α-L-fucosidase mutant and its application

Abstract

The present invention relates to the technical field of functional enzymes, and in particular to an α-L-fucosidase mutant and an application thereof. The amino acid sequence of the mutant has at least 80% homology to the amino acid sequence of fucosidase SEQ ID NO: 1; the mutant has at least one characteristic sequence of the fucosidase, and the amino acid sequence of the fucosidase is modified, substituted, deleted or added with one or more amino acids to obtain the amino acid sequence of the mutant; the mutant has an amino acid sequence encoded by a nucleotide sequence shown in SEQ ID NO: 2 or its complementary sequence, or a sequence that is different from the nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 2 or its complementary sequence due to the degeneracy of the genetic code. The mutant provided by the present invention is beneficial to reducing the cost and reaction time in the catalytic reaction of fucosylated compound synthesis.

Description

Technical Field

[0001] The present invention relates to the technical field of functional enzymes, in particular to an alpha-L-fucosidase mutant and applications thereof. Background Art

[0002] Fucosidases (FUCs) are a group of glycoside hydrolases that specifically catalyze the removal of non-reducing terminal L-fucose. To date, α-L-fucosidases from various sources have been recombinantly purified and functionally characterized. These enzymes are divided into two major groups based on substrate type: the first group is capable of hydrolyzing various types of fucosidic bonds as well as synthetic substrates (EC 3.2.1.51), while the second group is active only at α-(1-2) linkages (EC 3.2.1.63). α-L-fucosidases (EC 3.2.1.51), as glycoside hydrolases, primarily catalyze the hydrolysis of fucosides and / or the transfer of fucosyl residues. These enzymes have been characterized and modified to form trans-fucosylation patterns or further engineered as "fucosidases," enabling the enzymatic synthesis of bioactive glycans, including fucosylated human milk oligosaccharides (HMOs). Over half of human milk oligosaccharides (HMOs) are fucosylated, and they have attracted increasing attention due to their excellent physiological functions for breastfed infants. 2'-fucosyllactose (2'-FL), a common human milk oligosaccharide (HMO), has important biological functions in infant nutrition and adult health, including improving intestinal health, regulating the immune system, promoting cognitive development, protecting against pathogen infection, and improving metabolic function.

[0003] Fucosidases of various types and sources can be used to efficiently synthesize a wide range of commercially valuable fucosylated compounds. Fucosidases also show potential for the synthesis of valuable oligosaccharides, including fucosylated HMOs and antibody glycans, via transfucosylation. Summary of the Invention

[0004] In order to solve the problems mentioned in the background technology, the object of the present invention is to provide an α-L-fucosidase mutant.

[0005] The α-L-fucosidase mutant is any one of the amino acid sequences shown in 1), 2) or 3) below:

[0006] 1) The amino acid sequence of the mutant has at least 80%, at least 85%, at least 90% or at least 95% homology to the amino acid sequence of fucosidase as shown in SEQ ID NO: 1;

[0007] 2) having at least one characteristic sequence of the fucosidase, and the amino acid sequence of the fucosidase is modified, substituted, deleted or added with one or more amino acids to obtain the amino acid sequence of the mutant;

[0008] 3) The mutant has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2 or its complementary sequence, or a sequence that is different from the nucleotide sequence shown in SEQ ID NO: 2 or its complementary sequence due to the degeneracy of the genetic code.

[0009] Furthermore, the modification includes amidation, phosphorylation, methylation, acetylation, ubiquitination, glycosylation or carbonylation.

[0010] Furthermore, the substitution is the substitution of one or more amino acids at positions 4, 5, 237, 298, 308 and 313 of the fucosidase with the amino acid sequence of SEQ ID NO: 1.

[0011] Furthermore, the substitution is that the 4th amino acid of the fucosidase with the amino acid sequence of SEQ ID NO: 1 is changed from P to A and / or the 5th amino acid is changed from S to P and / or the 237th amino acid is changed from A to E and / or the 298th amino acid is changed from A to T and / or the 308th amino acid is changed from I to L and / or the 313th amino acid is changed from E to D.

[0012] Furthermore, the amino acid sequence of the mutant is shown in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9.

[0013] The nucleotide sequence encoding the mutant is also within the protection scope of the present invention, and the nucleotide sequence is shown in SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10.

[0014] The expression vector comprising the nucleotide sequence of the mutant is also within the protection scope of the present invention.

[0015] The host cells obtained by the expression vector are also within the protection scope of the present invention.

[0016] The use of the mutant, the expression vector or the host cell in the catalytic reaction of fucosylated compound synthesis also falls within the protection scope of the present invention.

[0017] The present invention has the following beneficial effects: Based on fucosidase AFUC01, the present invention provides a fucosidase mutant AFUC02 containing a double-site mutation of P4A+S5P, a fucosidase mutant AFUC03 containing a multiple-site mutation of P4A+S5P+A237E+A298T, a fucosidase mutant AFUC04 containing a multiple-site mutation of P4A+S5P+A237E+A298T+I308L, and a fucosidase mutant AFUC05 containing a multiple-site mutation of P4A+S5P+A237E+A298T+I308L+E313D. Compared with fucosidase AFUC01, the reaction rates are increased by 14%, 16%, 30%, and 33%, respectively, thereby facilitating the reduction of cost and reaction time in the catalytic reaction for the synthesis of fucosylated compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison table of the time required for the enzymatic reaction of different concentrations of Fuc01 and mutants to exhaust the substrate in Example 6 of the invention. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present invention.

[0020] The present invention utilizes conventional techniques and methods in the field of molecular biology. For the main techniques, reference may be made to the methods described in MOLEC ULARCLONING: A LABORATORY MANUAL, 3rd Ed. (Sambrook, 2001). These general references provide general definitions and methods known to those skilled in the art. However, those skilled in the art may employ other conventional methods, experimental protocols, and reagents in the art based on the technical solutions described in the present invention, without being limited to the specific examples of the present invention. For example, the present invention may utilize the following experimental materials and reagents:

[0021] Strains and vectors: E. coli TOP10, E. coli BL21 (DE3), and vector PET22b were purchased from Novagen. Enzymes and kits: PCR enzyme and ligase were from our own kits, and plasmid extraction kits and gel purification and recovery kits were purchased from Omega.

[0022] Example 1: Construction of AFUC01 strain

[0023] The target gene fragment AFU01 was obtained by gene synthesis from Xanthomonas albilineans (strain GPE PC73 / CFBP7063). The synthetic sequence included a 6×His purification tag and a TAA stop codon at the 3' end. Both the synthetic fragment and the pET-28a vector were double-digested with restriction endonucleases Xho I and Bam HI. The digestion system (20 μL) consisted of: 10 μL of the target gene fragment or plasmid, 2 μL of 10× Buffer, 1 μL of Bam HI, 1 μL of Xho I, and ddH O to 20 μL. Digestion was performed at 37°C for 1 hour. The digest was then analyzed by agarose gel electrophoresis. DNA was then recovered from the gel. After recovering the target gene fragment, ligation was performed using T4 DNA ligase at a molar ratio of vector to target gene fragment of 1:10. The ligation system (25 μL) consisted of 2.5 μL of 10× T4 DNA Ligase Buffer, 10 μL of DNA fragment, 4 μL of vector DNA, 1 μL of T4 DNA Ligase, and dH2O to a final volume of 25 μL. Ligation was performed overnight at 18°C. The constructed recombinant expression vector pET-28a(+)-AFU01 was transformed into E. coli TOP10 competent cells, plated on antibiotic-supplemented LB agar plates, and cultured overnight at 37°C. Three positive transformants were extracted and identified by double enzyme digestion. Positive transformants identified by double enzyme digestion were sent to Shanghai Jierui Biotechnology Co., Ltd. for sequencing.

[0024] A single positive colony that has been sequenced correctly was inoculated into competent E. coli BL21(DE3) cells, plated onto LA agar plates containing kanamycin, and cultured overnight at 37°C. White colonies were selected and cultured overnight at 37°C, 200 rpm. Under sterile conditions, the bacterial suspension was mixed with 50% glycerol at a 1:1 ratio to a final glycerol concentration of 25%.

[0025] Example 2: Construction of AFUC01 mutant expression strain

[0026] The gene sequences of the AFUO1 parent were all derived from AFU01, see SEQ ID NO. 1, obtained by gene synthesis, and cloned into the vector pET-28a to generate the pET-28a(+)-AFU01 plasmid.

[0027] Construction of AFU01 mutants Using plasmid pET-28a(+)-AFU01 as the original template, the AFU01 coding gene was subjected to one or more rounds of site-directed mutagenesis according to the PCR-mediated site-directed mutagenesis method. Mutation primers were designed using the QuikChange Primer Design website, and the AFU01 coding gene was subjected to site-directed mutagenesis PCR reaction system (50 μL) according to the PCR-mediated site-directed mutagenesis method. Specifically, the components in Table 1 were mixed on ice and then PCR amplified according to the following procedure: (1) pre-denaturation at 98°C for 3 minutes; (2) denaturation at 98°C for 20 seconds, annealing at 60°C for 30 seconds, and extension at 68°C for 6 minutes; (3) step 2 was performed for 30 cycles; (4) extension at 68°C for 10 minutes; 5) incubation at 4°C. After the reaction according to this procedure, a mixture of amplified product and template DNA was obtained. According to the instructions for use of DpnⅠ digestion enzyme, add 1μL DpnⅠ and 9μL corresponding buffer to 10μL of PCR amplification product, and incubate at 37℃ overnight to completely digest the template DNA molecules.

[0028] Table 1 Composition of site-directed mutagenesis PCR reaction system

[0029] Serial number Element Volume (μL) 1 5*buffer 5 2 DMSO 5 3 magnesium ions 3 4 A pair of forward and reverse primers 2 each (10mM) 5 pET-28a(+)-AFU01 1(20ng / μL) 6 KOD polymerase 1 7 water 31

[0030] The overnight digested product was initially purified using a DNA purification and recovery kit; refer to the manufacturer's instructions for specific steps. The purified PCR product was transformed into E. coli TOP10 competent cells and evenly plated on LB plates containing 50 μg / mL kanamycin. The plates were incubated upright until the liquid was completely absorbed, then inverted and incubated overnight at 37°C.

[0031] Six to ten single colonies were selected from each LB plate and inoculated into 50 μg / mL kanamycin-containing medium for overnight culture. Plasmids were extracted using Plasmid DNA Midiprep Kits. Plasmids were sequenced (the amino acid sequence of mutant FUC02 was shown as SEQ ID NO. 3, the amino acid sequence of mutant FUC03 was shown as SEQ ID NO. 5, the amino acid sequence of mutant FUC04 was shown as SEQ ID NO. 7), and the amino acid sequence of mutant FUC05 was shown as SEQ ID NO. 9). Sequencing results were aligned using SnapGene software. Correctly sequenced plasmids were transformed into E. coli BL21 (DE3) competent cells to obtain recombinant bacteria containing the target mutant gene.

[0032] A single positive colony that has been sequenced correctly was inoculated into competent E. coli BL21(DE3) cells, plated onto LA agar plates containing kanamycin, and cultured overnight at 37°C. White colonies were selected and cultured overnight at 37°C, 200 rpm. Under sterile conditions, the bacterial suspension was mixed with 50% glycerol at a 1:1 ratio to a final glycerol concentration of 25%.

[0033] Example 3: Recombinant expression of AUC01 and its mutant proteins:

[0034] Streak the frozen bacterial liquid corresponding to the strain to be expressed on an LB plate to activate the bacteria to ensure growth during the fermentation process.

[0035] A single colony of the recombinant expression bacteria BL21 (DE3) / Pet28a-target gene was inoculated into a centrifuge tube containing 10 mL of LB liquid medium and cultured in a shaking incubator at 37° C. and 200 rpm for 12-14 hours.

[0036] The bacterial solution was transferred to a flask containing 100 mL of LB liquid medium at a 1:100 inoculation ratio and cultured in a shaking incubator at 37°C and 200 rpm for 2 h until the OD600 reached approximately 0.6-0.8.

[0037] 1 M IPTG was added to control the final concentration of IPTG to 1 mM, and cultured at 30°C and 200 rpm for 3 h.

[0038] Example 4: Bacteria disruption and protein purification:

[0039] Pour the fermentation broth into a 50ml centrifuge tube, centrifuge at 8000rpm for 15 minutes and discard the supernatant. The tube can be inverted on absorbent paper. Then, resuspend the cells in distilled water and centrifuge at 8000rpm for 15 minutes and discard the supernatant. After washing twice, add Buffer A (20mM HEPES, 500mM NaCl, pH7.4, 5mM imidazole) to resuspend the cells and pipette evenly.

[0040] During the ultrasonic cell disruption process, the bacteria should be placed on ice. The ultrasonic cell disruptor conditions are set as follows: 6mm amplitude, 60min, 50mL bacterial suspension and ensure that the probe is close to but not touching the bottom.

[0041] Place the cell suspension in a pre-cooled centrifuge and centrifuge at 10,000 rpm at 4°C for 30 minutes. The supernatant should be stored at 4°C. The crude enzyme solution obtained from the centrifugation may still contain some impurities and should usually be placed in a pre-cooled centrifuge again and centrifuged at 10,000 rpm at 4°C for 60 minutes before loading onto the Ni column. For Ni column affinity chromatography, pre-soak the filter disc in 1M NaOH, sonicate for 5 hours, and then resuspend in 20% ethanol. The solution used during the loading process must be filtered, and the lid should be sealed with aluminum foil after opening. Gradient elution should be used for the first loading. Elute impurities from the Ni column using varying concentrations of Buffer B (20mM HEPES, 500mM NaCl, pH 7.4, 25-300mM imidazole). Collect samples based on UV peak patterns.

[0042] Example 5: Determination of protein concentration

[0043] The Bradford protein quantification kit was used for determination. The specific method is as follows:

[0044] (1) Take the protein standard BSA and dilute it with distilled water to a final concentration of 0.5 mg / mL.

[0045] (2) Add 2000, 1600, 1200, 800, 400, and 0 μL to the centrifuge tubes respectively. Add distilled water to make all the centrifuge tubes reach 2000 μL. Add 2000 μL of Bradford staining solution to each centrifuge tube and mix well.

[0046] (3) Take 200 μL of the mixed solution and add it to the ELISA plate, set up three parallel plates, and place it at room temperature for 3-5 minutes.

[0047] (4) Turn on the microplate reader to measure the A595 value of the standard and obtain the standard curve.

[0048] (5) Take a new centrifuge tube, add 2000 μL of sample, and then add 2000 μL of Bradford solution and mix well.

[0049] (6) Take 200 μL of the mixture and add it to the ELISA plate, set up three parallel plates, and let it stand at room temperature for 3-5 minutes.

[0050] (7) Turn on the microplate reader to measure the A595 of the sample and calculate the protein concentration in the sample based on the standard curve.

[0051] Example 6: Activity Assay

[0052] The enzyme kinetic curves of AFUO1 and mutants at different concentrations in the C1 buffer system were tested, and the time taken to reach the first-order reaction phase (substrate exhaustion) was compared.

[0053] The test system is shown in the following table:

[0054] Activity testing process:

[0055] 1. Main equipment and materials: microplate reader, 96-well plate

[0056] 2. Experimental Procedure

[0057] 1) Dilute FUC01 and its mutants to 5.6 mg / ml, 3.7 mg / ml, 2.5 mg / ml, and 1.7 mg / ml, respectively, using C1 Buffer (50 mM Hepes, 150 mM NaCl, pH 7.4, 5% glycerol).

[0058] 2) Prepare sufficient reaction solution according to the following table:

[0059] Reaction solution components and added concentration Addition amount μL Potassium phosphate buffer (pH 6.5, 200 mM) 50 FucDH (0.5 mg / ml) 5 <![CDATA[NADP + (15mM)]]> 5

[0060] 3) Use a pipette to add 10 μL of substrate 2'-FL (4 mM), 60 μL of enzyme reaction solution, and 30 μL of H1 homlog to the 96-well plate in sequence.

[0061] 4) Set the microplate reader parameters: kinetic mode, 340 nm, oscillation enabled, reaction time 1 hour, and assay interval 30 seconds.

[0062] The time required for the enzymatic reaction of different concentrations of Fuc01 and mutants to exhaust the substrate is as follows Figure 1 As shown, it can be seen that the reaction time of each mutant is significantly reduced compared with Fuc01.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An α-L-fucosidase mutant, characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO:

9.

2. A nucleic acid molecule encoding the mutant according to claim 1, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO:

10.

3. An expression vector, characterized in that Comprising any nucleotide sequence according to claim 2.

4. A host cell, characterized in that The method is obtained by transferring the expression vector according to claim 3 into cells.

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