A fluoridase mutant and a microbial cell displaying fluoridase on the cell surface and applications thereof

Abstract

The present invention provides a fluorinase mutant and a microbial cell displaying fluorinase on the cell surface and its application, belonging to the field of genetic engineering technology. In order to solve the problem of substrate transmembrane restriction in the whole cell catalysis process. The present invention provides a gene with a sequence as shown in SEQ ID NO: 3, and the Escherichia coli engineering bacteria is an Escherichia coli engineering bacteria obtained by using Escherichia coli as a starting strain and expressing an ice nuclease N-terminal encoding gene and a fluorinase encoding gene. The engineering bacteria can stably express the fluorinase gene in Escherichia coli and automatically display it on the cell surface, directly contacting the enzyme with the substrate, avoiding the restriction of the substrate passing through the cell membrane, maximizing the utilization rate of the enzyme, and reducing the influence of exogenous proteins on the host by surface display. A larger cell amount can be obtained, and the possibility of engineering application is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a fluoridase mutant and a microbial cell displaying fluoridase on the cell surface and applications thereof. Background Art

[0002] Fluoridase was first discovered in Streptomyces cattleya. It is the only known natural enzyme that introduces inorganic fluoride ions into organic matter, catalyzing the formation of CF bonds between adenosylmethionine and fluoride ions. This reaction belongs to SN2 reaction. Compared with chemical synthesis, the use of fluoridase to generate the corresponding fluorine-containing precursors is a greener approach. The enzyme catalysis method has inherent problems such as cumbersome enzyme purification and inactivation. In contrast, whole-cell catalysis is a more convenient biocatalytic method. Due to the low catalytic cost and the reduction of traditional enzyme catalysis steps such as cell lysis and protein concentration, it has shown unique advantages and great application prospects in the fields of pharmaceuticals, industrial synthesis of green renewable energy, etc. However, the permeability barrier of cells has become the main problem hindering the development of cell fluoridation catalysis. On the one hand, the transport of S-adenosylmethionine from in vitro to the inside of the cell is blocked by the cell membrane. On the other hand, due to the fluoride ion efflux of the CrcB channel protein of wild Escherichia coli, the intracellular fluoride concentration is very low. Summary of the invention

[0003] The purpose of the present invention is to solve the technical problem of substrate transmembrane limitation in the whole cell catalysis process.

[0004] The present invention provides a mutant of fluoridase, wherein the mutant is based on the amino acid sequence as shown in SEQ ID NO: 7, and at least one of the amino acids at positions 72 and 164 is mutated.

[0005] It is further defined that the mutant is a mutant in which the 72nd position of the amino acid sequence shown in SEQ ID NO: 7 is mutated to alanine, and the 164th position is mutated to alanine.

[0006] The present invention provides a gene encoding the mutant described in claim 1.

[0007] The present invention provides a recombinant microbial cell, which contains the coding gene according to claim 3.

[0008] It is further defined that the microbial cell is an engineered Escherichia coli bacterium that displays fluoridase on its cell surface, and the engineered Escherichia coli bacterium is an Escherichia coli engineered bacterium obtained by taking Escherichia coli as a starting strain and expressing an N-terminal encoding gene of an ice nucleation protein and the above-mentioned encoding gene, and the nucleotide sequence of the N-terminal encoding gene of the ice nucleation protein is shown in SEQ ID NO: 2.

[0009] The present invention provides a recombinant vector, which contains the above-mentioned coding gene and a recombinant expression vector of an ice nucleoprotein N-terminal coding gene.

[0010] The present invention provides the use of the mutant, the coding gene, the microbial cell or the recombinant vector in the production of fluoridase.

[0011] The method for producing the fluoridase is further defined as follows:

[0012] (1) The engineered E. coli bacteria described in the claim are transferred to 100 mL of LB medium at an inoculum rate of 1-2%, and when the bacterial solution grows to OD600 = 0.6, 0.2 mM IPTG is added, the temperature is 30°C, and the induction is carried out for 2h-35h. The cells in the induction process are collected, washed with Tris-HCl, and then resuspended;

[0013] (2) Collect the cells induced for 4 h and resuspend them in PBS. Use a high-pressure crusher to disrupt the cells with the pressure set to 30 Kpsi. Then, remove large cell fragments at 6000 rpm. Centrifuge the collected supernatant at 10,000 g for 1 h to obtain a membrane component precipitate. Remove the supernatant and resuspend the precipitate in PBS containing 2% Triton-X100. Centrifuge again at 10,000 g for 1 h to obtain a cell extracellular membrane precipitate.

[0014] The present invention provides the use of the mutant, the encoding gene, the microbial cell or the recombinant vector as a whole-cell catalyst, characterized in that the use is to catalyze S-adenosylmethionine and potassium fluoride to obtain 5'-fluoropyrimidine.

[0015] It is further defined that the method for obtaining 5'-fluoropyrimidine is to add the above-mentioned engineered Escherichia coli bacteria to an aqueous solution of S-adenosylmethionine and potassium fluoride, and carry out a catalytic reaction at 37°C.

[0016] Beneficial effects: The present invention constructs an Escherichia coli engineered bacterium that displays fluoridase on the cell surface through a genetic recombination method. The engineered bacterium can stably express the fluoridase gene in Escherichia coli and automatically display it on the cell surface. The engineered bacterium has the following advantages: (1) The enzyme is immobilized while expressing the target protein, which simplifies the steps of the traditional enzyme immobilization method, is easy to operate, and can obtain a fluoridase with biological activity. (2) The enzyme is directly contacted with the substrate, avoiding the limitation of the substrate passing through the cell membrane and maximizing the utilization rate of the enzyme. (3) Surface display reduces the impact of exogenous proteins on the host, a larger cell mass can be obtained, and the possibility of engineering application is increased.

[0017] The present invention provides an engineered Escherichia coli with fluoridase displayed on its cell surface obtained by the construction method. The cell surface of the Escherichia coli stably expresses the fluoridase. This method provides a whole-cell catalyst for catalyzing the fluorination of organic matter, which has good industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram of the construction of surface-displayed fluorinase;

[0019] Figure 2 is the map of the recombinant plasmid pET28a-INP-Faa;

[0020] Figure 3 It is the electrophoretic pattern of the fluoridase gene fragment Faa;

[0021] Figure 4 is the electrophoretic pattern of the vector fragment pET28a-INP;

[0022] Figure 5 This is the SDS-PAGE map of the components of the Escherichia coli engineered bacteria cells displaying fluoridase on the cell surface;

[0023] Figure 6 It is a map of the catalytic effect of the whole cell of the engineered Escherichia coli bacteria displaying fluoridase on the cell surface;

[0024] Figure 7 This is a graph showing the catalytic effect of the whole cell of the engineered E. coli bacteria displaying fluoridase on the cell surface as a function of induction time;

[0025] Figure 8 It is a map of the stability of the whole-cell catalyst of the engineered Escherichia coli bacteria displaying fluoridase on the cell surface;

[0026] Fig. 9 The time-dependent changes of 5'-FDA were prepared by using engineered Escherichia coli bacteria that displayed fluorinase on the cell surface as whole-cell catalysts. Specific embodiments

[0027] The NCBI sequence of the N-terminal coding sequence of ice nuclease protein is:

[0028] MNDDKVLVLRTCANNMADHCGQIWPVSGVVECKYWEPTRKLENGLAGLLWGKGASTHLNMQADARWVICEVAVSDIIFLDAQGGVKFPRAEVVHVGTRNSAAGYISANIASYASSTVALNETFVFPEVRTETKVDFPASPATADSTFDIDRHATIQGPQTLETAV (SEQ ID NO: 6).

[0029] The NCBI accession number of the coding sequence of fluorinase is: WP_103354124.1, which is the initial enzyme (WT): MAKPSRPIIAFMSDLGITDDSVAQCKGLMLSVCPDVTIVDVCHTMKPWDVEEGARYIVDLPRLFPEGTVFATTTYPATGTTTRSVALRIKQAAKGGARGQWAGSGAGFERAEGSYIYIAPNNGLLTSVIEEHGYVEAYEVSSTEVIPEQPEPTFYSREMVALPSAHLAAGFPLEKVGRPLADDEIVRFERAKPAQNDDGELVGVVTAIDHPFGNVWTNIHREDLEKLGAGYGTRLRITLDEVLPFDLPLSPTFADAGPIGTPVAYLSSRGYLALARNAASLAYPYNLNAGISVRVVAA (SEQ ID NO: 7).

[0030] The steps of constructing an Escherichia coli engineering bacterium displaying fluoridase on the cell surface of the present invention are as follows: Figure 1 The schematic diagram is described.

[0031] LB medium components: tryptone (10 g / L), yeast extract (5 g / L), sodium chloride (10 g / L).

[0032] S-adenosylmethionine (SAM), potassium fluoride (KF).

[0033] Example 1. Obtaining a gene encoding fluoridase

[0034] 1. Optimization of the fluorinase protein sequence: To address the problem of low initial catalytic efficiency of the fluorinase, the 72T and 164S amino acids were mutated by site-directed mutagenesis. The amino acid sequence after mutation is SEQ ID NO: 1: MAKPSRPIIAFMSDLGITDDSVAQCKGLMLSVCPDVTIVDVCHTMKPWDVEEGARYIVDLPRLFPEGTVFA A TTYPATGTTTRSVALRIKQAAKGGARGQWAGSGAGFERAEGSYIYIAPNNGLLTSVIEEHGYVEAYEVSSTEVIPEQPEPTFYSREMVALP AAHLAAGFPLEKVGRPLADDEIVRFERAKPAQNDDGELVGVVTAIDHPFGNVWTNIHREDLEKLGAGYGTRLRITLDEVLPFDLPLSPTFADAGPIGTPVAYLSSRGYLALARNAASLAYPYNLNAGISVRVVAA. (The underlined part is the mutation site), and the T72A / S164A mutant was obtained.

[0035] 2. The activity of the mutated enzyme was compared with that of the crude enzyme solution of the initial enzyme. The results showed that the catalytic efficiency of the enzyme was increased by about 1.7 times as shown in Table 1.

[0036] Table 1 Comparison of catalytic activity between mutant (T72A / S164A) and initial enzyme (WT)

[0037]

[0038] 3. Taking the above mutated amino acid sequence as the target, the corresponding nucleic acid sequence was codon optimized for the E. coli expression system, and the optimized sequence was SEQ ID NO: 2.

[0039] Example 2. Construction of a plasmid for displaying fluoridase on the cell surface

[0040] 1) The optimized N-terminal sequence of ice nuclease protein (SEQ ID NO: 2) was directly synthesized between the NcoI and BanmHI restriction sites of the pET28a vector to obtain the pET28a-INP vector.

[0041] (1) The nucleotide sequence encoding the N-terminal fragment of ice nucleoprotein after codon optimization is shown in SEQ ID NO: 2:

[0042] (ccatgggcATGAACGATGATAAAGTGCTGGTGCTGCGCACATGTGCAAATAATATGGCAGATCATTGCGGTCAGATCTGGCCAGTGAGCGGTGTTGTGGAATGCAAATATTGGGAACCGACCCGCAAACTGGAAAATGGCCTGGCAGGTCTGCTGTGGGGTAAAGGTGCAAGCACGCATCTGAATATGCAGGCAGATGCCCGTTGGGTTATTTGTGAAGTTGCGGTTAGCGATATTATTTTTCTGGATGCCCAGGGTGGTGTAAAATTTCCGCGTGCAGAAGTTGTTCATGTTGGTACACGTAATAGCGCAGCAGGTTATATTAGTGCAAATATTGCGAGTTATGCAAGCAGCACAGTTGCACTGAATGAAACATTTGTTTTTCCGGAAGTTCGTACCGAAACCAAAGTTGATTTTCCTGCCTCACCGGCAACAGCCGATTCAACCTTTGATATTGATCGTCATGCAACAATCCAGGGTCCTCAGACACTGGAAACCGCAGTTggatcc)。

[0043] (2) The nucleotide sequence of the fluorinase-encoding gene after codon optimization is shown in SEQ ID NO:3:

[0044] (ATGGCGAAACCTAGCCGCCCGATTATTGCGTTTATGAGCGATCTGGGCATTACCGATGATAGCGTGGCGCAATGCAAAGGCCTGATGTTAAGCGTGTGCCCGGATGTGACCATTGTGGATGTGTGCCATACCATGAAACCGTGGGATGTGGAAGAAGGCGCGCGCTATATTGTTGATCTGCCGCGCTTATTTCCTGAAGGCACCGTGTTTGCGgctACCACCTATCCTGCGACCGGTACCACTACTCGTTCAGTTGCGCTGCGCATTAAACAGGCGGCGAAAGGTGGTGCACGTGGTCAATGGGCAGGTAGCGGTGCGGGTTTTGAACGTGCGGAAGGCAGCTATATTTATATTGCGCCGAACAACGGCTTACTGACCAGCGTGATTGAAGAACATGGCTATGTGGAAGCGTATGAAGTGAGCAGCACCGAAGTGATTCCGGAACAGCCGGAACCGACCTTTTATAGCCGCGAAATGGTGGCATTACCGgctGCGCATTTAGCAGCGGGCTTTCCTCTGGAAAAAGTGGGTCGCCCTCTGGCGGATGATGAAATTGTGCGCTTTGAACGCGCAAAACCGGCGCAAAACGATGATGGCGAACTGGTGGGTGTTGTGACCGCGATTGATCATCCGTTTGGCAACGTGTGGACCAACATTCATCGCGAAGATCTGGAAAAACTGGGCGCGGGCTATGGTACTCGTCTGCGCATTACCCTGGATGAAGTGCTGCCGTTTGATCTGCCTCTGAGCCCTACTTTTGCAGATGCGGGCCCTATTGGTACTCCTGTGGCGTATTTAAGCAGCCGCGGCTATTTAGCGCTGGCACGTAATGCGGCGAGCTTAGCGTATCCGTATAACCTGAACGCGGGCATTAGCGTTCGCGTTGTTGCAGCG)。

[0045] 2) Using the optimized fluorinase sequence (SEQ ID NO: 3) as a template to obtain a gene fragment with SacI and HindIII restriction sites, the upstream primer: Faa-F having a nucleotide sequence as shown in SEQ ID NO: 4 (ggatcttccagagatgagctcATGGCGAAACCTAGCCGC); the downstream primer: Faa-R having a nucleotide sequence as shown in SEQ ID NO: 5 (ctgccgttcgacgataagcttACGCTGCAACAACGCGAA).

[0046] The fluoridase gene was cloned using PCR amplification primers, and then the product was recovered and purified. The PCR Enzyme was commercial PrimeSTAR Max DNA Polymerase, and the PCR program was: (98°C, 10s; 55°C, 5s; 72°C, 10s)×30 cycles.

[0047] 3) The pET28a-INP vector was digested with SacI and HindIII restriction endonucleases to obtain a linearized vector fragment, and the fluorinase gene sequence amplification product was digested with SacI and HindIII restriction endonucleases to obtain a linearized target gene fragment.

[0048] 4) The linearized vector fragment was connected with the target gene fragment to obtain pET28a-INP-Faa. Figure 2 It is a map of the recombinant plasmid pET28a-INP-Faa.

[0049] Example 3. Construction of engineered Escherichia coli bacteria displaying fluoridase on cell surface

[0050] 1. The recombinant vector obtained in Example 1 was transformed into the host strain Escherichia coli to obtain the Escherichia coli engineered bacteria displaying the fluoridase on the surface, the steps are as follows:

[0051] 2. The pET28a-INP-Faa recombinant vector obtained in Example 2 was transformed into the host strain Escherichia coli. The conditions of the heat shock method were as follows:

[0052] A. Add the expression vector pET28a-INP-Faa to 100 μl of competent E. coli, mix gently and place on ice for 20 min.

[0053] B. Place in a 42°C water bath for 90 seconds and immediately place on ice for 2 minutes.

[0054] C. Immediately add 800 μl of LB liquid and incubate at 37°C in a shaker for 1 hour. D. Take 100 μl of the culture solution and spread it on an LB plate containing kanamycin and incubate at 37°C overnight.

[0055] For plates with single colonies, colony PCR was used for verification. The primers used for PCR verification were Faa-F and Faa-R. The products were identified by 1% agarose gel. Figure 3 This is the electrophoresis pattern of the fluoridase gene fragment Faa. Figure 4 It is the electrophoresis pattern of the vector fragment pET28a-INP. The corresponding colonies with a mark of about 1.5Kbp were selected and sent to Qingdao Qingke Biotechnology Co., Ltd. for sequencing to obtain the correctly connected expression vector.

[0056] Example 4. Obtaining cell surface displayed fluoridase

[0057] 1. Pick a single colony of the recombinant Escherichia coli engineered strain BL21 (DE3) obtained in Example 3 and place it in 5 mL LB liquid containing kanamycin, and culture it in a 37° C. constant temperature shaker overnight to obtain a seed solution.

[0058] 2. Transfer the activated seed solution to a 100 mL LB flask at an inoculum volume of 1-2% and continue culturing. When the bacterial solution grows to an OD600 of 0.6, add IPTG at a final concentration of 0.2 mM and adjust the temperature to 30°C for induction for 2 h to 35 h. Collect the cells during the induction process, wash them with Tris-HCl (pH 7.8, 50 mM), and then resuspend them.

[0059] 3. Collect the cells induced for 4 hours and resuspend them in PBS (pH 8.0, 50mM). Use a high-pressure crusher to crush the cells with a pressure set to 30Kpsi, and then remove large cell fragments at 6000rpm. Centrifuge the collected supernatant at 10000g for 1 hour to obtain a membrane component precipitate, and the supernatant is the cytoplasm component. Resuspend the precipitate in PBS containing 2% Triton-X100 and centrifuge it again at 10000g for 1 hour to obtain a precipitate with extracellular membrane components and a supernatant component with intracellular membrane components.

[0060] 4. Mix the obtained whole cell fraction (cell), cytoplasmic fraction (om), intracellular membrane fraction (im), and extracellular membrane fraction (om) with protein loading buffer, and perform SDS-PAGE gel electrophoresis after boiling water bath for 2 minutes to analyze the location of the target protein. The theoretical size of fluorinase is about 33KDa, and the theoretical size of the N-terminus of ice nuclease is about 18KDa, so the fusion protein is about 51KDa. The results are as follows Figure 5 As shown, the target protein in the experimental group is obviously mainly present in the outer membrane component, and the bands in the inner membrane and cytoplasm components are not observable in the gel image, which indicates that the protein is successfully localized on the outer membrane of the cell.

[0061] Example 5: Fluoridase catalytic effect displayed on bacterial surface

[0062] 1. The cells collected after 4 hours of induction according to the method of steps 1-3 of Example 4 were resuspended in Tris-HCl (pH 7.8, 50 mM), and after high-pressure disruption, the disrupted liquid was separated by centrifuge at 6000 rpm to obtain the supernatant soluble component, and the precipitate was resuspended in an equal amount of Tris-HCl. The final cell OD 600 The concentration of 10 was 10, and 980 μl of whole cell suspension, cell disruption solution, disruption supernatant, and disruption precipitate suspension were taken respectively. 20 μl of substrate was added to prepare a mixed reaction solution with a final substrate concentration of SAM (1 mM) and KF (20 mM). Each sample was placed in a 37°C water bath for 1 hour, and then the reaction was terminated in a boiling water bath, and the corresponding product generation amount was measured. Taking into account the differences in protein expression between strains, the proportion of the catalytic capacity of the whole cell component to the catalytic capacity of the disruption component was used to represent the display effect of the fluoridase. The results of cells induced for 4 hours are shown in Figure 2. Figure 6 The catalytic capacity of the whole cell was not significantly improved compared with the control, while the catalytic capacity of the supernatant was significantly lower. Considering that the fluoridase formed precipitation with the large cell fragments and caused the loss of supernatant activity, it was speculated that the fluoridase had been localized in the cell membrane, but it took a certain amount of time to obtain active polymers. Then the changes in the catalytic capacity of the whole cell component to the proportion of the cell lysis solution at different induction times were measured. The results are as follows Figure 7 The percentage of whole-cell catalytic capacity to total catalytic capacity gradually increased with the induction time, which confirmed that the process from intracellular expression to cell surface display of fluorinase was not instantaneous. The surface display reached stability after 22 hours, with the highest efficiency of about 78%.

[0063] 2. Preparation of substrate stock solution: Accurately weigh 0.1 g SAM and dissolve it in 5 mL of KF (1 M) aqueous solution to prepare a substrate stock solution containing 1 M KF and 50 mM SAM. S-adenosylmethionine (SAM) and potassium fluoride (KF).

[0064] Example 6. Stability of the whole-cell catalyst of the engineered Escherichia coli bacteria displaying fluoridase on its surface

[0065] The cells were resuspended in Tris-HCl (pH 7.8, 50 mM) after 4 hours of induction according to the method of steps 1-3 of Example 4, and the OD600 was adjusted to about 10. The cells were stored at 4°C, and the whole cell catalyst was taken every 24 hours to prepare the reaction and determine the changes in the catalytic capacity of the whole cell catalyst at different storage times. The results are shown in FIG. Figure 8 Compared with the traditional crude enzyme solution (i.e., the crushed supernatant of the non-surface displayed control), the whole-cell catalyst of the Escherichia coli engineered bacteria displaying fluoridase on the surface has better stability and can maintain 120% of its initial activity during 7 days of storage.

[0066] Example 7. Whole-cell catalysis of engineered Escherichia coli bacteria displaying fluoridase on their surface

[0067] The cells were resuspended in Tris-HCl (pH 7.8, 50 mM) after 24 h of induction according to the method of steps 1-3 of Example 4. 800 μl of the whole cell suspension was taken and 200 μl of substrate was added to prepare a reaction solution with cell OD600 = 5 and substrates of SAM (1 mM) and KF (200 mM). The product generation was continuously measured at 37°C to identify the catalytic ability of the whole cell. The catalytic effect of the whole cell is shown in FIG. Fig. 9 As shown, the whole-cell catalytic rate was faster in the first 10 hours, and as the substrate concentration decreased, the rate decreased. After 37 hours of catalysis, the product 5'-FDA obtained 0.55 mM, and the yield for SAM was 55%.

[0068] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention. SEQUENCE LISTING <110> Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences <120> A fluoridase mutant and a microbial cell displaying fluoridase on the cell surface and applications thereof <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 298 <212> PRT <213> Synthesis <400> 1 Met Ala Lys Pro Ser Arg Pro Ile Ile Ala Phe Met Ser Asp Leu Gly 1 5 10 15 Ile Thr Asp Asp Ser Val Ala Gln Cys Lys Gly Leu Met Leu Ser Val 20 25 30 Cys Pro Asp Val Thr Ile Val Asp Val Cys His Thr Met Lys Pro Trp 35 40 45 Asp Val Glu Glu Gly Ala Arg Tyr Ile Val Asp Leu Pro Arg Leu Phe 50 55 60 Pro Glu Gly Thr Val Phe Ala Ala Thr Thr Tyr Pro Ala Thr Gly Thr 65 70 75 80 Thr Thr Arg Ser Val Ala Leu Arg Ile Lys Gln Ala Ala Lys Gly Gly 85 90 95 Ala Arg Gly Gln Trp Ala Gly Ser Gly Ala Gly Phe Glu Arg Ala Glu 100 105 110 Gly Ser Tyr Ile Tyr Ile Ala Pro Asn Asn Gly Leu Leu Thr Ser Val 115 120 125 Ile Glu Glu His Gly Tyr Val Glu Ala Tyr Glu Val Ser Ser Thr Glu 130 135 140 Val Ile Pro Glu Gln Pro Glu Pro Thr Phe Tyr Ser Arg Glu Met Val 145 150 155 160 Ala Leu Pro Ala Ala His Leu Ala Ala Gly Phe Pro Leu Glu Lys Val 165 170 175 Gly Arg Pro Leu Ala Asp Asp Glu Ile Val Arg Phe Glu Arg Ala Lys 180 185 190 Pro Ala Gln Asn Asp Asp Gly Glu Leu Val Gly Val Val Thr Ala Ile 195 200 205 Asp His Pro Phe Gly Asn Val Trp Thr Asn Ile His Arg Glu Asp Leu 210 215 220 Glu Lys Leu Gly Ala Gly Tyr Gly Thr Arg Leu Arg Ile Thr Leu Asp 225 230 235 240 Glu Val Leu Pro Phe Asp Leu Pro Leu Ser Pro Thr Phe Ala Asp Ala 245 250 255 Gly Pro Ile Gly Thr Pro Val Ala Tyr Leu Ser Ser Arg Gly Tyr Leu 260 265 270 Ala Leu Ala Arg Asn Ala Ala Ser Leu Ala Tyr Pro Tyr Asn Leu Asn 275 280 285 Ala Gly Ile Ser Val Arg Val Val Ala Ala 290 295 <210> 2 <211> 509 <212> DNA <213> Synthetic <400> 2 ccatgggcat gaacgatgat aaagtgctgg tgctgcgcac atgtgcaaat aatatggcag 60 atcattgcgg tcagatctgg ccagtgagcg gtgttgtgga atgcaaatat tgggaaccga 120 cccgcaaact ggaaaatggc ctggcaggtc tgctgtgggg taaaggtgca agcacgcatc 180 tgaatatgca ggcagatgcc cgttgggtta tttgtgaagt tgcggttagc gatattattt 240 ttctggatgc ccagggtggt gtaaaatttc cgcgtgcaga agttgttcat gttggtacac 300 gtaatagcgc agcaggttat attagtgcaa atattgcgag ttatgcaagc agcacagttg 360 cactgaatga aacatttgtt tttccggaag ttcgtaccga aaccaaagtt gattttcctg 420 cctcaccggc aacagccgat tcaacctttg atattgatcg tcatgcaaca atccagggtc 480 ctcagacact ggaaaccgca gttggatcc 509 <210> 3 <211> 894 <212> DNA <213> Synthetic <400> 3 atggcgaaac ctagccgccc gattattgcg tttatgagcg atctgggcat taccgatgat 60 agcgtggcgc aatgcaaagg cctgatgtta agcgtgtgcc cggatgtgac cattgtggat 120 gtgtgccata ccatgaaacc gtgggatgtg gaagaaggcg cgcgctatat tgttgatctg 180 ccgcgcttat ttcctgaagg caccgtgttt gcggctacca cctatcctgc gaccggtacc 240 actactcgtt cagttgcgct gcgcattaaa caggcggcga aaggtggtgc acgtggtcaa 300 tgggcaggta gcggtgcggg ttttgaacgt gcggaaggca gctatattta tattgcgccg 360 aacaacggct tactgaccag cgtgattgaa gaacatggct atgtggaagc gtatgaagtg 420 agcagcaccg aagtgattcc ggaacagccg gaaccgacct tttatagccg cgaaatggtg 480 gcattaccgg ctgcgcattt agcagcgggc tttcctctgg aaaaagtggg tcgccctctg 540 gcggatgatg aaattgtgcg ctttgaacgc gcaaaaccgg cgcaaaacga tgatggcgaa 600 ctggtgggtg ttgtgaccgc gattgatcat ccgtttggca acgtgtggac caacattcat 660 cgcgaagatc tggaaaaact gggcgcgggc tatggtactc gtctgcgcat taccctggat 720 gaagtgctgc cgtttgatct gcctctgagc cctacttttg cagatgcggg ccctattggt 780 actcctgtgg cgtatttaag cagccgcggc tatttagcgc tggcacgtaa tgcggcgagc 840 ttagcgtatc cgtataacct gaacgcgggc attagcgttc gcgttgttgc agcg 894 <210> 4 <211> 39 <212> DNA <213> Synthetic <400> 4 ggatcttcca gagatgagct catggcgaaa cctagccgc 39 <210> 5 <211> 39 <212> DNA <213> Synthetic <400> 5 ctgccgttcg acgataagct tacgctgcaa caacgcgaa 39 <210> 6 <211> 165 <212> PRT <213> Pseudomonas borealis <400> 6 Met Asn Asp Asp Lys Val Leu Val Leu Arg Thr Cys Ala Asn Asn Met 1 5 10 15 Ala Asp His Cys Gly Gln Ile Trp Pro Val Ser Gly Val Val Glu Cys 20 25 30 Lys Tyr Trp Glu Pro Thr Arg Lys Leu Glu Asn Gly Leu Ala Gly Leu 35 40 45 Leu Trp Gly Lys Gly Ala Ser Thr His Leu Asn Met Gln Ala Asp Ala 50 55 60 Arg Trp Val Ile Cys Glu Val Ala Val Ser Asp Ile Ile Phe Leu Asp 65 70 75 80 Ala Gln Gly Gly Val Lys Phe Pro Arg Ala Glu Val Val His Val Gly 85 90 95 Thr Arg Asn Ser Ala Ala Gly Tyr Ile Ser Ala Asn Ile Ala Ser Tyr 100 105 110 Ala Ser Ser Thr Val Ala Leu Asn Glu Thr Phe Val Phe Pro Glu Val 115 120 125 Arg Thr Glu Thr Lys Val Asp Phe Pro Ala Ser Pro Ala Thr Ala Asp 130 135 140 Ser Thr Phe Asp Ile Asp Arg His Ala Thr Ile Gln Gly Pro Gln Thr 145 150 155 160 Leu Glu Thr Ala Val 165 <210> 7 <211> 298 <212> PRT <213> Amycolatopsis sp. <400> 7 Met Ala Lys Pro Ser Arg Pro Ile Ile Ala Phe Met Ser Asp Leu Gly 1 5 10 15 Ile Thr Asp Asp Ser Val Ala Gln Cys Lys Gly Leu Met Leu Ser Val 20 25 30 Cys Pro Asp Val Thr Ile Val Asp Val Cys His Thr Met Lys Pro Trp 35 40 45 Asp Val Glu Glu Gly Ala Arg Tyr Ile Val Asp Leu Pro Arg Leu Phe 50 55 60 Pro Glu Gly Thr Val Phe Ala Thr Thr Thr Tyr Pro Ala Thr Gly Thr 65 70 75 80 Thr Thr Arg Ser Val Ala Leu Arg Ile Lys Gln Ala Ala Lys Gly Gly 85 90 95 Ala Arg Gly Gln Trp Ala Gly Ser Gly Ala Gly Phe Glu Arg Ala Glu 100 105 110 Gly Ser Tyr Ile Tyr Ile Ala Pro Asn Asn Gly Leu Leu Thr Ser Val 115 120 125 Ile Glu Glu His Gly Tyr Val Glu Ala Tyr Glu Val Ser Ser Thr Glu 130 135 140 Val Ile Pro Glu Gln Pro Glu Pro Thr Phe Tyr Ser Arg Glu Met Val 145 150 155 160 Ala Leu Pro Ser Ala His Leu Ala Ala Gly Phe Pro Leu Glu Lys Val 165 170 175 Gly Arg Pro Leu Ala Asp Asp Glu Ile Val Arg Phe Glu Arg Ala Lys 180 185 190 Pro Ala Gln Asn Asp Asp Gly Glu Leu Val Gly Val Val Thr Ala Ile 195 200 205 Asp His Pro Phe Gly Asn Val Trp Thr Asn Ile His Arg Glu Asp Leu 210 215 220 Glu Lys Leu Gly Ala Gly Tyr Gly Thr Arg Leu Arg Ile Thr Leu Asp 225 230 235 240 Glu Val Leu Pro Phe Asp Leu Pro Leu Ser Pro Thr Phe Ala Asp Ala 245 250 255 Gly Pro Ile Gly Thr Pro Val Ala Tyr Leu Ser Ser Arg Gly Tyr Leu 260 265 270 Ala Leu Ala Arg Asn Ala Ala Ser Leu Ala Tyr Pro Tyr Asn Leu Asn 275 280 285 Ala Gly Ile Ser Val Arg Val Val Ala Ala 290 295

Claims

1. A mutant of fluoridase, characterized in that: The amino acid sequence of the mutant is that the 72nd position of the amino acid sequence shown in SEQ ID NO: 7 is mutated to alanine, and the 164th position is mutated to alanine.

2. A gene encoding the mutant according to claim 1.

3. A recombinant microbial cell, characterized in that The microbial cell contains the encoding gene according to claim 2.

4. The microbial cell according to claim 3, characterized in that The microbial cell is an engineered Escherichia coli bacterium displaying fluoridase on its cell surface. The engineered Escherichia coli bacterium is obtained by taking Escherichia coli as a starting strain and expressing an ice nucleation protein N-terminal encoding gene and the encoding gene according to claim 2. The nucleotide sequence of the ice nucleation protein N-terminal encoding gene is shown in SEQ ID NO:

2.

5. A recombinant vector, characterized in that: The recombinant vector is a recombinant expression vector containing the coding gene according to claim 2 and the ice nuclease protein N-terminal coding gene.

6. Use of the mutant of claim 1, the encoding gene of claim 2, the microbial cell of claim 3 or 4, or the recombinant vector of claim 5 in producing fluoridase.

7. The use according to claim 6, characterized in that: The method for producing fluoridase is as follows: (1) Transfer the engineered E. coli described in claim 4 to 100 mL LB medium at a 1-2% inoculum volume, and wait for the bacterial solution to grow to an OD of 600 = 0.6, add 0.2 mM IPTG, the temperature is 30°C, induce for 2 h-35 h, collect the cells during the induction process, wash with Tris-HCl, and then resuspend; (2) Collect the cells induced for 4 h and resuspend them in PBS. Use a high-pressure crusher to disrupt the cells with the pressure set to 30 Kpsi. Then, remove large cell fragments at 6000 rpm. Centrifuge the collected supernatant at 10,000 g for 1 h to obtain the membrane component precipitate. Remove the supernatant and resuspend the precipitate in PBS containing 2% Triton-X100. Centrifuge again at 10,000 g for 1 h to obtain the cell extracellular membrane precipitate.

8. Use of the mutant according to claim 1, the coding gene according to claim 2, the microbial cell according to claim 3 or 4, or the recombinant vector according to claim 5 in preparing a whole-cell catalyst, characterized in that: The application is to catalyze the reaction of S-adenosylmethionine with potassium fluoride to obtain 5'-FDA.

9. The use according to claim 8, characterized in that: The method for obtaining 5'-FDA is to add the engineered Escherichia coli described in claim 4 to an aqueous solution of S-adenosylmethionine and potassium fluoride, and perform a catalytic reaction at 37°C.

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