N-acetylglucosamine-2-epimerase mutants and n-acetylneuraminate lyase mutants and uses thereof

Abstract

This application provides an N-acetylglucosamine-2-epimerase mutant and an N-acetylneuraminic acid lyase mutant, and their applications. The N-acetylglucosamine-2-epimerase mutant and N-acetylneuraminic acid lyase mutant of this application exhibit higher enzyme activity compared to the wild type, enabling the synthesis of Neu5Ac at a higher level and showing broad prospects for industrial applications.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to an N-acetylglucosamine-2-epimerase mutant and an N-acetylneuraminic acid lyase mutant and their applications. Background Technology

[0002] N-acetylneuraminic acid (NeuAc) is an acidic amino sugar containing 9 carbon atoms and having a pyranose structure. It plays an important role in biorecognition, cellular immunity and disease. As one of the most important monomers of sialylated human milk oligosaccharides (sialylated HMOs or sialyl lactose), it is currently legal for use in countries such as China, Europe, the United States and Japan.

[0003] In the biosynthesis of N-acetylneuraminic acid, N-acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid lyase (NAL) play crucial roles. AGE isomerizes the substrate N-acetyl-D-glucosamine (GlcNAc) to N-acetylmannosamine (ManNAc). N-acetylmannosamine then combines with the substrate sodium pyruvate under the action of N-acetylneuraminic acid lyase (NAL) to generate 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, both N-acetylglucosamine-2-epomerase and N-acetylneuraminic acid lyase are bidirectional enzymes in the catalytic synthesis of NeuAc. The reverse reaction leads to 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 improve their forward reaction activity is of great significance for increasing NeuAc production capacity and reducing production costs. Summary of the Invention

[0005] To address the problem of low catalytic efficiency of known N-acetylglucosamine-2-epomerase and N-acetylneuraminic acid lyase in the synthesis of N-acetylneuraminic acid, this application provides a mutant of N-acetylglucosamine-2-epomerase and a mutant of N-acetylneuraminic acid lyase, as well as their applications.

[0006] Specifically, this application relates to the following aspects:

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

[0008] A1) A mutant whose amino acid sequence contains at least one or both of the 251st and 252nd positions of the sequence shown in SEQ ID NO.4, resulting in a sequence obtained by mutation;

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

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

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

[0012] 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.

[0013] 4. Biomaterials, including any of the following:

[0014] B1) A nucleic acid molecule encoding an N-acetylneuraminic acid lyase mutant as described in any of items 1-3;

[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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms 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 whose amino acid sequence includes at least the sequence shown in SEQ ID NO.2 obtained by mutating position 172;

[0021] C2) is a protein obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of the mutant described in C1), which has more than 98% identity with the protein described in C1) and has the same function.

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

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

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

[0025] 7. Biomaterials, including any of the following:

[0026] D1) Encodes a nucleic acid molecule for an N-acetylglucosamine-2-epimerase mutant as described in item 5 or 6;

[0027] D2) An expression cassette containing the nucleic acid molecules 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) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms 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 an N-acetylneuraminic acid lyase mutant as described in any one of items 1-3 or the biological material as described in item 4, and / or

[0032] The N-acetylglucosamine-2-epimerase mutant described in item 5 or 6, or the biomaterial described in item 7;

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

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

[0035] 9. The use of any of the N-acetylneuraminic acid lyase mutants described in items 1-3, or the biomaterial described in item 4, wherein the use is selected from at least one of the following:

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

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

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

[0039] IV. Constructing recombinant microorganisms for the production of N-acetylneuraminic acid;

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

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

[0042] 10. The use of the N-acetylglucosamine-2-epimerase mutant as described in item 5 or 6, or the biomaterial as described in item 7, wherein said use is selected from at least one of the following:

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

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

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

[0046] IV. Constructing recombinant microorganisms for the production of N-acetylneuraminic acid;

[0047] V. Regulating the production of N-acetylneuraminic acid by utilizing 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 this application have higher enzyme activity than the wild type, enabling the synthesis of Neu5Ac at a higher level and showing broad prospects for industrial application. Detailed Implementation

[0050] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments 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 commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0052] In this document, the terms “polynucleotide,” “nucleotide sequence,” and “nucleic acid molecule” are used interchangeably. They refer to a polymer of nucleotides of any length, which may be deoxyribonucleotides or ribonucleotides, or analogs thereof, and the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0053] In this document, the term "vector" is used to describe nucleic acid molecules that can be engineered to contain one or more polynucleotides that can be amplified in host cells. Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing 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. Some vectors are capable of autonomous replication in the host cells in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, thereby replicating along with the host genome.

[0054] Furthermore, certain vectors are capable of directing the expression of those genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” or “recombinant vectors.” Recombinant vectors may contain the nucleic acids of the present invention in a form suitable for expression in host cells, meaning that recombinant expression vectors include one or more regulatory elements that can be selected based on the host cell for expression, which can be operatively linked to the nucleic acid sequence to be expressed.

[0055] In this document, the term “recombinant microorganism” includes microorganisms (e.g., bacteria, yeast, algae, fungi, etc.) or strains of microorganisms that have been genetically altered, modified, or engineered (e.g., genetically engineered) so that they exhibit altered, modified, or different genotypes and / or phenotypes compared to their origin, naturally occurring microorganisms, or “parental” microorganisms (e.g., when genetic modifications affect the coding nucleic acid sequence of the microorganism).

[0056] The term "expression cassette" refers to DNA capable of expressing the protease of this application, which has the function of N-acetylglucosamine-2-epimerase or N-acetylneuraminic acid lyase, in microorganisms. This DNA includes not only a promoter to initiate transcription of the target gene but also a terminator to terminate transcription of the target gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0057] The term "fusion protein" refers to a protein containing the genetic link between at least a first protein and at least a second protein. Fusion proteins are generated by linking two or more genes that initially encode different proteins. Fusion proteins may further include additional domains that do not participate in target binding, such as, but not limited to, polymerizing moieties, polypeptide tags, polypeptide linkers, or moieties that bind to targets other than PSMA. The protein tags may be Flag tags, His tags, MBP tags, HA tags, myc tags, GST tags, and / or SUMO tags, etc.

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

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

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

[0063] In some implementations, the mutations are 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 The mutation is one or more of the following: 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 the following: V251L, V251V, F252H, and F252M.

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

[0065] Those skilled in the art will understand that proteins with more than 98% identity and the same function obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of the above mutants are also included in the scope of the N-acetylneuraminic acid lyase mutants of this application.

[0066] In some embodiments, the mutants or proteins described above 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] This application also provides a biomaterial comprising any of the following:

[0068] B1) Encodes a nucleic acid molecule for any 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms 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 mutants or related biomaterials

[0074] This application also provides the application of the above-mentioned N-acetylneuraminic acid lyase mutant or related biological materials, wherein the application is selected from at least one of the following:

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

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

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

[0078] IV. Constructing recombinant microorganisms for the production of N-acetylneuraminic acid;

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

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

[0081] N-acetylglucosamine-2-epimerase mutants and related biological materials

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

[0083] In some embodiments, the amino acid sequence of the mutant includes the sequence obtained by mutating 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 the sequence obtained by mutating position 172 of the sequence shown in SEQ ID NO.2.

[0085] In some implementations, the mutations are A172C, A172D, A172E, A172F, A172G, A172I, A172K, A172L, A172N, A172P, A172R, A172S, A172T, A172Y, F115L, F115M, F115W, F115Y, F371D, and F37. 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 implementations, the mutation is A172S+C118A or A172S+N179L.

[0087] Those skilled in the art will understand that proteins with more than 98% identity and the same function obtained by substituting and / or deleting and / or adding amino acid residues from the above mutant sequence are also included in the scope of the N-acetylglucosamine-2-epimerase mutants of this application.

[0088] In some embodiments, the mutants or proteins described above 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] This application also provides a biomaterial comprising any of the following:

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

[0091] D2) An expression cassette containing the nucleic acid molecules 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) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms 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 mutants or related biomaterials

[0096] This application also provides the application of the above-mentioned N-acetylglucosamine-2-epimerase mutant or related biological materials, wherein the application is selected from at least one of the following:

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

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

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

[0100] IV. Constructing recombinant microorganisms for the production of N-acetylneuraminic acid;

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

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

[0103] Methods for producing N-acetylneuraminic acid

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

[0105] In some embodiments, the method includes using an N-acetylneuraminic acid lyase mutant or related biological material, and an N-acetylglucosamine-2-epimerase mutant or related biological material, wherein the combination of the N-acetylneuraminic acid lyase mutant and the N-acetylglucosamine-2-epimerase mutant is selected from at least one of 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] Taking AGE as an example, the DNA fragment to be assembled is designed with a 15-30 bp overlap region at its end. The starting AGE gene fragment is mixed with the pET28a vector and 2×ABclonal seamless assembly premix at a volume ratio of 1:1. Assembly is performed by incubation in a 50℃ water bath for 1 hour. The assembly product is then transformed into E. coli DH5α competent cells. Correct transformants are selected for plasmid extraction to obtain the pET28a-AGE wild-type plasmid. Similarly, the pET28a-NAL wild-type plasmid is obtained.

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

[0119] Site-directed mutagenesis (SEM PCR) was used, and primers were designed using Snap Gene software (see Tables 1 and 2). The mutated bases were incorporated into the upstream and downstream primers. Using pET28a-AGE as a template, PCR amplification was performed using the mutated primers. The PCR products were digested with DpnI enzyme in a 37°C water bath for 40 min to remove the influence of the template. The digested PCR products were then transformed into E. coli DH5α competent cells to obtain the pET28a-AGE mutant plasmid.

[0120] The pET28a-AGE mutant plasmid was transformed into competent cells of E. coli (BL21). 100 μL of the revived competent cells were spread on a resistant plate containing kanamycin and incubated 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℃ pre-denaturation for 5 min, 95℃ denaturation for 20 s, 55-62℃ annealing for 20 s, 72℃ extension for 2 min, 30 cycles, and 72℃ full extension for 10 min.

[0122] The PCR reaction system (50 μL) consisted of: 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, an appropriate amount of DNA template, and sterile distilled water to bring the total volume to 50 μL.

[0123] The pET28a-NAL mutant plasmid was obtained in the same manner, and the pET28a-NAL mutant plasmid was transformed into competent cells of E. coli (BL21). 100 μL of the recovered competent cells were plated 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 during shake-flask fermentation of mutants

[0131] Mutant shake-flask fermentation and preparation of crude enzyme solution: Single colonies were inoculated into 10 mL LB liquid medium containing 50 μg / mL ampicillin and cultured at 37°C with shaking for 12 h. Then, 1% of the colonies were transferred to 50 mL LB liquid medium containing 50 μg / mL ampicillin 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 the culture was continued at 30°C with shaking for 24 h, after which fermentation was stopped. After fermentation, the cells were collected by centrifugation at 9000 rpm for 10 min and then sonicated. 1 g of wet cells was weighed into a 50 mL centrifuge tube, resuspended in 10 mL of 0.01 M PBS buffer, and then sonicated. The sonication conditions were: 60% power, 5 s sonication, 5 s pause, 10 min sonication. The disrupted bacterial solution was centrifuged at 9000 rpm for 10 min, and the supernatant was collected; this supernatant was the crude enzyme solution.

[0132] Enzyme activity assay: The crude enzyme solution was diluted 100-fold and 200-fold, respectively, and the enzyme activity was then measured according to Tables 3 and 4. The reaction conditions were: water bath for 30 min, followed by termination of the reaction with 3% glacial acetic acid. After the reaction, the supernatant was collected and analyzed by HPLC, and the enzyme activity was calculated accordingly.

[0133] The preparation methods for solvents related to enzyme activity detection are as follows:

[0134] 100mM Tris-HCl buffer: Weigh 12.11g of tris(hydroxymethyl)aminomethane powder and dissolve it in about 900mL of water. Adjust the pH to 7.5 with concentrated hydrochloric acid, transfer to a volumetric flask, and add purified water to bring the volume to 1000mL. Store at 4℃. Shelf life is one month.

[0135] 600mM NAG solution: Weigh 13.27g N-acetyl-D-glucosamine powder, add 100mM Tris-HCl (pH 7.5) to dissolve, and bring the solution to a final volume of 100mL. Store at 4℃ for later use. Shelf life is one month.

[0136] 60mM MgCl2·6H2O solution: Weigh 1.22g of MgCl2·6H2O solid, add 100mM Tris-HCl (pH 7.5) to dissolve, and bring the solution to a final volume of 100mL. Store at 4℃ for later use. Shelf life is one month.

[0137] 15mM ATP solution: Weigh 0.83g of disodium adenosine triphosphate powder, add 100mM Tris-HCl (pH 7.5) to dissolve, and bring the solution to a final volume of 100mL. Store at 4℃ protected from light for later use. Shelf life is one month.

[0138] 400mM Man solution: Weigh 2.21g of N-acetyl-D-mannosamine powder, add 100mM Tris-HCl (pH 7.5) to dissolve, and bring the solution to a final volume of 25mL. Store at 4℃ for later use. Shelf life is one month.

[0139] 400mM PEP solution: Weigh 4.40g of sodium pyruvate solid, add 100mM Tris-HCl (pH 7.5) to dissolve it, and then bring the solution to a final volume of 100mL. Store at 4℃ for later use. 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] 400mM Man solution 250.0 400mM PEP solution 250.0 NAL crude enzyme solution 500.0 Total system (mL) 1.0

[0144] The enzyme activity results are shown in Tables 5 and 6. As shown in Tables 5 and 6, the enzyme activities of a few mutants were slightly increased compared to the original strain. Specifically, the crude enzyme activities of the AGE enzyme mutants A172S and A172G were 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 were increased by 14.6%, 3.0%, and 2.1% respectively compared to WT. The specific enzyme activities of most mutants did not change significantly, while the specific enzyme activities of some mutants were slightly decreased.

[0145] Table 5 Results of AGE unit point mutant enzyme activity assay

[0146] 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 for NAL unit point mutants.

[0148] 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] Since most enzyme activities did not change significantly after single-site mutation, we selected random combinations of single-site mutations to further mutate the enzymes into two-site mutations. The mutation combinations and enzyme activity assay results are shown in Tables 7 and 8. The enzyme activities of the two-site mutants of AGE and NAL enzymes showed a more significant increase compared to the single-site mutations. Among them, the enzyme activity of mutant combination B of AGE enzyme showed the most significant increase, increasing by 71.98% compared to the wild type; the enzyme activities of mutant combinations 1, 2, 3, and 4 of NAL enzyme increased by 20.51%, 32.52%, 34.78%, and 26.70%, respectively.

[0150] Table 7 Results of AGE enzyme dual-site mutation combination enzyme activity assay

[0151] WT 2742.48 A A172G+N179L 2650.47 B A172S+C118A 4716.51 C A172S+N179L 2906.05

[0152] Table 8. Results of NAL enzyme activity assay using a two-site mutant combination.

[0153] 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 yield

[0155] Recombinant *E. coli* expressing both enzymes were subjected to shake-flask fermentation. Seed culture was inoculated at a rate of 0.1% into 10 mL LB broth containing 50 μg / mL ampicillin and cultured overnight at 37°C with shaking. Then, the inoculum was transferred at a rate of 1% to 50 mL LB broth containing 50 μg / mL ampicillin 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 the culture was continued at 30°C with shaking for 8 hours. Fermentation was then stopped, and the OD was approximately 3-5. The cells were collected by centrifugation at 6000 rpm and washed 1-2 times with 0.8% NaCl buffer. The cells expressing AGE and NAL enzymes were then cultured according to their OD values. 600 The 1:1 ratio of MgCl2 to 50 mL of cell catalysis buffer was added. The cell catalysis buffer consisted 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 (the volume of the buffer). The reaction was carried out at 40 °C for 24 h. After the reaction, the supernatant was collected by centrifugation at 9000 rpm for 10 min, and the NeuAc content was determined by liquid chromatography.

[0156] The HPLC detection method is as follows:

[0157] The supernatant was filtered into a sample vial using a 0.22 μm filter membrane.

[0158] Liquid chromatography detection conditions: Column: Bio-Rad Aminex HPX-87H (300m×7.8mm) glycosyl column, 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 hours of whole-cell catalysis, the NeuAc yield in experimental group 3 increased by 19.6% compared to WT, and the NeuAc yield in group 4 increased by 27.7%.

[0161] Table 9. Unit point mutation combinations of AGE and NAL enzymes

[0162]

Claims

1. An N-acetylneuraminic acid lyase mutant, the amino acid sequence of which is the sequence obtained by mutating the sequence shown in SEQ ID NO.4, wherein the mutation is V251L, F252H+V251L, or V251L+I139V.

2. Biomaterials, including any of the following: B1) Encodes a nucleic acid molecule for the N-acetylneuraminic acid lyase mutant as described in claim 1; 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms 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).

3. A method for producing N-acetylneuraminic acid, comprising using the N-acetylneuraminic acid lyase mutant of claim 1 or the biomaterial of claim 2.

4. The application of the N-acetylneuraminic acid lyase mutant of claim 1, or the biomaterial of claim 2, wherein the application is selected from at least one of the following: I. Preparation of N-acetylneuraminic acid; II. Preparation of products containing N-acetylneuraminic acid; III. Increase the yield of N-acetylneuraminic acid produced by microorganisms.

Images