A beta-carrageenase mutant afcary123a and application thereof
By performing site-directed mutagenesis on β-carrageenase AfCar to form AfCarY123A, the problem of narrow product distribution of existing enzymes was solved, and the ability to prepare carrageenan oligosaccharides with various degrees of polymerization was realized.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
- Filing Date
- 2026-03-26
- Publication Date
- 2026-07-28
AI Technical Summary
Existing β-carrageenases have a narrow product distribution, which cannot meet the needs for preparing carrageenan oligosaccharides with different degrees of polymerization.
By performing a site-directed mutagenesis on the 123rd amino acid of β-carrageenase AfCar, changing tyrosine to alanine, a β-carrageenase mutant AfCarY123A was formed, expanding the types of oligosaccharides that can be prepared from it.
The range of oligosaccharides that can be prepared by wild-type enzymes has been successfully expanded. The mutant AfCarY123A can prepare κ-neocarrageenan tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides and dodecasaccharides to meet diverse oligosaccharide requirements.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of functional enzyme technology, specifically relating to a β-carrageenase mutant AfCarY123A and its applications. Background Technology
[0002] Carrageenan is a natural linear sulfated polysaccharide derived from the cell walls of red algae. It consists of repeating disaccharide units composed of D-galactose (D-Gal, G) and 3,6-anhydro-D-galactose (D-AHG, DA / D), linked alternately by β-1,4- and α-1,3-glycosidic bonds. Based on the number and type of sulfate groups, carrageenan polysaccharides are generally classified into kappa(κ)-, iota(ι)-, and lambda(λ)-carrageenan. The disaccharide units of κ-, ι-, and -λ-carrageenan contain 1, 2, and 3 sulfate groups, respectively, with disaccharide monomer structures of G4S-DA, G4S-DA2S, and G2S-D2,6-2S, respectively. In addition, mixed-structure carrageenan polysaccharides exist in nature. For example, the polysaccharide chain of β / κ-carrageenan contains two disaccharide monomers with different structures, G4S-DA and G-DA.
[0003] Carrageenan polysaccharides, due to their poor biosolubility and large molecular size, lack significant physiological activity; some studies have even used them to create inflammatory mouse models in mice. In contrast, low-molecular-weight carrageenan oligosaccharides exhibit good biosolubility and are easily absorbed and utilized by the body, demonstrating excellent physiological activity. These activities include antioxidant, antitumor, anti-inflammatory, antibacterial, and antiviral properties, showing broad application prospects in the food and pharmaceutical industries. Therefore, developing efficient and specific preparation technologies for carrageenan oligosaccharides is crucial for realizing the high-value utilization of carrageenan.
[0004] Currently, methods for preparing carrageenan oligosaccharides include physical, chemical, and biological methods, among which the most common are acid-based chemical methods and enzymatic biological methods. Acid hydrolysis has advantages such as rapid reaction and the ability to handle high substrate concentrations, but the products after acid hydrolysis are relatively mixed and difficult to separate. Compared with acid hydrolysis, enzymatic hydrolysis has advantages such as mild reaction, single product, and easy separation. Therefore, enzymatic methods are a green method with sustainable application prospects, and exploring carrageenase for the preparation of carrageenan oligosaccharides is of great significance.
[0005] β-carrageenase belongs to the glycoside hydrolase 16 (GH16) family and can hydrolyze both κ-carrageenan and β-carrageenan simultaneously, making it a multifunctional carrageenan hydrolase. Current research on carrageenase modification mainly focuses on improving enzyme catalytic activity and thermostability, while reports on carrageenase product modification are relatively scarce. This results in a narrow range of oligosaccharides that can be prepared using carrageenase, failing to meet the demand for simultaneously preparing carrageenan oligosaccharides with different degrees of polymerization. Summary of the Invention
[0006] In view of the above-mentioned prior art, the present invention provides a β-carrageenase mutant AfCarY123A and its application. By performing site-directed mutagenesis on the amino acids of the substrate channel of β-carrageenase AfCar, the product composition of the mutant is changed, successfully expanding the types of oligosaccharides that can be known, and has broad application prospects.
[0007] This invention is achieved through the following technical solution: A β-carrageenase mutant, AfCarY123A, has the amino acid sequence shown in SEQ ID NO.1, while the amino acid sequence of the wild-type β-carrageenase AfCar is shown in SEQ ID NO.2. Compared with β-carrageenase AfCar, a site-directed mutation was performed on amino acid position 123, changing tyrosine (Tyr) to alanine (Ala).
[0008] The amino acid sequence of the β-carrageenase mutant AfCarY123A (SEQ ID NO.1): MKSTTITVFILVLFFVSCQKSEHQNKIDTVEINKFEPASDLSNNGNWKFIEALSDEFDAVQLDESKWLIQGRNGVFQSNFKGRAPSQFSTENVKLEDGKLKLETRWQPNYNFNPKVDKDGDPAEYITTAAVITKKELLYGYIEVKSKAADAEVTSSF WATGNGSEFDFFEMFGDHKQAEKEANGKERELWWSIHDWSSAGGGKTTYTEYHDLGFRVADDFHVYGYEWSEEGVAIYIDGKLFRNVSKETINAHDDVKNNNGGNGPEDNFVITKPVKIWFDQETFPWHGVPDSKEEVGEDGAVDFEIEYIRVWQKK.
[0009] The amino acid sequence of wild-type β-carrageenase AfCar (SEQ ID NO.2): MKSTTITVFILVLFFVSCQKSEHQNKIDTVEINKFEPASDLSNNGNWKFIEALSDEFDAVQLDESKWLIQGRNGVFQSNFKGRAPSQFSTENVKLEDGKLKLETRWQPNYNFNPKVDKDGDPYEYITTAAVITKKELLYGYIEVKSKAADAEVTSSF WATGNGSEFDFFEMFGDHKQAEKEANGKERELWWSIHDWSSAGGGKTTYTEYHDLGFRVADDFHVYGYEWSEEGVAIYIDGKLFRNVSKETINAHDDVKNNNGGNGPEDNFVITKPVKIWFDQETFPWHGVPDSKEEVGEDGAVDFEIEYIRVWQKK.
[0010] The present invention also provides a gene encoding the β-carrageenase mutant AfCarY123A, the nucleotide sequence of which is shown in SEQ ID NO.3, and the nucleotide sequence of the wild-type β-carrageenase AfCar is shown in SEQ ID NO.4.
[0011] The nucleotide sequence of the gene encoding the β-carrageenase mutant AfCarY123A is shown below (direction 5'-3') (as shown in SEQ ID NO.3): 。
[0012] The nucleotide sequence of the wild-type β-carrageenase AfCar gene is shown below (direction 5'-3') (as shown in SEQ ID NO. 4): 。
[0013] An enzyme preparation containing the β-carrageenase mutant AfCarY123A.
[0014] The application of the enzyme preparation in the degradation of κ-carrageenan involves using the enzyme preparation to degrade carrageenan while simultaneously preparing κ-neocarrageenan tetrasaccharide, κ-neocarrageenan hexasaccharide, κ-neocarrageenan octasaccharide, κ-neocarrageenan decasaccharide and κ-neocarrageenan dodecasaccharide.
[0015] Further, the degradation conditions are as follows: κ-carrageenan substrate concentration of 5-10 g / L, enzyme dosage of 1-10 U / mL, preferably 5 U / mL, enzymatic hydrolysis temperature of 30-65 ℃, preferably 40 ℃, pH value of 6.0-8.0, preferably 6.0, and enzymatic hydrolysis time of 12 hours.
[0016] Application of the gene encoding the β-carrageenase mutant AfCarY123A in the preparation of the above-mentioned enzyme preparation.
[0017] A recombinant expression vector carrying a gene encoding a β-carrageenase mutant, AfCarY123A.
[0018] A recombinant engineered bacterium carries a gene encoding the β-carrageenase mutant AfCarY123A in its genome and can express the β-carrageenase mutant AfCarY123A.
[0019] The recombinant expression vector and recombinant engineered bacteria are used in the preparation of the β-carrageenase mutant AfCarY123A and in the preparation of the above-mentioned enzyme preparation.
[0020] This invention addresses the problem of narrow product distribution in existing β-carrageenases. Starting with β-carrageenase AfCar, it achieves site-directed mutagenesis of the key amino acid at the end of its substrate-binding channel. Specifically, it mutates amino acid position 123 from tyrosine (Tyr) to alanine (Ala). The results show that after the mutation, the product polymers changed from 2, 4, and 6 of the wild-type enzyme to 4, 6, 8, 10, and 12 of the mutant enzyme, successfully expanding the degree of polymerization range of oligosaccharides that can be prepared from the wild-type enzyme. This discovery lays the foundation for the preparation of carrageenan oligosaccharides with higher degrees of polymerization using β-carrageenase. Attached Figure Description
[0021] Figure 1 This is an SDS-PAGE electrophoresis image of the purified β-carrageenanase AfCar of the present invention; Figure 2 A graph showing the relative enzyme activities of β-carrageenase AfCar in the hydrolysis of different types of carrageenan; Figure 3The HPLC chromatogram shows the products of β-carrageenase AfCar hydrolysis of κ-carrageenan. Figure 4 A surface-format diagram showing the docking results of β-carrageenanase AfCar with the octasaccharide ligand; Figure 5 The diagram shows the docking results of β-carrageenase AfCar with the octasaccharide ligand. Figure 6 The HPLC chromatogram of the product of κ-carrageenan hydrolysis by the β-carrageenase mutant AfCarY123A; Figure 7 Figure showing the docking results of the β-carrageenase mutant AfCarY123A with the octasaccharide ligand; Figure 8 A comparison chart of root mean square deviation (RMSD) results from molecular dynamics simulations; Figure 9 A comparison chart of root mean square fluctuation (RMSF) results from molecular dynamics simulations; Figure 10 This is a schematic diagram of two loop regions in Y123A-octasaccharides that show significantly higher root mean square fluctuation (RMSF) values compared to AfCar-octasaccharides; the red line marks residues 70–82, and the blue line marks residues 114–125. Detailed Implementation
[0022] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.
[0023] Unless otherwise specified, the instruments, reagents, and materials involved in the following embodiments are all conventional instruments, reagents, and materials already existing in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods involved in the following embodiments are all conventional experimental methods and detection methods already existing in the prior art. The various terms and phrases used in this embodiment have their general meanings known to those skilled in the art.
[0024] The method of the present invention will be further illustrated below through specific examples.
[0025] Example 1 Cloning of β-carrageenase AfCar The inventors of this application obtained a source from the NCBI database. Aureibaculum flavumA β-carrageenase (GenBank ID: WP_198840367.1) potentially possessing carrageenan hydrolytic activity, with its amino acid sequence shown in SEQ ID NO.2, was named AfCar. The inventors optimized the DNA sequence of this gene fragment based on the codon preferences of the host *E. coli*, and the optimized gene sequence is shown in SEQ ID NO.5 (excluding the N-terminal 51 bp signal peptide coding sequence).
[0026] The nucleotide sequence of the artificially synthesized β-carrageenase AfCar gene is shown below (direction 5'-3') (as shown in SEQ ID NO.5): TGTCAGAAAAGCGAGCATCAGAATAAAATTGATACCGTTGAGATTAACAAATTCGAGCCGGCAAGCGATCTGAGCAATAATGGGAATTGGAAATTTATTGAGGCGCTGAGTGATGAGTTTGATGCAGTTCAGCTGGATGAAAGCAAATGGCTGATTCAGGGTCGTAACGGTGTGTTTCAGAGCAATTTTAAAGGCCGTGCACCGAGCCAGTTTTCGACCGAAAATGTTAAACTGGAGGATGGTAAACTGAAACTGGAAACCCGCTGGCAACCGAATTATAATTTCAATCCTAAAGTGGATAAAGACGGAGATCCTTATGAATATATCACCACGGCAGCAGTGATTACCAAAAAGGAACTGCTGTATGGATATATTGAAGTAAAAAGCAAAGCCGCAGATGCAGAAGTCACCAGTAGCTTCTGGGCAACCGGTAACGGTAGCGAATTCGACTTTTTTGAAATGTTTGGGGATCATAAACAGGCAGAAAAAGAAGCAAACGGAAAAGAACGTGAACTGTGGTGGAGTATTCATGATTGGAGCAGCGCAGGGGGCGGAAAAACCACCTATACCGAGTATCACGATCTGGGGTTTCGCGTAGCAGATGATTTTCATGTGTATGGCTATGAATGGTCTGAGGAGGGGGTTGCAATTTATATTGATGGAAAACTGTTCCGCAATGTTAGCAAAGAGACCATCAATGCACATGATGATGTGAAAAATAACAACGGTGGAAATGGACCGGAAGATAACTTTGTGATTACAAAACCGGTTAAAATCTGGTTCGATCAGGAAACCTTTCCGTGGCATGGCGTTCCGGATAGCAAAGAGGAAGTGGGCGAAGACGGTGCAGTGGATTTTGAGATTGAATATATTCGTGTTTGGCAGAAAAAGTAA。
[0027] Synthesize the gene fragment shown in SEQ ID NO.5 artificially. Using the artificially synthesized gene fragment as a template, perform PCR amplification. The specific primers used for PCR are shown as follows: Forward primer: 5'-TGTCAGAAAAGCGAGCATC-3', as shown in SEQ ID NO.6.
[0028] Reverse primer: 5'-CTTTTTCTGCCAAACACG-3', as shown in SEQ ID NO.7.
[0029] Example 2 Construction of an expression vector carrying the β-carrageenase gene AfCar The target fragment obtained from Example 1 was amplified and reacted with the linearized pCold II vector using a seamless splicing kit at 50°C for 5 minutes. The resulting fragment was then transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 100 μg / mL ampicillin. After overnight incubation at 37°C, single clones were picked for positive clone verification. Clones with the correct band size were sent to a sequencing company for sequencing. If the sequencing alignment was completely correct, the recombinant plasmid was obtained, named pCold-AfCar, and stored at -20°C for later use.
[0030] Example 3 Construction of engineered bacteria containing β-carrageenase gene The recombinant plasmid carrying the carrageenase gene obtained in Example 2 was transformed into Escherichia coli BL21(DE3) competent cells using a 42 °C heat shock transformation method. The transformed cells were plated on LB solid antibiotic plates containing 100 μg / mL ampicillin. After overnight incubation at 37 °C, single clones were picked for positive clone verification. Single clones with the correct band size were cultured overnight in liquid LB medium (containing 100 μg / mL ampicillin). The bacterial culture was preserved in 10% glycerol and stored at -80 °C for long-term use.
[0031] Example 4: Preparation and purification of β-carrageenase AfCar The bacterial culture preserved in Example 3 was activated by overnight incubation at 37°C in LB liquid medium (containing 100 μg / mL ampicillin), and then transferred to 100 mL LB Erlenmeyer flasks (containing 100 μg / mL ampicillin) and incubated at 37°C and 200 rpm until OD. 600 IPTG was added at a final concentration of 0.1 mM at approximately 0.6 g / L, and the mixture was incubated at 16 °C for 16 hours to express β-carrageenase.
[0032] After fermentation, the bacterial cells were collected by centrifugation at 8000 rpm for 5 minutes. A certain amount of sterile water was added to wash the cells, and the cells were collected again by centrifugation at 8000 rpm for 5 minutes. The cells were reconstituted in Tris-HCl buffer (pH 8.0) and then sonicated on ice (320 W, 3 seconds on, 3 seconds off, continuous sonication for 30 minutes). After complete sonication, the cells were centrifuged at 8000 rpm at 4 °C for 15 minutes, and the supernatant was collected as the crude enzyme. The expressed target gene contains a His purification tag, therefore we used Ni-NTA affinity chromatography for purification. The target protein was eluted using imidazole at different concentration gradients (10, 20, 50, 80, 120, 200, and 500 mM), and purified using an Akta purification system. SDS-PAGE results showed that 200 mM imidazole yielded a relatively single band of the target protein. Figure 1 The protein concentration of the purified enzyme solution was determined to be 0.05 g / L using the R250 Coomassie Brilliant Blue method. The enzyme solution was then concentrated using a 10 kDa ultrafiltration tube with ultrapure water as the replacement solvent until the protein concentration reached 1 g / L, yielding a pure enzyme for the determination of its enzymatic properties.
[0033] Example 5 Comparison of the hydrolytic activity of β-carrageenase AfCar for different types of carrageenan The AfCar pure enzyme obtained in Example 4 was tested for hydrolytic activity against four types of carrageenan (β, κ, λ, and ι) at 40°C and pH 6.0. The concentration of each of the four types of carrageenan was controlled at 3 g / L, and the hydrolytic enzyme activity was measured after reacting for 45 minutes under the same conditions.
[0034] Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of reducing sugar in 1 minute under optimal reaction conditions.
[0035] like Figure 2 As shown, AfCar exhibited the highest hydrolytic activity against κ-carrageenan, with a hydrolytic activity of 121 U / mg.
[0036] Example 6: Determination of hydrolysis products of AfCar hydrolyzed κ-carrageenan The purified AfCar enzyme obtained in Example 4 was reacted with 3 g / L κ-carrageenan at 40 °C and pH 6.0 for 12 hours, and the hydrolysis products were determined by HPLC. Figure 3As shown, compared with the standard, it can be seen that the product composition contains disaccharides and tetrasaccharides. At the same time, there is also an oligosaccharide peak at an earlier elution time. The principle of the gel chromatography column used is to separate the oligosaccharides based on their molecular weight. The larger the molecular weight, the earlier the elution time. κ-carrageenan is composed of repeating disaccharide units, so the products produced by carrageenase cleavage of κ-carrageenan are all even-numbered oligosaccharides. Based on this, reasonable analysis of the elution time and time interval shows that the oligosaccharide peak with the earlier elution time is a hexasaccharide. Therefore, it can be concluded that the product composition is disaccharides, tetrasaccharides and hexasaccharides, with the tetrasaccharide being the main product.
[0037] Example 7 Selection of Mutation Sites for AfCar Product Modification To identify key amino acid sites that could potentially influence the distribution of β-carrageenase AfCar products, we first predicted the tertiary structure of AfCar using ColabFold. Then, we performed molecular docking between AfCar and κ-neocarrageenan octasaccharide using AutoDock Vina. The docking results showed that the Tyr ligand at position 123 locked the -4 subunit of the octasaccharide ligand (…). Figure 4 It forms a strong hydrogen bond with the -4 subunit. Figure 5 This indicates that it may play a crucial role in controlling the number of monosaccharides that the product enters the enzyme substrate channel. Therefore, Tyr at position 123 was selected as the key mutation site to carry out the following implementation case.
[0038] Example 8 Construction of β-carrageenase mutant AfCarY123A Using the vector pCold-AfCar from Example 2 as a template, PCR amplification was performed with the sequence shown in SEQ ID NO.8 as the forward primer and the sequence shown in SEQ ID NO.9 as the reverse primer. After digestion with DpnI enzyme, the product was transformed into Escherichia coli BL21(DE3) to construct the β-carrageenanase mutant AfCarY123A, in which the tyrosine at position 123 was mutated to alanine.
[0039] The specific primers used for PCR are shown below: Forward primer: 5'- GCAGAATATATCACCACGGCAG-3', as shown in SEQ ID NO.8; Reverse primer: 5'-GCCGTGGTGATATATTCTGCAGGATCTCCGTCTTTATC-3', as shown in SEQ ID NO.9.
[0040] Example 9 Preparation and purification of β-carrageenase mutant AfCarY123A The β-carrageenanase mutant AfCarY123A recombinant bacteria were inoculated into LB liquid medium (containing 100 μg / mL ampicillin) and cultured overnight at 37°C for activation. After activation, the culture was transferred to 100 mL LB Erlenmeyer flasks (containing 100 μg / mL ampicillin) and cultured at 37°C and 200 rpm until OD500. 600 IPTG was added at a final concentration of 0.1 mM at approximately 0.6 g / L, and the mixture was incubated at 16 °C for 16 hours to express the β-carrageenase mutant.
[0041] After fermentation, the bacterial cells were collected by centrifugation at 8000 rpm for 5 minutes. A certain amount of sterile water was added to wash the cells, and the cells were collected again by centrifugation at 8000 rpm for 5 minutes. The cells were reconstituted in Tris-HCl buffer (pH 8.0) and then sonicated on ice (320 W, 3 seconds on, 3 seconds off, continuous sonication for 30 minutes). After complete sonication, the cells were centrifuged at 8000 rpm at 4 °C for 15 minutes, and the supernatant was collected as the crude enzyme. The expressed target gene contains a His purification tag, therefore, Ni-NTA affinity chromatography was used for purification. The target protein was eluted using imidazole at different concentration gradients (10, 20, 50, 80, 120, 200, and 500 mM), and purified using an Akta purification system to obtain pure enzyme. The protein concentration of the purified enzyme solution was determined to be 0.08 g / L by the R250 Coomassie Brilliant Blue method. The enzyme solution was concentrated to a protein concentration of 1 g / L using a 10 kDa ultrafiltration tube and ultrapure water as the replacement solvent, and then used for the determination of its enzymatic properties.
[0042] Example 10: Determination of hydrolysis products of AfCarY123A hydrolyzed κ-carrageenan The purified AfCarY123A enzyme obtained in Example 9 was reacted with 3 g / L κ-carrageenan at 40 °C and pH 6.0 for 12 hours, and the hydrolysis products were determined by HPLC. Figure 6 As shown, compared with the standard, it can be seen that the product composition contains disaccharides and tetrasaccharides. At the same time, there are four relatively obvious oligosaccharide peaks at earlier elution times. The principle of the gel chromatography column used here is to separate the oligosaccharides based on their molecular weight. The larger the molecular weight, the earlier the elution time. κ-carrageenan is composed of repeating disaccharide units, so the products produced by carrageenase cleavage of κ-carrageenan are all even-numbered oligosaccharides. Based on the reasonable analysis of the elution time and time interval, it can be seen that the four oligosaccharide peaks with earlier elution times are hexasaccharides, octasaccharides, decasaccharides, and dodecasaccharides. Thus, the product composition is tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, and dodecasaccharides, among which hexasaccharides, octasaccharides, and decasaccharides are the main products. Compared with wild-type AfCar, the degree of polymerization composition of the mutant AfCarY123A product has changed significantly. The mutant is more inclined to produce large-molecule carrageenan oligosaccharides.
[0043] Example 11 Molecular docking of mutant AfCarY123A with octasaccharide ligand To investigate the potential reasons for the altered product distribution in the mutant AfCarY123A, we first predicted the tertiary structure of AfCarY123A using ColabFold, and then performed molecular docking between AfCarY123A and κ-neocarrageenan octaose using AutoDock Vina. Figure 7 As shown, the mutated alanine at position 123 (Ala) is 10 Å away from the oligosaccharide subunit at position -4, indicating that more subunits can be accommodated in between, which helps explain why the mutant tends to produce oligosaccharides with a higher degree of polymerization.
[0044] Example 12 Molecular Dynamics Simulation To further explore the potential reasons for the altered product distribution of the mutant AfCarY123A, we performed molecular dynamics simulations on AfCar-octasaccharides and AfCarY123A-octasaccharides using Gromacs 2022.3 software. For small molecule pretreatment, AmberTools 22 was used to add a GAFF force field, and Gaussian 16W was used for hydrogenation and RESP potential calculations; the potential data were added to the molecular dynamics system topology file. Simulations were conducted at a static temperature of 300 K and atmospheric pressure (1 Bar), using Amber99sb-ildn as the force field and water molecules as the solvent (Tip3p water model). An appropriate amount of Na ions was added to neutralize the total charge of the simulation system. The molecular dynamics simulation system was first minimized using the steepest descent method, followed by 100,000 steps of isothermal and isochoric ensemble equilibrium and isothermal and isobaric ensemble equilibrium, with a coupling constant of 0.1 ps and a duration of 100 ps. Finally, a free molecular dynamics simulation was run, consisting of 5,000,000 steps at a step size of 2 fs, lasting a total of 100 ns. After the simulation, the software's built-in tools were used to analyze the trajectories and calculate the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of each amino acid's trajectory.
[0045] The RMSD value is a core parameter for monitoring structural changes. A lower RMSD value indicates that the simulated trajectory deviates little from the initial structure, suggesting the system is in equilibrium. Figure 8As shown, the average RMSD of the AfCar-octasaccharide system complex is 0.26 ± 0.02 nm, and the RMSD of the Y123A-octasaccharide system complex is 0.28 ± 0.02 nm. This indicates that the overall RMSD performance of both systems is excellent, and the structures of both complexes tend to be stable during the simulation, which can be used for further analysis.
[0046] RSMF is commonly used to assess the flexibility of protein residues, with lower RSMF values indicating higher rigidity and higher values indicating increased flexibility. For example... Figure 9 As shown, in the AfCar-octasaccharide system, the average RMSF of the protein was 0.11 ± 0.07 nm, indicating that most residues maintained low variability during simulation, especially those near the binding pocket. In contrast, the average RMSF of the Y123A-octasaccharide system was slightly higher, at 0.13 ± 0.08 nm, suggesting that mutations may have enhanced the flexibility of certain regions. Notably, the two loop regions (residues 70–82 and 114–125) had significantly higher RMSF values in the Y123A-octasaccharide than in the AfCar-octasaccharide. These loop regions form part of the AfCar substrate binding pocket and are involved in anchoring the -4 and -3 sites of the ligand in the AfCar-octasaccharide conformation. Figure 10 The increased flexibility of these regions in the Y123A mutant may allow the binding pocket to accommodate longer substrates, thus structurally explaining why the mutant enzyme produces a larger product compared to the wild type.
[0047] The above embodiments are only for illustrating the technical concept and features of the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make various modifications or alterations to the present invention, and these equivalent forms are also covered within the scope defined by the appended claims.
Claims
1. A β-carrageenase mutant AfCarY123A, characterized in that, The amino acid sequence of the mutant AfCarY123A is shown in SEQ ID NO.
1.
2. The gene encoding the β-carrageenase mutant AfCarY123A as described in claim 1.
3. An enzyme preparation, characterized in that, The enzyme preparation contains the β-carrageenase mutant AfCarY123A as described in claim 1.
4. The application of the enzyme preparation according to claim 3 in the degradation of κ-carrageenan, characterized in that, The application involves using the enzyme preparation to degrade κ-carrageenan while simultaneously preparing κ-neocarrageenan tetrasaccharide, κ-neocarrageenan hexasaccharide, κ-neocarrageenan octasaccharide, κ-neocarrageenan decasaccharide, and κ-neocarrageenan dodecasaccharide.
5. A recombinant expression vector, characterized in that, The recombinant expression vector carries the gene encoding the β-carrageenase mutant AfCarY123A as described in claim 1.
6. A recombinant engineered bacterium, characterized in that, Its genome carries a gene encoding the β-carrageenase mutant AfCarY123A as described in claim 1, and it is able to express the β-carrageenase mutant AfCarY123A.
7. The use of the recombinant expression vector of claim 5 or the recombinant engineered bacteria of claim 6 in the preparation of the β-carrageenase mutant AfCarY123A of claim 1.