Alpha-1,2-fucosyltransferase mutants and uses thereof

Abstract

The present application relates to the field of biotechnology, and more particularly to alpha-1, 2-fucosyltransferase mutant and application thereof. Traditionally, it is considered that lactose is not a natural substrate of alpha-1, 2-fucosyltransferase, and the alpha-1, 2-fucosyltransferase mutant provided in the present application is obtained by site-directed mutagenesis of alpha-1, 2-fucosyltransferase, and the gene engineering bacteria thereof are constructed and applied in efficient catalysis of fucosylated oligosaccharide production. The alpha-1, 2-fucosyltransferase can use lactose as a substrate by replacing multiple amino acid residues.

Description

[0001] This application is a divisional application filed on September 30, 2021, with application number 202111159897.9, entitled "α-1,2-fucosyltransferase mutant and its application". Technical Field

[0002] This invention relates to the field of biotechnology, and more particularly to α-1,2-fucosyltransferase mutants and their applications. Background Technology

[0003] α-1,2-Fucosyltransferases belong to the fucosyltransferase family and catalyze the transfer of guanosine diphosphate-activated fucosylate to a galactosyl-linked acceptor substrate, forming an α-1,2-glycosidic bond. For example, GDP-L-fucose and lactose can be converted into 2'-fucosylated lactose under the catalysis of α-1,2-fucosyltransferases, which plays a crucial role in promoting the development of a healthy gut microbiota, immune system development, prevention of allergic diseases, and brain function development.

[0004] Currently, methods for synthesizing fucosylated products include chemical and biological methods. Chemical methods include separation and extraction and de novo synthesis. Separation and extraction refers to the separation and purification of fucosylated oligosaccharides from samples (such as breast milk). This method is limited by the amount of sample and suffers from significant losses. De novo synthesis assembles each group by synthesizing chemical groups, but this often involves multiple reaction steps, complex and difficult-to-control conditions, and the involvement of toxic or harmful reagents. Biological methods include whole-cell fermentation and enzymatic synthesis. Due to their mild conditions and high specificity, these are currently the main methods used.

[0005] Currently, the synthesis of fucosylated products mainly relies on whole-cell fermentation, which involves constructing the desired metabolic pathway in genetically engineered strains and introducing α-1,2-fucosyltransferase genes to achieve product synthesis. Foreign companies have already commercialized fucosylated oligosaccharides using this method. However, in my country, this method has not yet received industry approval due to the involvement of genetically engineered bacteria. Furthermore, the whole-cell fermentation process also has limitations in terms of the relatively low yield of the final product, leaving significant room for improvement. This is because the activity exhibited by the currently discovered α-1,2-fucosyltransferases does not meet industrial requirements, greatly limiting their large-scale application.

[0006] With the increasing demand for fucosylated products, the use of genetic engineering techniques to improve and create enzyme molecules and their genetically engineered strains in order to obtain functionally enhanced α-1,2-fucosyltransferases has become a development trend in the field of biocatalysis. Currently, there are no reports on the modification of the catalytic activity of α-1,2-fucosyltransferases. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide α-1,2-fucosyltransferase mutants and their applications, in order to improve the activity of fucosyltransferase and expand its substrate recognition range.

[0008] The mutant of α-1,2-fucosyltransferase provided by this invention has the wild-type amino acid sequence shown in SEQ ID NO:1, and the mutant includes at least one of the following mutation sites:

[0009] N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.

[0010] In some embodiments, the number of mutation sites in the mutant is 1 to 11.

[0011] In some specific embodiments, the mutation sites in the mutant are N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, and L271. Any one of S, G273C, H274G, N276Y, V283N, K284F or V291L;

[0012] Or possibly H269W and G172Y;

[0013] Or it could be H269W, G172Y, or C175F;

[0014] Or it could be H269W, G172Y, C175F, or I179E;

[0015] Or it could be H269W, G172Y, C175F, I179E, or K206W;

[0016] Or it could be H269W, G172Y, C175F, I179E, K206W, or Y249C;

[0017] Or it could be H269W, G172Y, C175F, I179E, K206W, Y249C, or Y255F;

[0018] Or H269W, G172Y, C175F, I179E, K206W, Y249C, Y255F, and G266C;

[0019] Or H269W, G172Y, C175F, I179E, K206W, Y249C, Y255F, G266C and H274G;

[0020] Or H269W, G172Y, C175F, I179E, K206W, Y249C, Y255F, G266C, H274G, and D115F;

[0021] Alternatively, it could be H269W, G172Y, C175F, I179E, K206W, Y249C, Y255F, G266C, H274G, D115F, or P116N.

[0022] The present invention also provides a nucleic acid encoding the mutant described herein.

[0023] The present invention also provides an expression vector comprising nucleic acid encoding the mutant described herein.

[0024] In some embodiments, the backbone vector of the expression vector is selected from the pET series vectors. In some specific embodiments, the backbone vector of the expression vector is pET22b.

[0025] The present invention also provides host cells for transforming or transfecting the expression vector.

[0026] In some embodiments, the host cell is *Escherichia coli*. In some specific embodiments, the host cell is the genetically engineered *E. coli* BL21(DE3).

[0027] The method for constructing the host cell includes: transforming the constructed expression vector into the host cell. The transformation method is heat shock transformation.

[0028] The method for preparing the mutant of the present invention includes: culturing the host cells and inducing the expression of the mutant.

[0029] The application of the mutant, nucleic acid, expression vector, host cell, or product prepared by the method described in this invention in the synthesis of fucosylated products.

[0030] The present invention also provides a biological agent for promoting the synthesis of fucosylation products, comprising: the mutant, nucleic acid, expression vector, host cell, or product obtained by the preparation method.

[0031] In some embodiments, the raw materials for preparing the biopharmaceutical include the fermentation broth of the mutant.

[0032] This invention also provides a method for synthesizing fucosylated products, using lactose as a substrate and the biological agent described in this invention to promote the synthesis of fucosylated products. In some embodiments, the reaction conditions for the synthesis include: reacting at pH = 6.0–8.0 and 20–40°C for 10–120 min.

[0033] Lactose is traditionally considered not to be a natural substrate for α-1,2-fucosyltransferase. However, the α-1,2-fucosyltransferase mutant provided in this invention, obtained through site-directed mutagenesis, allows for the construction of genetically engineered bacteria that can be used in the efficient catalytic production of fucosylated oligosaccharides. The α-1,2-fucosyltransferase, by replacing multiple amino acid residues, can utilize lactose as a substrate. Detailed Implementation

[0034] This invention provides a fucosyltransferase mutant and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0035] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0036] The wild-type α-1,2-fucosyltransferase has an amino acid sequence length of 298, as shown in SEQ ID NO:1, specifically:

[0037] MAFKVVQICGGGLGNQMFQYAFAKSLQKHLNTPVLLDITSFDWSNRKMQLELFPIDLPYASAKEIAIAKMQHLPKLVRDTLKCMGFDRVSQEIVFEYEPGLLKPSRLTYFYGYFQDPRYFDAISPLIKQTFTLPPPENGNNKKKEEEYHR KLALILAAKNSVFVHVRRGDYVGIGCQLGIDYQKKALEYIAKRVPNMELFVFCEDLKFTQNLDLGYPFMDMTTRDKEEEAYWDMLLMQSCKHGIIANSTYSWWAAYLINNPEKIIIGPKHWLFGHENILCKEWVKIESHFEVKSKKYNA

[0038] The nucleic acid encoding wild-type α-1,2-fucosyltransferase, after codon optimization, totals 897 bp, and its sequence is shown in SEQ ID NO:2. Specifically:

[0039] atggcgttcaaggtggttcagatttgcggtggcctgggtaaccagatgttccaatatgcgtttgcgaaaagcctgcaaaagcacctgaacaccccggttctgctggacattaccagcttcgattggagcaaccgtaaaatgcagctggagctgtttccgatcgacctgccgtacgcgagcgcgaaagaaattgcgatcgcgaagatgcagcacctgccgaaactggtgcgtgacaccctgaagtgcatgggtttcgatcgtgttagccaagagatcgtgtttgagtatgaaccgggtctgctgaaaccgagccgtctgacctacttctatggctactttcaggacccgcgttacttcgatgcgattagcccgctgatcaagcaaacctttaccctgccgccgccggaaaacggcaacaacaagaagaaggaagaggagtatcaccgtaagctggcgctgattctggcggcgaaaaacagcgtgtttgttcacgtgcgtcgtggtgactacgttggtattggctgccagctgggcatcgattatcagaagaaggcgctggagtacatcgcgaagcgtgttccgaacatggagctgttcgtgttttgcgaagacctgaaattcacccagaacctggatctgggttatccgtttatggacatgaccacccgtgataaggaagaggaagcgtactgggatatgctgctgatgcaaagctgcaaacacggcatcattgcgaacagcacctattcctggtgggcggcgtacctgatcaacaacccggaaaagatcattatcggtccgaaacactggctgttcggccacgagaacattctgtgcaaagaatgggttaagatcgagagccactttgaagtgaaaagcaaaaagtacaacgcg tga

[0040] In this invention, the mutant represents a change in one or more amino acid residues in the amino acid sequence (SEQ ID NO: 1) of the wild-type α-1,2-fucosyltransferase. Studies have shown that any of the following mutations in the amino acid sequence of the wild-type α-1,2-fucosyltransferase enables it to use lactose as a substrate. The mutation sites include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. The mutation sites are: G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L. As the number of mutation sites increases, the enzyme activity also increases.

[0041] In some embodiments, the mutant includes at least one of the following mutation sites:

[0042] N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.

[0043] For example, in some embodiments, the mutant includes the following mutation sites:

[0044] N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L1 00F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y Any one of the following: C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0045] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. Any two of the following: G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0046] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. Any three of the following: G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0047] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. Any four of the following: G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0048] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. Any 5 of the following: G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0049] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. Any 6 of the following: G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0050] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. Any 7 of the following: G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0051] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. Any 8 of the following: G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0052] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. Any 9 of the following: G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0053] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 10 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0054] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 11 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0055] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 12 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0056] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 13 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0057] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 14 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0058] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 15 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0059] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 16 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0060] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 17 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0061] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 18 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0062] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 19 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0063] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 20 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0064] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 21 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0065] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 22 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0066] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 23 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0067] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 24 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0068] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 25 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0069] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 26 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0070] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 27 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0071] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 28 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0072] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 29 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0073] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 30 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0074] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 31 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0075] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 32 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0076] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 33 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0077] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 34 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0078] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 35 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0079] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 36 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0080] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 37 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0081] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 38 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0082] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 39 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0083] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 40 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0084] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 41 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0085] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G Any 42 of the following: 172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, or V291L.

[0086] Alternatively, the mutants include N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118 T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I2 64V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F and V291L.

[0087] In this embodiment of the invention, validation was performed for mutants with different numbers of mutation sites, for example:

[0088] The mutant with a mutation site number of 1 has a mutation site of H269W; the nucleic acid encoding the mutant is identical to SEQ ID NO:2 at other positions, but the codon encoding the 269th amino acid is tgg.

[0089] The mutant with two mutation sites has mutation sites H269W and G172Y. The nucleic acids encoding the mutant are identical to those in SEQ ID NO:2, but the codons encoding the two mutation sites are tgg and tac, respectively.

[0090] The mutant with 3 mutation sites has the mutation sites H269W, G172Y, and C175F. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 3 mutation sites are tgg, tac, and gcg, respectively.

[0091] The mutant with 4 mutation sites has the mutation sites H269W, G172Y, C175F, and I179E. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 4 mutation sites are tgg, tac, gcg, ctg, and aaa, respectively.

[0092] The mutant with 5 mutation sites has the mutation sites H269W, G172Y, C175F, I179E, and K206W. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 5 mutation sites are tgg, tac, gcg, ctg, aaa, and tgg, respectively.

[0093] The mutant with 6 mutation sites has the following mutation sites: H269W, G172Y, C175F, I179E, K206W, and Y249C. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 6 mutation sites are tgg, tac, gcg, ctg, aaa, tgg, and tgc, respectively.

[0094] The mutant with 7 mutation sites has the following mutation sites: H269W, G172Y, C175F, I179E, K206W, Y249C, and Y255F. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 7 mutation sites are tgg, tac, gcg, ctg, aaa, tgg, tgc, and ttc, respectively.

[0095] The mutant with 8 mutation sites has the following mutation sites: H269W, G172Y, C175F, I179E, K206W, Y249C, Y255F, and G266C. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 8 mutation sites are, in order, tgg, tac, gcg, ctg, aaa, tgg, tgc, ttc, and tgc.

[0096] The mutant with 9 mutation sites has the following mutation sites: H269W, G172Y, C175F, I179E, K206W, Y249C, Y255F, G266C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 9 mutation sites are, in order, tgg, tac, gcg, ctg, aaa, tgg, tgc, ttc, tgc, and gac.

[0097] The mutant with 10 mutation sites has the following mutation sites: H269W, G172Y, C175F, I179E, K206W, Y249C, Y255F, G266C, H274G, and D115F. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 10 mutation sites are, in order, tgg, tac, gcg, ctg, aaa, tgg, tgc, ttc, tgc, gac, and ttc.

[0098] The mutant with 11 mutation sites has the following mutation sites: H269W, G172Y, C175F, I179E, K206W, Y249C, Y255F, G266C, H274G, D115F, and P116N. The nucleic acids encoding this mutant are identical to those in SEQ ID NO:2, but the codons encoding the 11 mutation sites are, in order: tgg, tac, gcg, ctg, aaa, tgg, tgc, ttc, tgc, gac, ttc, aac.

[0099] The mutant with 12 mutation sites has the following mutation sites: D115F, P116N, G172Y, C175F, I179E, K206W, Y249C, Y255F, G266C, H269W, L271S, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 12 mutation sites are, in order, ttc, aac, tac, ttt, gag, tgg, tgc, ttt, tgc, tac, agc, tgc, and tac.

[0100] The mutant with 13 mutation sites has the following mutation sites: D115F, P116N, G172Y, C175F, I179E, K206W, Y249C, Y255F, G266C, H269W, L271S, G273C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 13 mutation sites are, in order: ttc, aac, tac, ttt, gag, tgg, tgc, ttt, tgc, tac, agc, tgc, ggt, and tac.

[0101] The mutant with 14 mutation sites has the following mutation sites: D115F, P116N, G172Y, C175F, I179E, K206W, Y249C, Y255F, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 14 mutation sites are, in order, ttc, aac, tac, ttt, gag, tgg, tgc, ttt, tgc, tac, tgg, agc, tgc, ggt, and tac.

[0102] The mutant with 15 mutation sites has the following mutation sites: D115F, P116N, G172Y, C175F, I179E, K206W, Y249C, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 15 mutation sites are, in order, ttc, aac, tac, ttt, gag, tgg, tgc, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, and tac.

[0103] The mutant with 16 mutation sites has the following mutation sites: D115F, P116N, G172Y, C175F, I179E, K206W, Y249C, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 16 mutation sites are, in order, ttc, aac, tac, ttt, gag, tgg, tgc, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, and tac.

[0104] The mutant with 17 mutation sites has the following mutation sites: D115F, P116N, G172Y, C175F, I179E, K206W, Y249C, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 17 mutation sites are, in order, ttc, aac, tac, ttt, gag, tgg, tgc, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, and tac.

[0105] The mutant with 18 mutation sites has the following mutation sites: D115F, P116N, G172Y, C175F, I179E, K206W, Y249C, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 18 mutation sites are, in order, ttc, aac, tac, ttt, gag, tgg, tgc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, and tac.

[0106] The mutant with 19 mutation sites has the following mutation sites: D115F, P116N, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 19 mutation sites are, in order, ttc, aac, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, and tac.

[0107] The mutant with 20 mutation sites has the following mutation sites: D115F, P116N, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 20 mutation sites are, in order, ttc, aac, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, and tac.

[0108] The mutant with 21 mutation sites has the following mutation sites: D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding the mutant at other positions are identical to those in SEQ ID NO:2, but the codons encoding the above 21 mutation sites are, in order: ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, and tac.

[0109] The mutant with 22 mutation sites contains the following mutation sites: Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 22 mutation sites are gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0110] The mutant with 23 mutation sites contains the following mutation sites: F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding this mutant at other positions are similar to SEQ ID NO. The IDNO:2 is the same, but the codons encoding the above 23 mutation sites are, in order, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0111] The mutant with 24 mutation sites contains the following mutation sites: Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 24 mutation sites are att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0112] The mutant with 25 mutation sites contains the following mutation sites: G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 25 mutation sites are, in order: aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0113] The mutant with 26 mutation sites contains the following mutation sites: F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. NO:2 is the same, but the codons encoding the above 26 mutation sites are att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0114] The mutant with 27 mutation sites contains the following mutation sites: L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 27 mutation sites are identical to NO:2, but are ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0115] The mutant with 28 mutation sites contains the following mutation sites: Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 28 mutation sites are, in order: agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0116] The mutant with 29 mutation sites contains the following mutation sites: E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. NO:2 is the same, but the codons encoding the above 29 mutation sites are, in order: att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0117] The mutant with 30 mutation sites contains the following mutation sites: F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. NO:2 is the same, but the codons encoding the above 30 mutation sites are, in order: gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0118] The mutant with 31 mutation sites contains the following mutation sites: S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 31 mutation sites are identical to NO:2, but are as follows: gcg, gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0119] The mutant with 32 mutation sites contains the following mutation sites: V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 32 mutation sites are identical to NO:2, but are as follows: aac, gcg, gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0120] The mutant with 33 mutation sites contains the following mutation sites: L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. NO:2 is the same, but the codons encoding the above 33 mutation sites are, in order, aaa, aac, gcg, gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0121] The mutant with 34 mutation sites contains the following mutation sites: Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 34 mutation sites are identical to NO:2, but are as follows: cac, aaa, aac, gcg, gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0122] The mutant with 35 mutation sites contains the following mutation sites: F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 35 mutation sites are identical to NO:2, but are as follows: gcg, cac, aaa, aac, gcg, gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0123] The mutant with 36 mutation sites contains the following mutation sites: F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The codons encoding the above 36 mutation sites are identical to NO:2, but are as follows: tat, gcg, cac, aaa, aac, gcg, gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.

[0124] The mutant with 37 mutation sites contains the following mutation sites: M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids encoding other positions of this mutant are similar to SEQ ID NO. The IDNO:2 is the same, but the codons encoding the above 37 mutation sites are, in order: acc, tat, gcg, cac, aaa, aac, gcg, gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac

[0125] The mutant with 38 mutation sites contains the following mutation sites: Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, and H274G. The nucleic acids at other positions encoding this mutant are similar to SEQ ID. The sequence is identical to NO:2, but the codons encoding the above 38 mutation sites are, in order: tgc, acc, tat, gcg, cac, aaa, aac, gcg, gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac

[0126] Among the mutants with 39 mutation sites, the mutation sites are N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, and R11. 7K, Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, the nucleic acids encoding this mutant at other positions are the same as SEQ ID NO. The ID NO:2 is the same, but the codons encoding the above 39 mutation sites are, in order: atg, tgc, acc, tat, gcg, cac, aaa, aac, gcg, gac, attc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac

[0127] Among the mutants with 40 mutation sites, the mutation sites are N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, and R117K. Y118T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, the nucleic acids encoding this mutant at other positions are similar to SEQ ID NO. The ID NO:2 is the same, but the codons encoding the above 40 mutation sites are, in order: atg, tgc, acc, tat, gcg, cac, aaa, aac, gcg, gac, attc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, aagc, tgc, ggt, tac, aac

[0128] The mutant with 41 mutation sites contains the following mutation sites: N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y11. 8T, G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, the nucleic acids encoding this mutant at other positions are similar to SEQ ID NO. The ID NO:2 is the same, but the codons encoding the above 41 mutation sites are, in order: atg, tgc, acc, tat, gcg, cac, aaa, aac, gcg, gac, attc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, aagc, tgc, ggt, tac, aac, ttt

[0129] Among the mutants with 42 mutation sites, the mutation sites are N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, and Y118T. G172Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, the nucleic acids encoding this mutant at other positions are similar to SEQ ID NO. The ID NO:2 is the same, but the codons encoding the above 42 mutation sites are, in order: atg, tgc, acc, tat, gcg, cac, aaa, aac, gcg, gac, attc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, aagc, tgc, ggt, tac, aac, ttt, ctg

[0130] Among the mutants with 43 mutation sites, the mutation sites are N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, and G17. 2Y, C175F, I179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, and V291L; the nucleic acids encoding this mutant are identical to those in SEQ ID NO:2 at other positions, but the codon encoding Met at position 14 is atg, and the codon encoding Cys at position 15 is tgc.

[0131] The codon for the 16th bit Thr is acc, and the codon for the 17th bit Tyr is tat.

[0132] The codon for the 21st Ala is gcg, and the codon for the 71st Ser is cac.

[0133] The codon for the 73rd bit Lys is aaa, and the codon for the 88th bit Asn is aac.

[0134] The codon for the 89th bit Ala is gcg, and the codon for the 94th bit Asp is gac.

[0135] The codon for the 95th bit Ile is att, and the codon for the 96th bit Ser is agc.

[0136] The codon for encoding the 100th bit Phe is ttc, and the codon for encoding the 109th bit Ile is att.

[0137] The codon for encoding the 111th bit Asn is aac, and the codon for encoding the 112th bit Ile is att.

[0138] The codon encoding the 113th Arg is cgt, and the codon encoding the 114th Val is gtg.

[0139] The codon for encoding the 115th bit Phe is ttc, and the codon for encoding the 116th bit Asn is aac.

[0140] The codon for the 117th bit Lys is aag, and the codon for the 118th bit Thr is acc.

[0141] The codon for encoding the 172nd bit Tyr is tac, and the codon for encoding the 175th bit Phe is ttt.

[0142] The codon encoding the 179th position Glu is gag, and the codon encoding the 206th position Trp is tgg.

[0143] The codon for the 249th bit Cys is tgc, and the codon for the 250th bit Tyr is acc.

[0144] The codon for the 251st bit Asn is aac, and the codon for the 252nd bit Ile is att.

[0145] The codon for encoding the 254th Phe is ttt, and the codon for encoding the 255th Phe is also ttt.

[0146] The codon encoding the 264th bit Val is gtt, and the codon encoding the 266th bit Cys is tgc.

[0147] The codon for the 267th bit Tyr is tac, and the codon for the 269th bit Trp is tgg.

[0148] The codon for the 271st Ser bit is agc, and the codon for the 273rd Cys bit is tgc.

[0149] The codon for the 274th bit Gly is ggt, and the codon for the 276th bit Tyr is tac.

[0150] The codon for encoding the 283rd bit Asn is aac, and the codon for encoding the 284th bit Phe is ttt.

[0151] The codon for the 291st character Leu is ctg.

[0152] This invention utilizes a one-step site-directed mutagenesis technique to mutate multiple amino acid sites of α-1,2-fucosyltransferase, altering its catalytic activity and thereby enhancing its ability to catalyze the synthesis of fucosylated products. The highest enzyme activity obtained with the same number of mutation sites, as described in this invention, refers to the highest enzyme activity detected among all mutants with the same number of mutation sites (e.g., all mutants containing 3 mutation sites, all mutants containing 5 mutation sites). In other words, with a certain number of mutation sites, this mutant exhibits the highest enzyme activity, while other mutants have relatively lower enzyme activities. This indicates that the mutation site configuration in the mutant with the highest enzyme activity is more conducive to the expression of enzyme activity.

[0153] This invention also provides a recombinant expression vector containing the α-1,2-fucosyltransferase gene; the recombinant expression vector preferably uses pET22b as the original expression vector; the α-1,2-fucosyltransferase gene is preferably inserted into the plasmid pET22b; this invention does not impose any special restrictions on the method of constructing the recombinant vector, and conventional methods in the art can be used.

[0154] In the specific implementation of this invention, the recombinant vector is constructed using the following method: using the encoding nucleotide sequence as a template, PCR is performed to obtain the recombinant expression vector pET22b-futC.

[0155] This invention also provides a recombinant bacterium comprising the recombinant expression vector described above; the recombinant bacterium preferably uses *Escherichia coli* as the host bacterium; the *E. coli* preferably includes *E. coli* BL21(DE3), NovaBlue, Origami, PlySs, T7 shuffle, and other DE3 series strains. This invention does not impose any special limitations on the construction method of the recombinant bacterium; conventional methods in the art can be used. In a specific implementation of this invention, the recombinant bacterium is prepared using the following method: the recombinant expression vector pET22b-futC is transformed into *E. coli* BL21 to obtain the recombinant bacterium, named pET22b-futC-E. coliBL21.

[0156] The α-1,2-fucosyltransferase mutant described in this invention is preferably prepared by the following method: culturing the recombinant bacteria and inducing the α-1,2-fucosyltransferase mutant; the culture medium for culturing the recombinant bacteria is preferably TB medium; the TB medium contains 1.2% (w / v) peptone, 2.4% (w / v) yeast extract, 0.4% (v / v) glycerol, 17 mM KH2PO4, and 72 mM K2HPO4. The culture temperature is preferably 37°C, and the culture time is based on the OD600 of the culture reaching 0.6-0.8. The induction method is preferably to add isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium at a final concentration of 0.1-1.0 mM and induce culture at 16-37°C for 4-28 h.

[0157] The induced fermentation broth was centrifuged to extract the cell fragments, and the supernatant was collected to promote the synthesis of fucosylation products. In the specific implementation of this invention, GDP-L-fucose, lactose, acetylated lactose, etc., were used as substrates. The α-1,2-fucosyltransferase mutant or its recombinant strain, without coenzymes, was reacted for 0.1-2 hours in an in vitro catalytic reaction system consisting of a buffer solution with a pH of 6.0-8.0 at 20-40°C to obtain a mixed enzymatic reaction system including the α-1,2-fucosyltransferase mutant.

[0158] In some embodiments, the fucosylation product is 2'-fucosylated lactose. In some specific embodiments, the preparation method of 2'-fucosylated lactose includes: adding the supernatant after lysing the fermentation cells to a TRIS-HCl buffered fermentation broth containing GDP-fucose and lactose, and incubating at 20-40°C for 10-120 min to obtain a reaction solution containing 2'-fucosylated lactose.

[0159] Previously, it was believed in the art that most enzymes could not complete enzymatic catalysis using lactose as a fucosylation acceptor. The wild-type α-1,2-fucosylation enzyme selected in this scheme also lacks the ability to use lactose as a fucosylation acceptor, which is one of the reasons for the extremely low activity of the wild-type enzyme. After mutation, its enzyme activity was significantly improved when lactose was still used as a substrate, that is, its ability to utilize lactose as a substrate was enhanced. As lactose is a common raw material in the food industry, it has many advantages such as being readily available, safe, and inexpensive. Therefore, it is even more necessary to develop enzymes that can efficiently convert lactose as a substrate.

[0160] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0161] Example 1

[0162] Based on the amino acid sequence of the mutant, the corresponding coding nucleic acid sequence was obtained through codon optimization (see Table 1).

[0163] The synthesized nucleic acid fragment was inserted into the pET22b vector to construct the recombinant vector pET22b-futC. This recombinant vector was transformed into *E. coli* DH5α competent cells, cultured overnight, and then transformed into *E. coli* BL21 competent cells. After recovery at 37°C for 1 hour, the cells were plated onto LB agar plates containing ampicillin at a final concentration of 0.1 mM and incubated at 37°C for 10–16 hours. Finally, single colonies from the ampicillin-resistant agar plates were selected for colony PCR verification, and sequencing confirmed the successful construction of the recombinant engineered bacteria containing the nucleic acid encoding the mutant.

[0164] Example 2: Expression and purification of α-1,2-fucosyltransferase in recombinant pET22b-futC-E.coli BL21

[0165] The recombinant strain pET22b-futC-E.coliBL21 constructed in Example 1 and the control strain expressing the unmutated original enzyme α-1,2-fucosyltransferase were separately inoculated into LB medium containing 0.1% ampicillin and cultured overnight at 37°C with shaking to prepare seed culture. The next day, 1% of the seed culture was transferred to 200 mL of TB medium containing ampicillin and cultured at 37°C for an appropriate time. After induction, an appropriate amount of IPTG was added and the culture was induced overnight at 16°C. After induction, the cells were collected by centrifugation at 8000 rpm at 4°C, and the cells were sonicated. The cell lysis supernatant (crude enzyme solution) was collected for subsequent purification.

[0166] Purification of α-1,2-fucosyltransferase or α-1,2-fucosyltransferase mutants was achieved using the histidine tag on the pET22b vector and Ni 2+ -NTA affinity chromatography was used for further purification. The crude enzyme solution was filtered through a 0.22 μm filter membrane and added at an appropriate flow rate to the pre-equilibrated Ni... 2+ In the column, wash with buffer 1 for 10 × column volume. Elute in stages with buffer 2 containing 10, 20, 50, 100, 200, 300, 400, and 500 mM imidazole, respectively. Collect the eluted samples at each stage and determine the protein concentration, molecular weight, and purity using Bradford and SDS-PAGE.

[0167] Buffer 1, preferably Tris-HCl buffer, 1, 5, 10, 20, 50 mM imidazole, pH 7.0-8.0.

[0168] Buffer 2, preferably Tris-HCl buffer, 10, 20, 50, 100, 200, 300, 400, 500 mM imidazole, pH 7.0-8.0.

[0169] Enzyme activity assay:

[0170] First, 20 mL of fermentation broth from each group was centrifuged at 12000 rpm for 15-20 min, and the cells were collected. After resuspending, the cells were disrupted by sonication and centrifuged at low temperature and high speed to obtain the cell disruption supernatant. The supernatant was purified by histidine tagging to obtain a relatively pure enzyme solution, and imidazole was removed by dialysis to adjust the buffer salt concentration. The final enzyme solution was stored for later use.

[0171] 100 μL of 100 μM GDP-L-fucose and 100 μL of 5 mM lactose were added, followed by the addition of 1 μg of enzyme. The reaction was incubated at 20-40 °C for 10-120 min to catalyze the formation of 2'-FL. The reaction was immediately stopped on ice after catalysis. Subsequently, the mixture was centrifuged at low temperature and ultrafiltered using a desalting column to remove impurities. The yield of 2'-FL and the remaining amount of substrate in the catalytic system were determined by high-performance liquid chromatography (HPLC).

[0172] The reaction solution obtained from the catalysis was filtered through a 0.22 μm filter membrane and set aside for later use. The mobile phases required for liquid chromatography detection were prepared: mobile phase A: acetonitrile, and mobile phase B: 0.5% aqueous solution of triethylamine acetate. An amide column (150 mm × 2.1 μm) was selected, and the detection conditions were: mobile phase ratio 80:20, flow rate 0.2 m / L, column temperature 35 °C, evaporative light scattering detector (ELSD) at 70 °C, and carrier gas flow rate 2.0 SLM. The product was quantified using the external standard method.

[0173] One unit of activity (U) is defined as the amount of enzyme required to convert 1 micromole of 2'-FL within 1 minute at 37°C. The recombinant enzyme was then subjected to in vitro enzyme catalysis verification under the above conditions. The specific activities of the purified enzyme proteins obtained at this time are shown in the table below:

[0174] Table 1

[0175]

[0176] Note: The table shows the highest enzyme activity obtained with the same number of mutation sites.

[0177] The results showed that mutations at specific sites in α-1,2-fucosyltransferase resulted in varying degrees of increased enzyme activity. Among them, mutant G266C exhibited the highest enzyme activity. Mutants containing two or more mutation sites showed stronger enzyme activity, and within a certain range, the more mutation sites present, the stronger the highest enzyme activity obtained at that number of mutation sites.

[0178] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of α-1,2-fucosyltransferase, characterized in that, The amino acid sequence of its wild type is shown in SEQ ID NO:1, and the mutation site of the mutant is H269W.

2. The nucleic acid encoding the mutant of claim 1.

3. An expression vector comprising a nucleic acid encoding the mutant of claim 1.

4. Transform or transfect the host cells of the expression vector according to claim 3.

5. The method for preparing the mutant according to claim 1, comprising: Culture the host cells according to claim 4 to induce the expression of the mutant.

6. The use of the mutant of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, the host cell of claim 4, or the product prepared by the method of claim 5 in the synthesis of fucosylated products.

7. A biological agent for promoting the synthesis of fucosylation products, characterized in that, include: The mutant of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, the host cell of claim 4, or the product prepared by the method of claim 5.

8. A method for synthesizing fucosylated products, characterized in that, Using lactose as a substrate, the bio-agent of claim 7 is used to promote the synthesis of fucosylated products.

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