GH29 fucoidase mutant and application thereof
By optimizing the sequence of GH29 fucoidanase and constructing a recombinant Escherichia coli expression system, the problem of low expression and purification efficiency of GH29 fucoidanase in Escherichia coli was solved, resulting in a significant improvement in enzyme activity, which is applicable to the food, pharmaceutical and bioenergy fields.
Patent Information
- Application Number
- CN202510956961.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-11-18
AI Technical Summary
In the existing technology, the heterologous expression level of GH29 fucoidanase in Escherichia coli is low, the enzyme activity is insufficient, and the purification efficiency is low, which limits its potential for industrial application.
The sequence of GH29 fucoidanase was optimized using molecular simulation and molecular dynamics to construct a recombinant Escherichia coli expression system. Signal peptide prediction and codon optimization were used to improve the solubility and expression level of the enzyme, and Ni column affinity chromatography was used for purification to improve enzyme activity.
It significantly improved the heterologous expression efficiency and enzyme activity of GH29 fucoidanase in Escherichia coli, with the highest enzyme activity reaching 28.26 U/mg, thus solving the problems of low enzyme activity and purification efficiency.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of enzyme engineering and microbial biotechnology, in particular to a GH29 fucanase mutant and application thereof. BACKGROUND
[0002] The alpha-L-fucosidase of GH29 family has important application value in degrading fucan, and widely exists in bacteria, fungi and part of marine microorganisms. The enzyme releases L-fucose by hydrolyzing alpha-1, 3 or alpha-1, 4 glycosidic bond, and has potential application value in the fields of food, medicine and biological energy. At present, heterologous expression of GH29 fucanase has been reported. For example, some studies express GH29 fucanase in E. coli Arctic Express (DE3) host bacteria, but the heterologous expression enzyme activity in the study is low, only 0.1 U / mg, which limits its potential in industrial application. In addition, due to the lack of natural secretion system in E. coli, most of the recombinant proteins form inclusion bodies, which is not conducive to downstream purification and application. Expression attempts in other microbial hosts such as Bacillus subtilis or yeast (Pichia pastoris) also have challenges, such as low expression level of exogenous protein, glycosylation modification affecting enzyme activity, etc. Therefore, improving the expression level of GH29 fucanase, optimizing the purification process and enhancing the enzyme activity performance are still the key research directions in this field. SUMMARY
[0003] In order to solve the technical problems in the prior art that the heterologous expression level of GH29 fucanase in E. coli is low, the enzyme activity is far from the level of industrial application, the solubility and expression amount of the enzyme are limited, the enzyme purification method is low in efficiency, and a high-efficiency and repeatable purification process cannot be provided, the present application provides a GH29 fucanase mutant and application thereof, which aims to optimize the gene expression system of GH29 fucanase, improve the heterologous expression efficiency of GH29 fucanase in E. coli, and optimize the sequence of GH29 fucanase based on the results of molecular simulation and molecular dynamics, so that the enzyme activity is significantly improved.
[0004] To achieve the above object, the technical scheme provided by the present application is as follows:
[0005] A GH29 fucanase mutant, the nucleotide sequence of which is shown in SEQ ID NO. 1.
[0006] Further, the amino acid sequence of the GH29 fucanase mutant is shown in SEQ ID NO. 2.
[0007] The recombinant E. coli is constructed by using the GH29 fucanase mutant as described above, and the construction method is that the nucleotide sequence gene of SEQ ID NO. 1 is cloned into the pET-22b(+) expression vector, and then transformed into the E. coli strain for induction expression.
[0008] Further, the induction expression condition is 16℃, and IPTG (600ul / L) is used as an inducer for induction expression for 18h.
[0009] The application of the GH29 fucanase mutant or the recombinant E. coli in producing high-activity GH29 fucanase and improving the heterologous expression amount of the GH29 fucanase.
[0010] Further, the GH29 fucanase is alpha-L-fucosidase, specifically alpha-1,3-fucosidase.
[0011] Compared with the prior art, the present application has the following beneficial effects:
[0012] The GH29 fucanase mutant obtained by the present application can effectively improve the heterologous expression efficiency of the GH29 fucanase in E. coli, and can significantly improve the activity of the obtained fucanase, and the highest activity can reach 28.26 U / mg; further, the construction method of the present application is based on molecular docking and molecular dynamics simulation, determines the key residues of the GH29 fucanase and optimizes, screens and optimizes the target gene, and improves the expression level of the GH29 fucanase in E. coli.
[0013] Deposit information
[0014] Celeribacter halophilus RS3 was deposited with the Guangdong Microbial Culture Collection Center (GDMCC) on November 26, 2023, and the deposit number is GDMCC No: 64072. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 is the heterologous expression of the GH29 fucanase mutant of the present application: (A) signal peptide prediction result; (B) soluble protein prediction result, (C) pEt 22(b)+|GH29_Fuc vector schematic diagram; (D) GH29 protein modeling diagram.
[0016] Figure 2 The heterologous expression of the GH29 fucanase mutant of the present application: (A) heterologous expression of crude enzyme freeze-dried powder; (B) DE3|pEt 22(b)+|GH29_Fuc ampicillin resistance plate growth result.
[0017] Figure 3(A) is the PCR gel electrophoresis diagram of the original enzyme gene sequence and three mutant vectors constructed on this basis; Figure 3 (B) is the SDS-PAGE result diagram of the original GH29 fucanase crude enzyme liquid and the purified enzyme liquid added with IPTG induction and without IPTG induction.
[0018] Figure 4 (A) is the molecular docking result of GH29 fucanase and glycosidic bond ligand; Figure 4 (B) is the score ratio column chart of each mutant protein-ligand complex / original enzyme protein-ligand complex.
[0019] Figure 5 is the sequencing result diagram of three GH29 fucanase mutant vectors; wherein (A) is pET 22b(+)|GH29Fuc||E288Q, (B) is pET 22b(+)|GH29Fuc|S289A, and (C) is pET 22b(+)|GH29Fuc|G294A.
[0020] Figure 6 is the SDS-PAGE result diagram of three GH29 fucanase mutants, A is GH29-Fuc288, B is GH29-Fuc289, and C is GH29-Fuc294; CL: crude enzyme liquid, WT: sample flow-through liquid, W3: last washing liquid, E1-E4: eluent, purified enzyme.
[0021] Figure 7 is the reducing sugar generation curve in the enzymatic hydrolysis process of three GH29 fucanase mutants.
[0022] Figure 8 is the comparison of enzyme activity of heterologous expression enzyme of three GH29 fucanase mutants and original enzyme.
[0023] Figure 9 is the protein standard curve.
[0024] Figure 10 is the fucose standard curve.
[0025] Figure 11 is the total energy diagram of original GH29 fucanase and three GH29 fucanase mutants; wherein, A is original GH29 fucanase, B is GH29-Fuc288 fucanase mutant, C is GH29-Fuc289 fucanase mutant, and D is GH29-Fuc294 fucanase mutant. DETAILED DESCRIPTION
[0026] The following detailed description, in conjunction with the accompanying drawings, outlines specific embodiments. However, it should be understood that the scope of protection of this invention is not limited to these specific embodiments. Unless otherwise specified, all raw materials and reagents used in the examples are commercially available.
[0027] Example 1
[0028] Target gene screening
[0029] A fast-growing halophilic bacterium (Celeribacter halophilus RS3) was screened from marine sediments, and whole-genome sequence analysis confirmed that this strain contains the GH29 family α-1,3 / 1,4-L-fucoidase gene. The specific methods are as follows:
[0030] (1) By comparing the 16S rDNA sequence, it was determined that strain RS3 was most closely related to Celeribacter halophilus strain DSM26270, with a similarity of 98.89%. It was named Celeribacter halophilus RS3 and deposited at Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No: 64072.
[0031] (2) The whole genome sequences of Celeribacter halophilus strain DSM26270 and Celeribacter indica strain P73 were downloaded from the GenBank database for gene alignment;
[0032] (3) Through amino acid sequence alignment and BLAST analysis, it was found that an unknown protein (GenBank: AAK76203.1) of Celeris indica strain P73 contains the AfuC functional domain, indicating that it has α-L-fucosidase activity.
[0033] Example 2
[0034] Gene cloning and expression vector construction
[0035] (1) Select the α-1,3 / 1,4-L-fucosidase sequence of the GH29 family (GenBank:ANW96108.1), the complete amino acid sequence is shown in SEQ ID NO.3;
[0036] (2) Signal peptide sequence was predicted using SignalP6, with the predicted signal peptide site being position 20-21 (GC). Figure 1 A), and the protein solubility was analyzed using Protein-Sol. The predicted value for soluble protein was 0.487, which is higher than the threshold of 0.45, indicating that the enzyme is unlikely to form embedded bodies after expression.Figure 1 B);
[0037] (3) Computer-aided semi-rational design of GH29 fucanase:
[0038] (i) GH29 fucanase modeling is completed using Alphafold tool, Figure 1 D) alpha-1, 3 / 1, 4-L-fucoside bond ligand modeling is completed using GLYCAM tool;
[0039] (ii) molecular docking of GH29 fucanase and glycoside bond ligand is performed;
[0040] (iii) based on the molecular docking results, Figure 4 A), key residues (288, 289, 294, 306, 491 and 264) of GH29 enzyme are determined, single mutation and combined mutation of each residue are performed based on the principles of semi-rational design, molecular docking of each enzyme receptor and glycoside bond ligand after mutation is completed, and DeepMice model is used to score each docking successful protein-ligand complex, Figure 4 B is the score ratio column chart of each mutant protein-ligand complex / original enzyme protein-ligand complex, several groups with the largest ratio of each residue mutation result are selected for molecular dynamics simulation, according to the total energy diagram of molecular dynamics simulation, the more stable the system is, the more stable the system is; according to the total energy Figure 11 The results show that the present application is reliable;
[0041] (iv) based on the above results, S289A is determined as a positive optimization, and the optimal enzyme sequence is selected to complete the construction of the optimized recombinant plasmid pET 22b(+)|GH29Fuc|S289A on the basis of the recombinant expression plasmid pET 22b(+)|GH29Fuc|Ori using point mutation method, Figure 3 A is the gel electrophoresis diagram of four kinds of plasmids: Ori represents the original sequence vector, S289A represents the vector in which the 289th serine is mutated to alanine on the basis of the original sequence, Figure 5 (B) is the sequencing result diagram of the mutant vector pET 22b(+)|GH29Fuc|S289A of the present application, proving that the recombinant plasmid is successfully constructed;
[0042] (4) codon optimization and addition of 6xHis tag, the optimized gene sequence is shown in SEQ ID NO. 1;
[0043] (5) after the gene synthesis is completed, it is cloned into pET-22b(+) expression vector Figure 1 C).
[0044] Heterologous expression
[0045] (1) Take E. coli DE3 as the host bacteria, transform the constructed recombinant expression plasmid, and after successful transformation, the growth results of the host bacteria on ampicillin resistance plates are shown in FIG. B. The final expression vector is pET 22b(+)|GH29Fuc|Ori (original sequence), pET 22b(+)|GH29Fuc|S289A, and a GH29-Fuc289 fucoidanase mutant (a GH29 fucoidanase mutant of the present application) is obtained; Figure 2 B, and the final expression vector is pET 22b(+)|GH29Fuc|Ori (original sequence), pET 22b(+)|GH29Fuc|S289A, and a GH29-Fuc289 fucoidanase mutant (a GH29 fucoidanase mutant of the present application) is obtained;
[0046] (2) Under the condition of 16℃, IPTG (600ul / L) is used as an inducer to induce expression for 18h.
[0047] The material obtained after inducing expression for 18h in step (2) is centrifuged and filtered at 5000rpm for 10min, and the precipitate is taken for standby use. The precipitate obtained from the GH29-Fuc289 fucoidanase mutant is marked as precipitate 289, and the precipitate obtained from the original GH29 fucoidanase is marked as precipitate ori.
[0048] Construction and expression of two other mutants with different positions:
[0049] Referring to the above-mentioned “gene cloning and expression vector construction” and “heterologous expression”, the enzyme sequence with E288Q and G294A as forward optimization and the selection result being optimal is constructed based on the recombinant expression plasmid pET 22b(+)|GH29Fuc|Ori, and the optimized recombinant plasmids pET 22b(+)|GH29Fuc|E288Q and pET 22b(+)|GH29Fuc|G294A are constructed by using point mutation method, Figure 3 A is the gel electrophoresis diagram of the four plasmids: Ori represents the original sequence vector, E288Q represents the vector in which glutamic acid at position 288 is mutated to asparagine based on the original sequence, G297A represents the vector in which glycine at position 297 is mutated to alanine based on the original sequence, Figure 5 is the sequencing result diagram of the mutant vectors, (A) is pET 22b(+)|GH29Fuc||E288Q, and (C) is pET 22b(+)|GH29Fuc|G294A, proving that the recombinant plasmid is successfully constructed;
[0050] Codon optimization and addition of 6×His tag, the gene sequence of E288Q after optimization is shown in SEQ ID NO. 4, and the gene sequence of G297A after optimization is shown in SEQ ID NO. 5;
[0051] After the gene synthesis is completed, it is respectively cloned into the pET-22b(+) expression vector Figure 1 C.
[0052] Heterologous expression
[0053] (1) Using E. coli DE3 as host bacteria, the constructed recombinant expression plasmid was transformed, and after successful transformation, the host bacteria grew on an ampicillin-resistant plate, as shown in Figure 2 B, and the final expression vector was determined as pET 22b(+)|GH29Fuc|E288Q, pET22b(+)|GH29Fuc|G294A, respectively, to obtain GH29-Fuc288 fucoidanase mutants and GH29-Fuc294 fucoidanase mutants;
[0054] (2) Under the condition of 16°C, IPTG (600ul / L) was used as an inducer to induce expression for 18h.
[0055] The material obtained after inducing expression for 18h in step (2) was centrifuged and filtered at 5000rpm for 10min, and the precipitate was taken for standby use. The precipitate obtained from the GH29-Fuc288 fucoidanase mutant was noted as precipitate 288, and the precipitate obtained from the GH29-Fuc294 fucoidanase mutant was noted as precipitate 294.
[0056] Example 3
[0057] Purification method
[0058] (1) Ni column affinity chromatography was used for purification, and His tag was used to improve the purity of the protein:
[0059] 1. Cell lysis and preparation of crude enzyme solution
[0060] 1) The standby precipitates 288, 289, 294, and ori obtained in Example 2 were resuspended with non-denaturing lysis solution (PBS solution containing protease inhibitors) respectively;
[0061] 2) Lysis enzyme (such as lysozyme) was added and left for 30 minutes;
[0062] 3) Ice ultrasonic lysis (intermittent treatment, 10s ice water bath ultrasonic treatment under 240W power, interval 10s, 6 rounds in total);
[0063] 4) If the liquid is thick, DNase I (5 μg / ml) can be added for improvement;
[0064] 5) Centrifugation at 4°C to remove insoluble substances, and the clear supernatant was collected for standby use. The supernatant was the crude enzyme solution (the crude enzyme solution can be treated by low-temperature freeze-drying: pre-frozen at −80°C and vacuum freeze-dried at −50°C to obtain freeze-dried powder, as shown in Figure 2 A).
[0065] 2. Ni column binding reaction
[0066] 1) Mix the crude enzyme solution with the nickel ion gel particles; the ratio is 4 ml of lysis solution: 0.5 ml of gel particles;
[0067] 2) Incubate gently on ice for 60 minutes to promote binding;
[0068] 3. Column loading and impurity elution
[0069] 1) Remove unbound substances by light centrifugation or column bottom liquid removal;
[0070] 2) Wash the column with non-denaturing washing solution (containing a small amount of imidazole to remove non-specific proteins); add 0.5-1 ml each time, repeat washing 5-6 times; the washing solution can be detected for protein content by the Bradford method.
[0071] 4. Protein elution
[0072] Elute with high-concentration imidazole (such as a buffer containing 250 mM imidazole); 0.5-1 ml each time, elute 6-10 times, collect the eluate from each tube, and you get the purified enzyme solution, labeled as enzyme solution 288, enzyme solution 289, enzyme solution 294, and enzyme solution ori.
[0073] 5. SDS-PAGE verification
[0074] Figure 3 B is the gel electrophoresis diagram of the crude enzyme solution and the purified GH29 fucanase without IPTG induction and with IPTG induction (the difference before and after induction is in the red box). Figure 6 A, B, and C are the SDS-PAGE gel electrophoresis diagrams of the purified enzyme solutions of GH29-Fuc288 (mutant site 288), GH29-Fuc289 (mutant site 289, mutant of GH29 fucanase of the present application), and GH29-Fuc294 (mutant site 294) fucanase mutants, respectively, from which Figure 6 It can be seen that the three mutants have been expressed and the purification effect is good.
[0075] Enzyme activity determination
[0076] (1) Use sargassum polysaccharide as the substrate and prepare a 10 mg / mL sargassum polysaccharide solution;
[0077] (2) Use an ultrafiltration membrane with a 10 kDa cutoff to separate, and collect the polysaccharide components larger than 10 kDa as the substrate;
[0078] (3) Use the DNS method to determine the enzyme activity, and the specific process is as follows:
[0079] a) Respectively take 0.5 mL purified enzyme solution (enzyme solution 288, enzyme solution 289, enzyme solution 294, enzyme solution ori obtained in Example 3), add 4.5 mL Gracilaria lemaneiformis polysaccharide solution (2 mg / mL), and enzymatically hydrolyze at 25°C for 12 h;
[0080] b) Take the inactivated crude enzyme solution as a control, i.e., inactivate after enzymatic hydrolysis by boiling in a water bath for 10 min;
[0081] c) Centrifuge at 4°C and 8000 rpm for 15 min, and take 2 mL supernatant;
[0082] d) Add 1.5 mL DNS reagent, and after boiling in a water bath for 10 min, make up to 10 mL;
[0083] e) Refer to the instruction manual of the Biyun Tian BCA protein content determination kit to draw protein standard curve and fucose standard curve (as shown in Figure 9 、 Figure 10 ), and use an enzyme marker to measure the absorbance at 540 nm (Table 2). According to the standard curve, the protein content of the enzyme sample is calculated (Table 1), and each sample has two parallel groups (288-1 refers to the first tube of sugar enzymatically hydrolyzed by GH29-Fuc288 mutant, and so on). The three GH29 fucanase mutants are used to enzymatically hydrolyze fucan, and the generation of reducing sugar in the enzymatic hydrolysis process is detected. The sample reducing sugar generation curve is shown in Figure 7 , the final generation of reducing sugar after enzymatic hydrolysis (Table 3, first column), and the enzyme activity is calculated according to the following formula:
[0084] The enzyme activity unit is defined as: the μmol number of reducing sugar generated per hour under the above conditions (U), and the enzyme activity calculation formula is:
[0085]
[0086] Wherein, m is the generation of reducing sugar (Table 3, second column, μg), M is the molar mass of reducing sugar (L-fucose is used as reducing sugar in the standard curve, and the molar mass is 164.16 g / mol), and T is the reaction time (h).
[0087] The total protein quantification kit (BCA method) is used to determine the protein content in the purified enzyme solution (Table 1), and then the specific enzyme activity of the purified enzyme solution is calculated according to the enzyme activity and protein content of the purified enzyme solution. The comparison results of the specific enzyme activities of the four enzymes are shown in Figure 8 .
[0088] The specific enzyme activity unit is defined as: the μmol number of reducing sugar generated per hour by unit mass (mg) of enzyme under the above conditions (U / mg), wherein V is the amount of enzyme added for each sample (all 0.5 mL)
[0089]
[0090] Table 1: Protein content of original enzyme and three GH29 fucanase mutants
[0091] ID Sample Absorbance 1 Absorbance 2 Protein content (mg / ml) 1 288-1 0.341 0.293 0.053183445 2 289-1 0.318 0.291 0.046882089 3 294-1 0.293 0.317 0.047134143 4 288-2 0.344 0.312 0.058728638 5 289-2 0.347 0.319 0.061249181 6 294-2 0.314 0.326 0.054695771 7 ori-1 0.328 0.319 0.05646015 8 ori-2 0.326 0.322 0.056712204
[0092] The absorbance of each sample was measured at 540 nm and brought into Figure 10 the standard curve to calculate the reducing sugar mass of each sample. The reducing sugar production in Table 3 was obtained by subtracting the reducing sugar mass of the NC group from the reducing sugar mass of all samples in Table 2 and then averaging.
[0093] Table 2: Absorbance of samples at 540 nm (NC refers to the inactivated purified enzyme solution group)
[0094] Sample Absorbance 288-1 289-1 294-1 288-2 289-2 294-2 NC 2.5ul system-1 0.417 0.467 0.443 0.442 0.42 0.445 0.323 10ul system-2 0.821 0.794 0.729 0.857 0.799 0.818 0.349
[0095] Table 3: Comparison of enzyme activity of three GH29 fucanase mutants and original enzyme
[0096] Name Reducing sugar production (ug) Enzyme activity (U) Specific enzyme activity (U / mg) GH29-Fuc288 713.66 0.73 26.46 GH29-Fuc289 751.64 0.76 28.26 GH29-Fuc294 730.81 0.59 22.81 GH29-FucOri 721.44 0.73 26.64
[0097] The results of Table 3 and Figure 8 show that the three GH29 fucanase mutants exhibit certain differences in specific enzyme activity compared with the original enzyme. Among them, the specific enzyme activity of GH29-Fuc289 is the highest (28.26 U / mg), which is significantly higher than that of the original enzyme (GH29-FucOri, 26.64 U / mg) and the other two mutants (GH29-Fuc288, 26.46 U / mg; GH29-Fuc294, 22.81 U / mg), indicating that the mutation of Fuc289 realizes the positive optimization of enzyme catalytic efficiency.
[0098] The foregoing description of specific exemplary embodiments of the application is intended to be illustrative only and is not intended to limit the application to the precise forms disclosed. Many modifications and variations are possible in light of the above teachings without departing from the spirit or essential characteristics of the application. The exemplary embodiments were chosen and described in order to explain the principles of the application and its practical application and to allow others skilled in the art to understand the application for various exemplary embodiments with various modifications being suited to the particular use contemplated. The scope of the application is intended to be defined by the claims and their equivalents.
Claims
1. A GH29 fucoidanase mutant, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.
1.
2. The GH29 fucoidanase mutant according to claim 1, characterized in that: The amino acid sequence of the GH29 fucoidan mutant is shown in SEQ ID NO.
2.
3. A recombinant *Escherichia coli* constructed using the GH29 fucoidanase mutant as described in claim 1, characterized in that: The construction method involves cloning the nucleotide sequence gene of SEQ ID NO.1 into an expression vector, and then transforming it into an Escherichia coli strain for induced expression.
4. The recombinant *Escherichia coli* constructed using the GH29 fucoidanase mutant according to claim 3, characterized in that: The induction conditions were 16℃, with IPTG as the inducer for 18 h.
5. The application of the GH29 fucoidanase mutant as described in claim 1 or 2 or the recombinant Escherichia coli as described in claim 3 or 4 in the production of highly active GH29 fucoidanase and the improvement of its heterologous expression level.
6. The application according to claim 5, characterized in that: The GH29 fucoidanase is α-L-fucosidase, specifically α-1,3-fucosidase.