CN113817701B - Alpha-1, 2-fucosyltransferase mutant and application thereof - Google Patents

Alpha-1, 2-fucosyltransferase mutant and application thereof Download PDF

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CN113817701B
CN113817701B CN202111159897.9A CN202111159897A CN113817701B CN 113817701 B CN113817701 B CN 113817701B CN 202111159897 A CN202111159897 A CN 202111159897A CN 113817701 B CN113817701 B CN 113817701B
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tgc
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tac
aac
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CN113817701A (en
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周洪波
刘雯娴
潘丽娜
朱玉玲
汪家琦
王玉光
陈祝
戴智勇
程海娜
颜卫彬
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Ausnutria Dairy China Co ltd
Changsha Yun Kang Bio Technology Co ltd
Central South University
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Ausnutria Dairy China Co ltd
Changsha Yun Kang Bio Technology Co ltd
Central South University
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12Y204/01069—Galactoside 2-alpha-L-fucosyltransferase (2.4.1.69)
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Abstract

The invention relates to the field of biotechnology, in particular to an alpha-1, 2-fucosyltransferase mutant and application thereof. Lactose is not a natural substrate of alpha-1, 2-fucosyltransferase in the traditional way, but the alpha-1, 2-fucosyltransferase mutant provided by the invention is transformed by site-directed mutagenesis to obtain the alpha-1, 2-fucosyltransferase mutant, and genetic engineering bacteria of the mutant are constructed and applied to efficient catalytic fucosylation oligosaccharide production. The alpha-1, 2-fucosyltransferase is capable of using lactose as a substrate through substitution of multiple amino acid residues.

Description

Alpha-1, 2-fucosyltransferase mutant and application thereof
Technical Field
The invention relates to the field of biotechnology, in particular to an alpha-1, 2-fucosyltransferase mutant and application thereof.
Background
Alpha-1, 2-fucosyltransferases belong to the family of fucosyltransferases, catalyzing the transfer of guanosine diphosphate activated fucosyl groups to galactosyl linked acceptor substrates, forming alpha-1, 2-glycosidic linkages. For example, GDP-L-fucose and lactose can produce 2' -fucosyllactose under the catalysis of alpha-1, 2-fucosyltransferase, which plays an extremely important role in promoting healthy intestinal flora construction, immune system development, allergic disease prevention, brain function development, etc.
Current methods for synthesizing fucosylated products include chemical and biological methods. Chemical methods include isolation extraction and de novo synthesis. Separation and extraction refers to the separation and purification of fucosylated oligosaccharides from a sample (e.g., breast milk), which is subject to substantial amounts of sample and significant losses. The de novo synthesis method realizes the assembly of each group through synthesizing chemical groups, but the method is often accompanied by the problems of a plurality of reaction steps, complex and poorly controlled conditions, participation of toxic and harmful reagents and the like. Biological methods comprise whole-cell fermentation and enzymatic synthesis, and are mainly adopted at present due to the advantages of mild conditions, strong specificity and the like.
Currently, the synthesis of fucosylated products is mainly achieved by whole cell fermentation, i.e. by constructing the desired metabolic pathway in genetically engineered strains and transferring the alpha-1, 2-fucosyltransferase gene. The commercialization of fucosylated oligosaccharides has been achieved by foreign companies in this way. In China, the method has not been approved for industrial application due to the participation of transgenic engineering bacteria. In addition, the disadvantage of whole cell fermentation processes is also represented by the fact that the yield of the final product is still at a low level, and there is a large space for improvement. This is because the activity exhibited by the alpha-1, 2-fucosyltransferases, which have been found to be insufficient for industrial purposes, has greatly limited their large-scale use.
With the increasing demands of people for fucosylation products, the application of genetic engineering technology to improve and create enzyme molecules and genetic engineering strains thereof to obtain function-enhanced alpha-1, 2-fucosyltransferase has become a development trend in the field of biocatalysis. Modification of the catalytic activity of alpha-1, 2-fucosyltransferase has not been reported.
Disclosure of Invention
In view of the above, the present invention has been made to solve the technical problems of providing an α -1, 2-fucosyltransferase mutant and its use, in order to increase the activity of fucosyltransferase and to expand the substrate recognition range thereof.
The invention provides a mutant of alpha-1, 2-fucosyltransferase, the wild amino acid sequence of which is shown as SEQ ID NO. 1, and the mutant comprises at least one of 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, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
In some embodiments, the number of mutation sites of the mutant is 1 to 11.
In some embodiments, the mutation site in the mutant is any one of N14 15 16 17 71 73 88 89 95 100 109 111 112 114 116 117 118 172 179 249 251 252 254 264 266 267 269 271 273 274 276 283F or V291L;
or H269W and V172Y;
or H269W, V Y and I175F;
or H269W, V172Y, I F and C179E;
or H269W, V172Y, I175F, C179E and K206W;
or H269W, V172Y, I175F, C179E, K W and Y249C;
or H269W, V172Y, I175F, C179E, K206W, Y249C and Y255F;
or H269W, V172Y, I175F, C179E, K W, Y249C, Y F and G266C;
or H269W, V172Y, I175F, C E, K W, Y249C, Y255F, G266C and H274G;
or H269W, V172Y, I175F, C E, K W, Y249C, Y255F, G266C, H274G and D115F;
or H269W, V172Y, I175F, C E, K W, Y249C, Y255F, G266C, H274G, D115F and P116N.
The invention also provides nucleic acids encoding the mutants of the invention.
The invention also provides an expression vector comprising a nucleic acid encoding the mutant of the invention.
In some embodiments, the backbone vector of the expression vector is selected from the pET series of vectors. In some embodiments, the backbone vector of the expression vector is pET22b.
The invention also provides host cells transformed or transfected with the expression vectors.
In some embodiments, the host cell is E.coli. In some embodiments, the host cell is E.coli BL21 (DE 3).
The construction method of the host cell comprises the following steps: the expression vector obtained by the construction is transformed into a host cell. The conversion method is heat shock conversion.
The preparation method of the mutant comprises the following steps: culturing said host cell, and inducing expression of said mutant.
The mutant, the nucleic acid, the expression vector, the host cell or the product prepared by the preparation method are applied to the synthesis of fucosylation products.
The present invention also provides a biological agent that promotes synthesis of a fucosylation product, comprising: the mutants, nucleic acids, expression vectors, host cells or products produced by the methods.
In some embodiments, the preparation feedstock for the biological agent comprises a fermentation broth of the mutant.
The invention also provides a method for synthesizing the fucosylation product, which takes lactose as a substrate and promotes the synthesis of the fucosylation product by using the biological agent. In some embodiments, the reaction conditions of the synthesis include: the reaction is carried out for 10 to 120 minutes at the temperature of 20 to 40 ℃ under the condition that the pH=6.0 to 8.0.
Lactose is not a natural substrate of alpha-1, 2-fucosyltransferase in the traditional way, but the alpha-1, 2-fucosyltransferase mutant provided by the invention is transformed by site-directed mutagenesis to obtain the alpha-1, 2-fucosyltransferase mutant, and genetic engineering bacteria of the mutant are constructed and applied to efficient catalytic fucosylation oligosaccharide production. The alpha-1, 2-fucosyltransferase is capable of using lactose as a substrate through substitution of multiple amino acid residues.
Detailed Description
The invention provides fucosyltransferase mutants and application thereof, and one skilled in the art can properly improve process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For definitions and terms in the art, the expert may refer specifically to CurrentProtocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
The length of the amino acid sequence of the wild alpha-1, 2-fucosyltransferase is 298, the sequence is shown as SEQ ID NO. 1, and the method specifically comprises the following steps:
MAFKVVQICGGLGNQMFQYAFAKSLQKHLNTPVLLDITSFDWSNRKMQLELFPIDLPYASAKEIAIAKMQHLPKLVRDTLKCMGFDRVSQEIVFEYEPGLLKPSRLTYFYGYFQDPRYFDAISPLIKQTFTLPPPENGNNKKKEEEYHRKLALILAAKNSVFVHVRRGDYVGIGCQLGIDYQKKALEYIAKRVPNMELFVFCEDLKFTQNLDLGYPFMDMTTRDKEEEAYWDMLLMQSCKHGIIANSTYSWWAAYLINNPEKIIIGPKHWLFGHENILCKEWVKIESHFEVKSKKYNA
the nucleic acid for encoding the wild alpha-1, 2-fucosyltransferase is codon optimized to total 897bp, and the sequence is shown as SEQ ID NO. 2, specifically:
atggcgttcaaggtggttcagatttgcggtggcctgggtaaccagatgttccaatatgcgtttgcgaaaagcctgcaaaagcacctgaacaccccggttctgctggacattaccagcttcgattggagcaaccgtaaaatgcagctggagctgtttccgatcgacctgccgtacgcgagcgcgaaagaaattgcgatcgcgaagatgcagcacctgccgaaactggtgcgtgacaccctgaagtgcatgggtttcgatcgtgttagccaagagatcgtgtttgagtatgaaccgggtctgctgaaaccgagccgtctgacctacttctatggctactttcaggacccgcgttacttcgatgcgattagcccgctgatcaagcaaacctttaccctgccgccgccggaaaacggcaacaacaagaagaaggaagaggagtatcaccgtaagctggcgctgattctggcggcgaaaaacagcgtgtttgttcacgtgcgtcgtggtgactacgttggtattggctgccagctgggcatcgattatcagaagaaggcgctggagtacatcgcgaagcgtgttccgaacatggagctgttcgtgttttgcgaagacctgaaattcacccagaacctggatctgggttatccgtttatggacatgaccacccgtgataaggaagaggaagcgtactgggatatgctgctgatgcaaagctgcaaacacggcatcattgcgaacagcacctattcctggtgggcggcgtacctgatcaacaacccggaaaagatcattatcggtccgaaacactggctgttcggccacgagaacattctgtgcaaagaatgggttaagatcgagagccactttgaagtgaaaagcaaaaagtacaacgcg tga
in the present invention, the mutant is altered with respect to one or more amino acid residues in the amino acid sequence (SEQ ID NO: 1) of the wild-type alpha-1, 2-fucosyltransferase. Studies have shown that any of the following mutations in the amino acid sequence of wild-type alpha-1, 2-fucosyltransferase may allow lactose to be used as a substrate. The mutation site includes N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L. And as the number of mutation sites increases, the enzyme activity increases.
In some embodiments, the mutant includes at least one of 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, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
For example, in some embodiments, the mutants include 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, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 2 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 3 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 4 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 5 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 6 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 7 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, a254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 8 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 9 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 10 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 11 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 12 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 13 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 14 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 15 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 16 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 17 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 18 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 19 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 20 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, a254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 21 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 22 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 23 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 24 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 25 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 26 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 27 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 28 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 29 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 30 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 31 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 32 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 33 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 34 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 35 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 36 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 37 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 38 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 39 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 40 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 41 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
Alternatively, the mutant includes any 42 of N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F or V291L.
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, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F and V291L.
In the embodiment of the invention, the verification is performed for mutants with different mutation site numbers, for example:
the mutant with the number of mutation sites being 1 has the mutation site of H269W; the nucleic acid encoding the mutant corresponds to SEQ ID NO. 2 at other positions, but the codon encoding amino acid 269 is tgg.
The mutant with the number of mutation sites of 2 has mutation sites of H269W and V172Y; the nucleic acid of other positions of the nucleic acid for encoding the mutant is identical to SEQ ID NO. 2, but the codons for encoding the 2 mutation sites are tgg and tac in sequence.
The mutant with the mutation site number of 3 is H269W, V172Y and I175F; the nucleic acid encoding the mutant is identical to SEQ ID NO. 2 at other positions, but the codons encoding the 3 mutation sites are tgg, tac and gcg in sequence.
The mutant with the number of 4 mutation sites has mutation sites of H269W, V172Y, I175F and C179E; the nucleic acid encoding the mutant has the same nucleic acid as SEQ ID NO. 2 at other positions, but the codons encoding the 4 mutation sites are tgg, tac, gcg, ctg and aaa in sequence.
The mutant with the mutation site number of 5 is H269W, V172Y, I175F, C179E and K206W; the nucleic acid encoding the mutant has the same nucleic acid at other positions as SEQ ID NO. 2, but the codons encoding the 5 mutation sites are tgg, tac, gcg, ctg, aaa and tgg in sequence.
The mutant with the mutation site number of 6 comprises mutation sites of H269W, V172Y, I175F, C179E, K206W and Y249C; the nucleic acid encoding the mutant has the same nucleic acid at other positions as SEQ ID NO. 2, but the codons encoding the 6 mutation sites are tgg, tac, gcg, ctg, aaa, tgg and tgc in sequence.
The mutant with the mutation site number of 7 comprises mutation sites of H269W, V172Y, I175F, C179E, K206W, Y249C and Y255F; the nucleic acid encoding the mutant has the same nucleic acid at other positions as SEQ ID NO. 2, but the codons encoding the 7 mutation sites are tgg, tac, gcg, ctg, aaa, tgg, tgc and ttc in sequence.
The mutant with the mutation site number of 8 is H269W, V172Y, I175F, C179E, K206W, Y249C, Y255F and G266C; the nucleic acid encoding the mutant has the same nucleic acid at other positions as SEQ ID NO. 2, but the codons encoding the 8 mutation sites are tgg, tac, gcg, ctg, aaa, tgg, tgc, ttc and tgc in sequence.
The number of mutation sites in the mutant with the mutation site number of 9 is H269W, V172Y, I175F, C179E, K206W, Y249C, Y255F, G266C and H274G; the nucleic acid encoding the mutant has the same nucleic acid at other positions as SEQ ID NO. 2, but the codons encoding the 9 mutation sites are tgg, tac, gcg, ctg, aaa, tgg, tgc, ttc, tgc and gac in sequence.
The number of mutation sites in the mutant with 10 mutation sites is H269W, V172Y, I175F, C179E, K206W, Y249C, Y255F, G266C, H274G, D115F; the nucleic acid encoding the mutant has the same nucleic acid at other positions as SEQ ID NO. 2, but the codons encoding the 10 mutation sites are tgg, tac, gcg, ctg, aaa, tgg, tgc, ttc, tgc, gac and ttc in sequence.
The number of mutation sites 11 was H269W, V172Y, I175F, C179E, K206W, Y249C, Y255F, G266C, H274G, D115F, P116N. The nucleic acid encoding the mutant has the same nucleic acid at other positions as SEQ ID NO. 2, but the codons encoding the 11 mutation sites are tgg, tac, gcg, ctg, aaa, tgg, tgc, ttc, tgc, gac, ttc and aac in sequence.
The mutant with the mutation site number of 12 comprises D115F, P116N, V172Y, I175F, C179E, K206W, Y249C, Y255F, G266C, H269W, L271S and H274G, wherein the nucleic acid encoding the other positions of the nucleic acid of the mutant is consistent with SEQ ID NO:2, but the codons encoding the 12 mutation sites are ttc, aac, tac, ttt, gag, tgg, tgc, ttt, tgc, tac, agc, tgc and tac in sequence.
The mutant with the mutation site number of 13 comprises D115F, P116N, V172Y, I175F, C179E, K206W, Y249C, Y255F, G266C, H269W, L271S, G273C, H274G, wherein the nucleic acid encoding the other positions of the nucleic acid of the mutant is consistent with SEQ ID NO. 2, but the codons encoding the 13 mutation sites are ttc, aac, tac, ttt, gag, tgg, tgc, ttt, tgc, tac, tgc, ggt and tac in sequence.
The number of mutation sites is 14, and the mutation sites in the mutant are D115F, P116N, V172Y, I175F, C179E, K206W, Y249C, Y255F, G266C, P267Y, H269W, L271S, G273C and H274G, and the nucleic acid encoding the other positions of the nucleic acid of the mutant are consistent with SEQ ID NO. 2, but the codons encoding the 14 mutation sites are ttc, aac, tac, ttt, gag, tgg, tgc, ttt, tgc, tac, tgg, agc, tgt, tac in sequence.
The number of mutation sites in the mutant with 15 is D115F, P116N, V172Y, I175F, C179E, K206W, Y249C, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, the nucleic acid encoding the mutant is identical to SEQ ID NO. 2 at other positions, but the codons encoding the 15 mutation sites are ttc, aac, tac, ttt, gag, tgg, tgc, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac in sequence.
The mutant with 16 mutation sites comprises D115F, P116N, V172Y, I175F, C179E, K206W, Y249C, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, and nucleic acid at other positions of the nucleic acid for coding the mutant is consistent with SEQ ID NO. 2, but the codons for coding the 16 mutation sites are ttc, aac, tac, ttt, gag, tgg, tgc, ttt, gtt, tgc, tgg, agc, tgc, ggt, tac.
The mutant with 17 mutation sites comprises D115F, P116N, V172Y, I175F, C179E, K206W, Y249C, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C and H274G, and the nucleic acid encoding the other positions of the nucleic acid of the mutant is consistent with SEQ ID NO. 2, but the codons encoding the 17 mutation sites are ttc, aac, tac, ttt, gag, tgg, tgc, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt and tac in sequence.
The mutant with 18 mutation sites comprises D115F, P116N, V172Y, I175F, C179E, K206W, Y249C, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C and H274G, wherein the nucleic acid encoding the other positions of the mutant is identical to SEQ ID NO. 2, but the codons encoding the 18 mutation sites are ttc, aac, tac, ttt, gag, tgg, tgc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt and tac in sequence.
The number of mutation sites in the mutant with 19 is D115F, P116N, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, the nucleic acid encoding the other positions of the mutant is identical to that of SEQ ID NO:2, but the codons encoding the 19 mutation sites are ttc, aac, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac in sequence.
The number of mutation sites in the mutant with 20 is D115F, P116N, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, the nucleic acid encoding the other positions of the nucleic acid of the mutant is consistent with SEQ ID NO:2, but the codons encoding the 20 mutation sites are ttc, aac, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, ttt, tgc, tgg, agc, tgc, tgg, tac in sequence.
The number of mutation sites in the mutant with the number of 21 is D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, the nucleic acid encoding the other positions of the nucleic acid of the mutant is consistent with SEQ ID NO:2, but the codons encoding the 21 mutation sites are ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.
The number of mutation sites of the 22 mutants is Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, and the nucleic acid encoding the other positions of the nucleic acid of the mutant is identical to SEQ ID NO:2, but the codons encoding the 22 mutation sites are gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, att, ttt, ttt, tgc, tac, tgg, agc, tgg, tac, etc. in sequence.
The mutant with 23 mutation sites comprises F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C and H274G, and the nucleic acid encoding the other positions of the nucleic acid of the mutant is consistent with SEQ ID NO:2, but the codons encoding the 23 mutation sites are cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, att, ttt, ttt, gtt, tgc, tac, tgg, tgc, ggt, tac.
The number of mutation sites in the mutant with the number of 24 is Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, nucleic acid encoding the other positions of the nucleic acid of the mutant is consistent with SEQ ID NO:2, but codons encoding the 24 mutation sites are att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, tgg, tgc, tgg, ggt, tac in sequence.
The number of mutation sites in the mutant with the mutation site number of 25 is G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, the nucleic acid encoding the other positions of the nucleic acid of the mutant is consistent with that of SEQ ID NO:2, but the codons encoding the above 25 mutation sites are aac, att, cgt, gtg, ttc, aac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, tgc, tagc, tgc.
The number of mutation sites of the mutant is 26, wherein the mutation sites are F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, and the nucleic acid encoding the other positions of the nucleic acid of the mutant is consistent with SEQ ID NO:2, but the codons encoding the 26 mutation sites are att, aac, att, cgt, gtg, ttc, aac, ttag, tac, gag, tgg, tgc, acc, aac, ttt, ttt, ttt, tgc, tac, ggt, tgc.
The number of mutation sites 27 is L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, and the nucleic acid encoding the mutant at other positions is as described in SEQ ID NO:2, but the codons encoding the 27 mutation sites are ttc, att, aac, att, cgt, gtg, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.
The number of mutation sites of the mutant was 28, the mutation sites were Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, the nucleic acids encoding the other positions of the nucleic acids of the mutant were identical to those of SEQ ID NO:2, but the codons encoding the 28 mutation sites were in this order agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, gagg, tac, ttt, ttt, ttt, ttg, tggc.
The number of mutation sites 29 of the mutant was E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, nucleic acid encoding the other positions of the nucleic acid of the mutant and SEQ ID NO:2, but the codons encoding the 29 mutation sites are 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 in sequence.
The number of mutation sites of the mutant was 30, and the mutation sites were F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, and the nucleic acid encoding the mutant was identical to that of SEQ ID NO:2, but the codons encoding the above 30 mutation sites were in this order gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, tac, ttgg, tgg, atc, ttt, ttttt, tgg.
The number of mutation sites of the mutant with the number of 31 is S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, and the nucleic acid encoding the other positions of the nucleic acid of the mutant is as follows: 2, but the codons encoding the above 31 mutation sites are 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 in sequence.
The number of mutation sites of the mutant with 32 is V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, nucleic acid encoding the other positions of the nucleic acid of the mutant and SEQ ID NO:2, but the codons encoding the 32 mutation sites are 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, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.
The number of mutation sites 33 was L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, and the nucleic acid encoding the other positions of the nucleic acid of the mutant was as described in SEQ ID NO:2, but the codons encoding the above 33 mutation sites are 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, ttt, gtt, tgc, tac, tgg, agc, tgc, ggt, tac.
The number of mutation sites of the 34 mutant was Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, nucleic acid encoding the other positions of the nucleic acid of the mutant and SEQ ID NO:2, but the codons encoding the 34 mutation sites are in turn 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.
The number of mutation sites of the mutant with 35 mutation sites is F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, and the nucleic acid encoding the other positions of the mutant is as follows: 2, but the codons encoding the 35 mutation sites are gcg, cat, 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, ggt, tac.
The number of mutation sites of the 36 mutant was F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, nucleic acid encoding the other positions of the nucleic acid of the mutant and SEQ ID NO:2, but the codons encoding the 36 mutation sites are sequentially 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.
The number of mutation sites of the mutant was 37, and the mutation sites were M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, and nucleic acids encoding other positions of the nucleic acids of the mutant were as described in SEQ ID NO:2, but the codons encoding the above 37 mutation sites are in that 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
The number of mutation sites 38 is Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, nucleic acid encoding the other positions of the nucleic acid of the mutant, and SEQ ID NO:2, but the codons encoding the 38 mutation sites are tgc, acc, tat, gcg, cac, aaa, aac, gcg, gac, att, agc, ttc, att, aac, att, cgt, gtg, ttc, aac, aag, aac, ttac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, ttt, gtt, tgc, tac, tgg, agc, ggt, tac
The number of mutation sites 39 is N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, nucleic acid encoding the other positions of the nucleic acid of the mutant and SEQ ID NO:2, but the codons encoding the above 39 mutation sites are sequentially atg, 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, ttt, gtt, tgc, tac, tgc, ggt, tac
The number of mutation sites 40 was N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, nucleic acid encoding the mutant at other positions and SEQ ID NO:2, but the codons encoding the 40 mutation sites are sequentially atg, 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, ttt, gtt, tgc, tac, tgc, ggt, gc, ggt, tac, aac
The number of mutation sites 41 of the mutant was N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, nucleic acid encoding the other positions of the mutant nucleic acid and SEQ ID NO:2, but the codons encoding the 41 mutation sites are sequentially atg, 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, ttt, gtt, tgc, tac, tgc, gg, ggt, tac, aac, ttt
The number of mutation sites 42 of the mutant was N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F, nucleic acid encoding the other positions of the mutant nucleic acid and SEQ ID NO:2, but the codons encoding the 42 mutation sites are sequentially atg, tgc, acc, tat, gcg, cac, aaa, aac, gcg, gac, att, agc, ttc, att, aac, att, cgt, tgt, ttc, aac, aag, acc, tac, ttt, gag, tgg, tgc, acc, aac, att, ttt, ttt, ttt, gtt, tgc, tac, tgc, ggt, gc, ggt, tac, aac, ttt, ctg
The number of mutation sites 43 was N14M, Q15C, M16T, F17Y, F21A, Q71S, L73K, V88N, S89A, F94D, E95I, Y96S, L100F, F109I, G111N, Y112I, F113R, Q114V, D115F, P116N, R117K, Y118T, V172Y, I175F, C179E, K206W, Y249C, S250T, W251N, W252I, A254F, Y255F, I264V, G266C, P267Y, H269W, L271S, G273C, H274G, N276Y, V283N, K284F and V291L; nucleic acid encoding the mutant at other positions along the nucleic acid sequence of seq id no:2, but the codon encoding Met at position 14 is atg, the codon encoding Cys at position 15 is tgc, the codon encoding Thr at position 16 is acc, the codon encoding Tyr at position 17 is tat, the codon encoding Ala at position 21 is gcg, the codon encoding Ser at position 71 is cac, the codon encoding Lys at position 73 is aaa, the codon encoding Asn at position 88 is aac, the codon encoding Ala at position 89 is gcg, the codon encoding Asp at position 94 is gac, the codon encoding Ile at position 95 is att, the codon encoding Ser at position 96 is agc, the codon encoding Phe at position 100 is ttc, the codon encoding Ile at position 109 is att, the codon encoding Asn at position 111 is aac, the codon encoding Ile at position 112 is att, the codon encoding Lys at position 113 is cag, the codon at position 114 is gtg, the codon at position 115 is Arg, the codon encoding Asn at position 116 is aac, the codon encoding Lys at position 117 is aag, the codon encoding Thr at position 118 is acc, the codon encoding Tyr at position 172 is tac, the codon encoding Phe at position 175 is ttt, the codon encoding Glu at position 179 is gag, the codon encoding Trp at position 206 is tgg, the codon encoding Cys at position 249 is tgc, the codon encoding Tyr at position 250 is acc, the codon encoding Asn at position 251 is aac, the codon encoding Ile at position 252 is att, the codon encoding Phe at position 254 is ttt, the codon encoding Phe at position 255 is ttt, the codon encoding Val at position 264 is gtt, the codon encoding Cys at position 266 is tgc, the codon encoding Tyr at position 267 is tac, the codon encoding Trp at position 269 is tgg, the codon at position 271 is Cys at position 273 is gct, the codon encoding Gly 274 is ggt, the codon encoding Tyr 276 is tac, the codon encoding Asn 283 is aac, the codon encoding Phe 284 is ttt, and the codon encoding Leu 291 is ctg.
The invention changes the catalytic activity of alpha-1, 2-fucosyltransferase by one-step site-directed mutagenesis to mutate multi-site amino acids of the alpha-1, 2-fucosyltransferase, thereby enhancing the ability of the enzyme to catalyze the synthesis of fucosylation products. The highest enzyme activity obtained under the same number of mutation sites in the present invention refers to the highest enzyme activity obtained by detection among all mutants having the same number of mutation sites (for example, all mutants having 3 mutation sites and all mutants having 5 mutation sites). In other words, at a certain number of mutation sites, the mutant has the highest enzyme activity, while the enzyme activities of other mutants are relatively low. The arrangement of mutation sites in the mutant with the highest enzyme activity is more beneficial to the exertion of the enzyme activity.
The invention also provides a recombinant expression vector containing the alpha-1, 2-fucosyltransferase gene; the recombinant expression vector preferably takes pET22b as an original expression vector; the α -1, 2-fucosyltransferase gene is preferably inserted on plasmid pET22 b; the method of constructing the recombinant vector of the present invention is not particularly limited, and conventional methods in the art may be employed.
In the specific implementation process of the invention, the recombinant vector is constructed by adopting the following method: and (3) taking the coding nucleotide sequence as a template, and performing PCR to obtain the recombinant expression vector pET22b-futC.
The invention also provides a recombinant bacterium containing the recombinant expression vector; the recombinant bacteria preferably take escherichia coli as host bacteria; the E.coli preferably comprises a strain of DE3 series such as E.coli BL21 (DE 3), novaBlue, origami, plyss, T7 shuffle, etc. The method for constructing the recombinant bacteria is not particularly limited, and conventional methods in the art can be adopted. In the specific implementation process of the invention, the recombinant bacteria are prepared by adopting the following method: e.coli BL21 is transformed by the recombinant expression vector pET22b-futC to obtain recombinant bacteria, and the recombinant bacteria are named as pET22b-futC-E.coli BL21.
The alpha-1, 2-fucosyltransferase mutant is preferably prepared by the following method: culturing the recombinant bacteria, and inducing to obtain an alpha-1, 2-fucosyltransferase mutant; the culture medium for culturing the recombinant bacteria is preferably a TB culture medium; the TB medium contains 1.2% (W/V) peptone, 2.4% (W/V) yeast extract, 0.4% (V/V) glycerol, 17mM KH 2 PO 4 ，72mM K 2 HPO 4 . The culture temperature is preferably 37deg.C, and the culture time is based on OD600 of the culture reaching 0.6-0.8, and the induction is preferably performed by adding isopropyl group with final concentration of 0.1-1.0mM into the culture medium-beta-D-thiogalactopyranoside (IPTG) and induction culture at 16-37℃for 4-28h.
Taking the induced fermentation liquor, centrifuging to obtain thalli, crushing, and taking supernatant for promoting the synthesis of fucosylation products. In the specific implementation process of the invention, GDP-L-fucose, lactose, acetyl lactose and the like are used as substrates, the alpha-1, 2-fucosyltransferase mutant or recombinant bacteria thereof has no coenzyme, and the alpha-1, 2-fucosyltransferase mutant is reacted for 0.1-2 hours in an in-vitro catalytic reaction system formed by buffer solution with the pH value of 6.0-8.0 at the temperature of 20-40 ℃ to obtain a mixed enzymatic reaction system comprising the alpha-1, 2-fucosyltransferase mutant.
In some embodiments, the fucosylation product is 2' -fucosyllactose. In some embodiments, the method for preparing 2' -fucosyllactose comprises: adding the supernatant after the fermentation thallus is cracked into TRIS-HCl buffer fermentation liquor containing GDP-fucose and lactose, and incubating for 10-120min at 20-40 ℃ to obtain reaction liquor containing 2' -fucosyllactose.
It was previously thought in the art that most enzymes were unable to perform enzymatic catalysis with lactose as a fucosyl acceptor. The wild-type alpha-1, 2-fucosyltransferase selected in the scheme does not have the capacity of taking lactose as a fucosyl receptor, and is one of reasons for extremely low activity of the wild-type enzyme. After mutation, when lactose is still used as a substrate, the enzyme activity is obviously improved, namely the capability of using lactose as the substrate is enhanced. Lactose, which is a common raw material in the food industry, has multiple advantages of easy availability, safety, low cost and the like, so that the development of an enzyme capable of efficiently converting lactose as a substrate is more required.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1
Based on the amino acid sequence of the mutants, the corresponding coding nucleic acid sequences were obtained by codon optimization (see Table 1).
And inserting the synthesized nucleic acid fragment into a pET22b vector, and constructing and obtaining a recombinant vector pET22b-futC. The recombinant vector is transformed into competent cells of escherichia coli DH5 alpha, and after overnight culture, the recombinant vector is transformed into competent cells of escherichia coli BL21, and the recombinant vector is resuscitated at 37 ℃ for 1h, coated with an ampicillin-resistant LB plate with the final concentration of 0.1mM, and cultured at 37 ℃ for 10-16h. And finally, selecting single colonies on an ampicillin resistance plate for colony PCR verification, and confirming that the construction of the recombinant engineering bacteria containing the nucleic acid for encoding the mutant is completed by sequencing.
Example 2: recombinant bacterium pET22b-futC-E.coli BL21 expression and purification of alpha-1, 2-fucosyltransferase
Recombinant pET22b-futC-E.coliBL21 constructed in example 1 and a control strain expressing unmutated original enzyme alpha-1, 2-fucosyltransferase are inoculated into LB medium containing 0.1% of ampicillin respectively, cultured overnight at 37 ℃ under shaking to prepare seed solution, transferred into 200mL of TB medium containing ampicillin at 1% of inoculum size the next day, cultured at 37 ℃ for a proper time, and then added with a proper amount of IPTG overnight at 16 ℃ for induction. After the induction was completed, the cells were collected by centrifugation at 8000rpm at 4℃and subjected to ultrasonic disruption, and the cell disruption supernatant (crude enzyme solution) was collected for subsequent purification.
The alpha-1, 2-fucosyltransferase or alpha-1, 2-fucosyltransferase mutant is purified by using histidine tag on pET22b vector and Ni 2+ -NTA affinity chromatography for further purification. Filtering the crude enzyme solution with 0.22 μm filter membrane, adding into balanced Ni at proper flow rate 2+ In the column, 10 Xthe column volume was washed with buffer 1. The phase elution was performed with buffer 2 containing 10, 20, 50, 100, 200, 300, 400, 500mM imidazole, respectively, and the phase eluted samples were collected and the protein concentration, molecular weight size and purity were measured by Bradford and SDS-PAGE.
Buffer 1, preferably Tris-HCl buffer, 1,5, 10, 20, 50mM imidazole, ph=7.0-8.0.
Buffer 2, preferably Tris-HCl buffer, 10, 20, 50, 100, 200, 300, 400, 500mM imidazole, ph=7.0-8.0.
Enzyme activity detection:
firstly, respectively taking 20mL of fermentation liquor from each group, centrifuging at 12000rpm for 15-20min, collecting thalli, re-suspending, crushing by ultrasonic waves, and centrifuging at a low temperature and high speed to obtain cell crushing supernatant. Purifying with histidine tag to obtain purer enzyme solution, and removing imidazole by dialysis to adjust salt concentration of buffer solution. And finally, the obtained enzyme solution is stored for standby.
100. Mu.M GDP-L-fucose 100. Mu.L and 5mM lactose 100. Mu.L were added, followed by 1. Mu.g enzyme to participate in the reaction, and incubation was carried out at 20-40℃for 10-120min to effect catalytic production of 2' -FL. Immediately after completion of the catalysis, the reaction was stopped by placing on ice. After subsequent high-speed centrifugation at low temperature and ultrafiltration through a desalting column, the yield of 2' -FL in the catalytic system and the residual amount of substrate were determined by High Performance Liquid Chromatography (HPLC).
The reaction solution obtained by the catalysis is passed through a filter membrane of 0.22 μm for standby. Preparing a mobile phase required for liquid chromatography detection, and a mobile phase A: acetonitrile, mobile phase b=0.5% aqueous triethylamine acetate. An amide chromatographic column (150 mm. Times.2.1 μm) was used under the following conditions: mobile phase ratio 80:20, flow rate 0.2m/L, column temperature 35 ℃, detector using evaporative light scattering detector, evaporation temperature 70 ℃, carrier gas flow rate 2.0SLM. The product was quantified by external standard.
The amount of enzyme required for conversion to 1. Mu. Mole of 2' -FL at 37℃in 1 minute was defined as one activity unit (U). In vitro enzyme catalysis verification is carried out on the recombinant enzyme according to the conditions, and the specific activities of the obtained groups of purified enzyme proteins are shown in the following table:
TABLE 1
Note that: the highest enzyme activities obtained at the same number of mutation sites are shown in the table.
The result shows that the enzyme activity of the obtained mutant is improved to different degrees by mutating specific sites in the alpha-1, 2-fucosyltransferase. Among them, mutant G266C has the highest enzyme activity. Mutants comprising two or more mutation sites have stronger enzyme activity, and the higher the number of mutation sites, the stronger the highest enzyme activity obtained at the number of mutation sites within a certain number range.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> university of south-middle school; australian milk industry (China) Limited; changsha fortune kang Biotechnology Co., ltd
<120> alpha-1, 2-fucosyltransferase mutants and uses thereof
<130> MP21018068
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 298
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Met Ala Phe Lys Val Val Gln Ile Cys Gly Gly Leu Gly Asn Gln Met
1 5 10 15
Phe Gln Tyr Ala Phe Ala Lys Ser Leu Gln Lys His Leu Asn Thr Pro
20 25 30
Val Leu Leu Asp Ile Thr Ser Phe Asp Trp Ser Asn Arg Lys Met Gln
35 40 45
Leu Glu Leu Phe Pro Ile Asp Leu Pro Tyr Ala Ser Ala Lys Glu Ile
50 55 60
Ala Ile Ala Lys Met Gln His Leu Pro Lys Leu Val Arg Asp Thr Leu
65 70 75 80
Lys Cys Met Gly Phe Asp Arg Val Ser Gln Glu Ile Val Phe Glu Tyr
85 90 95
Glu Pro Gly Leu Leu Lys Pro Ser Arg Leu Thr Tyr Phe Tyr Gly Tyr
100 105 110
Phe Gln Asp Pro Arg Tyr Phe Asp Ala Ile Ser Pro Leu Ile Lys Gln
115 120 125
Thr Phe Thr Leu Pro Pro Pro Glu Asn Gly Asn Asn Lys Lys Lys Glu
130 135 140
Glu Glu Tyr His Arg Lys Leu Ala Leu Ile Leu Ala Ala Lys Asn Ser
145 150 155 160
Val Phe Val His Val Arg Arg Gly Asp Tyr Val Gly Ile Gly Cys Gln
165 170 175
Leu Gly Ile Asp Tyr Gln Lys Lys Ala Leu Glu Tyr Ile Ala Lys Arg
180 185 190
Val Pro Asn Met Glu Leu Phe Val Phe Cys Glu Asp Leu Lys Phe Thr
195 200 205
Gln Asn Leu Asp Leu Gly Tyr Pro Phe Met Asp Met Thr Thr Arg Asp
210 215 220
Lys Glu Glu Glu Ala Tyr Trp Asp Met Leu Leu Met Gln Ser Cys Lys
225 230 235 240
His Gly Ile Ile Ala Asn Ser Thr Tyr Ser Trp Trp Ala Ala Tyr Leu
245 250 255
Ile Asn Asn Pro Glu Lys Ile Ile Ile Gly Pro Lys His Trp Leu Phe
260 265 270
Gly His Glu Asn Ile Leu Cys Lys Glu Trp Val Lys Ile Glu Ser His
275 280 285
Phe Glu Val Lys Ser Lys Lys Tyr Asn Ala
290 295
<210> 2
<211> 897
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atggcgttca aggtggttca gatttgcggt ggcctgggta accagatgtt ccaatatgcg 60
tttgcgaaaa gcctgcaaaa gcacctgaac accccggttc tgctggacat taccagcttc 120
gattggagca accgtaaaat gcagctggag ctgtttccga tcgacctgcc gtacgcgagc 180
gcgaaagaaa ttgcgatcgc gaagatgcag cacctgccga aactggtgcg tgacaccctg 240
aagtgcatgg gtttcgatcg tgttagccaa gagatcgtgt ttgagtatga accgggtctg 300
ctgaaaccga gccgtctgac ctacttctat ggctactttc aggacccgcg ttacttcgat 360
gcgattagcc cgctgatcaa gcaaaccttt accctgccgc cgccggaaaa cggcaacaac 420
aagaagaagg aagaggagta tcaccgtaag ctggcgctga ttctggcggc gaaaaacagc 480
gtgtttgttc acgtgcgtcg tggtgactac gttggtattg gctgccagct gggcatcgat 540
tatcagaaga aggcgctgga gtacatcgcg aagcgtgttc cgaacatgga gctgttcgtg 600
ttttgcgaag acctgaaatt cacccagaac ctggatctgg gttatccgtt tatggacatg 660
accacccgtg ataaggaaga ggaagcgtac tgggatatgc tgctgatgca aagctgcaaa 720
cacggcatca ttgcgaacag cacctattcc tggtgggcgg cgtacctgat caacaacccg 780
gaaaagatca ttatcggtcc gaaacactgg ctgttcggcc acgagaacat tctgtgcaaa 840
gaatgggtta agatcgagag ccactttgaa gtgaaaagca aaaagtacaa cgcgtga 897

Claims (8)

1. A mutant of alpha-1, 2-fucosyltransferase is characterized in that the wild amino acid sequence of the mutant is shown as SEQ ID NO. 1, and the mutation site of the mutant is G266C.
2. A nucleic acid encoding the mutant of claim 1.
3. An expression vector comprising a nucleic acid encoding the mutant of claim 1.
4. A host cell transformed or transfected with the expression vector of claim 3.
5. A method of preparing the mutant of claim 1, comprising: culturing the host cell of claim 4, and inducing expression of the mutant.
6. 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 of the preparation method of claim 5 in the synthesis of a fucosylated product.
7. A biological agent that facilitates synthesis of a fucosylated product, comprising: 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 of the production method of claim 5.
8. A method for synthesizing a fucosylation product, comprising using lactose as a substrate and promoting the synthesis of the fucosylation product by using the biological preparation according to claim 7.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106795484A (en) * 2014-05-15 2017-05-31 格里康辛有限责任公司 For α (1,2) the fucosyltransferase mutation used when fucosylation oligosaccharide is produced
CN112322565A (en) * 2020-11-09 2021-02-05 江南大学 Method for improving yield of 2' -fucosyllactose in recombinant escherichia coli
CN113056562A (en) * 2018-10-02 2021-06-29 齐米科技股份有限公司 Export of oligosaccharides using substrate import

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106795484A (en) * 2014-05-15 2017-05-31 格里康辛有限责任公司 For α (1,2) the fucosyltransferase mutation used when fucosylation oligosaccharide is produced
CN113056562A (en) * 2018-10-02 2021-06-29 齐米科技股份有限公司 Export of oligosaccharides using substrate import
CN112322565A (en) * 2020-11-09 2021-02-05 江南大学 Method for improving yield of 2' -fucosyllactose in recombinant escherichia coli

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Sanabria-Valentin,E.等.alpha-(1,2) fucosyltransferase [Helicobacter pylori].GenBank: ABO61750.1.2008,序列. *

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