An α-L-fucosidase mutant and its application

Abstract

The present application relates to the field of microbial genetic engineering technology, and in particular to an α-L-fucosidase mutant and its application. The present application provides an α-L-fucosidase mutant, the α-L-fucosidase mutant having at least 90% nucleotide sequence identity with SEQ ID NO.2, and comprising at least one of the following mutations, the mutation site including at least one of positions 292, 725, 847, and 1452 of SEQ ID NO.1 or a corresponding position, the mutation site replacing, inserting or deleting one or more amino acid residues at the mutation site. The present application utilizes α-L-fucosidase mutants to effectively increase the fucose production in recombinant cells, and the shake flask fermentation yield of fucose can reach up to 4.332 g / L, which is 28.70% higher than that of the chassis strain FUC001.

Description

Technical Field

[0001] The present application relates to the technical field of microbial genetic engineering, and in particular to an α-L-fucosidase mutant and its application. Background Art

[0002] Fucose (FUC), also known as 6-deoxygalactose, is a ubiquitous natural sugar. L-fucose is one of the five monosaccharides that make up human milk oligosaccharides and one of the eight essential sugars for the human body. It is widely found in animals, plants, and algae, playing important physiological roles. L-fucose can regulate intestinal flora and maintain intestinal health; it can bind to viruses, bacteria, and toxins, preventing them from infecting cells and thus enhancing the body's immune system. L-fucose has skin moisturizing properties, delaying skin aging, stimulating fibroblast proliferation, and protecting fibroblasts from damage caused by radiation and ultraviolet rays. Currently, commercial production of FUC primarily involves extraction and purification of fucoidan using alginate hydrolysis, chemical conversion using hexose as a raw material, and enzymatic hydrolysis of the extracellular polysaccharides of fucose-producing bacteria. These L-fucose production methods suffer from low economic efficiency, environmental impact, and limited application scenarios. Therefore, the development of new L-fucose production methods has potential application value. The biosynthesis method only requires cheap carbon sources and renewable donors within cells as raw materials, and can achieve high economic output at a low environmental cost, so it has broader application prospects.

[0003] Currently, the yield of Fucoxanthin (Fuc) produced by microorganisms remains low, unable to meet the needs of large-scale industrial production. Previous studies have identified α-L-fucosidase as a key enzyme in the synthesis of Fucoxanthin. However, low activity of α-L-fucosidase (AfcA) has hindered the bioproduction of Fucoxanthin. Therefore, its modification is crucial. Summary of the Invention

[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide an α-L-fucosidase mutant and its application that improves α-L-fucosidase activity to increase the conversion of 2'-FL into FUC in cells and thus increase FUC production.

[0005] To achieve the above objectives, the technical solutions adopted in this application are:

[0006] In the first aspect, the present application provides an α-L-fucosidase mutant, which has at least 90% nucleotide sequence identity with SEQ ID NO.2 and comprises at least one of the following mutations, wherein the mutation site of the α-L-fucosidase mutant includes at least one of positions 292, 725, 847, and 1452 of SEQ ID NO.2 or the corresponding positions, and one or more amino acid residues are substituted, inserted or deleted at the mutation site.

[0007] The present application discovered that the amino acids 577-1474 of α-L-fucosidase afcA are the region with 1,2-α-L-fucosidase activity. Experiments have found that point mutations in at least one of positions 292, 725, 847 and 1452 of AfcA or corresponding positions can effectively increase the activity of AfcA.

[0008] As a preferred embodiment of the mutant described in the present application, the mutation is a substitution mutation, comprising substitution of at least one of the following amino acid residues:

[0009] Arginine at position 292 is substituted with lysine; or

[0010] Arginine at position 292 is substituted with histidine; or

[0011] Aspartic acid at position 725 is substituted by glutamic acid; or

[0012] Lysine 847 is substituted with phenylalanine; or

[0013] Aspartic acid at position 1452 was substituted by leucine.

[0014] As a preferred embodiment of the mutant described in this application, the mutation is selected from any of the following combinations:

[0015] 1) comprising a substitution of arginine at position 292 by lysine and aspartic acid at position 725 by glutamic acid; or

[0016] 2) comprising a substitution of arginine at position 292 by lysine and a substitution of lysine at position 847 by phenylalanine; or

[0017] 3) comprising a substitution of arginine at position 292 by lysine and aspartic acid at position 1452 by leucine; or

[0018] 4) comprising a substitution of arginine at position 292 by lysine, aspartic acid at position 725 by glutamic acid, and lysine at position 847 by phenylalanine; or

[0019] 5) comprising a substitution of arginine at position 292 by lysine, aspartic acid at position 725 by glutamic acid, and aspartic acid at position 1452 by leucine; or

[0020] 6) comprising a substitution of arginine at position 292 by lysine, lysine at position 847 by phenylalanine, and aspartic acid at position 1452 by leucine; or

[0021] 7) comprising a substitution of arginine at position 292 by lysine, aspartic acid at position 725 by glutamic acid, lysine at position 847 by phenylalanine, and aspartic acid at position 1452 by leucine; or

[0022] 8) comprising a substitution of arginine at position 292 by histidine and aspartic acid at position 725 by glutamic acid; or

[0023] 9) comprising a substitution of arginine at position 292 by histidine and lysine at position 847 by phenylalanine; or

[0024] 10) comprising a substitution of arginine at position 292 by histidine and aspartic acid at position 1452 by leucine; or

[0025] 11) comprising a substitution of arginine at position 292 by histidine, aspartic acid at position 725 by glutamic acid, and lysine at position 847 by phenylalanine; or

[0026] 12) comprising a substitution of arginine at position 292 by histidine, aspartic acid at position 725 by glutamic acid, and aspartic acid at position 1452 by leucine; or

[0027] 13) comprising a substitution of arginine at position 292 by histidine, lysine at position 847 by phenylalanine, and aspartic acid at position 1452 by leucine; or

[0028] 14) comprising a substitution of arginine at position 292 by histidine, aspartic acid at position 725 by glutamic acid, lysine at position 847 by phenylalanine, and aspartic acid at position 1452 by leucine; or

[0029] 15) comprising a substitution of aspartic acid at position 725 by glutamic acid and a substitution of lysine at position 847 by phenylalanine; or

[0030] 16) comprising a substitution of aspartic acid at position 725 by glutamic acid and a substitution of aspartic acid at position 1452 by leucine; or

[0031] 17) comprising a substitution of aspartic acid at position 725 by glutamic acid, a substitution of lysine at position 847 by phenylalanine, and a substitution of aspartic acid at position 1452 by leucine; or

[0032] 18) comprising a substitution of phenylalanine for lysine at position 847 and a substitution of leucine for aspartic acid at position 1452.

[0033] The present application codon-optimized the obtained AfcA nucleotide sequence and obtained an α-L-fucosidase mutant through point mutation, which can promote the synthesis of fucose in recombinant cells.

[0034] In a second aspect, the present application provides a polynucleotide encoding the aforementioned α-L-fucosidase mutant.

[0035] In a third aspect, the present application provides an expression vector comprising the above-mentioned polynucleotide.

[0036] In a fourth aspect, the present application provides a recombinant cell comprising the above-mentioned polynucleotide or the above-mentioned expression vector.

[0037] As a preferred embodiment of the recombinant cell described in the present application, the cell includes Escherichia coli.

[0038] In a fifth aspect, the present application provides the use of the above mutant in the production of fucose.

[0039] In a sixth aspect, the present application provides the use of the above-mentioned recombinant cells in the production of fucose.

[0040] In a seventh aspect, the present application provides a process for producing fucose, comprising the following steps: subjecting the above-mentioned recombinant cells to fed-batch fermentation to obtain fucose.

[0041] Compared with the prior art, this application has the following beneficial effects:

[0042] The present application provides an α-L-fucosidase mutant and its application. The α-L-fucosidase mutant can effectively increase the fucose production in recombinant cells. The shake flask fermentation yield of fucose can reach up to 4.332 g / L, which is an increase of 28.70% compared with the chassis strain FUC001. DETAILED DESCRIPTION

[0043] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below in conjunction with specific embodiments.

[0044] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0045] In the following examples, the gene information used is shown in Table 1.

[0046] Table 1 Genes and their information used in the following examples

[0047]

[0048] In the following examples and comparative examples, unless otherwise specified, the methods used for transferring plasmids into competent cells or for transformation were all chemical transformations; the resistance plates used were kanamycin resistance plates; and the positive transformants screened were identified by PCR detection and cloned fragment sequencing.

[0049] In the following examples and comparative examples, unless otherwise specified, the cloning described herein refers to cloning using a ligase-independent single-fragment one-step cloning kit, and the operation was performed according to the kit instructions. The ligase-independent single-fragment one-step cloning kit was provided by Nanjing Novozymes Biotech Co., Ltd., with the product number C112-02.

[0050] The fermentation medium used in the shake flask fermentation experiment contained 40 g / L glycerol, 17.9 g / L Na2HPO4·12H2O, 3.1 g / L KH2PO4, 2.0 g / L NH4Cl, 1 g / L (NH4)3PO4, 2.2 g / L trisodium citrate dihydrate, 2 g / L yeast powder, 15 g / L tryptone, 10 g / L MgSO4·7H2O, 15 mg / LCaCl2, 0.3 mL / L Triton X-100, 10 mg / L vitamin B1 and 10 mL / L trace element solution.

[0051] The trace element solution mainly consists of 13.74 g / L nitrilotriacetic acid sodium salt, 5.6 g / L ammonium ferric citrate, 0.9 g / L ZnSO2·7H2O, 0.2 g / L CoCl2·6H2O, 1.0 g / L MnCl2·4H2O, 0.10 g / L CuCl2·2H2O, 0.2 g / L H3BO3, and 0.2 g / L Na2MoO4·2H2O, and is sterilized by filtration using a 0.22 μm pore size filter membrane.

[0052] The fermentation medium used in the fed-batch fermentation experiment contained 10 g / L initial glycerol, 4.0 g / L (NH4)2SO4, 9.2 g / L K2HPO4, 8.2 g / L KH2PO4, 0.3 g / L citric acid, 6.0 g / L tryptone, 2.0 g / L yeast extract, 10 mg / L thiamine, 2.0 g / L MgSO4·7H2O, 0.02 g / L CaCl2 and 10 mL / L trace element solution.

[0053] The feed solution used in the fed-batch fermentation experiment included 800 g / L carbon source and 5 g / LMgSO4·7H2O. The carbon source was glucose and glycerol, and the mass ratio of glucose to glycerol was glucose:glycerol=4:6.

[0054] The pEcCas vector, pSPIN plasmid, pCDFDuet plasmid, pRSFDuet plasmid, pETDuet plasmid, and pEcgRNA plasmid were all provided by Addgene.

[0055] The techniques not described in detail in the following embodiments and effect examples are all commonly used techniques in the art. Please refer to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Publishing House), "Molecular Biology Experiment (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiment" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0056] In the following examples, comparative examples, and effect examples, "D" refers to aspartic acid (Asp), "E" refers to glutamic acid (Glu), "R" refers to arginine (Arg), "K" refers to lysine (Lys), "H" refers to histidine (His), "W" refers to tryptophan (Trp), "F" refers to phenylalanine (Phe), "L" refers to leucine (Leu), "Y" refers to tyrosine (Tyr), and "I" refers to isoleucine (Ile).

[0057] Example 1

[0058] This example provides a wild-type strain containing α-L-fucosidase AfcA and a construction method thereof, wherein the construction method comprises the following steps:

[0059] 1.1 The lacZ, fucIK, and wcaj genes in Escherichia coli star BL21(DE3) were knocked out using a modified CRISPR-Cas9 technique to generate strain A. The knockout vector used was the pEcCas vector containing Cas9 and λ-Red recombinase, and targeted gene editing was performed using a pEcgRNA containing an sgRNA sequence and an N20-specific sequence.

[0060] 1.2 Amplify manB, manC, GDP-mannose 4,6-dehydrogenase gmd, and wcaG from Escherichia coli str. K-12 substr. MG1655 by PCR, insert them into the vector pRSFDuet-1 through the corresponding restriction sites, and obtain the recombinant plasmid pRSFDuet-manC-manB-gmd-wcaG. Transform the recombinant plasmid into strain A obtained in step 1.1, and screen the positive transformants using resistance plates. The positive transformants are strain B.

[0061] 1.3 Based on the wbgL sequence in Escherichia coli O126, the sequence of the α-1,2-fucosyltransferase gene wbgL* was obtained through codon optimization and chemical synthesis. The wbgL gene fragment was amplified by PCR using the wbgL-F / R primer pair (see Table 1) and inserted into the pETDuet-1 vector through the corresponding restriction sites to obtain the recombinant vector pETDuet-wbgL.

[0062] 1.4 The afcA-specific sequence was obtained through codon optimization and chemical synthesis. The expression cassette fragment containing afcA was cloned into the pETDuet-wbgL plasmid obtained in step 1.3 to obtain the pETDuet-wbgL-afcA plasmid. This plasmid was transformed into strain B obtained in step 1.2. Positive transformants were obtained by resistance plate screening. The resulting positive transformants were designated as strain C, which was designated as FUC001.

[0063] The primer sequences used in the above steps are shown in Table 2.

[0064] Table 2 Primers used to construct E. coli FUC001

[0065]

[0066]

[0067] Example 2

[0068] This embodiment provides an α-L-fucosidase mutant, and the method for constructing the α-L-fucosidase mutant comprises the following steps:

[0069] 2.1 The pETD-wbgL-afcA plasmid obtained in step 1.3 of Example 1 was codon optimized. The optimized afcA nucleic acid sequence is shown in SEQ ID NO. 2;

[0070] 2.2 A point mutation was performed on position 725 of the optimized afcA amino acid sequence, replacing D with E (Asp→Glu), to obtain the point-mutated pETD-wbgL-afcA* plasmid;

[0071] 2.3 The pETD-wbgL-afcA* plasmid obtained in step 2.2 was transformed into Escherichia coli FUC001 obtained in Example 1 to obtain the mutant strain D725E. The α-L-fucosidase AfcA D725E expressed by the strain is the α-L-fucosidase mutant.

[0072] Examples 3-6

[0073] Examples 3-6 respectively provide an α-L-fucosidase mutant. The construction method of the α-L-fucosidase mutant is similar to that of Example 2, except that:

[0074] The mutation site described in Example 3 is to replace R with K (Arg→Lys) at position 292 of the afcA amino acid sequence, and the resulting mutant strain is named R292K;

[0075] The mutation site described in Example 4 is to replace R with H (Arg→His) at position 292 of the afcA amino acid sequence, and the resulting mutant strain is named R292H;

[0076] The mutation site described in Example 5 is to replace K with F (Lys→Phe) at position 847 of the afcA amino acid sequence, and the resulting mutant strain is named K847F;

[0077] The mutation site in Example 6 is to replace D at position 1452 of the afcA amino acid sequence with L (Asp→Leu), and the resulting mutant strain is named D1452L.

[0078] Example 7

[0079] This embodiment provides an α-L-fucosidase mutant, and the method for constructing the α-L-fucosidase mutant comprises the following steps:

[0080] 7.1 Based on the mutant strain D725E obtained in Example 2, a point mutation was performed on position 292 of the afcA amino acid sequence, replacing R with H (Arg→His), to obtain the point-mutated pETD-wbgL-afcA* plasmid;

[0081] 7.2 The pETD-wbgL-afcA* plasmid obtained in step 7.1 was transformed into the mutant strain D725E obtained in Example 2 to obtain the mutant strain FUCEH. The α-L-fucosidase AfcA D725ER292H expressed by this strain is the α-L-fucosidase mutant.

[0082] Example 8

[0083] This embodiment provides an α-L-fucosidase mutant, and the method for constructing the α-L-fucosidase mutant comprises the following steps:

[0084] 8.1 Based on the mutant strain K847F obtained in Example 5, a point mutation was performed on position 1452 of the afcA amino acid sequence, replacing D with L (Asp→Leu), to obtain the point-mutated pETD-wbgL-afcA* plasmid;

[0085] 8.2 The pETD-wbgL-afcA* plasmid obtained in step 8.1 was transformed into the mutant strain K847F obtained in Example 5 to obtain the mutant strain FUCFL. The α-L-fucosidase AfcA K847FD1452L expressed by this strain is the α-L-fucosidase mutant.

[0086] Example 9

[0087] This embodiment provides an α-L-fucosidase mutant, and the method for constructing the α-L-fucosidase mutant comprises the following steps:

[0088] 9.1 Based on the mutant strain FUCEH obtained in Example 7, a point mutation was performed on the amino acid sequence of afcA at position 847, replacing K at position 847 with F (Lys→Phe), to obtain the point-mutated pETD-wbgL-afcA* plasmid;

[0089] 9.2 The pETD-wbgL-afcA* plasmid obtained in step 9.1 was transformed into the mutant strain FUCEH obtained in Example 7 to obtain the mutant strain FUCEHF. The α-L-fucosidase AfcA D725E R292H K847F expressed by this strain is the α-L-fucosidase mutant.

[0090] Example 10

[0091] This embodiment provides an α-L-fucosidase mutant, and the method for constructing the α-L-fucosidase mutant comprises the following steps:

[0092] 10.1 Based on the mutant strain FUCEH obtained in Example 7, a point mutation was performed on the amino acid sequence of afcA at position 1452, replacing the D at position 1452 with L (Asp→Leu), to obtain the point-mutated pETD-wbgL-afcA* plasmid;

[0093] 10.2 The pETD-wbgL-afcA* plasmid obtained in step 10.1 was transformed into the mutant strain FUCEH obtained in Example 7 to obtain the mutant strain FUCEHL. The α-L-fucosidase AfcA D725E R292H D1452L expressed by this strain is the α-L-fucosidase mutant.

[0094] Example 11

[0095] This embodiment provides an α-L-fucosidase mutant, and the method for constructing the α-L-fucosidase mutant comprises the following steps:

[0096] 11.1 Based on the mutant strain FUCEH obtained in Example 7, point mutations were made at positions 847 and 1452 of the afcA amino acid sequence, replacing K at position 847 with F (Lys→Phe) and D at position 1452 with L (Asp→Leu), to obtain the point-mutated pETD-wbgL-afcA* plasmid.

[0097] 11.2 The pETD-wbgL-afcA* plasmid obtained in step 11.1 was transformed into the mutant strain FUCEH obtained in Example 7 to obtain the mutant strain FUCEHFL. The α-L-fucosidase AfcA D725E R292H K847FD1452L expressed by this strain is the α-L-fucosidase mutant.

[0098] Comparative Example 1

[0099] This comparative example provides an α-L-fucosidase mutant. The construction method of the α-L-fucosidase mutant is similar to that of Example 2, except that the mutation site is to replace K at position 847 of the afcA amino acid sequence with W (Lys→Trp), and the resulting mutant strain is named K847W.

[0100] Comparative Example 2

[0101] This comparative example provides an α-L-fucosidase mutant. The construction method of the α-L-fucosidase mutant is similar to that of Example 2, except that the mutation site is to replace K at position 847 of the afcA amino acid sequence with Y (Lys→Tyr), and the resulting mutant strain is named K847Y.

[0102] Comparative Example 3

[0103] This comparative example provides an α-L-fucosidase mutant. The construction method of the α-L-fucosidase mutant is similar to that of Example 2, except that the mutation site is to replace K at position 847 of the afcA amino acid sequence with I (Lys→Ile), and the resulting mutant strain is named K847I.

[0104] Comparative Example 4

[0105] This comparative example provides an α-L-fucosidase mutant. The construction method of the α-L-fucosidase mutant is similar to that of Example 2, except that the mutation site is to replace K at position 847 of the afcA amino acid sequence with L (Lys→Leu), and the resulting mutant strain is named K847L.

[0106] Comparative Example 5

[0107] This comparative example provides an α-L-fucosidase mutant. The construction method of the α-L-fucosidase mutant is similar to that of Example 8, except that in steps 8.1 and 8.2, the mutant strain K847F is replaced by the mutant strain K847W obtained in Comparative Example 1, and the obtained mutant strain is named FUCWL.

[0108] Effect Examples

[0109] The mutant strains of Examples 1-11 and Comparative Examples 1-5 were subjected to shake flask fermentation and fed-batch fermentation experiments, and the yield of fucose in the fermentation broth was detected by high performance liquid chromatography.

[0110] 1. Shake flask fermentation experiment

[0111] (1) Take the thawed bacterial strain and streak it onto an antibiotic-free LB solid plate and culture it at 37°C for 12-16 hours to obtain the activated mutant strain;

[0112] (2) Pick a single colony of the activated mutant strain obtained in step (1) and inoculate it into 5 mL of LB liquid medium without antibiotics, and culture it at 37°C and 230 rpm for 12-16 hours to obtain a first-level seed solution:

[0113] (3) The first-level seed solution obtained in step (2) was inoculated into 50 mL of LB liquid medium without antibiotics at an inoculum size of 1%, and cultured at 37°C and 230 rpm for 6 h to obtain a second-level seed solution;

[0114] (4) The secondary seed solution obtained in step (3) was inoculated into 100 mL of fermentation medium at a 1% inoculum size and cultured at 37°C until OD 600 =0.6-0.8, add lactose with a final concentration of 6 g / L and IPTG with a final concentration of 1 mM, and culture at 30°C and 230 rpm for 48 h to obtain a shake flask fermentation broth.

[0115] 2. Post-treatment of fermentation broth and HPLC detection

[0116] 1 mL of fermentation broth was placed in a boiling water bath for 10 min to kill the strain, and then centrifuged at 12000 g for 10 min. The supernatant was filtered using a 0.22 μm filter, and the filtrate was used to detect FUC and 2'-FL.

[0117] FUC and 2'-FL in the samples were determined using an HPX.87H column (Bio-Rad) and a high-performance liquid chromatograph (LC-16, Shimadzu, Japan). The FUC and 2'-FL contents in the samples were calculated using the external standard method. The chromatographic conditions were a mobile phase of 5 mM aqueous sulfuric acid, a flow rate of 0.5 mL / min, a column temperature of 60°C, and an injection volume of 10 μL.

[0118] A FUC standard with a gradient concentration of 0-2.0 g / L was taken, and a standard curve was drawn with FUC concentration as the horizontal axis and liquid chromatography peak area as the vertical axis; a 2'-FL standard curve was drawn according to the FUC method.

[0119] The calculated FUC and 2'-FL concentrations in different fermentation broths were converted to the yields obtained by fermentation in a 5 L fermenter. The results are shown in Table 3.

[0120] 3. Fed-batch fermentation experiment

[0121] (1) The mutant strain FUCEHFL obtained in Example 9 was cultured in a 1 L shake flask containing 150 mL of LB medium for 6 h to obtain a secondary seed solution;

[0122] (2) 150 mL of the seed solution obtained in step (1) was transferred to a 5 L fermentor containing 2.5 L of fermentation medium, and 28% (v / v) NH4OH was automatically added to adjust the pH to 6.8. The dissolved oxygen was maintained at 30-50% by automatically controlling the stirring speed. The culture temperature was 37°C, the ventilation rate was 2 VVM, and the rotation speed was 900 rpm;

[0123] (3) When the initial glycerol in the fermentation medium was completely consumed, the feed solution was added to the fermenter at a constant rate of 4 g / L / h, the fermentation temperature was adjusted to 29.5°C, and the fermentation was continued for 4 h;

[0124] (4) Isopropyl β-d-1-thiogalactopyranoside was added to a final concentration of 0.2 mM and 40 g of lactose was added, and 17.78 g / L / h of glycerol solution was supplemented. After 90 h of culture, the fermentation was terminated to obtain a fermentation broth, and the FUC production and 2'-FL residue were detected in the fermentation broth according to the above steps.

[0125] Table 3 Calculation results of FUC and 2'-FL production of different mutant strains

[0126]

[0127]

[0128] As shown in Table 1, the 725th residue of the codon-optimized afcA amino acid sequence was mutated, and when D was replaced by E, strain D725E was obtained. The shake flask yield of this mutant strain reached 3.624 g / L, which was 13.42% higher than that of strain FUC001. The 867th residue of the codon-optimized afcA amino acid sequence was mutated, and when R was replaced by K or H, strains R292K / H were obtained. The shake flask yields of this mutant strain reached 3.526 g / L and 3.848 g / L, respectively, which were 10.25% and 20.43% higher than those of strain FUC001, respectively. The strains D725E and R292H were combined to obtain strain FUCEH, which had a shake flask yield of 3.912 g / L, which was 22.44% higher than that of strain FUC001.

[0129] The codon-optimized afcA residue at position 847 was mutated, replacing K with a hydrophobic amino acid to enhance the stability of the enzyme-substrate intermediate. Aromatic amino acids Y, F, and W, and aliphatic amino acids I and L were selected as alternatives. As shown in Table 1, the results showed that the most effective mutation was found when K at position 847 was replaced with W and F. The amino acid at position 1452 of afcA was mutated, replacing D with a hydrophobic amino acid to enhance the stability of the enzyme-substrate intermediate. Aromatic amino acids Y, F, and W, and aliphatic amino acids I and L were selected as alternatives. The results showed that the most effective mutation was found when D at position 1452 was replaced with L.

[0130] Based on the mutant strain K847F, the amino acid D at position 1452 of the enzyme afcA was mutated to L. The resulting strain, later named FUCFL, achieved a shake flask yield of 4.112 g / L, a 28.70% increase over strain FUC001. Combining the mutation sites of FUCFL with those of FUCEH resulted in a mutant strain named FUCEHFL, which achieved a FUC shake flask yield of 4.332 g / L, a 35.58% increase over strain FUC001.

[0131] Finally, a 5L fed-batch fermentation experiment was conducted on the mutant strain FUCEHFL. The results showed that after 90 hours of fermentation, the FUC production of the strain was 36.17 g / L and the 2'-FL residual content was 5.6 g / L.

[0132] In summary, the α-L-fucosidase mutant of the present application can effectively increase the activity of AfcA, thereby improving the efficiency of converting 2'-FL to FUC, thereby increasing the yield of FUC.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. An α-L-fucosidase mutant, characterized in that The amino acid sequence of the α-L-fucosidase mutant is: any one of the following mutations occurs in the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.2: 1) Arginine at position 292 is substituted with lysine; or 2) arginine at position 292 is substituted with histidine; or 3) substitution of arginine at position 292 by histidine and substitution of aspartic acid at position 725 by glutamic acid; or 4) Arginine at position 292 was substituted by histidine, aspartic acid at position 725 was substituted by glutamic acid, and lysine at position 847 was substituted by phenylalanine.

2. A polynucleotide, characterized in that The polynucleotide encodes the amino acid sequence of the α-L-fucosidase mutant according to claim 1.

3. An expression vector, characterized in that The expression vector comprises the polynucleotide according to claim 2.

4. A recombinant cell, characterized in that The cell comprises the polynucleotide of claim 2 or the expression vector of claim 3.

5. The recombinant cell according to claim 4, wherein The cells include Escherichia coli. Use of the α-L-fucosidase mutant according to claim 1 in producing fucose.

7. Use of the recombinant cell according to claim 4 in producing fucose.

8. A process for producing fucose, characterized in that: The following steps are involved: The recombinant cell according to claim 4 is subjected to fed-batch fermentation to obtain fucose.

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