

[0111] Stata 17.0 statistical software was used to perform multiple regression analysis on the experimental results in the above table (molecular weight of degradation product Y, enzyme addition amount X1, reaction time X2), and the fitting equation obtained was Log 10 (Y)=3.190863-0.0082655X1-0.1347936X2+0.0054209X2 2The model significance results show that Prob>F=0.0000<0.0001, indicating that the regression equation is statistically significant with a confidence level of 99.99%; R-squared=0.9224, indicating a good fitting effect. The model can be applied to the controlled degradation of iota-carrageenan, and the molecular weight can be quickly and easily predicted according to the enzymatic hydrolysis conditions to obtain iota-carrageenan with different molecular weights.

[0112] Example 11: Based on the enzymatic hydrolysis model, accurate preparation of 300 kDa carrageenan was achieved by controlling the reaction conditions

[0113] Taking the precise preparation of 300 kDaι-carrageenan as an example, the enzymatic hydrolysis time was controlled to be 2 h, 4 h, and 6 h, respectively, and the enzymatic hydrolysis model in Example 8 was substituted, and the enzyme solution obtained in Example 1 was added according to the predicted enzyme addition amount. After the reaction was completed, the molecular weight of carrageenan was monitored by HPSEC-MALLS-RI, and the mobile phase conditions were the same as in Example 10. The molecular weight detection results are shown in Table 3. The results show that the relative errors of the measured values ​​and the predicted values ​​of the molecular weight are within 5%, indicating that the precise preparation of 300 kDaι-carrageenan can be achieved at different reaction times.

[0114] Table 3: Precision preparation monitoring results of 300 kDa ι-carrageenan

[0115]

[0116]

[0117] Example 12: Preparation of iota-carrageenan with different molecular weights by controlling the amount of enzyme added based on the enzymatic hydrolysis model

[0118] The enzymatic hydrolysis time was controlled to be 4h, and the predicted molecular weights of 400kDa, 300kDa, 200kDa, and 100kDa were substituted into the enzymatic hydrolysis model in Example 8, and the enzyme solution obtained in Example 1 was added according to the predicted enzyme addition amount. After the reaction was completed, the molecular weight of carrageenan was monitored by HPSEC-MALLS-RI, and the mobile phase conditions were the same as in Example 10. The molecular weight detection results are shown in Table 4. The results show that the relative errors between the measured values ​​and the predicted values ​​of the molecular weights are within 10%, indicating that the preparation of ι-carrageenan with different molecular weights can be achieved based on the enzymatic hydrolysis model.

[0119] Table 4 Molecular weight monitoring results of iota-carrageenan with different molecular weights

[0120]

[0121] Example 13: κ-carrageenan with different molecular weights can be prepared by controlling the amount of enzyme added

[0122] 0.1, 0.2, 0.4U, 0.6U, 0.8U, 1U of the enzyme solution obtained in Example 1 was added to 20mL of 5mg / mL κ-carrageenan solution at pH 7.0, reacted at 55°C for 1h, and 600μL was taken for inactivation. After the reaction, the molecular weight of κ-carrageenan was monitored by HPSEC-MALLS-RI, and the mobile phase conditions were the same as those in Example 10. The molecular weight detection results are shown in Figure 5 As shown, by controlling the amount of enzyme added, κ-carrageenan with different molecular weights of 20 kDa-400 kDa can be obtained.

[0123] Example 14: κ-carrageenan with different molecular weights can be prepared by controlling the reaction time

[0124] 1U of the enzyme solution obtained in Example 1 was added to 20mL of 5mg / mL κ-carrageenan solution at pH 7.0, and reacted at 55°C for 10min, 20min, 30min, 40min, 50min, and 60min, and 600μL was taken for inactivation. After the reaction, the molecular weight of κ-carrageenan was monitored by HPSEC-MALLS-RI, and the mobile phase conditions were the same as those in Example 10. The molecular weight detection results are shown in Figure 6 As shown, κ-carrageenan with different molecular weights ranging from 20 kDa to 400 kDa can be obtained within 1 hour.

[0125] Based on Examples 8-14, by controlling the amount of enzyme added and the reaction time, iota-carrageenan and κ-carrageenan with different molecular weights and iota-carrageenan oligosaccharides and κ-carrageenan oligosaccharides with different polymerization degrees can be prepared, which lays a foundation for the study of the structure-activity relationship of iota-carrageenan.

[0126] Finally, it should be noted that although the above embodiments describe the specific implementation of the present invention, they are not intended to limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. All modifications or equivalent replacements should be included in the protection scope of the present invention.

Claims

1. A random bifunctional carrageenase, whose amino acid sequence is SEQ ID NO.1 and an enzyme derived from 1 which has the enzymatic activity of 1 after substitution, deletion or addition of one or more amino acids, and other sequences such as SEQ ID NO.2 and SEQ ID NO.3 having a homology with 1 greater than 70%.

2. Use of the carrageenase according to claim 1 in enzymatic hydrolysis of kappa-carrageenan and iota-carrageenan.

3. The use according to claim 2, characterized in that: The reaction temperature for ι-carrageenan is 45-60°C, and the optimum reaction temperature is 55°C; the reaction temperature for κ-carrageenan is 40-55°C, and the optimum reaction temperature is 50°C.

4. The use according to claim 2, characterized in that: The optimal reaction pH for iota-carrageenan and kappa-carrageenan is 7.

0.

5. The use according to claim 2, characterized in that: Based on the random bifunctional carrageenanase described in claim 1, a molecular weight controllable enzymatic hydrolysis model of iota-carrageenan or κ-carrageenan is established; and the enzymatic hydrolysis time and the amount of enzyme added are controlled to obtain iota-carrageenan or κ-carrageenan with different molecular weights.

6. The use according to claim 5, characterized in that: The fitting equation of the controllable enzymatic hydrolysis model of iota-carrageenan molecular weight is Log 10 (Y)=3.190863-0.0082655X1-0.1347936X2+0.0054209X2 2 ; Among them, the molecular weight of the degradation product is Y, the amount of enzyme added is X1, and the reaction time is X2.

7. A gene encoding the random bifunctional carrageenase according to claim 1, wherein the corresponding nucleotide sequence is shown in SEQ ID NO.4 and the entire sequence of SEQ ID NO.1 can be translated.

8. The use of the gene corresponding to the random bifunctional carrageenase in claim 7, characterized in that: The characterized protein can be used to degrade iota-carrageenan and kappa-carrageenan to obtain carrageenan oligosaccharides with various polymerization degrees.

9. A method for preparing the random bifunctional carrageenase according to claim 1, characterized in that: The enzyme was heterologously expressed in Escherichia coli, Bacillus subtilis, and Pichia pastoris systems to achieve overexpression and large-scale preparation of random bifunctional carrageenase.

10. A controllable enzymatic hydrolysis technology for iota-carrageenan or kappa-carrageenan based on the random bifunctional carrageenanase according to claim 1, characterized in that: According to the target molecular weight, the amount of enzyme added and the reaction time are designed and controlled to achieve the precise preparation of iota-carrageenan or κ-carrageenan of a specific molecular weight.

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