Acid-resistant alpha-L-fucosidase IDSFuc29-2 and application thereof

Abstract

The invention discloses acid-resistant alpha-L-fucosidase IDSFuc29-2 and application thereof, and belongs to the field of protease, the amino acid sequence of the alpha-L-fucosidase IDSFuc29-2 is as shown in SEQ ID No.2, the alpha-L-fucosidase IDSFuc29-2 is derived from a metagenome of bottom mud of the East China Sea, and recombinase with definite catalytic activity can be obtained through a prokaryotic expression system. The enzyme shows good reaction characteristics under the acidic condition, the optimum reaction pH of the enzyme is about 4.0, high relative enzyme activity can still be kept within the pH range of 4.0-6.0, and meanwhile, the enzyme has acceptable stability under the condition of 20-40 DEG C. The application conditions of the GH29 family enzyme in degradation and related application of fucoidin are expanded, and the GH29 family enzyme has important research significance and application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of proteases, and particularly relates to an acid-tolerant α-L-fucosidase IDSFuc29-2 and application thereof. BACKGROUND

[0002] Fucoidan is a kind of structural and functional polysaccharide widely existing in nature, mainly distributed in the cell walls of marine algae such as brown algae, and also exists in some microbial polysaccharides and animal sugar chain structures. Fucoidan usually takes α-L-fucose as the main component unit, and is connected to form a complex structure through different types of glycosidic bonds, and has multiple physiological functions in organisms. In the fields of food processing, biological medicine, functional oligosaccharide preparation and polysaccharide structure analysis, fucoidan and its degradation products have attracted widespread attention due to their potential biological activity. However, due to the complex molecular structure of fucoidan, poor solubility and degradability, the mild and efficient biological degradation process of fucoidan still faces certain challenges in practical application.

[0003] α-L-fucosidase is a kind of glycoside hydrolase (GHs) that can specifically hydrolyze α-L-fucosidic bond, and is an important biological catalyst for realizing selective degradation of fucoidan and release of fucose. In existing research, α-L-fucosidase of GH29 family is widely studied, and has been applied in artificial substrate hydrolysis, polysaccharide structure analysis and the like. However, the optimal reaction conditions of the α-L-fucosidase of GH29 family reported at present are mainly concentrated in the weakly acidic to neutral range, and the common optimal pH value is mainly distributed around pH 5–6, and the reaction capacity and applicability under more acidic conditions are relatively limited, which to some extent limits the application range of the existing α-L-fucosidase.

[0004] CN202110254306.X discloses α-L-fucosidase OUCJdch-16 and application thereof, which discloses an α-L-fucosidase derived from Flavobacterium algicola. In the technology, the optimal reaction conditions of the α-L-fucosidase are mainly concentrated in the low temperature and weakly acidic to near neutral pH range, and the enzymatic property research and application examples mainly revolve around the pH 5–6 condition, and the enzyme activity under more acidic conditions is low.

[0005] CN201911231875.1 discloses an alpha-L-fucosidase and its related biological material and application, which discloses an alpha-L-fucosidase derived from Pedobacter sp., its enzymatic property research and application are mainly concentrated in weakly acidic to near neutral pH conditions, and the reported optimal reaction pH is near pH 5.0. Under more acidic reaction conditions, the catalytic activity of the enzyme is significantly reduced, and the adaptability to the acidic environment in the reaction system is limited, so its application range is still limited in the application scenarios that require enzyme catalytic reaction under acidic conditions.

[0006] Therefore, it is of important research significance and application value to screen and obtain an alpha-L-fucosidase suitable for catalytic reaction under acidic conditions, so as to expand the use conditions of GH29 family enzymes in fucoidan degradation and related applications. SUMMARY

[0007] The technical problem to be solved by the present application is to provide an alpha-L-fucosidase suitable for catalytic reaction under acidic conditions.

[0008] The technical solution of the present application is: the alpha-L-fucosidase IDSFuc29-2, the amino acid sequence of which is shown in SEQ ID No. 2.

[0009] The gene encoding the above-mentioned alpha-L-fucosidase IDSFuc29-2.

[0010] Further, the nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0011] The expression vector containing the above-mentioned gene.

[0012] The genetically engineered bacteria containing the above-mentioned expression vector.

[0013] The above-mentioned gene or expression vector or genetically engineered bacteria for preparing the above-mentioned alpha-L-fucosidase IDSFuc29-2.

[0014] The above-mentioned alpha-L-fucosidase IDSFuc29-2 is used for degrading polysaccharides containing alpha-L-fucosidic bonds.

[0015] Further, the degradation conditions are: temperature 40℃, pH 4.0.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The application provides an alpha-L-fucosidase IDSFuc29-2 derived from a macrogenome of East China sea mud, and the recombinant enzyme with clear catalytic activity can be obtained through a prokaryotic expression system. The enzyme shows good reaction characteristics under acidic conditions, and the optimal reaction pH is about 4.0, and the relative enzyme activity can still be maintained at a high level in the pH range of 4.0-6.0, and the enzyme has acceptable stability under the condition of 20-40 DEG C. In addition, different metal ions have differential effects on the enzyme activity of IDSFuc29-2, indicating that the enzyme has a certain adaptability to common ions in the reaction system. The above characteristics make IDSFuc29-2 provide a new enzymatic selection for the application of GH29 family alpha-L-fucosidase in an acidic reaction system, and has potential value for further research and application. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a SDS-PAGE analysis diagram of the recombinant protein alpha-L-fucosidase IDSFuc29-2.

[0019] Figure 2 It is the effect of temperature on the enzyme activity of the recombinant protein IDSFuc29-2.

[0020] Figure 3 It is the temperature stability analysis result of the recombinant protein IDSFuc29-2.

[0021] Figure 4 It is the effect of pH on the enzyme activity of the recombinant protein IDSFuc29-2.

[0022] Figure 5 It is the pH stability analysis result of the recombinant protein IDSFuc29-2.

[0023] Figure 6 It is the effect of different metal ions on the enzyme activity of the recombinant protein IDSFuc29-2; the final concentration of metal ions added in the reaction system is 1 mM (gray) and 5 mM (black), respectively, and the enzyme activity of the control group (CK) is defined as 100%. The enzyme activity is expressed as relative activity (%), and the data is the average value ± standard deviation (n = 3). The difference between all treatment groups and CK was statistically analyzed by unpaired two-tailed Student's t test. * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and ns represents no significant difference. DETAILED DESCRIPTION

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0025] Preparation of Example 1 α-L-fucosidase IDSFuc29-2

[0026] I. Collection of marine sediment samples in the East China Sea, extraction and sequencing of metagenomic DNA

[0027] 1. Collect marine sediment samples at different water depths at typical stations in the coastal and continental shelf waters of the East China Sea using a sterile sampler. Immediately after collection, store the samples in a low-temperature environment and send them back to the laboratory for use.

[0028] 2. Take an appropriate amount of sediment sample and use a marine sediment-specific DNA extraction kit to extract total DNA from the sample. Detect the integrity of the DNA using 1% agarose gel electrophoresis, and determine its concentration and purity using a UV spectrophotometer.

[0029] 3. Use the qualified total DNA to construct a metagenomic library, follow the standard procedures of the high-throughput sequencing platform to construct the library, and then perform double-end high-throughput metagenomic sequencing to obtain raw sequencing data.

[0030] II. Analysis of metagenomic data and screening of IDSFuc29-2 gene

[0031] 1. Perform quality control on the raw data obtained from metagenomic sequencing, remove adapter sequences and low-quality reads, and obtain high-quality sequencing data.

[0032] 2. Use assembly software to assemble and assemble the cleaned data to obtain metagenomic assembly sequences.

[0033] 3. Perform open reading frame (ORF) prediction on the assembled sequences to obtain a non-redundant gene set.

[0034] 4. Align and annotate the predicted gene sequences with the CAZy database to screen for candidate gene sequences with the characteristic conserved domain of GH29 family α-L-fucosidase.

[0035] 5. Perform conserved motif analysis and functional domain prediction on the amino acid sequences of the candidate genes, select a gene sequence with a complete open reading frame and conserved key catalytic site, and name it IDSFuc29-2. The nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2.

[0036] III. Synthesis of IDSFuc29-2 gene and construction of recombinant plasmid

[0037] 1. According to the screened IDSFuc29-2 gene sequence, design the nucleotide sequence according to the codon bias of the E. coli expression system.

[0038] 2, The optimized IDSFuc29-2 gene fragment is entrusted to a commercial company for full gene synthesis, and the synthesized IDSFuc29-2 gene is directionally cloned into the pET28a expression vector to construct the recombinant plasmid pET28a / IDSFuc29-2 (the amino acid sequence of the expressed recombinant protein is shown in SEQ ID No. 3, which has a multifunctional fusion tag at the N-terminus and a 6×His tag at the C-terminus).

[0039] 3, The constructed recombinant plasmid is sequenced and verified to confirm the correctness of the inserted sequence and the integrity of the reading frame.

[0040] Four, Construction of Recombinant BL21 / pET28a / IDSFuc29-2 and Protein Expression

[0041] 1, Heat shock transformation of recombinant plasmid into E. coli

[0042] (1) Take 1 μl of recombinant plasmid with a concentration of 100 ng / μl and mix carefully with E. coli competent BL21 (DE3) in ice bath for 30 min;

[0043] (2) Place the centrifuge tube in a 42 ℃ water bath for 90 s, immediately take out and ice bath for 2 min, do not shake the centrifuge tube during this process;

[0044] (3) Add 900 μl of 37 ℃ preheated sterile LB medium (without antibiotics) to the centrifuge tube, mix well and incubate at 37 ℃ on a shaker at 200 rpm for 1 h;

[0045] (4) Take 100 μl of the transformed competent cells and spread on LB solid medium containing kanamycin (the amount of spreading can be adjusted according to the actual situation, if the expected number of clones is small, centrifuge at 4000 rpm for 2 min and remove part of the culture liquid). Place the plate at room temperature until the liquid is absorbed, invert the plate and incubate at 37 ℃ for 12-16 h to obtain positive recombinant engineering bacteria BL21 / pET28a / IDSFuc29-2 single colonies

[0046] 2, Induction expression and purification of positive strain

[0047] (1) Induction expression of positive strain

[0048] ① Pick positive recombinant engineering bacteria BL21 / pET28a / IDSFuc29-2 single colonies and place them in 5 mL LB liquid medium containing 50 μg / mL Kan, 37 ℃, 200 rpm for 12-16 h;

[0049] ② Transfer into 500 mL LB medium with 1% inoculation, 37 ℃, 200 rpm shaking culture until OD600 0.6-1.0;

[0050] ③Add final concentration of 0.5 mmol / L IPTG, 16 ℃, 100 rpm induction culture for 16 h;

[0051] ④6000 rpm, 4 ℃ centrifugal 10 min to collect cells, wash cells with 100 mL 1×PBS buffer (137 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na2HPO4, 2 mmol / L KH2PO4, pH 7.4), 6000 rpm centrifugal 10 min. Discard the supernatant, resuspend the bacteria in 50 mL 1×PBS buffer;

[0052] ⑤Flow rate 6 L / h, pressure 810 MPa, 6 ℃, broken in FB-2010 homogenizer (Shanghai Liti Company) for 5 min;

[0053] ⑥10000 rpm, 4 ℃ centrifugal 15 min, collect the supernatant as crude enzyme solution of recombinant protein.

[0054] (2) Affinity purification of recombinant protein and electrophoresis analysis

[0055] ①Column packing: 2 mL Ni-NTA agarose filler is added to DE01 column;

[0056] ②Equilibrium: 20 mmol / L imidazole phosphate buffer is used to equilibrate the column at a flow rate of 0.5 mL / min for 1 h;

[0057] ③Sample loading: 75 mL protein supernatant is loaded at a flow rate of 0.5 mL / min;

[0058] ④Re-equilibrium: 20 mmol / L imidazole phosphate buffer is used to equilibrate the column at a flow rate of 0.5 mL / min for 1 h;

[0059] ⑤Elution: 20 mmol / L (A liquid) and 2 mol / L (B liquid) mixed phosphate buffer gradient elution, set the final proportion of B liquid to 13%, collect the eluate;

[0060] ⑥Washing: 20 mmol / L imidazole phosphate buffer is used to wash the column at a flow rate of 0.5 mL / min for 1 h;

[0061] ⑦The purified protein is used for SDS-PAGE electrophoresis (4% concentrated gel, 15% separation gel) analysis, and the target protein with an expected size of 54.4 kDa is obtained Figure 1 ).

[0062] Example 2 Enzymatic property analysis of α-L-fucosidase IDSFuc29-2

[0063] The enzyme activity of α-L-fucosidase IDSFuc29-2 was determined using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as substrate. The amount of enzyme required to release 1 μmol of p-nitrophenol (pNP) per min was defined as 1 enzyme activity unit (U), and the enzyme activity of the purified IDSFuc29-2 was determined to be 3.66 U / mg. The protein concentration was determined using the Bradford method.

[0064] (1) Optimum temperature

[0065] 30 μL of purified IDSFuc29-2 enzyme solution (about 3 μg, same below) was mixed with 20 μL of 10 mM pNP-Fuc substrate and incubated at 20, 30, 40, 50, 60, 70, and 80 °C, respectively, for 15 min. A blank control group was also set up, in which boiled inactivated enzyme solution was added. After the reaction, 100 μL of 1 M Na2CO3 was immediately added to terminate the reaction, and the mixture was incubated at room temperature for 5 min in the dark, after which 100 μL was taken and placed in a 96-well plate, and the absorbance was measured at 405 nm using an enzyme marker. The relative enzyme activity at different temperatures was calculated based on the temperature corresponding to the highest enzyme activity as 100%, and three replicates were set up for the experiment.

[0066] The results showed that the optimum reaction temperature of recombinant enzyme IDSFuc29-2 was about 40 °C. Figure 2

[0067] (2) Thermal stability

[0068] 30 μL of purified IDSFuc29-2 enzyme solution was incubated at 20, 30, 40, 50, and 60 °C, respectively, and sampled at 0, 5, 10, 30, and 60 min. At each time point, 30 μL of enzyme solution was taken and added with 20 μL of 10 mM pNP-Fuc substrate, and the reaction was carried out under the optimum reaction conditions for 15 min. After the reaction, 100 μL of 1 M Na2CO3 was immediately added to terminate the reaction, and the mixture was incubated at room temperature for 5 min in the dark, after which 100 μL was taken and placed in a 96-well plate, and the absorbance was measured at 405 nm using an enzyme marker. The residual enzyme activity of IDSFuc29-2 under different temperature and treatment time conditions was calculated based on the enzyme activity of the untreated enzyme solution as 100%, and three replicates were set up for the experiment.

[0069] The results showed that the optimum reaction temperature of recombinant enzyme IDSFuc29-2 was about 40 °C. Figure 3 ​), the residual activity of recombinant enzyme IDSFuc29-2 was not affected after incubation at 20, 30 ℃ for 60 min, and was rapidly inactivated after incubation at 50 ℃ for 5 min.

[0070] (3) Determination of optimal reaction pH

[0071] 30 μL of purified IDSFuc29-2 enzyme solution was mixed with 20 μL of 10 mM pNP-Fuc substrate prepared with different pH buffers (sodium bicarbonate / citric acid buffer pH 3.0-8.0; Tris-HCl buffer pH 8.0-9.0; glycine-sodium hydroxide buffer pH 9.0-12.0), and reacted for 15 min under optimal temperature conditions. The reaction was terminated and detected as described above. The relative enzyme activity under each pH condition was calculated based on the pH corresponding to the highest enzyme activity as 100%, and three sets of repeats were set for the experiment.

[0072] The results show that (Fig. 4) Figure 4 The optimal reaction pH of recombinant enzyme IDSFuc29-2 is about 4.0.

[0073] (4) pH stability analysis

[0074] 30 μL of purified IDSFuc29-2 enzyme solution was mixed with 10 μL of different pH buffers (pH 3.0-10.0) and incubated at 4 ℃ for 30 min. Then 10 μL of 20 mM pNP-Fuc substrate was added and reacted for 15 min under optimal reaction conditions, and the enzyme activity was determined as described above. The residual enzyme activity under different pH conditions was calculated based on the enzyme activity of the enzyme solution without pH treatment as 100%, and three repeats were set for each experiment.

[0075] The results show that (Fig. 5) Figure 5 Recombinant enzyme IDSFuc29-2 has good stability in the pH range of 4.0-6.0.

[0076] (6) Effect of metal ions on enzyme activity

[0077] 30 μL of purified IDSFuc29-2 enzyme solution was mixed with 10 μL of metal ion solution (Na⁺, Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺, Zn²⁺, etc.) with a final concentration of 1 mM and 5 mM, respectively, and incubated at 4 ℃ for 30 min. After incubation, 10 μL of 20 mM pNP-Fuc substrate was added and reacted for 15 min under optimal conditions, and the enzyme activity was determined. The relative enzyme activity under each metal ion condition was calculated based on the reaction system without metal ions as 100%, and three repeats were set for each experiment.

[0078] The results show that (Fig. 6)Figure 6 ) 1 mM / 5 mM of Mn2+and Na+and 1 mM of Fe 3 The rest of metal ions have different degrees of inhibition on the enzyme activity.

Claims

1. α-L-fucosidase IDSFuc29-2, the amino acid sequence of which is shown in SEQ ID No.

2.

2. The gene encoding the α-L-fucosidase IDSFuc29-2 as described in claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. An expression vector containing the gene of claim 2 or 3.

5. Genetically engineered bacteria containing the expression vector of claim 4.

6. The use of the gene according to any one of claims 2-3, the expression vector according to claim 4, or the genetically engineered bacteria according to claim 5 in the preparation of the α-L-fucosidase IDSFuc29-2 according to claim 1.

7. The application of the α-L-fucosidase IDSFuc29-2 according to claim 1 in the degradation of polysaccharides containing α-L-fucosidic bonds.

8. The application according to claim 7, characterized in that, The degradation conditions were: temperature 40 ℃, pH 4.0.

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