Method for preparing florfenicol intermediate through double-enzyme system

Abstract

The invention relates to the field of biological catalysis and enzyme engineering, in particular to a method for preparing a florfenicol intermediate through a double-enzyme system. The invention provides a preparation method of a florfenicol intermediate, which comprises the following steps: by taking p-methylsulfonyl benzaldehyde and threonine as raw materials and L-threonine transaldolase and alcohol dehydrogenase as catalysts, reacting in a water solution system containing a cosolvent to obtain the florfenicol intermediate (2S, 3R)-p-methylsulfonyl phenyl serine. The invention creatively provides a novel double-enzyme system of transaldolase and alcohol dehydrogenase for catalytic synthesis of (2S, 3R)-p-methylsulfonyl phenyl serine. Compared with the existing synthesis route, the production cost of the (2S, 3R)-p-methylsulfonylphenylserine is obviously reduced, the synthesis process is simplified, byproducts such as acetaldehyde are not accumulated, and the (2S, 3R)-p-methylsulfonylphenylserine better meets the requirements of current green industrial production.

Description

Technical Field

[0001] The present application relates to the fields of biocatalysis and enzyme engineering, and in particular to a method for preparing a florfenicol intermediate by a dual enzyme system. Background Art

[0002] Florfenicol is a widely used new generation of chloramphenicol veterinary antibiotics, which has the advantages of rapid absorption, wide distribution in the body, long half-life, no side effects of aplastic anemia, not easy to develop drug resistance, no residue, no cross-resistance, etc. It is mainly used to treat bacterial diseases and mycoplasma infections in cattle, pigs, chickens, ducks, fish and other animals. In recent years, the global demand for florfenicol has increased, and its current market size has exceeded 3 billion yuan.

[0003] (2S,3R)-p-Methylsulfonylphenylserine is a key chiral intermediate in the synthesis of florfenicol, and its structural formula is shown below:

[0004]

[0005] At present, the main methods for large-scale production of (2S, 3R)-p-methylsulfonylphenylserine at home and abroad are chemical synthesis and bio-enzyme catalysis. The chemical synthesis method uses p-methylsulfonylbenzaldehyde and glycine as raw materials. In the presence of copper sulfate and ammonia water, p-methylsulfonylphenylserine is generated through an aldol condensation reaction. After esterification, p-methylsulfonylphenylserine ethyl ester is obtained, and then D-tartaric acid is used to resolve (2S, 3R)-p-methylsulfonylphenylserine. The defects of the chemical synthesis process are: (1) The reaction conditions are severe, strong acids and strong bases are used, and a large amount of copper sulfate wastewater is produced, which makes wastewater treatment difficult. (2) Expensive chiral resolution reagent D-tartaric acid is required. These two defects lead to high costs for chemical synthesis, environmental unfriendliness, and non-green production.

[0006] There are two main routes for the preparation of (2S, 3R)-p-methylsulfonylphenylserine by bioenzyme catalysis. One of the routes is to use L-threonine aldolase to catalyze the condensation reaction of p-methylsulfonylbenzaldehyde and glycine. The reaction formula is as follows:

[0007]

[0008] However, this method has the disadvantages of low conversion rate, large amount of glycine, poor stereoselectivity, and difficulty in separating and purifying the product. The unreacted substrate needs to be recovered and reused, which increases the complexity of the process.

[0009] Patents CN 110540977 B, CN 109836362 A, and CN 110951799 A all adopt another route, that is, using p-methylsulfonylbenzaldehyde and L-threonine as raw materials, synthesizing (2S, 3R)-p-methylsulfonylphenylserine through whole cell or bioenzyme catalysis. The reaction equation is as follows:

[0010]

[0011] The current defects of this route are: (1) To achieve a conversion rate of more than 90%, an overnight reaction is required, which takes a long time. (2) The reaction produces acetaldehyde as a byproduct. In order to reduce the inhibitory effect of acetaldehyde on the reaction, acetaldehyde reductase and NADH coenzyme cycle are required to reduce acetaldehyde to ethanol, which is a three-enzyme system for synthesizing (2S, 3R)-p-methylsulfonylphenylserine, increasing the cost. Summary of the invention

[0012] In view of the shortcomings of the prior art mentioned above, in order to further improve the efficiency of preparing (2S, 3R)-p-methylsulfonylphenylserine, the present application utilizes a dual enzyme system and obtains four highly active transaldehyde enzymes through gene mining, with a conversion rate of more than 99%. Through literature research, an alcohol dehydrogenase that can simultaneously and efficiently catalyze acetaldehyde reduction and NADH regeneration was discovered, and a dual enzyme catalytic system was established, which greatly reduced the enzyme dosage and the production cost of the entire process.

[0013] To achieve the above-mentioned purpose and other related purposes, the first aspect of the present application provides a method for preparing a florfenicol intermediate, which uses p-methylsulfonylbenzaldehyde and threonine as raw materials, L-threonine transaldolase and alcohol dehydrogenase as catalysts, and reacts in an aqueous solution system containing a cosolvent to obtain a florfenicol intermediate (2S, 3R)-p-methylsulfonylphenylserine.

[0014] The second aspect of the present application provides a (2S, 3R)-p-methylsulfonylphenylserine, which is prepared by the aforementioned preparation method.

[0015] The third aspect of the present application provides the aforementioned preparation method or use of the aforementioned (2S, 3R)-p-methylsulfonylphenylserine in the preparation of florfenicol.

[0016] In a fourth aspect, the present application provides an L-threonine aldolase, the amino acid sequence of the L-threonine aldolase is shown in SEQ ID NO:5.

[0017] The fifth aspect of the present application provides the use of the aforementioned L-threonine aldolase in the preparation of a florfenicol intermediate.

[0018] Compared with the prior art, the beneficial effects of this application are:

[0019] 1. This application innovatively provides a new dual enzyme system of transaldolase and alcohol dehydrogenase to catalyze the synthesis of (2S, 3R)-p-methylsulfonylphenylserine. Compared with the current existing technology such as the three-enzyme system synthesis route reported by Suzhou Pilot Company, the production cost of (2S, 3R)-p-methylsulfonylphenylserine is significantly reduced, the synthesis process is simplified, and it also makes it more in line with the needs of today's green industrial production.

[0020] 2. This application does not accumulate by-products such as acetaldehyde, and generates (2S, 3R)-p-methylsulfonylphenylserine in one step, which is more environmentally friendly.

[0021] 3. Compared with the route using glycine in the chemical synthesis method, the (2S, 3R)-p-methylsulfonylphenylserine prepared in the present application has a high conversion rate.

[0022] 4. The preparation method of the present application has a short reaction time and can prepare the target product more quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the mining of new enzymes for L-threonine aldolase. Red represents the template, green and blue represent the newly mined enzymes, and blue represents the most active new enzyme.

[0024] Figure 2 The mass spectrum of the product in Example 5. The molecular weight of (2S, 3R)-p-methylsulfonylphenylserine is 259.05, positive ion mode, M+1, 2M+1. DETAILED DESCRIPTION

[0025] In order to make the invention purpose, technical scheme and beneficial effect of the present application clearer, the present application is further described below in conjunction with the examples. It should be understood that the examples are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present application from the contents disclosed in this description.

[0026] After extensive exploration and research, the inventors of the present application discovered a method for preparing florfenicol intermediates using a dual enzyme system, and completed the present application on this basis.

[0027]

[0028] The first aspect of the present application provides a method for preparing a florfenicol intermediate, using p-methylsulfonylbenzaldehyde and threonine as raw materials, L-threonine transaldolase and alcohol dehydrogenase as catalysts, and reacting in an aqueous solution system containing a cosolvent to obtain a florfenicol intermediate (2S, 3R)-p-methylsulfonylphenylserine. In the reaction, L-threonine transaldolase is used to catalyze the synthesis of p-methylsulfonylbenzaldehyde and threonine (2S, 3R)-p-methylsulfonylphenylserine, and this process will produce a byproduct acetaldehyde, and alcohol dehydrogenase simultaneously catalyzes the NADH cycle regeneration and the consumption of acetaldehyde. The present application pioneeringly provides a new transaldolase and alcohol dehydrogenase dual enzyme system to catalyze the synthesis of (2S, 3R)-p-methylsulfonylphenylserine. Compared with the current prior art three-enzyme system synthesis route, the production cost of (2S, 3R)-p-methylsulfonylphenylserine is significantly reduced, the synthesis process is simplified, and it also makes it more in line with the needs of today's green industrial production. The present invention does not accumulate byproducts such as acetaldehyde, and generates (2S, 3R)-p-methylsulfonylphenylserine in one step, which is more environmentally friendly.

[0029] In the preparation method provided in the present application, the catalyst also includes a coenzyme. The coenzyme includes pyridoxal phosphate (PLP) and nicotinamide adenine dinucleotide (NADH); further, the molar ratios of pyridoxal phosphate and nicotinamide adenine dinucleotide to p-methylsulfonyl benzaldehyde are 0.0005-0.0030:1 and 0.0002-0.0020:1, respectively. In some embodiments, the molar ratio of pyridoxal phosphate to p-methylsulfonyl benzaldehyde can be 0.0005-0.0010:1, 0.0010-0.0015:1, 0.0015-0.0020:1, or 0.0020-0.0030:1, etc. In some embodiments, the molar ratio of nicotinamide adenine dinucleotide to p-methylsulfonylbenzaldehyde can be 0.0002-0.0005:1, 0.0005-0.0010:1, 0.0010-0.0015:1, or 0.0015-0.0020:1, etc.

[0030] In the preparation method provided in the present application, the cosolvent includes isopropanol. The mass fraction of isopropanol in the aqueous solution is 5-15% (w / w); specifically, it can be 5-8% (w / w), 8-10% (w / w), or 10-15% (w / w).

[0031] In the preparation method provided by the present application, the amino acid sequence of L-threonine aldolase is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0032] In some embodiments, the L-threonine transaldolase of the present application is based on the L-threonine transaldolase (SEQ ID No: 1) reported in the literature as a template, and Blast is performed through the databases NCBI and UniProt. In the NCBI database, sequences with identity>50% with the template sequence are downloaded to the local database, and sequences with a length deviation of less than 30 amino acids from the template sequence and identity>40% with the template sequence are selected from the UniProt database and downloaded to the local database. The gene sequences downloaded from the two databases are de-redundant and merged into one database and saved in .fasta format. A phylogenetic tree is constructed by MEGA-X ( Figure 1 ), a total of 20 new genes were discovered by comprehensively considering factors such as kinship, bacterial origin, active sites, conserved sequences, isoelectric points, and the number of cysteine ​​residues in the evolutionary tree.

[0033] Specifically, the amino acid sequence shown in SEQ ID No: 1 is as follows:

[0034] MSNVKQQTAQIVDWLSSTLGKDHQYREDSLSLTANENYPSALVRLTSGSTAGAFYHCSFPFEVPAGEWHFPEPGHMNAIADQVRDLGKTLIGAQAFDWRPNGGSTAEQALMLAACKPGEGFVHFAHRDGGHFALESLAQKMGIEIFHLPVNPTSLLIDVAKLDEMVRRNPHIRIVILDQSFKLRWQPLAEIRSVLPDSCTLTYDMSHDGGLIMGGVFDSPLSCGADIVHGNTHKTIPGPQKGYIGFKSAQHPLLVDTSLWVCPHLQSNCHAEQLPPMWVAFKEMELFGRDYAAQIVSNAKTLARHLHELGLDVTGESFGFTQTHQVHFAVGDLQKALDLCVNSLHAGGIRSTNIEIPGKPGVHGIRLGVQAMTRRGMKEKDFEVVARFIADLYFKKTEPAKVAQQIKEFLQAFPLAPLAYSFDNYLDEELLAAVYQGAQR*。

[0035] The nucleotide sequence (SEQ ID NO: 22) corresponding to the amino acid shown in SEQ ID NO: 1 is as follows:

[0036]

[0037] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 2 is as follows:

[0038] MNQHKHNAGPLIEWLAQTLDQDYQYRQDTLSLTANENYPSELVRLTSGSTAGAFYHCSFPFPMGEWHFPEPGQMNEIADDLRGLARRMLGAQAFDWRPNGGSPAEQAL MLAACKPGEGFVHFAHRDGGHFALEQLASKMGIEIFHLPVDPSSLLIDVARLDDLVRRNPHIRIVILDQSFKLRWQPLAEIRAVLPESCTLTYDMSHDGGLILGGVFDSPL ACGADVVHGNTHKTIPGPQKGYIAFKSAQHPLLVDTSMWVCPHLQSNCHAELLPSMWAAFKEMEVFGMAYAQQMVRNAKALANQLHELGLEVSGESFGFTETHQVHFAVG TLQQALAMCVDSLHAGGIRSTNIEIPGKPGVHGIRLGVQAMTRRGMREDDFRQVAGLVADLYFKRTEPARVAARVKELLAGFPLAPLAFSFDGHIDEACRHLIERGIQR*.

[0039] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 3 is as follows:

[0040] MKQDESNVGPVIDWLAQTLGQDYKYRQDTLSLTANENYPSELVRLTSGSTAGAFYHCSFPFPVPLGEWHFPEPGQMNEIADDLRGLAKRMMGAQAFDWRPNGGSPAEQALMLAACKQGEGFVHFAHRDGGHFALEQLATKMGIEIFHLPVDPQSLLIDVAKLDDMVRRNPHIRIVILDQSFKLRWQPLAEIRAILPDSCTLTYDMSHDGGLILGGVFDSPLACGADIAHGNTHKTIPGPQKGFIAFKSAQHPLLVETSLWVCPHLQSNCHAELLPSMWAAFKEMEAFGPAYAHQMVRNAKALANQLHELGLNVSGESFGFTETHQVHFAVGDLQQALSMCVDSLHAGGIRSTNIEIPGKPGMHGIRLGVQAMTRRGMKEDDFRRVAGLIADLYFKRTEPARVASKVKELLGDFPLAPLAYSFDQQIDESRRRLLERGIQR*。

[0041] The nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 3 (SEQ ID NO: 23) is as follows:

[0042]

[0043] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 4 is as follows:

[0044] MSNVKQQTVQIVDWLSSTLEKDHQYREDSLSLTANENYPSALVRLTSGSTAGAFYHCSFPFAVPAGEWHFPEPGHMNEIADQVRALGKSLIGAQAFDWRPNGGSTAEQAL MLAACKPGEGFVHFAHRDGGHFALESLAQKMGIEIFHLPVDPTSLLIDVAKLDHLVRRNPHIRIVILDQSFKLRWQPLDEIRSVLPDSCTTLYDMSHDGGLIMGGVFDSPL NCGADVVHGNTHKTIPGPQKGYIGFKSDQHPLLVDTSLWVCPHLQSNCHAEQLPPMWVAFKEMEVFGRDYAAQIVSNAKTLARHLHELGLDVTGESFGFTQTHQVHFAVG DLQKALDLCVNSLHAGGIRSTNIEIPGKPGVHGIRLGVQAMTRRGMKEKDFEVVARFIADLYFKKTEPARVALQIKEFLQAFPLAPLAYSFDNYLDEELLATVYRGTQR*.

[0045] The nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 4 (SEQ ID NO: 24) is as follows:

[0046]

[0047] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 5 is as follows:

[0048] MNQDKHNAGPLIEWLAQTLDQDYQYRQDTLSLTANENYPSELVRLTSGSTAGAFYHCSFPFPVPLGEWHFPEPGQMNEIADDLRGLARRMLGAQAFDWRPNGGSPAEQAL MLAACKPGEGFVHFAHRDGGHFALEQLASKMGIEIFHLPVDPRSLLIDVAKLDDMVRRNPHIRIVILDQSFKLRWQPLAEIRAVLPESCALTYDMSHDGGLILGGVFDSPL ACGADAVHGNTHKTIPGPQKGYIAFKSAQHPLLVDTSMWVCPHLQSNCHAELLPSMWAAFKEMEVFGMAYAQQMVKNAKSLAQQLHELGLEVSGESFGFTETHQVHFAVG TLQQALAMCVDSLHAGGIRSTNIEIPGKPGVHGIRLGVQAMTRRGMREDDFRQVAGLVADLYFKRTEPARVANRVKELLAGFPLAPLAYSFDGQIDEAHRHLIERGIQR*.

[0049] The nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 5 (SEQ ID NO: 25) is as follows:

[0050]

[0051] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 6 is as follows:

[0052] MTRTTPQARHVVERLNSVLGQDYRYREDCLSLTANENYPSALVRLAGSATAGAFYHCSFPFEVPPGEWYFPESGRMGELAQQLNELGRSLLGAGTFDWRPNGGSPAEQAL MLAACKHGEGMVHFAHRDGGHFALENLAQKAGIDIFHLPVDPQTLLIDVARLDELVRRNPQIRIVILDQSFKLRWQPLAAIRKVLPPSCTLTYDTSHDGGLIMGGVFDSPL HCGADVIHGNTHKTVPGPQKGYIAFKSAEHPLLVDTSLWLCPHLQSNCHAELLPPMWVAFKEMEAFGHDYAPQVARNAKALAGHLHRLGFEVSGEAFGFTETHQVHFAVG DLQQALDLCMNTLHRGGIRSTNIEIPGKPGIQGIRLGVQAMTRRGLREDDFEQVARFIADLHFRKADPAGVAAQVAEFLRAFPLAPLHYSFDQELDHELLQSLIGEALR*.

[0053] The nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 6 (SEQ ID NO: 26) is as follows:

[0054]

[0055] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 7 is as follows:

[0056] MKQEPTGAFEVATVLNDIFSADHRYREATLSLTANENYPSELVRVTSGSTAGAFYHVSFPFDVPDGEWHFPEPGHMHAVADKVRSLGKSLLHAQTFDWRPNGGSAAEQAL MLAACQPGDGFVHFAHGDGGHFALEALASKAGIEIFHLPVDPDTLLIDVDRLAALVDAHPRIRIVILDQSFKLRWQPLRAIRDALPAHCTLTYDASHDGGLVMGGWFDSPL RCGADIVHGNTHKTIAGPQKAYIAFGSAEHPLLADTSIWVCPNIQSNCHAEQLPSMWVALKEIEVYGLAYASQVVRNAAALARALHARGLDVSGESFGFTETHQVHFSVG TPEDALLTCRDILHRGGIRTTNIELPGKPGMHGIRLGVQAMTRRGMVERDFETAADFIAALCTRKRTPEEVAPDVATFLGDFPLSPLAFSFDSGMTDALRAALRQGVMR*.

[0057] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 8 is as follows:

[0058] MTDFTQAVVNPFVDEQRKGRLAEKISNIFDVLHSDFALDNLYRASHLSLTASENYPSKFVRTMGAGMQGGFYEFAPPYAANPGEWYFPDSGAQGSLVEKLASLGKQLFEANSFDWRPNGGSAAEQAVLLGTCARGDGFVHFAHKDGGHFALEELAQKIGVSIFHLPIEEKSLLIDVNRLASLIKDNPHIKLVILDQSFKLRWQPLLQIRQALPESIVLSYDASHDGGLIIGECLPQPLLFGADIVHGNTHKTIPGPQKGYIAFKDADHPAMKQVSDWVCPHLQSNSHAELIAPMYIALVEMSLYGRSYAEQVIKNAKALAHALHAEGARVSGESFGFTETHQVHVVVGAERKALELVTGTLALAGIRCNNIEIPGANGLFGLRLGVQALTRRGIKEHGMAEVARFLVRLILKNEAPTAIRDEIASFLESYPINTLHYSLDAHYYTPSGIKLMEEVIA*。

[0059] The nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 8 (SEQ ID NO: 27) is as follows:

[0060]

[0061] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 9 is as follows:

[0062] MRAAEQDSGVGLVDDGAQAAVEAAEEIIEEASGGDGGVFAPAWSDDRVAHLLERLTSDFVLDNTYREQHLSLTASENYPSKLVRMLGSALQGGFYEFAPPYPTEAGEWAFPDSGANASLV GKLTGIGRQLFEAATFDWRPNGGSVAEQAVLLGTCGRGDGFVHFAHKDGGHFALESLAAAAGVNTYHLPMVDRTLLIDVDRLATLCAEHPEIKLVILDQSFKLRWQPLAQIRAALPEGVFL AYDASHDGALIAGGALPQPTLLGADAVHGNTHKTIAGPQKAYIAFRDADHPRLRAVSDWVCPQMQSNSHAELIAPMYVALSEIALYGHDYARQILANAKALAHALHEEGVRVSGESFGFT ETHQVHLVTGSAADALRLALGDLAHAGIRTTNIEVPGANGLHGLRLGVQAMTRRGLRETEMREVARLVAKVVLRRAEPTTVRAEVADLLQSHPLDRLAFSFDSHVESPAAARLLGEVFR*.

[0063] The nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 9 (SEQ ID NO: 28) is as follows:

[0064]

[0065] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 10 is as follows:

[0066] MRAAEQDSGIGLVDDEAQAAVVAAEGIIEKASGGDSGGEFGPVWSDDRVAHLLERLASDFVLDNTYREQHLSLTASENYPSKLVRMLGSGLQGGFYEFAPPYPAEAGEWAFPDSGANASL VGKLTGIGRQLFEAATFDWRPNGGSVAEQAVLLGTCGRGDGFVHFAHKDGGHFALEALAGAAGVNTYHLPMVDRTLLVDVDRLATLCAEHPEIKLVILDQSFKLRWQPLAQIRAALPEGVF LAYDASHDGALIAGGALPQPTLLGADAVHGNTHKTIAGPQKAYIAFRDADHPKLRAVSDWVCPQMQSNSHAELIAPMYVALSEIALYGHDYARQILANSKALAHALHEEGVRVSGESFGFT ETHQVHVITGSAADALRLSLGDLAHAGIRTTNIEVPGANGLHGLRLGVQAMTRRGLREAEMREVARLVAKVVLRRAEPTTVRAEVADLLQDHALDRLAYSFDAYVDSPAAARLLGEVFR*.

[0067] The nucleotide sequence (SEQ ID NO: 29) corresponding to the amino acid shown in SEQ ID NO: 10 is as follows:

[0068]

[0069] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 11 is as follows:

[0070] MGLEAVEAEAEAIVEDAVDAEGVWAGDRVAQVLERLASDFVLDNTYREQHLSLTASENYPSKLVRMLGAGLQGGFYEFAPPYPAEAGEWAFPDSGANASLVGKLTGIGRQLFEAA TFDWRPNGGSVAEQAVLLGTCGRGDGFVHFAHKDGGHFALESLAGAAGINTYHLPMVDRTLLIDVDRLATLCAEHPEIKLVILDQSFKLRWQPLAQLRAALPAGVFLAYDASHDGA LIAGGALPQPTLLGADAVHGNTHKTIAGPQKAYIAFRDAEHPKLKAVSDWVCPQMQSNSHAELIAPMYVALSEVALYGHAYARQILANAQALAHGLHEEGVRVSGESFGFTETHQV HVVTGSAADALRLSLGELARAGIRTTNIEVPGANGLHGLRLGVQAMTRRGLREPQMREVARLVAKVVLRRAEPAAVRAEVADLLQHHPLDRLAYSFDSYVDSPAAARLLGEVFR*.

[0071] The nucleotide sequence (SEQ ID NO: 30) corresponding to the amino acid shown in SEQ ID NO: 11 is as follows:

[0072]

[0073] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 12 is as follows:

[0074] MVDDGTVLVDDEAKAAVEAAEGIIEEASGSDDGTFGEAAWADDRVAHLLERLTSDFVLDNTYREQHLSLTASENYPSRLVRMLGSGLQGGFYEFAPPYQAEAGEWAFPDSGANASLVGK LTGIGRQLFEAATFDWRPNGGSVAEQAVLLGTCGRGDGFVHFAHKDGGHFALESLAAAAGVNTYHLPMVDRTLLIDVDRLATLCAEHPEIKLVILDQSFKLRWQPLAQIRAALPEGVFLA YDASHDGALIAGGALPQPTLLGADAVHGNTHKTIAGPQKAYIAFRDAEHPKLKAVSDWVCPQMQSNSHAELIAPMYVALSEIALYGHDYARQILANAKALAHGLHEEGVRVSGESFGFTE THQVHVVTGSAADALRLSLGDLAHAGIRTTNIEVPGANGLHGLRLGVQAMTRRGLREAEMREVARLVAKVVLRRAEPTAVRAEVADLLQHHTLDQLAYSFDAYVDSPAAARLLGEVFR*.

[0075] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 13 is as follows:

[0076] MAVYDRAGASSLRSARADQLPTETHAGDGKLLGWLAGVLAQDRHYHTVMATLTASENYPSAVVRAAAAFFGGEHYFFDAPSPGHCGEWSFPRSATLPALQTKFLAQMKRLFAADIADWRPNGGSMCEQAVVMAACKGGDAFVELAPEDGGHFGASVLAERLGIESFAFPMHGDLIDVEGTVRLVKEHPSIRLLLVQPSHCRRPQPIEELASAMPRQIVLAVDVSHTAGLIAAGLLPQPLLQGADILTFNTHKTLPGPNKGVIAFADGDHPLAHTMWETVCPQLQSNSHAECLPGLVIALEEIANFGRAYGEQVIANARTLAHVLDSNGLSVPGMEYGGTETHQVHVRLGAAEVANRVANELLPTCGIRTNSVLIPGTGGEFGLRLGTQALTRRGLTEIEFAKLGRLLARAVRWTADVGQIRHEVAELLADHPLFPLRFSFDESAYGQQLERMLGVVLR*。

[0077] The nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 13 (SEQ ID NO: 31) is as follows:

[0078]

[0079] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 14 is as follows:

[0080] MKISTKTNNKGSVYNRTTPVKSSSLEPKIAKFFGAVTQHLASDNLYRKDMLSLTASENYQSFLVRMLSSSHNSSFYSFIPPYNVESAGEEWFFPDSDSRKEITALLRETGKAMFHS QTFDERPNGGSGAEQAVLLATCNRGDGWLHFSAQDGGHFGNEALSIKAGLEEHHFPVDPTTGLIKIEELNTILKKHQNIKLLLLDQSHKLRYQPLAEIKKLLQPDVLLAYDCSHDM GLIVGGSLPQPLLEGADVLIGNTHKTFPSVNKAIIAFADKNHPLFKPVSDWVCPFLQSNSHHELTLPLLISLMEMKVFGKDYAEQTVKNAKAFAKELREQGFNVSGEHFCYTKTHQ VHLILDSRQEALKAVSELLPSAGIRGNNIMIPGHNAFGLLRGTQAITRRGMKEEEMREVAKFLAKVLLKKENPTKIKAEVKELLMDFSLFPLAYSFDFLVDDDLGKSLLMEVLSK*.

[0081] The nucleotide sequence corresponding to the amino acid shown in SEQ ID NO: 14 (SEQ ID NO: 32) is as follows:

[0082]

[0083] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 15 is as follows:

[0084] MKFDETKNNLIENNYSLFDELSGLFAIDFNYRQNTLSLTASENYPSLFVRILGSALCGGFYYFDPPYKADPGEWFFPDSGIMDAVAEKLKTLGSVLFEGQTFDWRPNGGSSA EQAVLLGICERGDGFVHFAHKDGGHFGLEELAKKIGINIYHFPMIEKTLLIDVERLKIMIQDHPDIKLVIFDQSFKLRWQPILKISEELPDIITLSYDCSHDGGLIAGNLFP QPLCQGIHILHGNTHKTIPGPQKGFISFADQNHPRLASVSNWVCPYMQSNSHAELIAPMLIAFSELAYFGKNYAKQITKNAKAFAIALAAEGFFVSGESFGYTETHQVHLIL GSKEKSLNLVSSLLPEAGIRVNNVEIPGSNGSFGLRFGTQAMTRRGMKEDDFQEVARILSDLILKNKSPLEVRYEVREMLKKFLLFPLKYSFDDLLYNELGNQLLREVLK*.

[0085] In some embodiments, the amino acid sequence as shown in SEQ ID NO: 16 is as follows:

[0086] MLSLTDIEDSPTEVSKMESVCDALAADFSRDNCYRSAHLSMTASENYPSKFVRAMGGAMQGAFYEFAPPYSTA--EGEWHFPDSGAQAALVQKLAQLGCQMFQAQTFDWRPNGGAAAEQAVLLGTCSRGDAFVHFGHQDGGHSSLEELARKIGIGIFHIPVQEDSLLIDVQRLAELLAAQPEIKLVILDQSFKLRWQPLAEIREIMPPTVTLAYDASHDGGLILGGVLPQPLAAGADIVLGNTHKTIPGPQKGYVAFFNARHPVLKPVSDWICPYMQSNSHAELLVPFYMALVEISLFGRPYAQQIVKNAKALALCMHAEGLRVVGESFGYTETHQVLVSIGSPGEALHAAAHLLALAGIRCNNIEIPGSGGRHGLRFGVQALTRRGIQEIDMAVIARFIARVLVRKEPPELVRNEVALFLRSFPLHPLAFSLDSQYEEPHAIRLREE*。

[0087] In some embodiments, the amino acid sequence as set forth in SEQ ID NO: 17 is as follows:

[0088] MRVAEQDRGAGPKPEAVGLEAVEAEVEAIVEDAAGAEGVWADDRVAQVLERLASDFVLDNTYREQHLSLTASENYPSKLVRMLGAGLQGGFYEFAPPYPAEAGEWAFPDSGANASLVGKLTGIGRQLFEAATFDWRPNGGSVAEQAVLLGSCGRGDGFVHFAHKDGGHFALESLAGAAGINTYHLPMVDRTLLIDVDRLATLCAEHPEIKLVILDQSFKLRWQPLAQIRAALPEGVFLAYDASHDGALIAGGALPQPTLLGADAVHGNTHKTIAGPQKAYIAFRDAEHPKLKAVSDWVCPQMQSNSHAELIAPMYVALSEVALYGHAYARQILANAQALAHGLHEEGVRVSGESFGFTETHQVHVVTGSAADALRLSLGELARAGIRTTNIEVPGANGLHGLRLGVQAMTRRGLREPQMREVARLVAKVVLRRAEPAAVRAEVADLLQHHPLDRLAYSFDSYVDSPAAARLLGEVFR*。

[0089] In some embodiments, the amino acid sequence shown in SEQ ID NO: 18 is as follows:

[0090] MRVAEQDRGAGPKAEAAAVEVEAEAIVEDAAGAEGVWVDDRVAQVLERLASDFVLDNTYREQHLSLTASENYPSKLVRMLGAGLQGGFYEFAPPYPAEAGEWAFPDSGANASLVGKLTGIGRQLFEAATFDWRPNGGSVAEQAVLLGSCGRGDGFVHFAHKDGGHFALESLAGAAGINTYHLPMVDRTLLIDVDRLATLCAEHPEIKLVILDQSFKLRWQPLAQIRAALPAGVFLAYDASHDGALIAGGALPQPTLLGADAVHGNTHKTIAGPQKAYIAFRDAEHPKLKAVSDWVCPQMQSNSHAELIAPMYVALSEVALYGHAYARQILANAQALAHGLHEEGLRVSGESFGFTETHQVHVVTGSAADALRLSLGELARAGIRTTNIEVPGANGLHGLRLGVQAMTRRGLREPQMREVARLVAKVVLRRAEPAAVRAEVADLLQHHPLDRLAYSFDSYVDSPAAARLLGEVFR*。

[0091] In some embodiments, the amino acid sequence set forth in SEQ ID NO: 19 is as follows:

[0092] MRVAEQDRGAGPKPEAVGLEAVEAEVEAIVEDVAGAEGVWAGDRVAQVLERLASDFVLDNTYREQHLSLTASENYPSKLVRMLGAGLQGGFYEFAPPYPAEAGEWAFPDSGANASLVGKLTGIGRQLFEAATFDWRPNGGSVAEQAVLLGTCGRGDGFVHFAHKDGGHFALESLAGAAGVNTYHLPMVDRTLLIDVDRLATLCAEHPEIKLVILDQSFKLRWQPLAQIRAALPEGVFLAYDASHDGALIAGGVLPQPTLLGADAVHGNTHKTIAGPQKAYIAFRDAEHPKLRAVSDWVCPQMQSNSHAELIAPMYVALSEVALYGHAYARQILANAQALAHGLHEEGVRVSGESFGFTETHQVHVVTGSAADALRLSLGELAQAGIRTTNIEVPGANGLHGLRLGVQAMTRRGLREPQMREVARLVAKVVLRRAEPAAVRAEVADLLQHHPLDQLAYSFDSYVDSPAAARLLGEVFR*。

[0093] In some embodiments, the amino acid sequence shown in SEQ ID NO: 20 is as follows:

[0094] MQVAEQGRDIGLADAEARAAAAEAEEIVEEAA-EEHGDVLGPVRPDDRVAHLLERLASDFVLDNTYREQHLSLTASENYPSRLVRMLGSGLQGGFYEFAPPYPSE--AGEWAFPDSGANASLVGKLTGIGRQLFEAATFDWRPNGGSVAEQAVLLGACGRGDGFVHFAHKDGGHFALESLAAAAGVNTYHLPMVNRTLLIDVDRLATLCAEHPEIKLVILDQSFKLRWQPLAQIRAALPEGVFLAYDASHDGALIAGGALPQPALLGADAVHGNTHKTIAGPQKAYIAFRDADHPKLRAISDWVCPRMQSNSHAELIAPMYVALSEIALYGHDYARQIVANATALAHALHEEGVRVSGESFGFTETHQVHIVTGSAADALRLSLGDLARAGIRTTNIEVPGANGLHGLRLGVQAMTRRGMREAEMREVARLVAKVVLRRAEPAAVRAEVADLLRHHPLDRLAYSFDAHVDSPAAARLLGEVFR*。

[0095] In some embodiments, the amino acid sequence set forth in SEQ ID NO: 21 is as follows:

[0096] MRAAEQDSDIGVVDDEAQAAVVAAEEIIDEASGGDGGVFGHAWSDDRVAHLLERLASDFVLDNTYREQHLSLTASENYPSKLVRMLGSGLQGGFYEFAPPYPAEAGEWAFPDSGANASLVGKLTGIGRQLFEAATFDWRPNGGSVAEQAVLLGTCGRGDGFVHFAHKDGGHFALESLAAAAGINTYHLPMVDRTLLIDVDRLATLCGEHPEIKLVILDQSFKLRWQPLAQIRAALPEGVFLAYDSSHDGALIAGGVLPQPTLLGADAVHGNTHKTIAGPQKAYIAFRDADHPKLRAVSDWVCPQMQSNSHAELIAPMYVALSEIALHGHDYARQIVANAKVLAQALHEEGVRVSGESFGFTETHQVHVITGSAADALRLSLGDLAHAGIRTTNIEVPGANGLHGLRLGVQAMTRRGLREAEMREVARLVAKVVLRRAEPTAVRAEVADLLRHHPLDELAYSFDAHVDSPAAARLLGEVFR*。

[0097] In the preparation method provided by this application, the amino acid sequence of alcohol dehydrogenase is as shown in SEQ ID NO: 33. Specifically, the sequence shown in SEQ ID No: 33 is as follows:

[0098] MKSLQYTRIGAGPELTEIPKPEPGPGEVLLEVTAAGVCHSDLFIMSLPEEQYTYGLPLTLGHEGAGRVAAVGEGVEGLDIGTNVVVYGPWGCGNCWHCSQGLENYCSRAQELGINPPGLGAPGAMAEFMIVDSPRHLVPIGDLDPVKTVPLTDAGLTPYHAIKRSLPKLRGGSYAVVIGIGGLGHVAIQLLRHLSAATVIALDVSADKLELATKVGAHEVVLSDKDAAENVRKITGSQGAALVLDFVGYQPTIDTAMAVAGVGSDVTIVGIGDGQAHAKVGFFQSPYEASVTVPYWGARNELIELIDLAHAGIFDISVETFSLDNGVEAYRRLAAGTLSGRAVVVPGL。

[0099] In the preparation method provided in the present application, the reaction temperature is 25-35°C; specifically, it can be 25-30°C, 30-33°C, or 33-35°C, etc.

[0100] In the preparation method provided in the present application, the reaction time is 15 to 24 hours; specifically, it can be 15 to 18 hours, 18 to 20 hours, or 20 to 24 hours, etc. The preparation method of the present application has a short reaction time and can prepare the target product more quickly.

[0101] In the preparation method provided in the present application, the molar ratio of p-methylsulfonylbenzaldehyde to threonine is 1.0-1.6:1.0; specifically, it can be 1.0-1.2:1.0, 1.2-1.4:1.0, or 1.4-1.6:1.0, etc.

[0102] In the preparation method provided in the present application, the weight proportion of L-threonine aldolase is 1-2% (w / w) based on the weight of the aqueous solution system; specifically, it can be 1-1.2% (w / w), 1.2-1.5% (w / w), or 1.5-2% (w / w), etc. In a specific embodiment of the present application, L-threonine aldolase is first fermented to prepare bacterial mud, and then the bacterial mud is resuspended to prepare a crude enzyme solution, and part of the crude enzyme solution is taken out for actual preparation. Based on the weight of the crude enzyme solution of L-threonine aldolase, the crude enzyme solution of L-threonine aldolase accounts for 10-20% (w / w); specifically, it can be 10-12% (w / w), 12-15% (w / w), or 15-20% (w / w), etc. The role of L-threonine aldolase is to catalyze L-threonine and p-methylsulfonyl benzaldehyde to generate (2S, 3R)-p-methylsulfonyl phenylserine with two chiral centers.

[0103] In the preparation method provided in the present application, the weight proportion of alcohol dehydrogenase is 0.5-1.5% (w / w) based on the weight of the aqueous solution system; specifically, it can be 0.5-0.8% (w / w), 0.8-1.0% (w / w), 1.0-1.2% (w / w), or 1.2-1.5% (w / w), etc. In a specific embodiment of the present application, alcohol dehydrogenase is first fermented to prepare bacterial mud, and then the bacterial mud is resuspended to prepare a crude enzyme solution, and part of the crude enzyme solution is taken out for actual preparation. Based on the weight of the crude enzyme solution of alcohol dehydrogenase, the crude enzyme solution of alcohol dehydrogenase accounts for 5-15% (w / w); specifically, it can be 5-8% (w / w), 8-10% (w / w), or 10-15% (w / w), etc. In a specific embodiment of the present application, when the cosolvent is isopropanol, the alcohol dehydrogenase is selected from isopropanol dehydrogenase. Isopropanol dehydrogenase can catalyze the reduction of acetaldehyde and the dehydrogenation of isopropanol at the same time, collectively referred to as alcohol dehydrogenase; specifically, 1. Isopropanol dehydrogenase catalyzes the reduction reaction of acetaldehyde to produce ethanol, which requires the consumption of NADH to ensure that acetaldehyde is completely converted; 2. Isopropanol dehydrogenase catalyzes isopropanol to produce acetone and NADH, which can be bubbled away in the system, and NADH can be used for the conversion of acetaldehyde. No byproducts such as acetaldehyde are produced, and (2S, 3R)-p-methylsulfonylphenylserine is produced in one step, which is more green and scientific.

[0104] The second aspect of the present application provides a (2S, 3R)-p-methylsulfonylphenylserine, which is prepared by the above-mentioned preparation method. Compared with the chemical synthesis method and the glycine synthesis route, the (2S, 3R)-p-methylsulfonylphenylserine prepared by the method of the present application has a high conversion rate.

[0105] The third aspect of the present application provides the aforementioned preparation method or the use of the aforementioned (2S, 3R)-p-methylsulfonylphenylserine in the preparation of florfenicol. Florfenicol can be widely used as a new generation of chloramphenicol veterinary antibiotics, which has the advantages of rapid absorption, wide distribution in the body, long half-life, no side effects of aplastic anemia, not easy to produce drug resistance, no residue, no cross-resistance, etc., and is mainly used to treat bacterial diseases and mycoplasma infections in animals such as cattle, pigs, chickens, ducks, and fish.

[0106] In a fourth aspect, the present application provides an L-threonine aldolase, the amino acid sequence of which is shown in SEQ ID NO: 5. The present application unexpectedly discovered through gene mining that the amino acid sequence shown in SEQ ID NO: 5 also has the function of L-threonine aldolase, and can catalyze p-methylsulfonylbenzaldehyde and threonine to generate (2S, 3R)-p-methylsulfonylphenylserine.

[0107] The fifth aspect of the present application provides a biomaterial selected from any of the following:

[0108] 1) The biological material is a polynucleotide encoding the amino acid of the aforementioned L-threonine aldolase;

[0109] 2) The biological material is a nucleic acid construct, and the nucleic acid construct contains the polynucleotide of 1);

[0110] 3) The biological material is a host cell, which includes the construct of 2) or the polynucleotide of 1) integrated into its genome.

[0111] The polynucleotides of the present application include polynucleotides obtained by mutating and / or modifying the bases at one or more positions on the basis of SEQ ID NO: 5. Such mutations or modifications include codon optimization, degenerate base design, methylation modification, etc. The approaches to achieve such modifications or mutations are well known in the art, including but not limited to: insertion, deletion, error-prone PCR, reconnection of different sequences, directed evolution of different parts of the sequence with homologous sequences from other sources, and chemical reagent mutagenesis.

[0112] The recombinant vector of the present application includes a recombinant expression vector or a recombinant cloning vector. The vector involved in the recombinant expression vector may be a vector for gene expression known in the art, including but not limited to pEAQ-HT, pSuper-1300, pET-28a, pET30a, pET24b, pESC-Ura, pESC-Trp, pESC-Leu, pESC-His, pGEX2T, pTAex3, and pYMB03.

[0113] Wherein expression refers to any step involved in the production of a polypeptide, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion. An expression vector refers to a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to a regulatory sequence that provides for its expression.

[0114] The nucleic acid constructs involved in the present application include the polynucleotides of the present application operably linked to one or more regulatory sequences, which direct the expression of the coding sequence in a suitable host cell. The regulatory sequence and the polynucleotide may be homologous or heterologous.

[0115] A regulatory sequence refers to a nucleic acid sequence that is necessary for the expression of a polynucleotide encoding a polypeptide. The regulatory sequence and the polynucleotide encoding the polypeptide may be homologous (i.e., both are from the same gene) or heterologous (i.e., both are from different genes). Such regulatory sequences include, but are not limited to, leader sequences, promoters, signal peptide sequences, transcription terminators, enhancers, and the like. At a minimum, the regulatory sequence includes a promoter, and transcription and translation termination signals. The control sequence may be connected to the polynucleotide encoding the polypeptide via a linker.

[0116] The application also relates to recombinant host cells, these host cells comprise the polynucleotide of the application that are operably connected to one or more regulating and controlling sequences, and these one or more control sequences regulate the generation of the L-threonine transaldolase of the application.Construct or the vector that will comprise the polynucleotide are introduced into the host cell, and construct or the vector are either integrated on the host chromosome, or autonomously replicated without integration.These host cells also comprise any parental cell offspring that is not identical with the parental cell due to sudden change during replication.The selection of host cell will depend on gene and source thereof of the polypeptide encoding.

[0117] The host cell can be any microbial cell that can be used to produce the polypeptide of the present invention. Such as a prokaryotic cell or a eukaryotic cell. The prokaryotic host cell can be any Gram-positive or Gram-negative bacteria. Microbial cells include, but are not limited to, Streptomyces, Pseudomonas, Bacillus, yeast cells, and Escherichia coli.

[0118] Methods for introducing expression vectors into host cells are known in the art and include, but are not limited to, electroporation, polyethylene glycol (PEG) transformation, lipofection, heat shock, calcium phosphate precipitation, virus-mediated, and microinjection.

[0119] The present application includes a method for producing the L-threonine aldolase of the present application, which comprises the following steps: under conditions conducive to the expression of L-threonine aldolase, a recombinant host cell containing a polynucleotide encoding the L-threonine aldolase of the present application is expressed to obtain the L-threonine aldolase.

[0120] The present application includes a method for producing the L-threonine aldolase of the present application, which includes culturing the recombinant host cell of the present application under conditions conducive to the production of L-threonine aldolase to obtain an L-threonine aldolase peptide. The method for producing the L-threonine aldolase of the present application may also include a step of recovering the L-threonine aldolase.

[0121] Can be by shake flask culture or in laboratory or industrial fermenter small-scale or large-scale fermentation (comprising continuous, batch, fed-batch or solid-state fermentation) culture cell, this culture is carried out in suitable substratum and under the condition that allows to express and / or separate L-threonine transaldolase.If L-threonine transaldolase is secreted in this nutrient medium, can directly reclaim this L-threonine transaldolase from this substratum so.If L-threonine transaldolase is not secreted, it can be reclaimed from cell lysate so.

[0122] L-threonine aldolase can be detected by methods known in the art, including but not limited to: specific antibody detection and enzyme activity detection.

[0123] The sixth aspect of the present application provides the use of the aforementioned L-threonine aldolase in the preparation of a florfenicol intermediate. The present application has found the amino acid sequence shown in SEQ ID NO: 5 through gene mining, and for the first time discloses that after expression, it has the function of L-threonine aldolase, and the enzyme activity is higher than the existing L-threonine aldolase with the amino acid sequence shown in SEQ ID NO: 1, which can catalyze the conversion of p-methylsulfonylbenzaldehyde and threonine into the florfenicol intermediate (2S, 3R)-p-methylsulfonylphenylserine.

[0124] The present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0125] Example 1

[0126] Gene mining.

[0127] The L-threonine transaldolase (SEQ ID No: 1) reported in the literature was used as a template for Blast analysis using the NCBI and UniProt databases. Sequences with identity > 50% with the template sequence in the NCBI database were downloaded to the local database. Sequences with a length deviation of less than 30 amino acids and identity > 40% with the template sequence in the UniProt database were downloaded to the local database. The gene sequences downloaded from the two databases were de-redundant and merged into one database and saved in .fasta format. A phylogenetic tree was constructed using MEGA-X ( Figure 1 ), a total of 20 new genes were discovered by comprehensively considering factors such as kinship, bacterial origin, active sites, conserved sequences, isoelectric points, and the number of cysteine ​​residues in the evolutionary tree.

[0128] Example 2

[0129] Strain construction and expression.

[0130] In the present invention, enzyme 1 mined from the database is derived from Pseudomonas (SEQ ID NO: 2), enzyme 2 is derived from Pseudomonas sp. Irchel s3a18 (SEQ ID NO: 3), enzyme 3 is derived from Pseudomonas mandelii (SEQ ID NO: 4), enzyme 4 is derived from Pseudomonas sp. SK (SEQ ID NO: 5), enzyme 5 is derived from Chitiniphilus shinanonensis (SEQ ID NO: 6), enzyme 6 is derived from Burkholderia multivorans (SEQ ID NO: 7), enzyme 7 is derived from Burkholderia lata (SEQ ID NO: 8), enzyme 8 is derived from Streptomyces sp. DI166 (SEQ ID NO: 9), enzyme 9 is derived from Streptomyces davaonensis (SEQ ID NO: 10), enzyme 10 is derived from Streptomyces sp. CS227 (SEQ ID NO: 11), enzyme 11 is derived from Streptomyces sp. SID13726 (SEQ ID NO: 12), and enzyme 12 is derived from Solirubrobacterales bacterium (SEQ ID NO: 13), enzyme 13 is derived from Oscillatoriales cyanobacterium USR001 (SEQ ID NO: 14), enzyme 14 is derived from Limnospira fusiformis SAG 85.79 (SEQ ID NO: 15), enzyme 15 is derived from Xylophilus rhododendri (SEQ ID NO: 16), enzyme 16 is derived from Streptomyces albidoflavus (SEQ ID NO: 17), enzyme 17 is derived from Streptomyces albidoflavus (SEQ ID NO: 18), enzyme 18 is derived from Streptomyces (SEQ ID NO: 19), enzyme 19 is derived from Streptomyces spp. PsTaAH-137 (SEQ ID NO: 20), enzyme 20 is derived from Streptomyces aureus (SEQ ID NO: 21), enzyme 21 is derived from Streptomyces spp. PsTaAH-137 (SEQ ID NO: 22), enzyme 21 is derived from Streptomyces spp. PsTaAH-137 (SEQ ID NO: 23), enzyme 22 is derived from Streptomyces spp. PsTaAH-137 (SEQ ID NO: 24), enzyme 23 is derived from Streptomyces spp. PsTaAH-137 (SEQ ID NO: 25), enzyme 24 is derived from Streptomyces spp. PsTaAH-137 (SEQ ID NO: 26), enzyme 25 is derived from Streptomyces spp. PsTaAH-137 (SEQ ID NO: 27), enzyme 26 is derived from Streptomyces spp. PsTaAH-137 (SEQ ID NO: 28), enzyme 27 is derived from Streptomyces spp. PsTaAH-137 (SEQ ID NO: 29), enzyme 28 NO: 21), all genes were obtained by gene synthesis, and each gene was connected between NcoI and XhoI in the vector pET28a. The constructed plasmid was transferred into the Escherichia coli BL21 (DE3) strain and cultured on plates. Finally, a single clone was selected to verify the correct sequence and then liquid culture was performed step by step.First, transfer to 3 ml of LB culture medium (37°C) containing 50 μM kanamycin for overnight culture, and then transfer 1% to 250 ml of LB culture medium containing 50 μM kanamycin. When the cell OD is about 0.6, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 30°C to induce protein expression for 20 hours; collect the bacteria by centrifugation at 6000 rpm for 20 min.

[0131] Example 3

[0132] Enzyme activity detection and strain screening.

[0133] Enzyme activity is calculated from HPLC test results. Enzyme activity definition: The amount of enzyme required to convert 1 μmol of substrate in 1 min at 30°C is 1 U.

[0134] Preparation of 2× transaldehyde reaction solution: weigh 589.47 mg of p-methylsulfonylbenzaldehyde, dissolve it in DMSO and make it dilute to 20 mL to prepare solution A; weigh 79.04 mg PLP and 381.184 mg L-threonine, use 100 mM pH 6.5 PBS Buffer to make it dilute to 380 mL to prepare solution B; mix the above solution A and solution B in a ratio of 1:19 to obtain the 2× transaldehyde reaction solution.

[0135] The concentrations of p-methylsulfonylbenzaldehyde and L-threonine are both 8mM. The aldehyde transfer reaction solution should be prepared before use and dispensed into the reaction as soon as possible after preparation. If left unused, it will easily precipitate and affect the test results.

[0136] Preparation of crude enzyme solution: accurately weigh 40 mg of bacterial mud after induction expression of L-threonine transaldehyde enzyme (i.e., the bacterial body after centrifugation in Example 2), add 2 mL 100 mM pH 6.5 PBS Buffer to resuspend (bacterial mud concentration 20 mg / mL), then dilute 4 times, the bacterial body concentration is 5 mg / mL, take 1 mL of the above resuspended bacterial solution under ice bath conditions for 3 min (ultrasonic crushing on / off = 1 / 4 s), 4 ° C, 12000 rpm centrifugation for 2 min, the supernatant is the crude enzyme solution to be detected. Reaction system construction: take a 2 mL round-bottom ep tube, add 0.2 ml of crude enzyme solution supernatant, 0.5 mL of 2 × transaldehyde reaction solution, 0.3 mL of 100 mM pH 6.5 PBS Buffer, the total reaction volume is 1 mL, three groups of parallel controls, shake and react for 30 min at 30 ° C and 220 rpm in a shaker. After the reaction, 0.2 mL of 3 M HCl was added to the sample and shaken for 10 min to quench the reaction, followed by adding 0.2 mL of 3 M NaOH to adjust the pH, and finally adding 0.6 mL of 100 mM pH 6.5 PBS Buffer to a total volume of 2 mL. The sample was centrifuged and filtered and then detected by HPLC, and the specific enzyme activity was calculated based on the amount of product detected.

[0137] The 20 enzymes after induced expression were tested according to the enzyme activity determination method as described above, and 4 recombinant bacteria with activities greater than 10 U / g bacterial mud were obtained. The results are shown in Table 1.

[0138] Table 1. Bacteria strains with detectable activity and their unit bacterial activity

[0139]

[0140]

[0141] Example 4

[0142] Fermentation amplification of dominant strains

[0143] 3 μl of the L-threonine aldolase bacterial cell (E1) shown in SEQ ID NO: 5 was taken, transferred into 3 ml of LB culture solution (37° C.) containing 50 μM kanamycin for overnight culture, 1% was transferred into 250 ml of LB culture solution containing 50 μM kanamycin, and finally transferred into a 10 L culture fermenter for culture; when the cell OD was ~ 15, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added at 30° C. to induce protein expression for 20 hours, and finally the cells were collected by high-speed centrifugation (6000 rpm, 15 min) to obtain 150-180 g of wet cells, which were stored at -20° C. for use.

[0144] 3 μl of the alcohol dehydrogenase glycerol bacteria (E2, the host is BL21 (DE3), which can catalyze acetaldehyde reduction and isopropanol dehydrogenation reactions) shown in SEQ ID NO: 33 was taken and transferred into 3 ml of LB culture solution containing 50 μM kanamycin (37° C.) for overnight culture, and 1% was transferred into 250 ml of LB culture solution containing 50 μM kanamycin, and finally transferred into a 10L culture fermenter for culture; when the cell OD was ~ 15, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added at 30° C. to induce protein expression for 20 hours, and finally the cells were collected by high-speed centrifugation (6000 rpm, 15 min) to obtain 150-180 g of wet cells, which were stored at -20° C. for use.

[0145] Example 5

[0146] Enzyme catalysis process optimization

[0147] Catalytic reaction of the novel enzyme (SEQ ID NO: 5)

[0148] Preparation of E1 enzyme solution: Weigh 10 g of E1 bacterial sludge in Example 4 of SEQ ID NO: 5, add 100 ml of (PBS) buffer (50 mM, pH 7.0), stir for 30 min to mix evenly, use a homogenizer to homogenize and break the cells at 600 bar, add 3 ml of PEI flocculant, centrifuge at 4000 rpm for 15 min, and obtain 100 ml of supernatant as E1 enzyme solution.

[0149] Preparation of E2 enzyme solution: Weigh 10 g of the E2 bacterial sludge in Example 4, add 100 ml of (PBS) buffer (50 mM, pH 7.0), stir for 30 min to mix evenly, use a homogenizer to homogenize and break the cells at 600 bar, add 2 ml of PEI flocculant, centrifuge at 4000 rpm for 15 min, and obtain 100 ml of supernatant as E2 enzyme solution.

[0150] Add 184g water and 25.5g isopropanol to a 500ml three-necked flask, add 20mg pyridoxal phosphate, 43mg nicotinamide adenine dinucleotide, and 11.7g threonine under stirring, stir for 5min to dissolve, add 15g p-methylsulfonylbenzaldehyde, 45.0ml E1 enzyme solution, and 22.5ml E2 enzyme solution. Stir and react, control the temperature at 33℃ and pH=6.4, use a small fish pump to blow air, react for 18h, the molar conversion rate is >95%, the diastereomeric excess de value is 97.8, and the mass spectrometry results are shown in Figure 2 The molar conversion rate is calculated by measuring the sample by HPLC using the external standard method, calculating the mass of the product according to the HPLC result, and calculating the mass of the product and the mass of the added substrate; specifically, it refers to M(product) / 259.28 / (M(p-methylsulfonylbenzaldehyde) / 184.21)*100%.

[0151] Comparative Example 1

[0152] Wild-type enzyme catalyzed reactions

[0153] Preparation of E1 enzyme solution: Weigh 10 g (SEQ ID NO: 1) of bacterial sludge prepared according to the culture method of Example 4, add 100 ml (PBS) buffer (50 mM, pH 7.0), stir for 30 min to mix evenly, use a homogenizer to homogenize and break the cells at 600 bar, add 3 ml of PEI flocculant, centrifuge at 4000 rpm for 15 min, and obtain 100 ml of supernatant as E1 enzyme solution.

[0154] Preparation of E2 enzyme solution: Weigh 10 g of the E2 bacterial sludge in Example 4, add 100 ml of (PBS) buffer (50 mM, pH 7.0), stir for 30 min to mix evenly, use a homogenizer to homogenize and break the cells at 600 bar, add 2 ml of PEI flocculant, centrifuge at 4000 rpm for 15 min, and obtain 100 ml of supernatant as E2 enzyme solution.

[0155] Add 184g water and 25.5g isopropanol to a 500ml three-necked flask, add 20mg pyridoxal phosphate, 43mg nicotinamide adenine dinucleotide, and 11.7g threonine under stirring, stir for 5 minutes to dissolve, add 15g p-methylsulfonylbenzaldehyde, 45.0ml E1 enzyme solution, and 22.5ml E2 enzyme solution. Stir and react, control the temperature at 33℃ and pH=6.4, and use a small fish pump to blow air. After 18 hours, the reaction conversion rate is 65%, and the diastereomeric excess de value is 94.3.

[0156] In summary, under the same conditions, the conversion rate of (2S, 3R)-p-methylsulfonylphenylserine generated by the new enzyme (SEQ ID NO: 5) of the present application and the alcohol dehydrogenase catalytic reaction is much higher than the conversion rate of (2S, 3R)-p-methylsulfonylphenylserine generated by the existing L-threonine transaldehyde enzyme (SEQ ID No: 1) and the alcohol dehydrogenase catalytic reaction. The dual enzyme system of the present application significantly reduces the production cost of (2S, 3R)-p-methylsulfonylphenylserine, simplifies the synthesis process, and also makes it more in line with the needs of today's green industrial production.

[0157] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present application.

Claims

1. A method for preparing a florfenicol intermediate, characterized in that: The preparation method uses p-methylsulfonylbenzaldehyde and threonine as raw materials, L-threonine transaldolase and alcohol dehydrogenase as catalysts, and reacts in an aqueous solution system containing a cosolvent to obtain the florfenicol intermediate (2S, 3R)-p-methylsulfonylphenylserine.

2. The preparation method according to claim 1, characterized in that The catalyst also includes a coenzyme.

3. The preparation method according to claim 2, characterized in that: The coenzyme comprises pyridoxal phosphate and nicotinamide adenine dinucleotide; further, the molar ratios of pyridoxal phosphate and nicotinamide adenine dinucleotide to p-methylsulfonylbenzaldehyde are 0.0005-0.0030:1 and 0.0002-0.0020:1 respectively.

4. The preparation method according to claim 1, characterized in that: The co-solvent includes isopropyl alcohol.

5. The preparation method according to claim 4, characterized in that: The mass fraction of the isopropanol in the aqueous solution is 5-15% (w / w).

6. The preparation method according to claim 1, characterized in that: The amino acid sequence of the L-threonine aldolase is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21; And / or, the amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO:

33.

7. The preparation method according to claim 1, characterized in that: The reaction temperature is 25-35° C.; and / or the reaction time is 15-24 h.

8. The preparation method according to claim 1, characterized in that: Also includes one or more combinations of the following features: a) the molar ratio of L-threonine to p-methylsulfonylbenzaldehyde is 1.0-1.6:1.0; b) based on the weight of the aqueous solution system, the weight proportion of the L-threonine aldolase is 1-2% (w / w); c) Based on the weight of the aqueous solution system, the weight percentage of the alcohol dehydrogenase is 0.5-1.5% (w / w).

9. (2S, 3R)-p-methylsulfonylphenylserine, prepared by the preparation method according to any one of claims 1 to 8.

10. The preparation method according to any one of claims 1 to 8 or use of (2S, 3R)-p-methylsulfonylphenylserine according to claim 9 in the preparation of florfenicol.

11. An L-threonine aldolase, characterized in that The amino acid sequence of the L-threonine aldolase is shown in SEQ ID NO:

5.

12. Use of the L-threonine transaldolase according to claim 11 in preparing a florfenicol intermediate.

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