N-acetylglucosamine-2-epimerase mutant, N-acetylneuraminic acid lyase mutant and application of N-acetylglucosamine-2-epimerase mutant and N-acetylneuraminic acid lyase mutant

Abstract

The invention provides an N-acetyl glucosamine-2-epimerase mutant, an N-acetylneuraminic acid lyase mutant and application of the N-acetyl glucosamine-2-epimerase mutant and the N-acetylneuraminic acid lyase mutant. Compared with a wild type, the N-acetyl glucosamine-2-epimerase mutant and the N-acetylneuraminic acid lyase mutant disclosed by the invention have higher enzyme activity, can realize synthesis of Neu5Ac at a higher level, and have a wide industrial application prospect.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to an N-acetylglucosamine-2-epimerase mutant and an N-acetylneuraminic acid lyase mutant and applications thereof. Background Art

[0002] N-acetylneuraminic acid (NeuAc) is a type of acidic amino sugar containing 9 carbon atoms and having a pyranose structure. It plays an important role in biological recognition, cellular immunity and disease. As one of the most important monomers of sialylated human milk oligosaccharides (sialylated HMOs or sialyllactose), it is currently legally used in China, Europe, the United States, Japan and other countries.

[0003] In the biosynthesis of N-acetylneuraminic acid, N-acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid lyase (NAL) play a key role. AGE isomerizes the substrate N-acetyl-D-glucosamine (GlcNAc) to form N-acetylmannosamine (ManNAc). N-acetylmannosamine further combines with the substrate sodium pyruvate under the action of N-acetylneuraminic acid lyase (NAL) to form NeuAc. Therefore, the catalytic efficiency of N-acetylglucosamine-2-epimerase and N-acetylneuraminic acid lyase is key to improving the yield of NeuAc synthesis.

[0004] Furthermore, N-acetylglucosamine-2-epimerase and N-acetylneuraminic acid lyase are bidirectional enzymes in catalyzing the synthesis of NeuAc. The reverse reaction of the enzymes results in the accumulation of the substrate GlcNAc, reducing the efficiency of N-acetylneuraminic acid (NeuAc) production and thus limiting NeuAc production. Therefore, rational modification and directed evolution of AGE and NAL to enhance their forward enzymatic activity is crucial for increasing NeuAc production and reducing production costs. Summary of the Invention

[0005] In order to solve the problem of low catalytic efficiency of N-acetylglucosamine-2-epimerase and N-acetylneuraminic acid lyase known in the prior art in the synthesis process of N-acetylneuraminic acid, the present application provides an N-acetylglucosamine-2-epimerase mutant and an N-acetylneuraminic acid lyase mutant and their applications.

[0006] Specifically, this application involves the following aspects:

[0007] 1. An N-acetylneuraminic acid lyase mutant comprising any one of the following:

[0008] A1) A mutant, the amino acid sequence of which comprises at least one or both of positions 251 and 252 of the sequence shown in SEQ ID NO. 4 being mutated;

[0009] A2) is a protein obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the mutant described in A1) and having 98% or more identity with the mutant described in A1) and having the same function;

[0010] A3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant in A1) and / or the protein in A2).

[0011] 2. The N-acetylneuraminic acid lyase mutant according to item 1, wherein the mutation comprises at least one or two or more of V251L, V251V, F252H, and F252M.

[0012] 3. The N-acetylneuraminic acid lyase mutant according to item 2, wherein the mutation comprises at least F252H+V251L, F252M+V251T, V251L+I139V or F252M+V251P.

[0013] 4. Biomaterial comprising any of the following:

[0014] B1) a nucleic acid molecule encoding the N-acetylneuraminic acid lyase mutant according to any one of items 1 to 3;

[0015] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0016] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0017] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0018] B5) A whole-cell catalyst containing the nucleic acid molecule described in B1), or a whole-cell catalyst containing the expression cassette described in B2), or a whole-cell catalyst containing the recombinant vector described in B3).

[0019] 5. An N-acetylglucosamine-2-epimerase mutant comprising any one of the following:

[0020] C1) a mutant, the amino acid sequence of which comprises at least a sequence obtained by mutation at position 172 of the sequence shown in SEQ ID NO. 2;

[0021] C2) is a protein having 98% or more identity with the protein described in C1) and having the same function as the protein described in C1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of the mutant described in C1);

[0022] C3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant in C1) and / or the protein in C2).

[0023] 6. The N-acetylglucosamine-2-epimerase mutant according to item 5, wherein the mutation comprises at least A172S and / or A172G;

[0024] Preferably, the mutation comprises at least A172S+C118A or A172S+N179L.

[0025] 7. Biomaterial comprising any of the following:

[0026] D1) A nucleic acid molecule encoding the N-acetylglucosamine-2-epimerase mutant according to item 5 or 6;

[0027] D2) an expression cassette containing the nucleic acid molecule described in D1);

[0028] D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0029] D4) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

[0030] D5) A whole-cell catalyst containing the nucleic acid molecule described in D1), or a whole-cell catalyst containing the expression cassette described in D2), or a whole-cell catalyst containing the recombinant vector described in D3).

[0031] 8. A method for producing N-acetylneuraminic acid, comprising using the N-acetylneuraminic acid lyase mutant according to any one of items 1 to 3 or the biomaterial according to item 4, and / or

[0032] The N-acetylglucosamine-2-epimerase mutant according to item 5 or 6 or the biomaterial according to item 7;

[0033] Preferably, the combination of the N-acetylneuraminic acid lyase mutant and the N-acetylglucosamine-2-epimerase mutant is at least one selected from the following:

[0034] F252M and A172G, V251L and A172S, F252M and A172S, F252H+V251L and A172S+C118A, F252M+V251T and A172S+C118A.

[0035] 9. Use of the N-acetylneuraminic acid lyase mutant according to any one of items 1 to 3, or the biomaterial according to item 4, wherein the use is at least selected from any one of the following:

[0036] 1. Preparation of N-acetylneuraminic acid;

[0037] II. Preparation of products containing N-acetylneuraminic acid;

[0038] III. Improve the activity of N-acetylneuraminic acid lyase;

[0039] IV. Construction of a recombinant microorganism for producing N-acetylneuraminic acid;

[0040] V. Regulating the production of N-acetylneuraminic acid by microorganisms;

[0041] VI. Improve the purity of N-acetylneuraminic acid produced by microorganisms.

[0042] 10. Use of the N-acetylglucosamine-2-epimerase mutant according to Item 5 or 6, or the biomaterial according to Item 7, wherein the use is at least selected from any one of the following:

[0043] 1. Preparation of N-acetylneuraminic acid;

[0044] II. Preparation of products containing N-acetylneuraminic acid;

[0045] III. Improve the activity of N-acetylglucosamine-2-epimerase;

[0046] IV. Construction of a recombinant microorganism for producing N-acetylneuraminic acid;

[0047] V. Regulating the production of N-acetylneuraminic acid by microorganisms;

[0048] VI. Improve the purity of N-acetylneuraminic acid produced by microorganisms.

[0049] The N-acetylglucosamine-2-epimerase mutant and N-acetylneuraminic acid lyase mutant of the present application have higher enzyme activity than the wild type, can achieve the synthesis of Neu5Ac at a higher level, and have broad industrial application prospects. DETAILED DESCRIPTION

[0050] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0051] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0052] As used herein, the terms "polynucleotide," "nucleotide sequence," and "nucleic acid molecule" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; or RNA, such as guide RNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0053] As used herein, the term "vector" is used to describe a nucleic acid molecule that can be engineered to contain a polynucleotide or multiple polynucleotides that can be cloned and amplified in a host cell. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other polynucleotide species known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome.

[0054] In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or "recombinant vectors." Recombinant vectors can contain a nucleic acid of the present invention in a form suitable for expressing the nucleic acid in a host cell, meaning that the recombinant expression vector includes one or more regulatory elements, which can be selected based on the host cell used for expression and to which the nucleic acid sequence to be expressed can be operably linked.

[0055] As used herein, the term "recombinant microorganism" includes a microorganism (e.g., bacteria, yeast, algae, fungi, etc.) or microbial strain that has been genetically altered, modified, or engineered (e.g., genetically engineered) so that it exhibits an altered, modified, or different genotype and / or phenotype (e.g., when the genetic modification affects the encoding nucleic acid sequence of the microorganism) compared to the naturally occurring microorganism or "parent" microorganism from which it was derived.

[0056] The term "expression cassette" refers to DNA capable of expressing the protease having N-acetylglucosamine-2-epimerase or N-acetylneuraminic acid lyase function of the present application in a microorganism. The DNA includes not only a promoter for initiating transcription of the target gene, but also a terminator for terminating transcription of the target gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0057] The term "fusion protein" refers to a protein comprising at least a first protein genetically linked to at least a second protein. A fusion protein is produced by linking two or more genes that originally encode different proteins. A fusion protein may further comprise additional domains not involved in target binding, such as, but not limited to, a multimerization moiety, a polypeptide tag, a polypeptide linker, or a moiety that binds to a target other than PSMA. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and / or a SUMO tag, among others.

[0058] N-acetylneuraminic acid lyase mutants and related biomaterials

[0059] The present application provides an N-acetylneuraminic acid lyase mutant.

[0060] In some embodiments, the amino acid sequence of the mutant comprises a sequence obtained by mutation of one or more of positions 139, 165, 251 and 252 of the sequence shown in SEQ ID NO.4.

[0061] In some embodiments, the amino acid sequence of the mutant comprises a sequence obtained by mutation of one or both of positions 251 and 252 of the sequence shown in SEQ ID NO.4.

[0062] In some embodiments, the amino acid sequence of the mutant is a sequence obtained by mutation of one or both of positions 251 and 252 of the sequence shown in SEQ ID NO.4.

[0063] In some embodiments, the mutation is F252D, F252E, F252G, F252H, F252M, F252N, F252P, F252Q, F252R, F252V, F252W, F252Y, I139A, I139C, I139E, I139G, I139L, I139M, I139N, I139P, I139Q, I139R, I139S, I139T, I139V, I139W, K 165A, K165D, K165G, K165P, V251A, V251D, V251E, V251F, V251H, V251I, V251N, V251P, V251Q, V251S, V251T, V251V, V251W, V251Y, V251L, and V251M. Preferably, the mutation is one or more of V251L, V251V, F252H, and F252M.

[0064] In some embodiments, the mutation is F252H+V251L, F252M+V251T, V251L+I139V, or F252M+V251P.

[0065] Those skilled in the art will understand that proteins having more than 98% identity and the same function as the mutants obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of the mutants are also included in the scope of the N-acetylneuraminic acid lyase mutants of the present application.

[0066] In some embodiments, the mutants or proteins may also be linked to a tag protein at their N-terminus and / or C-terminus to obtain a fusion protein with the same function.

[0067] The present application also provides a biomaterial comprising any of the following:

[0068] B1) a nucleic acid molecule encoding any one of the above-mentioned N-acetylneuraminic acid lyase mutants;

[0069] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0070] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0071] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0072] B5) A whole-cell catalyst containing the nucleic acid molecule described in B1), or a whole-cell catalyst containing the expression cassette described in B2), or a whole-cell catalyst containing the recombinant vector described in B3).

[0073] Application of N-acetylneuraminic acid lyase mutant or related biomaterials

[0074] The present application also provides the application of the above-mentioned N-acetylneuraminic acid lyase mutant or its related biomaterials, wherein the application is at least selected from any one of the following:

[0075] 1. Preparation of N-acetylneuraminic acid;

[0076] II. Preparation of products containing N-acetylneuraminic acid;

[0077] III. Improve the activity of N-acetylneuraminic acid lyase;

[0078] IV. Construction of a recombinant microorganism for producing N-acetylneuraminic acid;

[0079] V. Regulating the production of N-acetylneuraminic acid by microorganisms;

[0080] VI. Improve the purity of N-acetylneuraminic acid produced by microorganisms.

[0081] N-acetylglucosamine-2-epimerase mutants and related biomaterials

[0082] The present application also provides an N-acetylglucosamine-2-epimerase mutant.

[0083] In some embodiments, the amino acid sequence of the mutant includes sequences obtained by mutations at positions 172, 115, 371, 176, 171, 179, 304, 53, 370, and 118 of the sequence shown in SEQ ID NO.2.

[0084] In some embodiments, the amino acid sequence of the mutant is a sequence obtained by mutation at position 172 of the sequence shown in SEQ ID NO.2.

[0085] In some embodiments, the mutation is A172C, A172D, A172E, A172F, A172G, A172I, A172K, A172L, A172N, A172P, A172R, A172S, A172T, A172Y, F115L, F115M, F115W, F115Y, F371D, F372 1H, F371Q, I176A, L171W, N179A, N179F, N179G, N179H, N179I, N179L, N179P, N179Q, N179S, N179T, N179W, W304R, W53D, C370A, C118A, preferably, the mutation is A172S or A172G.

[0086] In some embodiments, the mutation is A172S+C118A or A172S+N179L.

[0087] Those skilled in the art will understand that the mutant obtained by replacing and / or deleting and / or adding amino acid residues in the amino acid sequence of the above-mentioned mutant has a protein with more than 98% identity and the same function and is also included in the scope of the N-acetylglucosamine-2-epimerase mutant of the present application.

[0088] In some embodiments, the mutants or proteins may also be linked to a tag protein at their N-terminus and / or C-terminus to obtain a fusion protein with the same function.

[0089] The present application also provides a biomaterial comprising any of the following:

[0090] D1) a nucleic acid molecule encoding any one of the above-mentioned N-acetylglucosamine-2-epimerase mutants;

[0091] D2) an expression cassette containing the nucleic acid molecule described in D1);

[0092] D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0093] D4) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

[0094] D5) A whole-cell catalyst containing the nucleic acid molecule described in D1), or a whole-cell catalyst containing the expression cassette described in D2), or a whole-cell catalyst containing the recombinant vector described in D3).

[0095] Application of N-acetylglucosamine-2-epimerase mutant or related biomaterials

[0096] The present application also provides the application of the above-mentioned N-acetylglucosamine-2-epimerase mutant or its related biomaterials, wherein the application is at least selected from any one of the following:

[0097] 1. Preparation of N-acetylneuraminic acid;

[0098] II. Preparation of products containing N-acetylneuraminic acid;

[0099] III. Improve the activity of N-acetylglucosamine-2-epimerase;

[0100] IV. Construction of a recombinant microorganism for producing N-acetylneuraminic acid;

[0101] V. Regulating the production of N-acetylneuraminic acid by microorganisms;

[0102] VI. Improve the purity of N-acetylneuraminic acid produced by microorganisms.

[0103] Method for producing N-acetylneuraminic acid

[0104] The present application also provides a method for producing N-acetylneuraminic acid, comprising using any one of the above-mentioned N-acetylneuraminic acid lyase mutants or related biological materials, and / or any one of the above-mentioned N-acetylglucosamine-2-epimerase mutants or related biological materials.

[0105] In some embodiments, the method comprises using an N-acetylneuraminic acid lyase mutant or a related biological material thereof, and an N-acetylglucosamine-2-epimerase mutant or a related biological material thereof, wherein the combination of the N-acetylneuraminic acid lyase mutant and the N-acetylglucosamine-2-epimerase mutant is at least one selected from the following:

[0106] F252M and A172G, V251L and A172S, F252M and A172S, F252H+V251L and A172S+C118A, F252M+V251T and A172S+C118A.

[0107] Example 1 Construction and screening of mutants

[0108] N-acetylglucosamine-2-epimerase (PDB: 2GZ6, amino acid sequence shown in SEQ ID NO: 2) and N-acetylneuraminic acid lyase (PDB: 3LBM, amino acid sequence shown in SEQ ID NO: 4) were screened from the Protein Data Bank. The codon-optimized nucleotide sequences are SEQ ID NO: 1 and SEQ ID NO: 3, respectively.

[0109] SEQ ID NO:1:

[0110]

[0111] SEQ ID NO:2:

[0112] MGKNLQALAQLYKNALLNDVLPFWENHSLDSEGGYFTCLDRQGKVYDTDKFIWLQNRQVWTFSMLCNQLEKRENWLKIARNGAKFLAQHGRDDEGNWYFALTRGGEPLVQPYNIFSDCFAAMAFSQYALASGEEWAKDVAMQAYNNVLRRKDNPKGKYTKTYPGTRPMKALAVPMILANLTLEMEWLLPQETLENVLAATVQEVMGDFLDQEQGLMYENVAPDGSHIDCFEGRLINPGHGIEAMWFIMDIARRKNDSKTINQAVDVVLNILNFAWDNEYGGLYYFMDAAGHPPQQLEWDQKLWWVHLESLVALAMGYRLTGRDACWAWYQKMHDYSWQHFADPEYGEWFGYLNRRGEVLLNLKGGKWKGCFHVPRAMYLCWQQFEALS

[0113] SEQ ID NO:3:

[0114] Atggcaacgaatttacgtggcgtaatggctgcactcctgactccttttgaccaacaacaagcactggataaagcgagtctgcgtcgcctggttcagttcaatattcagcagggcatcgacggtttatacgtgggtggttcgaccggcgaggcctttgtacaaagcctttccgagcgtgaacaggtactggaaatcgtcgccgaagaggcgaaaggtcagattaaactcatcgcccacgtcggttgcgtcagcaccgccgaaagccaacaacttgcggcatcggctaaacgttatggcttcgatgccgtctccgccgtcacgccgttctactatcctttcagctttgaagagcactgcgatcactatcgggcaattattgattcggcggatggtttgccgatggtggtgtacaacattccagccctgagtggggtaaaactgaccctggatcagatcaacacacttgttacgttgcctggcgtaggtgcgctgaaacagacctctggcgatctctatcagatggagcagatccgtcgtgaacatcctgatctggtgctctataacggttacgacgaaatcttcgcctctggtcttctggcgggcgctgatggtggtatcggtagtacctacaacatcatgggctggcgttatcaggggattgtgaaggcgctgaaagaaggcgatatccagaccgcgcagaagctgcaaaccgaatgtaataaagtcattgatttactgatcaaaacgggcgtattccgcggcctgaaaactgtcctgcattatatggatgtcgtttctgtgccgctgtgccgcaaaccgtttggtccggtagatgaaaaatatctgccagaactgaaggcgctggcccagcagttgatgcaagagcgcgggtga

[0115] SEQ ID NO:4:

[0116] MATNLRGVMAALLTPFDQQQALDKASLRRLVQFNIQQGIDGLYVGGSTGEAFVQSLSEREQVLEIVAEEAKGQIKLIAHVGCVSTAESQQLAASAKRYGFDAVSAVTPFYYPFSFEEHCDHYRAIIDSADGLPMVVYNIPALSGVKLT LDQINTLVTLPGVGALKQTSGDLYQMEQIRREHPDLVLYNGYDEIFASGLLAGADGGIGSTYNIMGWRYQGIVKALKEGDIQTAQKLQTECNKVIDLLIKTGVFRGLKTVLHYMDVVSVPLCRKPFGPVDEKYLPELKALAQQLMQERG

[0117] For example, design the ends of the DNA fragments to be assembled to contain 15-30bp of overlapping sequences. Mix the starting AGE gene fragment with the pET28a vector at a 1:1 volume ratio and 2× ABclonal seamless assembly premix. Incubate in a 50°C water bath for 1 hour to allow assembly. Transform the assembled product into E. coli DH5α competent cells and select the correct transformants for plasmid extraction to obtain the wild-type pET28a-AGE plasmid. Similarly, obtain the wild-type pET28a-NAL plasmid.

[0118] AutoDock Vina and Discovery Studio 2019 were used to evaluate the docking conformations of the substrate and receptor proteins, as well as the bond affinity simulations after saturation mutation of key amino acids. Sites with low affinity values ​​were selected for enzyme activity verification. Based on the simulation results, mutations were made at positions 172, 118, 179, and 370 of the AGE enzyme and at positions 252, 139, and 251 of the NAL enzyme.

[0119] Site-directed mutagenesis (SEM) PCR was performed using SnapGene software to design the relevant primers (see Tables 1 and 2). The mutated bases were incorporated into the upstream and downstream primers. PCR amplification was performed using pET28a-AGE as a template and the mutant primers. The PCR product was digested with DpnI in a 37°C water bath for 40 minutes to remove template influence. The digested PCR product was then transformed into E. coli DH5α competent cells to generate the pET28a-AGE mutant plasmid.

[0120] The pET28a-AGE mutant plasmid was transformed into the competent cells of E. coli (BL21). 100 μL of the revived competent cells was aspirated and spread on a resistant plate containing kanamycin. The cells were cultured at 37°C for 24 h. After colonies grew, single colonies were picked for fermentation verification.

[0121] The PCR reaction program was as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 20 s, 55-62°C annealing for 20 s, 72°C extension for 2 min, 30 cycles, and full extension at 72°C for 10 min.

[0122] The PCR reaction system (50 μL) is: 25 μL 2× Buffer, 1 μL dNTP, 1 μL upstream primer (10 μmol / L), 1 μL downstream primer (10 μmol / L), 1 μL DNA polymerase, appropriate amount of DNA template, and sterilized distilled water is added to make up to 50 μL.

[0123] The NAL mutant was obtained in the same way as the pET28a-NAL mutant plasmid, and the pET28a-NAL mutant plasmid was transformed into the competent E. coli (BL21). 100 μL of the revived competent cells was aspirated and spread on a resistant plate containing kanamycin and cultured at 37°C for 24 h. After colonies grew, single colonies were picked for fermentation verification.

[0124] Table 1 Primers used for AGE mutants

[0125]

[0126]

[0127] Table 2 Primers used for NAL mutants

[0128]

[0129]

[0130] Example 2 Determination of enzyme activity of mutant shake flask fermentation

[0131] Mutant shake flask fermentation and crude enzyme preparation: A single colony was inoculated into 10 mL of LB liquid medium containing 50 μg / mL ampicillin and cultured with shaking at 37°C for 12 h. A 1% inoculum was then transferred to 50 mL of LB liquid medium containing 50 μg / mL ampicillin and cultured with shaking at 37°C until the OD reached 0.6-0.8. IPTG (final concentration: 0.3 mmol / L) was added and cultured with shaking at 30°C for 24 h before fermentation was terminated. After fermentation, the cells were centrifuged at 9000 rpm for 10 min, harvested, and ultrasonically disrupted. 1 g of wet cells was weighed into a 50 mL centrifuge tube, resuspended in 10 mL of 0.01 M PBS buffer, and ultrasonically disrupted. Ultrasonication conditions were: 60% power, 5 s of sonication, 5 s of pause, and 10 min of sonication. The disrupted cell suspension was centrifuged at 9000 rpm for 10 min, and the supernatant was collected. This was the crude enzyme solution.

[0132] Enzyme activity assay: The crude enzyme solution was diluted 100-fold and 200-fold, respectively, and then the enzyme activity was assayed according to Tables 3 and 4. The reaction conditions were: incubate in a water bath for 30 min, and terminate the reaction by adding glacial acetic acid to a final concentration of 3%. After the reaction, the supernatant was assayed by HPLC and the enzyme activity was calculated accordingly.

[0133] The preparation method of enzyme activity detection related solvents is as follows:

[0134] 100mM Tris-HCl buffer: Weigh 12.11g of tris(hydroxymethyl)aminomethane powder and dissolve it in approximately 900mL of water. Adjust the pH to 7.5 with concentrated hydrochloric acid. Transfer to a volumetric flask and add purified water to make up to 1000mL. Store in a refrigerator at 4°C. The shelf life is one month.

[0135] 600mM NAG solution: Weigh 13.27g N-acetyl-D-glucosamine powder, dissolve in 100mM Tris-HCl (pH 7.5), and dilute to 100mL. Store at 4°C until use. The expiration date is one month.

[0136] 60mM MgCl2·6H2O solution: Weigh 1.22g of MgCl2·6H2O solid, dissolve it in 100mM Tris-HCl (pH 7.5), and dilute the solution to 100mL. Store at 4°C until use. The shelf life is one month.

[0137] 15mM ATP solution: Weigh 0.83g of adenosine disodium triphosphate powder, dissolve it in 100mM Tris-HCl (pH 7.5), and dilute the solution to 100mL. Store at 4°C in the dark until use. The shelf life is one month.

[0138] 400 mM Man solution: Weigh 2.21 g of N-acetyl-D-mannosamine powder, dissolve in 100 mM Tris-HCl (pH 7.5), and dilute to 25 mL. Store at 4°C until use. The expiration date is one month.

[0139] 400 mM PEP solution: Weigh 4.40 g of sodium pyruvate solid, add 100 mM Tris-HCl (pH 7.5) to dissolve and dilute the solution to 100 mL. Store at 4°C until use. The shelf life is one month.

[0140] Table 3 AGE enzyme activity assay system (1 mL)

[0141]

[0142] Table 4 NAL enzyme activity assay system (1 mL)

[0143] Reagents Addition amount (μL) 400 mM Man solution 250.0 400 mM PEP solution 250.0 NAL crude enzyme solution 500.0 Total system (mL) 1.0

[0144] Enzyme activity results are shown in Tables 5 and 6. As shown in Tables 5 and 6, a few of the verified mutants showed slight increases in enzyme activity compared to the original strain. The crude enzyme activities of the AGE enzyme mutants A172S and A172G increased by 3.5% and 3.7%, respectively, compared to the wild-type strain (WT). The crude enzyme activities of the NAL enzyme mutants V251L, F252H, and F252M increased by 14.6%, 3.0%, and 2.1%, respectively, compared to the WT. The specific enzyme activities of most mutants remained unchanged, while some showed slight decreases.

[0145] Table 5 AGE single site mutation enzyme activity determination results

[0146] mutant U / g WT 2742.48 A172S 2839.77 A172C 2574.44 A172N 2495.30 A172G 2845.11 N179G 2652.52 N179H 2152.60 N179A 2712.82 N179Q 1993.24 C118A 2534.33 C370A 2568.95

[0147] Table 6 Results of crude enzyme activity assay of NAL single-site mutants

[0148] mutant U / g WT 234.56 F252G 230.96 F252H 241.64 F252M 239.59 F252P 205.00 F252Q 193.88 F252V 180.84 F252W 207.49 V251I 201.21 V251L 268.80 V251M 180.33 V251P 222.07 V251Q 178.60 V251T 187.14 I139L 190.67 I139M 194.44

[0149] Because most enzyme activities did not significantly change after single-site mutations, we further performed double-site mutations by randomly combining single-site mutations. The results of the mutation combinations and enzyme activity assays are shown in Tables 7 and 8. The double-site mutants of the AGE and NAL enzymes showed significant increases in activity compared to the single-site mutations. The most significant increase was observed in AGE enzyme mutant combination B, which increased activity by 71.98% compared to the wild-type. The NAL enzyme mutant combinations 1, 2, 3, and 4 showed increases in activity of 20.51%, 32.52%, 34.78%, and 26.70%, respectively.

[0150] Table 7 AGE enzyme double-site mutation combination enzyme activity determination results

[0151] serial number AGE mutants U / g WT 2742.48 A A172G+N179L 2650.47 B A172S+C118A 4716.51 C A172S+N179L 2906.05

[0152] Table 8 NAL enzyme double-site mutation combination enzyme activity determination results

[0153] serial number NAL mutants U / g WT 234.56 1 F252M+V251P 282.67 2 F252M+V251T 310.84 3 F252H+V251L 316.15 4 V251L+I139V 297.19

[0154] Example 3 Whole-cell catalysis verification of NeuAc production

[0155] The recombinant E. coli expressing the two enzymes were fermented in shake flasks respectively. The seeds were inoculated at a 0.1% inoculum into 10 mL LB liquid medium containing 50 μg / mL ampicillin, and cultured at 37°C with shaking overnight. Then, the inoculum was transferred to 50 mL LB liquid medium containing 50 μg / mL ampicillin at a 1% inoculum and cultured at 37°C with shaking until the OD reached 0.6-0.8. IPTG (final concentration: 0.3 mmol / L) was added and cultured at 30°C with shaking for 8 h. The fermentation was stopped and the OD was about 3-5. The cells were collected by centrifugation at 6000 rpm, and the cells were washed again with 0.8% NaCl buffer 1-2 times. The cells expressing AGE and NAL enzymes were separated according to the OD. 600 Add the solution to 50 mL of catalytic buffer at a 1:1 ratio. The catalytic buffer consists of 10 mM MgCl2, 1.4-1.6 M sodium pyruvate, 0.4 M GlcNAc, 10 mM Tris-HCl (pH 7.5), and 0.3% Triton X-100 by volume. Incubate at 40°C for 24 h. Centrifuge at 9000 rpm for 10 min, and collect the supernatant for liquid chromatography-mass spectrometry analysis of NeuAc content.

[0156] The HPLC detection method is as follows:

[0157] The supernatant was filtered through a 0.22 μm filter membrane into a sample bottle.

[0158] Liquid phase detection conditions: chromatographic column: sugar column Bio-Rad Aminex HPX-87H (300m×7.8mm), mobile phase: 5mmol / L sulfuric acid, flow rate: 0.5mL / min, injection volume: 20μL, column temperature: 65℃, differential detector, detector temperature: 40℃.

[0159] The combinations and results are shown in Table 9.

[0160] As shown in Table 9, after 24 h of whole-cell catalysis, the NeuAc production in experimental group 3 increased by 19.6% compared with the WT, and the NeuAc production in group 4 increased by 27.7%.

[0161] Table 9 AGE and NAL enzyme single site mutation combinations

[0162]

Claims

1. An N-acetylneuraminic acid lyase mutant comprising any one of the following: A1) a mutant, the amino acid sequence of which at least comprises a sequence obtained by mutation of one or both of positions 251 and 252 of the sequence shown in SEQ ID NO. 4; A2) is a protein obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the mutant described in A1) and having more than 98% identity with the mutant described in A1) and having the same function; A3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant in A1) and / or the protein in A2). 2 . The N-acetylneuraminic acid lyase mutant according to claim 1 , wherein the mutation comprises at least one or two or more of V251L, F252H, and F252M.

3. The N-acetylneuraminic acid lyase mutant according to claim 2, wherein the mutation comprises at least F252H+V251L, F252M+V251T, V251L+I139V or F252M+V251P.

4. Biomaterial comprising any of the following: B1) a nucleic acid molecule encoding the N-acetylneuraminic acid lyase mutant according to any one of claims 1 to 3; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A whole cell catalyst containing the nucleic acid molecule described in B1), or a whole cell catalyst containing the expression cassette described in B2), or a whole cell catalyst containing the recombinant vector described in B3).

5. An N-acetylglucosamine-2-epimerase mutant comprising any one of the following: C1) a mutant, the amino acid sequence of which at least includes a sequence obtained by mutation at position 172 of the sequence shown in SEQ ID NO.2; C2) is a protein having more than 98% identity with the protein described in C1) and having the same function as the protein described in C1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of the mutant described in C1); C3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the mutant in C1) and / or the protein in C2).

6. The N-acetylglucosamine-2-epimerase mutant according to claim 5, wherein the mutation comprises at least A172S and / or A172G; Preferably, the mutation comprises at least A172S+C118A or A172S+N179L.

7. Biomaterial comprising any of the following: D1) a nucleic acid molecule encoding the N-acetylglucosamine-2-epimerase mutant according to claim 5 or 6; D2) an expression cassette containing the nucleic acid molecule described in D1); D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) A whole cell catalyst containing the nucleic acid molecule described in D1), or a whole cell catalyst containing the expression cassette described in D2), or a whole cell catalyst containing the recombinant vector described in D3).

8. A method for producing N-acetylneuraminic acid, comprising using the N-acetylneuraminic acid lyase mutant according to any one of claims 1 to 3 or the biomaterial according to claim 4, and / or The N-acetylglucosamine-2-epimerase mutant according to claim 5 or 6 or the biomaterial according to claim 7; Preferably, the combination of the N-acetylneuraminic acid lyase mutant and the N-acetylglucosamine-2-epimerase mutant is at least selected from the following: F252M and A172G, V251L and A172S, F252M and A172S, F252H+V251L and A172S+C118A, F252M+V251T and A172S+C118A.

9. Use of the N-acetylneuraminic acid lyase mutant according to any one of claims 1 to 3, or the biomaterial according to claim 4, wherein the use is at least selected from any one of the following:

1. Preparation of N-acetylneuraminic acid; II. Preparation of products containing N-acetylneuraminic acid; III. Improve the activity of N-acetylneuraminic acid lyase; IV. Construction of a recombinant microorganism for producing N-acetylneuraminic acid; V. Regulating the production of N-acetylneuraminic acid using microorganisms; VI. Improve the purity of N-acetylneuraminic acid produced by microorganisms.

10. Use of the N-acetylglucosamine-2-epimerase mutant according to claim 5 or 6, or the biomaterial according to claim 7, wherein the use is at least selected from any one of the following:

1. Preparation of N-acetylneuraminic acid; II. Preparation of products containing N-acetylneuraminic acid; III. Improve the activity of N-acetylglucosamine-2-epimerase; IV. Construction of a recombinant microorganism for producing N-acetylneuraminic acid; V. Regulating the production of N-acetylneuraminic acid using microorganisms; VI. Improve the purity of N-acetylneuraminic acid produced by microorganisms.

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