Alpha-L-fucosidase mutant and application thereof

Abstract

The invention discloses an alpha-L-fucosidase mutant and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the alpha-L-fucosidase mutant and the application thereof disclosed by the invention, based on sequence analysis, a geobacterium alpha-L-fucosidase female parent gene is subjected to rational design, primers are designed, and ten mutants are obtained by adopting a site-directed mutagenesis PCR (Polymerase Chain Reaction) technology; the method comprises the following steps of: performing random mutation on a female parent gene by using error-prone PCR (Polymerase Chain Reaction), constructing a random mutation library, performing high-throughput screening on the mutation library, screening six mutation sites capable of improving the heat resistance of alpha-L-fucosidase through two schemes, and integrating the six mutation sites onto a coding sequence through a complete sequence synthesis method, thereby obtaining the alpha-L-fucosidase. And obtaining the combined mutant. The thermal stability of the combined mutant is obviously improved compared with that of a female parent, and the combined mutant shows potential application value in industries such as biological energy, feed, health food and medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and enzyme engineering, and more particularly to an alpha-L-fucosidase mutant and application thereof. Background Art

[0002] α-L-fucosidase is a neutral exoglycoside hydrolase that selectively hydrolyzes or catalyzes transglycosidic reactions to synthesize fucosidic bonds. 2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide in breast milk and has multiple functional activities, including regulating the intestinal microbiome, preventing pathogen adhesion, immunomodulating, and promoting nervous system development and repair. Geobacter α-L-fucosidase (EC 3.2.1.51) not only catalyzes the release of L-fucose residues from oligosaccharides and glycoconjugates but also exhibits transglycosylation activity, catalyzing the synthesis of 2'-FL from 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) and D-lactose. Thermal stability is a crucial parameter in the industrial application of enzymes. Highly stable enzymes reacting at high temperatures can increase reaction rate and reactant solubility, reduce enzyme input, and thus reduce costs. Directed protein evolution is a core modification strategy in enzyme engineering. It achieves targeted optimization of enzyme molecular function by simulating natural evolutionary mechanisms. This "gene randomization-phenotypic directed selection" model transcends the traditional rational design's reliance on structural information and exhibits unique advantages in industrial enzyme modification. Furthermore, rational design, as an important method in enzyme engineering, offers significant advantages over traditional directed evolution strategies, such as smaller mutation libraries and greater targeted precision, and is therefore widely used in enzyme modification. Natural α-L-fucosidases generally suffer from poor thermal stability, which manifests itself in industrial production as decreased catalytic efficiency, shortened half-life, and low reuse rates at high temperatures. Applying existing technologies to further modify α-L-fucosidase and improve its thermal stability will promote its wider application in related fields.

[0003] Therefore, providing an α-L-fucosidase mutant and its application is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides an α-L-fucosidase mutant and applications thereof.

[0005] The present invention utilizes rational design and random mutation to perform molecular improvement on α-L-fucosidase derived from Pedobacter sp. CAU209, thereby obtaining an α-L-fucosidase mutant with improved thermal stability.

[0006] Based on the previously obtained α-L-fucosidase coding sequence, a rational design approach was used to successfully screen for a mutant site with significantly improved thermostability. Simultaneously, a round of error-prone PCR was used to randomly mutagenize the parent gene to establish a mutant library. Using a high-throughput screening system, five α-L-fucosidase mutant sites with improved thermostability were identified. These six mutant sites were then integrated into a single coding sequence through full-sequence synthesis, resulting in the combined mutant M6, which exhibits significantly improved thermostability.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] Rationally Designed Site-Directed and Random Mutation Library Construction and Screening: Previously, an α-L-fucosidase gene (gene accession number: ON152707.1) was cloned from Pedobacter sp. CAU209 and named PbFuc (see SEQ ID NO. 1). A recombinant plasmid, pET28a-PbFuc, was constructed and successfully expressed in E. coli BL21, yielding a molecular weight of approximately 55 kDa. Wild-type PbFuc served as the parental vector for the following genetic manipulations.

[0009] The amino acid sequence of Geobacter α-L-fucosidase was submitted as a FASATA file to the website http: / / kazlab.umn.edu / for potential mutation prediction. Site-directed mutagenesis was performed on sites where at least 70% of similar proteins shared the same amino acid. Primers corresponding to the codons of the 10 selected amino acid mutation sites were designed based on the codon preference of Escherichia coli and synthesized by Sangon Biotech (Shanghai). The entire plasmid was amplified using the pET28a-PbFuc plasmid as a template, and the PCR product was verified by agarose gel electrophoresis. The parent plasmid was eliminated by the addition of DpnI endonuclease, and 10 μL of the product was added to E. coli DH5α competent cells for transformation. Several single colonies were selected for each mutation site and cultured. The overnight culture was then sent to Sangon Biotech (Shanghai) for sequencing. Plasmids from correctly sequenced transformants were extracted using a plasmid extraction kit and transformed into E. coli BL21. After induction of expression, the cells were harvested, disrupted, and the supernatant was collected. The thermostability of the mutant strain was determined using the residual enzyme activity of the wild-type enzyme as a control. A thermostable α-L-fucosidase mutant was obtained and named PbFuc-T71H.

[0010] The gene encoding the parental PbFuc was randomly mutated using error-prone PCR. A mutant library was constructed using pET28a. Recombinant plasmids containing the mutant genes were then transformed into E. coli BL21. Single mutant clones and a control strain from the parental strain were cultured in 96-well plates and induced to express α-L-fucosidase. The cells were centrifuged and resuspended in acetic acid-sodium acetate buffer. After incubation at an appropriate temperature for a specified time, the reaction was initiated by adding pNP-Fuc solution. After completion, the reaction was terminated by adding sodium carbonate solution. Strains with residual activity higher than the control strain were transferred to new 96-well plates for repeated screening. Recombinant strains with improved heat tolerance were cultured, their plasmids extracted, and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. Through the construction and screening of the random mutation library, five α-L-fucosidase mutants with different degrees of improved heat resistance caused by mutations in single amino acid residues were obtained and named as PbFuc-G53C, PbFuc-M54I, PbFuc-N59S, PbFuc-R125C, and PbFuc-S199P.

[0011] Integration of six mutant sites: Six mutation sites were integrated through full-sequence gene synthesis. The synthesized gene fragment was ligated into the pET28a vector and expressed in E. coli BL21 (combined mutant α-L-fucosidase, designated M6, amino acid sequence see SEQ ID NO. 9). Comparison of the heat resistance of the purified parent and M6 mutants revealed that the heat tolerance of the mutant M6, which incorporates all six mutation sites, was significantly improved compared to the parent. M6 retained approximately 40% of its enzyme activity after treatment at 60°C for 30 minutes, whereas wild-type Geobacter α-L-fucosidase lost its activity after treatment at 45°C for 30 minutes. The optimal reaction temperature of the Geobacter α-L-fucosidase mutant M6 was 5°C higher than that of the wild-type Geobacter α-L-fucosidase. The improved heat resistance of the α-L-fucosidase mutant demonstrates the potential application of this enzyme in industries such as bioenergy, feed, health foods, and pharmaceuticals.

[0012] An α-L-fucosidase mutant, based on the α-L-fucosidase amino acid sequence shown in SEQ ID NO. 2, having one of the following amino acid sequences after mutation:

[0013] (1) The glycine at position 53 was mutated to cysteine, the methionine at position 54 was mutated to isoleucine, the asparagine at position 59 was mutated to serine, the threonine at position 71 was mutated to histidine, the arginine at position 125 was mutated to cysteine, and the serine at position 199 was mutated to proline;

[0014] (2) mutating glycine at position 53 to cysteine;

[0015] (3) mutating the methionine at position 54 to isoleucine;

[0016] (4) mutating the asparagine at position 59 to serine;

[0017] (5) mutating the threonine at position 71 to histidine;

[0018] (6) mutating the arginine at position 125 to cysteine;

[0019] (7) The serine at position 199 was mutated to proline.

[0020] Furthermore, the α-L-fucosidase mutant is used to improve thermal stability.

[0021] Furthermore, the α-L-fucosidase mutant is used to increase the yield of 2'-fucosyllactose.

[0022] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a mutant of α-L-fucosidase and its application. Based on sequence analysis, the parent gene of Geobacter α-L-fucosidase was rationally designed and primers were designed. Ten mutants were obtained using site-directed mutagenesis PCR technology. At the same time, the parent gene was randomly mutated using error-prone PCR, a random mutation library was constructed, and the mutation library was subjected to high-throughput screening. Through the two schemes, a total of 6 mutation sites that improve the heat resistance of α-L-fucosidase were screened. These 6 mutation sites were integrated into a coding sequence through a full sequence synthesis method to obtain a combined mutant. This combined mutant has significantly improved thermal stability compared to the parent gene, showing potential application value in industries such as bioenergy, feed, health food, and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0024] Figure 1 is the residual enzyme activity of the site-directed mutant of the present invention;

[0025] Figure 2 is the residual enzyme activity of the Geobacter α-L-fucosidase and its mutants of the present invention;

[0026] Figure 3The enzymatic properties of the wild-type PbFuc of the present invention; wherein, a: optimal reaction temperature; b: temperature stability; c: optimal reaction pH; d: pH stability;

[0027] Figure 4 The enzymatic properties of the mutant M6 of the present invention; wherein, a: optimal reaction temperature; b: temperature stability; c: optimal reaction pH; d: pH stability;

[0028] Figure 5 : The thermal inactivation curves of the wild-type PbFuc and the mutant M6 of the present invention; wherein, a: the thermal inactivation curve of the wild-type PbFuc; b: the thermal inactivation curve of the mutant M6;

[0029] Figure 6 The TLC diagram of the wild-type PbFuc and mutant M6 transglycosylation products of the present invention; wherein, M: standard mixture; 1: wild-type transglycosylation product; 2: mutant M6 transglycosylation product;

[0030] Figure 7 HPLC chart of the wild-type PbFuc transglycosylation product of the present invention; wherein, a: mixture of pNP-Fuc and D-lactose standard; b: L-fucose standard; c: 2'-FL standard; d: wild-type PbFuc transglycosylation product;

[0031] Figure 8 HPLC chart of the transglycosylation product of mutant M6 of the present invention; wherein, a: mixture of pNP-Fuc and D-lactose standard; b: L-fucose standard; c: 2'-FL standard; d: transglycosylation product of mutant M6. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] Example 1 Expression and purification of α-L-fucosidase

[0034] The Geobacter α-L-fucosidase gene (gene accession number: ON152707.1) was codon-optimized according to the codon preference of Escherichia coli and synthesized, named PbFuc (as shown in SEQ ID NO.1). It was successfully ligated with the pET28a plasmid with restriction sites of BamHI and NotⅠ at both ends to obtain the recombinant plasmid pET28a-PbFuc.

[0035] Wild-type α-L-fucosidase PbFuc gene sequence:

[0036]

[0037] Transform the recombinant plasmid pET28a-PbFuc into E.coli DH5α competent cells, pick the transformants and place them in liquid LB medium containing 50μg / ml kanamycin, and culture them at 37℃, 200rpm overnight. Extract the plasmid using a plasmid extraction kit and transform it into E.coli BL21 competent cells. Pick the E.coli BL21 transformants and place them in 20ml LB medium containing 50μg / ml kanamycin and culture them at 37℃, 200rpm overnight to prepare seed solution. Pipette 1mL of seed solution into 100ml LB medium containing 50μg / ml kanamycin and culture at 37℃, 200rpm to obtain the OD value. 600 To 0.7, 1 mM IPTG inducer was added, and then the culture was induced at 16 ° C and 200 rpm for 16 h. The bacteria were collected by centrifugation at 4000 rpm, and the bacteria were resuspended in 10 mL of binding buffer (50 mM pH 8.0 potassium phosphate buffer, 300 mM sodium chloride, 10 mM imidazole). The bacterial suspension was broken by ultrasonic disruptor and the supernatant was collected by centrifugation at 12000 rpm as crude enzyme solution.

[0038] The crude enzyme solution was mixed with nickel ion affinity chromatography resin and shaken on ice for 60 minutes to allow the target protein to fully bind to the nickel ion affinity chromatography resin. The mixture was poured into a chromatography column and washed with rinsing buffer (50mM pH 8.0 potassium phosphate buffer, 300mM sodium chloride, 20mM imidazole) to remove contaminants. After the contaminants were completely eluted, the target protein was eluted with elution buffer (50mM pH 8.0 potassium phosphate buffer, 300mM sodium chloride, 250mM imidazole) and collected. The purification of the target protein was observed by polyacrylamide gel electrophoresis (SDS-PAGE).

[0039] Example 2 Rational design of site-directed mutagenesis and screening of α-L-fucosidase

[0040] α-L-fucosidase PbFuc amino acid sequence:

[0041] QQNTNPEQGKFNTTDESLKQYKYPEWFRDAKFGIWSHWGPQAVPRQGDWYARGMYLQGNDQNKYHVEHYGTPSKFGYKDIIPLWKAEKWNPEQLMALYKKAGAKYFVSMGSHHDMFFLWNSK IHRWNSVKMGPHKDVVGLWQKAAKKEGLRFGISEHLAASFNWFQPSHGSDKTGEFAGVPYDGHDPQYADLYHLPADSSNIKEWLTNNPAWHKKWLAYVTELIDNYHPDLLYSDSKVPFEEYG RKMVAHYYNQDLAKNKGKLEAVYTPKEPSGGKWAQDVERGVLDSISPFPWQTDTSIGDWYYRTGQRYKNANEIAQLLIDVVSKNGNLLINVVQTPEGDLEPDVIKIVTELGEWTKTYGEGIY ATRPWKVYGEGPSTIKSNQKKGDFGGLTDTRGYEATDIRYTKKGSNLYAFCMSIPQTEIKMELLGKNSKYLEKPISSVTLMGSKEKLEWTQTDGSLVIKKPKNYPAWAVTGFKIEFKK;SEQ ID NO.2.

[0042] The amino acid sequence of Geobacter α-L-fucosidase (shown in SEQ ID NO. 2) was submitted as a FASATA file to the website http: / / kazlab.umn.edu / for potential mutation prediction. Ten sites, containing the same amino acid as at least 70% of similar proteins, were selected for site-directed mutagenesis: T71H, M115N, I154V, E176P, A278V, I333V, Y361N, R397E, Y406F, and M447L. Primers were designed for the codons corresponding to the selected amino acid mutations based on the codon preference of Escherichia coli and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Table 1).

[0043] Table 1 Site-directed mutagenesis primer design

[0044]

[0045]

[0046] Note: The underlined part is the mutation site.

[0047] Full-plasmid amplification was performed using the pET28a-PbFuc plasmid as a template. The reaction system consisted of 1 μL of plasmid template, 2 μL of upstream primer, 2 μL of downstream primer, 25 μL of 2× Pfu PCRMasterMix (Solarbio, Beijing), and ddH2O to 50 μL. Reaction conditions were: initial denaturation at 94°C for 3 min; 20 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 7.5 min; and a final extension at 72°C for 5 min.

[0048] After adding DpnⅠ endonuclease to the PCR amplification product to eliminate the parent plasmid, E. coli DH5α competent cells were transformed. Transformants were picked and cultured overnight. The bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The plasmid of the transformant with correct sequencing was extracted and then transformed into E. coli BL21. The crude enzyme solution was induced and broken according to the method of Example 1 to obtain a crude enzyme solution. The appropriately diluted crude enzyme solution was treated at 45°C for 20 minutes and immediately cooled on ice. 10 μL of the treated enzyme solution was taken, and a pNP-FUC solution with a final concentration of 1 mM was added to the reaction volume of 100 μL. After reacting at pH 5.5 and 35°C for 10 minutes, 100 μL of 2M sodium carbonate solution was added to terminate the reaction. The OD value was measured at 405 nm and the residual enzyme activity was calculated to determine its thermal stability ( Figure 1 and Table 2). Residual enzyme activity = (enzyme activity after treatment / enzyme activity before treatment) × 100%.

[0049] Table 2 Residual enzyme activities of site-directed mutants

[0050]

[0051] Using the residual enzyme activity of the wild-type enzyme as a control, a mutant with a residual enzyme activity 1.5 times that of the control was obtained and named PbFuc-T71H (as shown in SEQ ID NO. 3). The characteristics are as follows:

[0052] PbFuc-T71H: The DNA sequence was changed from ACC to CAT, and the threonine at position 71 of the enzyme was mutated to histidine.

[0053] Amino acid sequence of mutant PbFuc-T71H:

[0054] QQNTNPEQGKFNTTDESLKQYKYPEWFRDAKFGIWSHWGPQAVPRQGDWYARGMYLQGNDQNKYHVEHYGHPSKFGYKDIIPLWKAEKWNPEQLMALYKKAGAKYFVSMGSHHDMFFLWNSK IHRWNSVKMGPHKDVVGLWQKAAKKEGLRFGISEHLAASFNWFQPSHGSDKTGEFAGVPYDGHDPQYADLYHLPADSSNIKEWLTNNPAWHKKWLAYVTELIDNYHPDLLYSDSKVPFEEYG RKMVAHYYNQDLAKNKGKLEAVYTPKEPSGGKWAQDVERGVLDSISPFPWQTDTSIGDWYYRTGQRYKNANEIAQLLIDVVSKNGNLLINVVQTPEGDLEPDVIKIVTELGEWTKTYGEGIY ATRPWKVYGEGPSTIKSNQKKGDFGGLTDTRGYEATDIRYTKKGSNLYAFCMSIPQTEIKMELLGKNSKYLEKPISSVTLMGSKEKLEWTQTDGSLVIKKPKNYPAWAVTGFKIEFKK;SEQ ID NO.3.

[0055] Example 3 Error-prone PCR Method for Construction of an α-L-fucosidase Mutant Library: Error-prone PCR was performed using the Gene Morph II Random Mutagenesis Kit purchased from Agilent using pET28a-PbFuc as a template according to the kit instructions. The gene fragment and plasmid were connected using a one-step cloning method. Primers containing homology arms (underlined portions) were designed:

[0056] Pb-pd-F: 5'-GCAAATGGGTCGCGGATCCCAGCAGAACACCAACCC-3'; SEQ ID NO.31;

[0057] Pb-pd-R: 5'-GTGCTCGAGTGCGGCCGCTTATTTTTTAAATTCGATTTTG-3'; SEQ ID NO. 32.

[0058] The reaction conditions were: pre-denaturation at 95°C for 2 minutes, followed by 25 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 3.5 minutes, followed by a complete extension at 72°C for 10 minutes. After amplification, the randomly mutated gene fragments were recovered using a gel recovery kit.

[0059] Primers 28a-yw-F: 5'-GCGGCCGCACTCGAGCAC-3' (SEQ ID NO. 33) and 28a-yw-R: 5'-GGATCCGCGACCCATTTGC-3' (SEQ ID NO. 34) were designed. Using pET28a as a template, a pET28a linear fragment was amplified. The randomly mutated PbFuc gene fragment was ligated to the amplified pET28a linear fragment using the Biomed seamless cloning kit. The ligation product was recovered using a gel recovery kit and transformed into Escherichia coli DH5α by electroporation to obtain approximately 1×10 4 The mutation library of each clone was constructed. The colonies on the mutation library plate were eluted with liquid LB medium and then cultured for 2-3 hours to extract the plasmids. The random mutation library of Geobacter α-L-fucosidase was established.

[0060] Example 4 Screening of α-L-fucosidase mutant library

[0061] After collecting the mutant library clones from Example 3, plasmids were extracted and transformed into E. coli BL21 competent cells. The cells were then plated with LB plates containing 50 μg / ml kanamycin. After overnight culture, 100 μL of LB liquid medium containing 50 μg / ml kanamycin was added to each well of a 96-well plate. A single colony was picked with a sterile toothpick and transferred to the 96-well plate. The plate was sealed with parafilm and incubated at 37°C and 200 rpm for 3 h. Then, 100 μL of LB liquid medium containing 50 μg / ml kanamycin and 1 mM IPTG was added and induced at 16°C and 200 rpm for 16 h. After induction, the cells were collected by centrifugation at 3000 rpm, and 100 μL of acetic acid-sodium acetate buffer at pH 5.5 was added to resuspend the cells. After treatment at 45°C for 20 minutes, the cells were placed in a 4°C refrigerator to cool. Then, 50 μL of 2 mol / L pNP-Fuc solution was added to each well. After reacting at 37°C for 10 minutes, 100 μL of 2M sodium carbonate solution was added to terminate the reaction. The OD value was measured at 405 nm to determine the residual activity of the mutant. Strains with higher residual activity than that of the parent (wild type) were picked up in a new 96-well culture plate for repeated screening. Five mutants with improved thermal stability were screened ( Figure 2 and Table 3, containing a mutant T71H with a site-directed mutation). After sequencing verification, these five strains all had one amino acid mutation and were named: PbFuc-G53C (as shown in SEQ ID NO.4), PbFuc-M54I (as shown in SEQ ID NO.5), PbFuc-N59S (as shown in SEQ ID NO.6), PbFuc-R125C (as shown in SEQ ID NO.7) and PbFuc-S199P (as shown in SEQ ID NO.8), with the following characteristics:

[0062] PbFuc-G53C: The DNA sequence changes from GGT to TGT, and the glycine at position 53 of the enzyme mutates to cysteine.

[0063] PbFuc-M54I: The DNA sequence changes from ATG to ATT, and the methionine at position 54 of the enzyme mutates to isoleucine.

[0064] PbFuc-N59S: The DNA sequence mutated from AAC to AGC, and the asparagine at position 59 of the enzyme mutated to serine.

[0065] PbFuc-R125C: The DNA sequence mutated from CGT to TGT, and the 125th position of the enzyme mutated from arginine to cysteine.

[0066] PbFuc-S199P: The DNA sequence mutated from TCT to CCT, and the 199th position of the enzyme mutated from serine to proline.

[0067] Table 3 Residual enzyme activity of Geobacter α-L-fucosidase and its mutants

[0068]

[0069]

[0070] Amino acid sequence of mutant PbFuc-G53C:

[0071] QQNTNPEQGKFNTTDESLKQYKYPEWFRDAKFGIWSHWGPQAVPRQGDWYAR CMYLQGNDQNKYHVEHYGTPSKFGYKDIIPLWKAEKWNPEQLMALYKKAGAKYFVSMGSHHDMFFLWNSKIHRWNSVKMGPHKDVVGLWQKAAKKEGLRFGISEHLAASFNWFQPSHGSDKTGEFAGVPYDGHDPQYADLYHLPADSSNIKEWLTNNPAWHKKWLAYVTELIDNYHPDLLYSDSKVPFEEYGRKMVAHYYNQDLAKNKGKLEAVYTPKEPSGGKWAQDVERGVLDSISPFPWQTDTSIGDWYYRTGQRYKNANEIAQLLIDVVSKNGNLLINVVQTPEGDLEPDVIKIVTELGEWTKTYGEGIYATRPWKVYGEGPSTIKSNQKKGDFGGLTDTRGYEATDIRYTKKGSNLYAFCMSIPQTEIKMELLGKNSKYLEKPISSVTLMGSKEKLEWTQTDGSLVIKKPKNYPAWAVTGFKIEFKK; SEQ ID NO.4。

[0072] Amino acid sequence of mutant PbFuc-M54I:

[0073] QQNTNPEQGKFNTTDESLKQYKYPEWFRDAKFGIWSHWGPQAVPRQGDWYARG IYLQGNDQNKYHVEHYGTPSKFGYKDIIPLWKAEKWNPEQLMALYKKAGAKYFVSMGSHHDMFFLWNSKIHRWNSVKMGPHKDVVGLWQKAAKKEGLRFGISEHLAASFNWFQPSHGSDKTGEFAGVPYDGHDPQYADLYHLPADSSNIKEWLTNNPAWHKKWLAYVTELIDNYHPDLLYSDSKVPFEEYGRKMVAHYYNQDLAKNKGKLEAVYTPKEPSGGKWAQDVERGVLDSISPFPWQTDTSIGDWYYRTGQRYKNANEIAQLLIDVVSKNGNLLINVVQTPEGDLEPDVIKIVTELGEWTKTYGEGIYATRPWKVYGEGPSTIKSNQKKGDFGGLTDTRGYEATDIRYTKKGSNLYAFCMSIPQTEIKMELLGKNSKYLEKPISSVTLMGSKEKLEWTQTDGSLVIKKPKNYPAWAVTGFKIEFKK; SEQ ID NO.5.

[0074] Amino acid sequence of mutant PbFuc-N59S:

[0075] QQNTNPEQGKFNTTDESLKQYKYPEWFRDAKFGIWSHWGPQAVPRQGDWYARGMYLQG SDQNKYHVEHYGTPSKFGYKDIIPLWKAEKWNPEQLMALYKKAGAKYFVSMGSHHDMFFLWNSKIHRWNSVKMGPHKDVVGLWQKAAKKEGLRFGISEHLAASFNWFQPSHGSDKTGEFAGVPYDGHDPQYADLYHLPADSSNIKEWLTNNPAWHKKWLAYVTELIDNYHPDLLYSDSKVPFEEYGRKMVAHYYNQDLAKNKGKLEAVYTPKEPSGGKWAQDVERGVLDSISPFPWQTDTSIGDWYYRTGQRYKNANEIAQLLIDVVSKNGNLLINVVQTPEGDLEPDVIKIVTELGEWTKTYGEGIYATRPWKVYGEGPSTIKSNQKKGDFGGLTDTRGYEATDIRYTKKGSNLYAFCMSIPQTEIKMELLGKNSKYLEKPISSVTLMGSKEKLEWTQTDGSLVIKKPKNYPAWAVTGFKIEFKK; SEQ ID NO.6。

[0076] Amino acid sequence of mutant PbFuc-R125C:

[0077] QQNTNPEQGKFNTTDESLKQYKYPEWFRDAKFGIWSHWGPQAVPRQGDWYARGMYLQGNDQNKYHVEHYGTPSKFGYKDIIPLWKAEKWNPEQLMALYKKAGAKYFVSMGSHHDMFFLWNSKIH CWNSVKMGPHKDVVGLWQKAAKKEGLRFGISEHLAASFNWFQPSHGSDKTGEFAGVPYDGHDPQYADLYHLPADSSNIKEWLTNNPAWHKKWLAYVTELIDNYHPDLLYSDSKVPFEEYGRKMVAHYYNQDLAKNKGKLEAVYTPKEPSGGKWAQDVERGVLDSISPFPWQTDTSIGDWYYRTGQRYKNANEIAQLLIDVVSKNGNLLINVVQTPEGDLEPDVIKIVTELGEWTKTYGEGIYATRPWKVYGEGPSTIKSNQKKGDFGGLTDTRGYEATDIRYTKKGSNLYAFCMSIPQTEIKMELLGKNSKYLEKPISSVTLMGSKEKLEWTQTDGSLVIKKPKNYPAWAVTGFKIEFKK; SEQ ID NO.7.

[0078] Amino acid sequence of mutant PbFuc-S199P:

[0079] QQNTNPEQGKFNTTDESLKQYKYPEWFRDAKFGIWSHWGPQAVPRQGDWYARGMYLQGNDQNKYHVEHYGTPSKFGYKDIIPLWKAEKWNPEQLMALYKKAGAKYFVSMGSHHDMFFLWNSKIHRWNSVKMGPHKDVVGLWQKAAKKEGLRFGISEHLAASFNWFQPSHGSDKTGEFAGVPYDGHDPQYADLYHLPAD P SNIKEWLTNNPAWHKKWLAYVTELIDNYHPDLLYSDSKVPFEEYGRKMVAHYYNQDLAKNKGKLEAVYTPKEPSGGKWAQDVERGVLDSISPFPWQTDTSIGDWYYRTGQRYKNANEIAQLLIDVVSKNGNLLINVVQTPEGDLEPDVIKIVTELGEWTKTYGEGIYATRPWKVYGEGPSTIKSNQKKGDFGGLTDTRGYEATDIRYTKKGSNLYAFCMSIPQTEIKMELLGKNSKYLEKPISSVTLMGSKEKLEWTQTDGSLVIKKPKNYPAWAVTGFKIEFKK; SEQ ID NO.8.

[0080] Integration of α-L-fucosidase mutation sites in Example 5

[0081] The six beneficial mutation sites obtained by site-directed mutagenesis and random mutagenesis screening were integrated and modified on the wild-type PbFuc gene sequence. The whole gene was synthesized by Qingke Biotechnology and the recombinant plasmid pET-28a-PbFuc-M6 with BamHⅠ and NotⅠ restriction sites at both ends was constructed. The mutant containing six mutation sites was named M6 (DNA sequence is shown in SEQ ID NO.9, amino acid sequence is shown in SEQ ID NO.10).

[0082] DNA sequence of the combined mutant M6:

[0083] CAGCAGAACACCAACCCGGAACAGGGTAAATTCAACACCACCGATGAAAGCCTGAAACAGTACAAATACCCGGAATGGTTCCGTGACGCTAAATTCGGCATCTGGTCCCACTGGGGTCCGCAGGCGGTTCCGCGTCAGGGTGATTGGTACGCGCGT TGTATT TACCTGCAGGGC AGC GACCAGAACAAATACCACGTTGAACACTACGGCCATCCGTCTAAATTCGGTTATAAAGATATTATCCCGCTGTGGAAAGCTGAAAAAATGGAACCCGGAACAGCTGATGGCGCTGTATAAAAAAGCGGGCGCGAAATACTTCGTTAGCATGGGTAGCCACCACGACATGTTTTTCCTGTGGAACAGCAAAATCCAC TGT TGGAACAGCGTTAAAATGGGTCCGCATAAAGATGGTGGTTGGCCTGTGGCAGAAAGCAGCGAAAAAAGAAGGCCTGCGCTTCGGCATCTCTGAACACCTGGCAGCGAGCTTCAACTGGTTCCAGCCGTCCCACGGTTCTTGATAAAACCGGCGAATTTGCTGGTGTTCCGTACGACGGCCACGACCCGCAGTACGCGGATCTGTACCACCTGCCGGCTGAT CCTAGCAACATCAAAGAATGGCTGACCAACAACCCGGCGTGGCACAAAAAATGGCTGGCGTACGTGACCGAACTGATCGATAACTATCATCCGGACCTGCTGTACAGCGACTCCAAAGTTCCGTTCGAAGAATATGGCCGTAAAATGGTGGCGCACTACTACAACCAGGATCTGGCAAAAAACAAAGGCAAACTGGAAGCGGTTTACACCCCGAAAGAACCGAGCGGTGGCAAATGGGCGCAGGATGTTGAACGTGGTGTTCTGGATTCTATCTCTCCGTTCCCGTGGCAGACCGATACCTCCATCGGCGATTGGTACTACCGTACCGGCCAGCGTTACAAAAACGCTAACGAAATCGCGCAGCTGCTGATCGATGTTGTTTCTAAAAACGGTAACCTGCTGATTAACGTGGTGCAGACCCCGGAAGGTGATCTGGAACCGGATGTTATCAAAATCGTGACCGAACTGGGTGAATGGACCAAAACCTATGGTGAAGGTATCTACGCAACCCGTCCGTGGAAAGTTTACGGTGAAGGCCCGTCCACCATCAAAAGCAACCAGAAAAAAGGCGATTTCGGCGGTCTGACCGACACTCGCGGTTACGAAGCGACCGATATCCGTTACACCAAAAAAGGTAGCAACCTGTACGCTTTCTGCATGAGCATTCCGCAGACCGAAATTAAAATGGAACTGCTGGGCAAAAACTCTAAATACCTGGAAAAACCGATCTCTAGCGTGACCCTGATGGGTTCTAAAGAAAAACTGGAATGGACCCAGACCGATGGCTCTCTGGTTATCAAAAAACCGAAAAACTACCCGGCGTGGGCGGTGACCGGTTTCAAAATCGAATTTAAAAAATAA; SEQ ID NO.9。

[0084] Amino acid sequence of the combined mutant M6:

[0085] QQNTNPEQGKFNTTDESLKQYKYPEWFRDAKFGIWSHWGPQAVPRQGDWYAR CIYGD S DQNKYHVEHYG H PSKFGYKDIIPLWKAEKWNPEQLMALYKKAGAKYFVSMGSHHDMFFLWNSKIH C WNSVKMGPHKDVVGLWQKAAKKEGLRFGISEHLAASFNWFQPSHGSDKTGEFAGVPYDGHDPQYADLYHLPAD P SNIKEWLTNNPAWHKKWLAYVTELIDNYHPDLLYSDSKVPFEEYGRKMVAHYYNQDLAKNKGKLEAVYTPKEPSGGKWAQDVERGVLDSISPFPWQTDTSIGDWYYRTGQRYKNANEIAQLLIDVVSKNGNLLINVVQTPEGDL SEQ ID NO.10.

[0086] The recombinant plasmid pET-28a-PbFuc-M6 was transformed into E. coli Top 10 competent cells. The E. coli Top 10 strain containing the combined mutant recombinant plasmid was streaked on LB solid medium (containing 50 μg / ml kanamycin). A single colony was picked and transferred to liquid LB medium containing 50 μg / ml kanamycin. The cells were cultured overnight at 37°C and 200 rpm. The plasmid was extracted from the bacterial solution using a plasmid extraction kit and the extracted plasmid was transferred into E. coli BL21 for subsequent expression.

[0087] Example 6 Enzymatic Properties Analysis of Wild-Type PbFuc and Combined Mutant M6

[0088] According to the protocol of Example 1, the wild-type PbFuc and the combined mutant M6 were induced, expressed, and purified, and their enzymatic properties were determined.

[0089] 1) Determination of optimal reaction temperature

[0090] Pipette 10 μL of the appropriately diluted crude enzyme solution and add pNP-Fuc solution with a final concentration of 1 mM to a reaction volume of 100 μL. Mix well and react at pH 5.5 and 20-50°C for 10 minutes. Add 100 μL of 2M sodium carbonate solution to terminate the reaction. Use the untreated crude enzyme solution as the control group, measure the OD value at 405 nm, calculate the enzyme activity, and determine the optimal reaction temperature. The optimal reaction temperature for wild-type PbFuc is 35°C ( Figure 3 a), the optimal reaction temperature of mutant M6 is 40℃( Figure 4 a), the optimal reaction temperature increased by 5°C compared to the wild type.

[0091] 2) Thermal stability determination

[0092] Appropriately diluted crude enzyme solutions were treated at different temperatures for 30 minutes and immediately cooled on ice. Aspirate 10 μL of the treated crude enzyme solution and add pNP-FUC solution (final concentration: 1 mM) to a reaction volume of 100 μL. The reaction was incubated at pH 5.5, 35°C for 10 minutes, and then terminated with 100 μL of 2 M sodium carbonate solution. Using untreated crude enzyme solution as a control, the OD value was measured at 405 nm and the residual enzyme activity was calculated to determine thermal stability.

[0093] In the thermal stability test, the residual enzyme activity of wild-type PbFuc treated at 45℃ for 30min was 0 ( Figure 3 b), the residual enzyme activity of mutant M6 after treatment at 20-55℃ for 30min can maintain more than 80% of the activity, and after treatment at 60℃, it still maintains about 40% of the residual enzyme activity ( Figure 4 b).

[0094] 3) Determination of optimal reaction pH

[0095] The pNP-Fuc solution was prepared using pH 3.0-6.0 (acetic acid-sodium acetate), pH 6.5-8.0 (potassium phosphate), and pH 8.5-9.0 (Tris-HCl) buffers, and the crude enzyme solution was appropriately diluted; 10 μL of the diluted crude enzyme solution was aspirated, and a pNP-Fuc solution with the same pH and a final concentration of 1 mM was added to a reaction volume of 100 μL. The reaction was carried out at 35°C for 10 minutes, and 100 μL of 2M sodium carbonate solution was added to terminate the reaction. The untreated crude enzyme solution was used as the control group, and the OD value was measured at 405 nm and the enzyme activity was calculated to determine its optimal reaction pH. The wild-type PbFuc was most active at pH 5.5 ( Figure 3 c), the optimal reaction pH of mutant M6 is also pH 5.5 ( Figure 4 c), consistent with the wild-type strain.

[0096] 4) pH stability determination

[0097] The crude enzyme solution was appropriately diluted with pH 2.0 (glycine-hydrochloric acid), pH 3.0-6.0 (acetic acid-sodium acetate), pH 6.5-8.0 (potassium phosphate), pH 8.5-9.0 (Tris-Hcl) and pH 10.0-12.0 (Kcl-NaOH) buffer solutions, and the diluted crude enzyme solution was treated at 25°C for 30 minutes and immediately cooled on ice. 10 μL of the treated crude enzyme solution was aspirated and a pNP-Fuc solution with a final concentration of 1 mM was added to a reaction volume of 100 μL. After reacting at pH 5.5 and 35°C for 10 minutes, 100 μL of 2M sodium carbonate solution was added to terminate the reaction. The untreated crude enzyme solution was used as the control group, and the OD value was measured at 405 nm and the residual enzyme activity was calculated to determine its pH stability. The residual enzyme activity of wild-type PbFuc after treatment in the pH 3.0-10.0 range can be maintained at more than 80% ( Figure 3 d); The pH stability of mutant M6 decreased compared with the wild-type strain in the entire pH range and was completely inactivated at pH < 3.0 and pH > 10.0 ( Figure 4 d).

[0098] 5) Thermal inactivation curve determination

[0099] The crude enzyme solution of Geobacter α-L-fucosidase was treated at 35℃, 40℃ and 45℃ for 60 minutes. Samples were taken at different time points and cooled on ice. The residual enzyme activity of the samples was then measured at pH 5.5 and 35℃. The results of the thermal inactivation curve showed that the wild-type enzyme could maintain more than 60% and 80% of its enzyme activity after treatment at 35℃ and 40℃ for 60 minutes, respectively. After treatment at 45℃ for 15 minutes, its residual enzyme activity was about 15% ( Figure 5 a). Mutant M6 can maintain 70% and 80% of its activity after being treated at 50℃ and 55℃ for 180min, respectively. It can still maintain about 40% of its activity after being treated at 60℃ for 30min, but is completely inactivated after 60min. Figure 5 b).

[0100] 6) Determination of enzyme kinetic parameters, concentration and specific activity

[0101] 10-80 mM pNP-FUC solutions were prepared in 50 mM acetic acid-sodium acetate buffer (pH 5.5). 10 μL of the appropriately diluted crude enzyme solution was added to a 100 μL reaction volume. After incubation at 35°C for 5 min, the reaction was immediately terminated by adding 100 μL of 2 M sodium carbonate solution. The initial enzyme activity was determined by measuring the OD at 405 nm. The Michaelis constant (Km) and maximum reaction rate (Vmax) were calculated using GraphPad Prism 7.0 software. The concentration of the purified Geobacter α-L-fucosidase was determined using a protein concentration assay kit from Sangon Biotech (Shanghai). Use acetic acid-sodium acetate solution with pH 5.5 to prepare 10mM pNP-Fuc solution, take 10μL of appropriately diluted crude enzyme solution, add pNP-Fuc solution with a final concentration of 1mM to the reaction volume of 100μL, mix well, react at 35℃ for 10min, add 100μL 2M sodium carbonate solution to terminate the reaction, measure the OD value at 405nm, and substitute it into the standard curve to calculate the enzyme activity. The specific activity of the enzyme is obtained by dividing the enzyme activity per minute by the mass of the enzyme used for the reaction. The Km value of the wild-type enzyme is 27.72±0.80mM, the Vmax value is 127.70±1.418, and the specific activity is 15.35±0.75U / mg. The Km value of the mutant M6 is 24.66±0.41, Vmax value is 127.70±1.418, and the specific activity is 15.35±0.75U / mg. max The value was 123.9±0.57, and the specific activity was 19.56±0.31U / mg.

[0102] Example 7 Analysis of 2'-FL Synthesis by Wild-Type PbFuc and Combined Mutant M6

[0103] 1) Reaction conditions for the synthesis of 2'-FL

[0104] Prepare 60 mM pNP-Fuc solution 1, 2 mg / mL L-fucose solution 2, 4 mg / mL 2'-FL solution 3, and 100 mM D-lactose solution 4 using acetic acid-sodium acetate solution at pH 5.5. Transfer equal amounts of pNP-Fuc and D-lactose solutions to a centrifuge tube, add 0.1 U / mL crude enzyme solution, mix thoroughly, and react at 35°C for 3 hours. Stop the reaction by boiling in water for 10 minutes, filter through a 0.22 μm filter, and reserve the reaction solution for later use.

[0105] 2) Thin layer chromatography (TLC) detection of transglycosylation products

[0106] Solutions 1, 2, 3, and 4 from step 1) and the reaction solution were used as standards and samples, and transglycosylation products were detected by thin-layer chromatography. The TLC sample volume was: 1 μL standard and 1 μL sample. The chromatographic developing solvent was n-butanol:acetic acid:water (4:1:1). After development, methanol containing 5% sulfuric acid was sprayed as the color developer, and color was developed at 120°C. The transglycosylation activity of the mutant strain M6 was determined using the same method.

[0107] 3) Detection of transglycosylation products by HPLC

[0108] Solutions 1, 2, 3, and 4 from step 1) and the reaction solution were used as standards and samples. The results were analyzed using an Agilent high-performance liquid chromatograph under the following conditions: mobile phase: 5 mM sulfuric acid solution, sample volume: 10 μL, column temperature: 60°C, detector temperature: 35°C, and flow rate: 0.6 ml / min.

[0109] TLC Figure 6 ) and HPLC( Figure 7 、 8 ) The results of the test showed that both the wild type and mutant M6 could synthesize the product 2'-FL under the same conditions using pNP-Fuc as the donor and D-lactose as the acceptor, accompanied by the production of the product L-fucose. Under the same conditions, the 2'-FL conversion rate of the wild type was 1.53%, and the 2'-FL conversion rate of M6 was 1.80%, an increase of about 17% compared with the wild type.

[0110] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An α-L-fucosidase mutant, characterized in that Based on the amino acid sequence of α-L-fucosidase shown in SEQ ID NO.2, after mutation, the amino acid sequence has one of the following mutation sites: (1) The glycine at position 53 was mutated to cysteine, the methionine at position 54 was mutated to isoleucine, the asparagine at position 59 was mutated to serine, the threonine at position 71 was mutated to histidine, the arginine at position 125 was mutated to cysteine, and the serine at position 199 was mutated to proline; (2) mutating glycine at position 53 to cysteine; (3) mutating the methionine at position 54 to isoleucine; (4) mutating the asparagine at position 59 to serine; (5) mutating the threonine at position 71 to histidine; (6) mutating the arginine at position 125 to cysteine; (7) The serine at position 199 was mutated to proline.

2. Use of the α-L-fucosidase mutant according to claim 1 in improving thermal stability.

3. Use of the α-L-fucosidase mutant according to claim 1 in increasing the yield of 2'-fucosyllactose.

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