In-vitro two-step enzymatic process for efficiently synthesizing GDP-L-fucose and application

Abstract

The invention relates to the technical field of biology, in particular to an in-vitro two-step enzymatic process for efficiently synthesizing GDP-L-fucose and application. According to the invention, a bifunctional enzyme (Uniprot: Q58T34) with an amino acid sequence as shown in SEQ ID NO: 1 is used as a template to carry out site-directed mutagenesis modification, and a variant M07 with high L-Fucose kinase activity and a variant M10 with high GDP-L-Fucose pyrophosphorylase activity are respectively obtained. The GDP-L-Fucose is prepared from the variant by means of a two-step enzyme method in the presence of FKP enzyme and PPA enzyme through an ATP regeneration system, and the GDP-L-Fucose is high in yield and purity, low in production cost and energy consumption, suitable for large-scale production and wide in application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an in vitro two-step enzymatic process for efficiently synthesizing GDP-L-fucose and its application. Background Art

[0002] GDP-L-fucose (guanosine 5'-diphosphate (GDP)-L-Fucose), a nucleotide sugar, is an important substrate in the synthesis of fucosylated compounds, especially an essential intermediate in the biosynthesis of fucosylated human milk oligosaccharides (HMOs).

[0003] The existing process for the in vitro enzymatic preparation of GDP-L-fucose is to use a de novo synthesis or direct remedy method; the de novo synthesis is to obtain GDP-L-fucose by catalysis of a multi-enzyme cascade reaction, specifically: using cheap guanosine diphosphate and sucrose as substrates, GDP-L-fucose is generated by catalysis of sucrose synthase, CDP-tyvetose 2-epimerase, GDP-mannose dehydratase and isomeroreductase. The direct remedy is to use the bifunctional enzyme FKP enzyme to catalyze the synthesis of GDP-L-fucose using the exogenous substrate L-fucose, ATP and GTP in a one-pot method, and the reaction formula is as follows: Figure 3 shown.

[0004] De novo synthesis is a multi-enzyme cascade with low cost, but this type of method has a long preparation route, involves many types of enzymes, and uses a large amount. The direct remedial pathway synthesis method brings difficulties to the design of reaction conditions due to the inconsistent enzyme properties of the two active regions of the bifunctional enzyme FKP enzyme. At present, the existing technology mostly uses one reaction (simultaneous feeding, one pH condition) to achieve milligram-level reactions. The reaction system is small and the reaction time is relatively long, and the conversion rate is also low, at 10%~30%; if the reaction temperature is low, the conversion is incomplete; if the reaction temperature is high, there will be problems such as GDP-L-fucose being unstable and decomposing. This directly leads to a time-consuming preparation process, low overall yield, and difficulty in separating and purifying the final product. Therefore, it is urgent to develop a converting enzyme and catalytic method that can quickly and stably obtain GDP-L-fucose with the possibility of scale-up. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an in vitro two-step enzymatic process for efficiently synthesizing GDP-L-fucose and its application.

[0006] The present invention provides L-fucokinase / GDP-L-fucose pyrophosphorylase variants, which include L-fucokinase variants and / or GDP-L-fucose pyrophosphorylase variants;

[0007] The L-fucokinase variant includes an L-fucokinase / GDP-L-fucose pyrophosphorylase with an amino acid sequence as shown in SEQ ID NO: 1 having the following mutations:

[0008] The glutamine Q at position 623 is mutated to serine S; and / or

[0009] The glutamine Q at position 627 is mutated to glycine G; and / or

[0010] The tyrosine Y at position 629 is mutated to serine S; and / or

[0011] Lysine K at position 631 is mutated to arginine R; and / or

[0012] Aspartic acid D at position 644 was mutated to alanine A;

[0013] The GDP-L-fucose pyrophosphorylase variant includes an L-fucokinase / GDP-L-fucose pyrophosphorylase with an amino acid sequence as shown in SEQ ID NO: 1 and having the following mutations:

[0014] The arginine R at position 592 is mutated to lysine K; and / or

[0015] The proline P at position 711 is mutated to alanine A; and / or

[0016] The glycine G at position 713 is mutated to alanine A; and / or

[0017] Serine S at position 714 is mutated to Threonine T; and / or

[0018] The leucine L at position 722 was mutated to alanine A.

[0019] Furthermore, in the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention,

[0020] The L-fucokinase variants include:

[0021] The glutamine Q at position 623 of the L-fucokinase / GDP-L-fucose pyrophosphorylase described in SEQ ID NO: 1 is mutated to serine S and the glutamine Q at position 627 is mutated to glycine G;

[0022] and / or the tyrosine at position 629 of the L-fucokinase / GDP-L-fucose pyrophosphorylase as described in SEQ ID NO: 1 is mutated to serine S and the lysine K at position 631 is mutated to arginine R;

[0023] and / or the L-fucokinase / GDP-L-fucose pyrophosphorylase as described in SEQ ID NO: 1, wherein the tyrosine at position 629 is mutated to serine S, the lysine at position 631 is mutated to arginine R, and the aspartic acid at position 644 is mutated to alanine A;

[0024] and / or the L-fucokinase / GDP-L-fucose pyrophosphorylase as described in SEQ ID NO: 1, wherein glutamine Q at position 623 is mutated to serine S, glutamine Q at position 627 is mutated to glycine G, tyrosine Y at position 629 is mutated to serine S, and lysine K at position 631 is mutated to arginine R;

[0025] The GDP-L-fucose pyrophosphorylase variants include:

[0026] and / or the L-fucokinase / GDP-L-fucose pyrophosphorylase described in SEQ ID NO: 1 wherein the arginine R at position 592 is mutated to lysine K and the proline P at position 711 is mutated to alanine A;

[0027] and / or the L-fucokinase / GDP-L-fucose pyrophosphorylase as described in SEQ ID NO: 1, wherein the proline P at position 711 is mutated to alanine A, the glycine G at position 713 is mutated to alanine A, and the serine S at position 714 is mutated to threonine T;

[0028] And / or the L-fucokinase / GDP-L-fucose pyrophosphorylase as described in SEQ ID NO: 1 has arginine R at position 592 mutated to lysine K, proline P at position 711 mutated to alanine A, serine S at position 714 mutated to threonine T and / or leucine L at position 722 mutated to alanine A.

[0029] The present invention mutates a bifunctional enzyme BFFKP (Uniprot: Q5LC59) derived from Bacteroides fragilis and uses it for the synthesis of GDP-L-fucose; the bifunctional enzyme BFFKP has both L-fucokinase and GDP-L-fucose pyrophosphorylase activities, and its amino acid sequence is shown in SEQ ID NO: 1.

[0030] The present invention constructs variants M01-M10 by means of truncation and point mutation with a bifunctional enzyme BFFKP as a template, wherein variants M04-M07 have higher L-fucokinase activity, and M07-M10 have higher GDP-L-fucose pyrophosphorylase activity; experimental results show that among the variants M04-M07 having L-fucokinase activity, the activity of M07 is significantly higher than that of other variants; among the variants M07-M10 having GDP-L-fucose pyrophosphorylase activity, the activity of M10 is significantly better than that of other variants.

[0031] In a specific embodiment of the present invention, the mutation sites corresponding to the variants M04 to M10 are shown in Table 1.

[0032] The present invention provides a composition comprising composition 1 and / or composition 2:

[0033] The composition 1 comprises the L-fucokinase variant and polyphosphate kinase (PPK enzyme) in the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention;

[0034] The composition 2 comprises the GDP-L-fucose pyrophosphorylase variant among the L-fucokinase / GDP-L-fucose pyrophosphorylase variants of the present invention and pyrophosphatase (PPA enzyme).

[0035] The present invention provides a biomaterial comprising at least one of the following A) to D):

[0036] A), a nucleic acid encoding the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention or the composition of the present invention;

[0037] B), a recombinant vector containing the nucleic acid described in A);

[0038] C), transforming or transfecting host cells with the recombinant vector described in B);

[0039] D) culturing the mixture obtained by culturing the host cells described in C).

[0040] Furthermore,

[0041] The backbone of the recombinant vector is pET28a.

[0042] The host cell is Escherichia coli.

[0043] The nucleic acid described in the present invention can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. Nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids can be linear or circular in topology. Nucleic acids can be part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0044] In the present invention, the nucleic acid may be codon-optimized or non-codon-optimized, and these optimizations include but are not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0045] The present invention also provides a transcription unit containing the nucleic acid, wherein the transcription unit refers to a DNA sequence starting from a promoter and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of the promoter, a poly (A) signal, etc.

[0046] The present invention provides a recombinant vector, which comprises a vector backbone and the nucleic acid of the present invention.

[0047] Furthermore, the sources of the vector backbone described in the present invention include plants, animals, bacteria, fungi, phages or viruses, which are not limited in the present invention. The bacterial vectors include but are not limited to pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vectors include but are not limited to pYES2, pYES3, pYES6, pAUR23, etc.

[0048] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence or element that is essential for the expression of an operably connected coding gene in a specific host organism. Known prokaryotes utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" can sometimes be interchanged and used, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors and / or are only potential genome inserts. In a specific embodiment, the vector of the present invention is pET28a.

[0049] In the present invention, the host cell includes but is not limited to plants, animals, bacteria, fungi, bacteriophages or viruses, and the present invention is not limited to this; specifically, it is a bacterial cell, more specifically Escherichia coli. In a specific embodiment of the present invention, the cell is E. coli (BL21).

[0050] The present invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, so that the transformed host cells have the ability to replicate protein-encoding vectors or express desired proteins.

[0051] In the present invention, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, and the like.

[0052] The present invention provides the use of at least one of the following i) to iii) in the synthesis of GDP-L-Fucose:

[0053] i), the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention;

[0054] ii), the composition of the present invention;

[0055] iii) The biomaterial of the present invention.

[0056] The present invention provides a method for preparing GDP-L-Fucose, which comprises synthesizing GDP-L-Fucose using at least one of the following I) to IV):

[0057] I), the L-fucokinase / GDP-L-fucose pyrophosphorylase variant of the present invention;

[0058] II), the composition of the present invention;

[0059] III), the biomaterial described in the present invention.

[0060] Furthermore, the preparation method of the present invention comprises the following steps:

[0061] Step 1, L-fucose kinase variant catalyzes L-fucose to generate Fuc-1-P in the presence of auxiliary 1;

[0062] Step 2, the GDP-L-fucose pyrophosphorylase variant catalyzes Fuc-1-P to generate GDP-L-fucose in the presence of auxiliary 2;

[0063] The auxiliary material 1 includes an auxiliary material 1a and / or an auxiliary material 1b;

[0064] The auxiliary substance 1a includes Tris-HCl, ATP, manganese chloride and polyphosphate kinase (PPK enzyme);

[0065] The auxiliary substance 1b includes Tris-HCl, ATP, and manganese chloride;

[0066] The auxiliary 2 includes GTP, magnesium chloride and pyrophosphatase (PPA enzyme).

[0067] Furthermore,

[0068] In step 1, the pH for catalyzing L-fucose to generate Fuc-1-P is 8.5-9.0, and the reaction time is 1.5-3 hours, specifically 2 hours;

[0069] In step 2, the pH for catalyzing Fuc-1-P to generate GDP-L-fucose is 6.5-7.0, and the reaction time is 3-5 h.

[0070] The present invention optimizes the method for preparing GDP-L-fucose (GDP-L-Fucose) by using the variant; in a specific embodiment of the present invention, the preparation method includes preparing GDP-L-Fucose by a one-pot enzymatic method, preparing GDP-L-Fucose by an enzyme-catalyzed cascade reaction of a two-step enzymatic method and PPA, and preparing GDP-L-Fucose by using an ATP regeneration system in the presence of FKP enzyme and PPA enzyme by means of the two-step enzymatic method; the experimental results of the present invention show that the yield and purity of GDP-L-Fucose prepared by using an ATP regeneration system in the presence of FKP enzyme and PPA enzyme by means of the two-step enzymatic method are higher.

[0071] Specifically, in some embodiments of the present invention, when the auxiliary substance 1 is auxiliary substance 1a, the preparation method is to prepare GDP-L-Fucose by using an ATP regeneration system in the presence of FKP enzyme and PPA enzyme by means of a two-step enzymatic method, which comprises the following steps:

[0072] Step 1, L-fucose catalyzes the formation of Fuc-1-P by L-fucose in the presence of auxiliary 1a;

[0073] Step 2, the GDP-L-fucose pyrophosphorylase variant catalyzes Fuc-1-P to generate GDP-L-fucose in the presence of auxiliary 2;

[0074] At this time, the concentration of the bacterial cells containing the L-fucokinase variant is 0.01g / mL~0.025g / mL (10g / L~25g / L), the concentration of Tris-HCl is 40mM~60mM, the concentration of ATP is 5mM~6mM, the concentration of manganese chloride is 40mM~60mM, and the concentration of polyphosphate kinase (PPK) is 1500U / L~2500U / L; the concentration of the bacterial cells containing the GDP-L-fucose pyrophosphorylase variant is 0.008g / mL~0.009g / mL (8g / L~9g / L), the concentration of GTP is 150mM~250mM, the concentration of magnesium chloride is 40mM~60mM, and the concentration of pyrophosphatase (PPA) is 1500U / L~2500U / L;

[0075] Specifically, as in Example 5, the amount of the bacterial cells containing the L-fucose kinase variant is 10 g / L, the concentration of Tris-HCl is 50 mM, the concentration of ATP is 5.4 mM, the concentration of manganese chloride is 50 mM, and the concentration of polyphosphate kinase (PPK) is 2000 U / L; the amount of the bacterial cells containing the GDP-L-fucose pyrophosphorylase variant is 9 g / L, the concentration of GTP is 200 mM, the concentration of magnesium chloride is 50 mM, and the concentration of pyrophosphatase (PPA) is 2000 U / L;

[0076] Specifically, in other embodiments of the present invention, when the auxiliary substance 1 is auxiliary substance 1b, the preparation method is a two-step enzymatic method and PPA for enzyme-catalyzed cascade reaction to prepare GDP-L-Fucose, which comprises the following steps:

[0077] Step 1, L-fucose kinase variant catalyzes L-fucose to generate Fuc-1-P in the presence of auxiliary 1b;

[0078] Step 2, the GDP-L-fucose pyrophosphorylase variant catalyzes Fuc-1-P to generate GDP-L-fucose in the presence of auxiliary 2;

[0079] At this time, when the auxiliary substance 1 is auxiliary substance 1b, the concentration of the bacterial cells containing the L-fucokinase variant is 0.01g / mL~0.025g / mL (10g / L~25g / L), the concentration of Tris-HCl is 40mM~60mM, the concentration of ATP is 150mM~250mM, the concentration of manganese chloride is 40mM~60mM, the concentration of the bacterial cells containing the GDP-L-fucose pyrophosphorylase variant is 0.05g / mL~1.5g / mL, the concentration of GTP is 150mM~250mM, the concentration of magnesium chloride is 40mM~60 mM, and the concentration of pyrophosphatase is 1500U / L~2500U / L;

[0080] Specifically, as in Example 4, when the auxiliary substance 1 is auxiliary substance 1b, in the 10 mL system in step 1, the concentration of the bacteria containing the L-fucokinase variant is 0.009 g / mL (9 g / L), the concentration of Tris-HCl is 50 mM, the concentration of ATP is 160 mM, and the concentration of manganese chloride is 50 mM; in step 2, the reaction substances are continued to be added on the basis of the system in step 1, and the total reaction system is 11.5 mL, wherein the concentration of the bacteria containing the GDP-L-fucose pyrophosphorylase variant is 0.0082 g / mL (8.2 g / L), the concentration of GTP is 181 mM, the concentration of magnesium chloride is 43.4 mM, and the concentration of pyrophosphatase is 1739 U / L;

[0081] In the present invention, when the auxiliary substance is auxiliary substance 1a or auxiliary substance 1b, the L-fucokinase variant and the GDP-L-fucose pyrophosphorylase variant need to be prepared into pure enzyme and / or crude enzyme solution when used; in a specific embodiment of the present invention, it is a crude enzyme solution, and the crude enzyme solution is obtained by mixing Escherichia coli (BL21) cells containing the enzyme variant with a buffer at a mass ratio of 1:10 and then crushing; the buffer is Tris-HCl buffer, specifically 50mM Tris-HCl (pH7.5); in step 1, the addition volume of the crude enzyme solution of the L-fucokinase variant is 10% to 25% of the total reaction system volume, and the corresponding bacterial cell used is 0.01g / mL to 0.025g / mL; in step 2, the addition volume of the crude enzyme solution of the GDP-L-fucose pyrophosphorylase variant is 8% to 10% of the total volume of the first step reaction system, and the corresponding bacterial cell used is 0.008g / mL to 0.009g / mL.

[0082] More specifically, in the preparation method of the present invention, the combination of enzymes in the preparation method is optimized; in a specific embodiment of the present invention, the L-fucokinase variant in step 1 may be any one of M04 to M07, and the GDP-L-fucose pyrophosphorylase in step 2 may be at least one of M08 to M10; or the L-fucokinase variant in step 1 may be M07, and the GDP-L-fucose pyrophosphorylase in step 2 may be M07; or the L-fucokinase variant in step 1 may be M10, and the GDP-L-fucose pyrophosphorylase in step 2 may be M10;

[0083] The test results of the present invention show that the yield and purity of GDP-L-fucose are best when the L-fucokinase variant in step 1 is M07 and the GDP-L-fucose pyrophosphorylase in step 2 is M10.

[0084] The present invention designs a two-step method to react at different pH values; considering that the L-fucose kinase of FKP is inhibited by GTP and ADP, a step-by-step method is adopted, GTP is not introduced first, and only ATP is added. In addition, the use of an ATP circulation system will also alleviate the inhibition of the kinase. After the phosphate-activated Fuc-1P is generated, GTP is added to generate the target product. Because it is a step-by-step reaction, the optimal metal ions can be used for the two-step enzyme reaction: Mn 2+ and Mg 2+ Therefore, in the present invention, the enzymes, raw materials and reaction conditions used cooperate with each other and work synergistically to achieve high yield and high purity of GDP-L-fucose.

[0085] The present invention uses a bifunctional enzyme (Uniprot: Q58T34) with an amino acid sequence as shown in SEQ ID NO: 1 as a template for site-directed mutagenesis, and obtains a variant M07 with high L-Fucose kinase activity and a variant M10 with high GDP-L-Fucose pyrophosphorylase activity. The variant is prepared into GDP-L-Fucose by a two-step enzymatic method in the presence of FKP enzyme and PPA enzyme using an ATP regeneration system, and the GDP-L-Fucose has high yield and purity, low production cost and energy consumption, is suitable for large-scale production, and has a wide range of application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Shown is the comparison of the L-Fucose kinase reaction activities of the mutants (WT enzyme activity is 100%, and the activities of other mutants are relative activity values ​​%);

[0087] Figure 2Shows the comparison of GDP-Fucose pyrophosphorylase activity (WT enzyme activity is 100%, and the activities of other mutants are relative activity values ​​%);

[0088] Figure 3 The reaction formula for preparing GDP-L-Fucose by one-pot enzymatic method using natural enzyme BFFKP is shown;

[0089] Figure 4 The results of one-pot enzymatic preparation of GDP-L-Fucose by natural enzyme BFFKP are shown;

[0090] Figure 5 The reaction formula for preparing GDP-L-Fucose by enzyme-catalyzed cascade reaction using two-step enzyme method (WT+WT, M07+M07, M10+M10, M07+M10) is shown;

[0091] Figure 6 The yield comparison of GDP-L-Fucose prepared by two-step reaction of single enzyme (wild WT, M07 or M10) and double enzyme (M07+M10) is shown;

[0092] Figure 7 It is shown that the FKP mutant M07 catalyzes the "fucose + ATP" reaction for 2 hours;

[0093] Figure 8 It shows that the FKP mutant M07+M10 catalyzes the synthesis of GDP-L-Fucose for 2+4 hours;

[0094] Fig. 9 The reaction formula of preparing GDP-L-Fucose by using 1L system - FKP enzyme (M07+M10) - step-by-step enzymatic method, ATP regeneration system and PPA to hydrolyze pyrophosphate;

[0095] Fig.10 Liquid phase detection after the reaction is completed;

[0096] Fig.11 shows the HPLC spectrum of GDP-fucose after purification;

[0097] Fig.12 The mass spectrum of the product GDP-fucose is shown. DETAILED DESCRIPTION

[0098] The present invention provides an in vitro two-step enzymatic process and application for the efficient synthesis of GDP-L-fucose. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0099] Enzyme related information:

[0100] In this patent: L-fucokinase / GDP-L-fucose pyrophosphorylase is a bifunctional enzyme derived from Bacteroides fragilis (Uniprot: Q5LC59), numbered BFFKP in this patent; in order to obtain a high enzyme activity monomer that can perform two-step reactions, BFFKP was used as a template for modification, and the specific mutation site information is shown in Table 1.

[0101] Polyphosphate kinase (PPK), first expressed for enzyme catalysis: Polyphosphate kinase from a salmon bacteria: Renibacterium salmoninarum, Uniprot ID: A9WPD3, numbered PPK in the present invention.

[0102] Pyrophosphatase (PPA): Inorganic pyrophosphatase derived from Escherichia coli (E Coli K12), Uniprot: P0A7A9, numbered as PPA in the present invention.

[0103] Amino acid sequence of BFFKP: MQKLLSLPSNLVQSFHELERVNRTDWFCTSDPVGKKLGSGGGTSWLLEECYNEYSDGATFGEWLEKEKRILLHAGGQSRRLPGYAPSGKILTPVPVFRWERGQHLGQNLLSLQLPLYEKIMSLAPDKLHTLIASGDVYIRSEKPLQSIPEADVVCYGLWVDPSLATHHGVFASDRKHPEQLDFMLQKPSLAELESLSKTHLFLMDIGIWLLSDRAVEILMKRSHKESSEELKYYDLYSDFGLALGTHPRIEDEEVNTLSVAILPLPGGEFYHYGTSKELISSTLSVQNKVYDQRRIMHRKVKPNPAMFVQNAVVRIPLCAENADLWIENSHIGPKWKIASRHIITGVPENDWSLAVPAGVCVDVVPMGDKGFVARPYGLDDVFKGDLRDSKTTLTGIPFGEWMSKRGLSYTDLKGRTDDLQAVSVFPMVNSVEELGLVLRWMLSEPELEEGKNIWLRSEHFSADEISAGANLKRLYAQREEFRKGNWKALAVNHEKSVFYQLDLADAAEDFVRLGLDMPELLPEDALQMSRIHNRMLRARILKLDGKDYRPEEQAAFDLLRDGLLDGISNRKSTPKLDVYSDQIVWGRSPVRIDMAGGWTDTPPYSLYSGGNVVNLAIELNGQPPLQVYVKPCKDFHIVLRSIDMGAMEIVSTFDELQDYKKIGSPFSIPKAALSLAGFAPAFSAVSYASLEEQLKDFGAGIEVTLLAAIPAGSGLGTSSILASTVLGAINDFCGLAWDKNEICQRTLVLEQLLTTGGGWQDQYGGVLQGVKLLQTEAGFAQSPLVRWLPDHLFTHPEYKDCHLLYYTGITRTAKGILAEIVSSMFLNSSLHLNLLSEMKAHALDMNEAIQRGSFVEFGRLVGKTWEQNKALDSGTNPPAVEAIIDLIKDYTLGYKLPGAGGGGYLYMVAKDPQAAVRIRKILTENAPNPRARFVEMTLSDKGFQVSRS (SEQ ID NO:1);

[0104] Amino acid sequence of PPK: MSLGRPAMASKHGFKKSPAELLVVGPEFNLDQVDTSGAPGFSGKKRDAEQVLAEGERKLSVLQEQLFAQSKFGGKHSVLLVLQAMDTAGKGGIVSHVVGSVDPQGMQLHAFKAPTDEEKQHDFLWRIEKGVPKAGMLGVFDRSHYEDVLIHRVHGWADETELERRYQAINDFEEQLTSNGTKIVKVMLDISPETQKERLTARLAEPTKHWKYSPTDVTEREFWPQYMEAYQLAFEKTSTKIAPWHVVPADKKWYARLAVQELLLDAFESLDLSWPEGDYDLALERKRVAAS (SEQ ID NO:2);

[0105] Amino acid sequence of PPA: MGSSHHHHHHSSGLVPRGSHMSLLNVPAGKDLPEDIYVVIEIPANADPIKYEIDKESGALFVDRFMSTAMFYPCNYGYINHTLSLDGDPVDVLVPTPYPLQPGSVIRCRPVGVLKMTDEAGEDAKLVAVPHSKLSKEYDHIKDVNDLPELLKAQIAHFFEHYKDLEKGKWVKVEGWENAEAAKAEIVASFERAKNK (SEQ ID NO:3);

[0106] Amino acid sequence of BFFKP-M07: MQKLLSLPSNLVQSFHELERVNRTDWFCTSDPVGKKLGSGGGTSWLLEECYNEYSDGATFGEWLEKEKRILLHAGGQSRRLPGYAPSGKILTPVPVFRWERGQHLGQNLLSLQLPLYEKIMSLAPDKLHTLIASGDVYIRSEKPLQSIPEADVVCYGLWVDPSLATHHGVFASDRKHPEQLDFMLQKPSLAELESLSKTHLFLMDIGIWLLSDRAVEILMKRSHKESSEELKYYDLYSDFGLALGTHPRIEDEEVNTLSVAILPLPGGEFYHYGTSKELISSTLSVQNKVYDQRRIMHRKVKPNPAMFVQNAVVRIPLCAENADLWIENSHIGPKWKIASRHIITGVPENDWSLAVPAGVCVDVVPMGDKGFVARPYGLDDVFKGDLRDSKTTLTGIPFGEWMSKRGLSYTDLKGRTDDLQAVSVFPMVNSVEELGLVLRWMLSEPELEEGKNIWLRSEHFSADEISAGANLKRLYAQREEFRKGNWKALAVNHEKSVFYQLDLADAAEDFVRLGLDMPELLPEDALQMSRIHNRMLRARILKLDGKDYRPEEQAAFDLLRDGLLDGISNRKSTPKLDVYSDQIVWGRSPVRIDMAGGWTDTPPYSLYSGGNVVNLAIELNGSPPLGVSVRPCKDFHIVLRSIDMGAMEIVSTFDELQDYKKIGSPFSIPKAALSLAGFAPAFSAVSYASLEEQLKDFGAGIEVTLLAAIPAGSGLGTSSILASTVLGAINDFCGLAWDKNEICQRTLVLEQLLTTGGGWQDQYGGVLQGVKLLQTEAGFAQSPLVRWLPDHLFTHPEYKDCHLLYYTGITRTAKGILAEIVSSMFLNSSLHLNLLSEMKAHALDMNEAIQRGSFVEFGRLVGKTWEQNKALDSGTNPPAVEAIIDLIKDYTLGYKLPGAGGGGYLYMVAKDPQAAVRIRKILTENAPNPRARFVEMTLSDKGFQVSRS (SEQ ID NO: 4);

[0107] Amino acid sequence of BFFKP-M10: MQKLLSLPSNLVQSFHELERVNRTDWFCTSDPVGKKLGSGGGTSWLLEECYNEYSDGATFGEWLEKEKRILLHAGGQSRRLPGYAPSGKILTPVPVFRWERGQHLGQNLLSLQLPLYEKIMSLAPDKLHTLIASGDVYIRSEKPLQSIPEADVVCYGLWVDPSLATHHGVFASDRKHPEQLDFMLQKPSLAELESLSKTHLFLMDIGIWLLSDRAVEILMKRSHKESSEELKYYDLYSDFGLALGTHPRIEDEEVNTLSVAILPLPGGEFYHYGTSKELISSTLSVQNKVYDQRRIMHRKVKPNPAMFVQNAVVRIPLCAENADLWIENSHIGPKWKIASRHIITGVPENDWSLAVPAGVCVDVVPMGDKGFVARPYGLDDVFKGDLRDSKTTLTGIPFGEWMSKRGLSYTDLKGRTDDLQAVSVFPMVNSVEELGLVLRWMLSEPELEEGKNIWLRSEHFSADEISAGANLKRLYAQREEFRKGNWKALAVNHEKSVFYQLDLADAAEDFVRLGLDMPELLPEDALQMSRIHNRMLRARILKLDGKDYRPEEQAAFDLLRDGLLDGISNRKSTPKLDVYSDQIVWGRSPVKIDMAGGWTDTPPYSLYSGGNVVNLAIELNGQPPLQVYVKPCKDFHIVLRSIDMGAMEIVSTFDELQDYKKIGSPFSIPKAALSLAGFAPAFSAVSYASLEEQLKDFGAGIEVTLLAAIAAGTGLGTSSIAASTVLGAINDFCGLAWDKNEICQRTLVLEQLLTTGGGWQDQYGGVLQGVKLLQTEAGFAQSPLVRWLPDHLFTHPEYKDCHLLYYTGITRTAKGILAEIVSSMFLNSSLHLNLLSEMKAHALDMNEAIQRGSFVEFGRLVGKTWEQNKALDSGTNPPAVEAIIDLIKDYTLGYKLPGAGGGGYLYMVAKDPQAAVRIRKILTENAPNPRARFVEMTLSDKGFQVSRS (SEQ ID NO:5);

[0108]

[0109] Nucleotide sequence of PPK: atgagcctgggccgtccggcaatggccagtaaacatggctttaaaaaaagtccggcagaactgctggtggttggcccggaatttaatctggatcaggttgataccagtggtgcacctggttttagcggcaaaaaacgtgatgcagaacaggttctggccgaaggcgaacgtaaactgagcgttctgcaggaacagctgtttgcccagagtaaatttggtggcaaacatagtgtgctgctggtgctgcaggcaatggataccgccggcaaaggcggtattgtgagccatgtggtgggcagtgttgatccgcagggtatgcagctgcatgcatttaaagcacctaccgatgaagaaaaacagcatgattttctgtggcgcattgaaaaaggcgttccgaaagcaggtatgctgggcgtttttgatcgtagccattatgaagatgtgctgattcatcgtgttcatggttgggcagatgaaaccgaactggaacgtcgttatcaggccattaatgattttgaagaacagctgaccagcaatggtaccaaaattgtgaaagtgatgctggatattagcccggaaacccagaaagaacgtctgaccgcccgcctggcagaaccgaccaaacattggaaatatagtccgaccgatgtgaccgaacgcgaattttggccgcagtatatggaagcctatcagctggcctttgaaaaaaccagcaccaaaattgcaccgtggcatgttgttccggccgataaaaaatggtatgcccgcctggcggttcaggaactgctgctggatgcctttgaaagcctggatctgagttggccggaaggtgattatgatctggcactggaacgtaaacgcgttgcagcaagttaa (SEQ ID NO:7);

[0110] Nucleotide sequence of PPA: (SEQ ID NO: 8);

[0111] In the present invention, the preparation of the crude enzyme solution in Examples 3 to 5 is the same as that in Example 2, wherein the bacterial cells and 50 mM Tris-HCl (pH 7.5) solution are uniformly mixed at a mass ratio of 1:10, and the cells are broken by homogenization to prepare the crude enzyme solution.

[0112] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0113] Example 1 Construction of mutants

[0114] 1. Enzyme mutation construction

[0115] In order to obtain a monofunctional enzyme, the present invention modified the enzyme by truncation and point mutation, and obtained mutants as shown in Table 1 after modification:

[0116] Table 1. Mutants

[0117]

[0118] 1.1 Construction of mutants

[0119] The target gene BFFKP was synthesized in a gene synthesis company. The amino acid sequence of BFFKP is shown in SEQ ID NO: 1. The other mutants were introduced with corresponding point mutations using BFFKP as a template by PCR. Then, the template fragments or point mutation fragments were digested with NdeI and XhoI restriction endonucleases, respectively, and then connected and inserted into the pET28a expression vector to form pET28a-BFFKP, pET28a-M01, pET28a-M02, pET28a-M03, pET28a-M04, pET28a-M05, pET28a-M06, pET28a-M07, pET28a-M08, pET28a-M09, and pET28a-M10 recombinant products.

[0120] Take 4 μL of the above recombinant product and transfer it into 30 μL DH5α competent cells. After ice bathing for 30 minutes, heat shock at 42℃ for 45 seconds, ice bath for 2-3 minutes, add 500 μL SOC, culture on a shaking table for 1.5 hours, centrifuge the whole plate, and culture in a 37 ℃ incubator overnight for 12-16 hours. Pick a single clone, confirm the correct construction of the above vector by sequencing, and store it for future use.

[0121] 1.2 Enzyme fermentation production

[0122] The plasmid successfully constructed in 1.1 was transferred into E. coli (BL21) competent cells for plate culture and single clone screening, as well as small-scale liquid culture of single clones. The bacteria with correct protein expression were finally scaled up for liquid culture.

[0123] The specific steps are as follows: a single colony is inoculated into 5 mL of LB culture medium containing 50 μM kanamycin (37 oC) Culture, when the cells grow to the logarithmic phase, inoculate into 250mL LB culture medium containing the same antibiotics, and when they grow to the logarithmic phase, transfer to a 5L culture fermenter for culture and final protein expression. In the 5L fermenter, when the cell OD is 20, add 0.3mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 25 o C was used to induce protein expression for 6 hours, and finally the cells were collected by high-speed centrifugation (4000rpm, 20min) to obtain 25g~50g of enzyme overexpression wet cells. A small amount of cells was first mixed evenly with tris-HCl buffer (50mM, pH 8.0) on an ice basin, and then the cells were broken by freeze-thaw method, and the cell walls were removed by high-speed centrifugation to obtain a clear liquid, which was subjected to SDS-PAGE gel electrophoresis. It was confirmed by the experiment that BFFKP and its variant proteins were successfully expressed.

[0124] The LB medium consists of: 1wt% tryptone, 0.5wt% yeast powder, 1wt% NaCl, 1wt% dipotassium hydrogen phosphate, 1wt% dipotassium hydrogen phosphate and 5wt% glycerol.

[0125] 2. Screening of mutants with L-Fucose kinase activity and GDP-Fucose pyrophosphorylase activity

[0126] The host cells containing the above mutation vector were lysed, and the lysate was as shown in Table 2.

[0127] Table 2. Lysis buffer

[0128]

[0129] The cleavage operation is as follows:

[0130] Take out the cultured deep-well plate containing only the bacteria from the -20°C refrigerator, add 100 μL of lysis solution to each well, and thoroughly blow and suspend the bacteria and then break them at 25°C, 800 rpm, 2h, to obtain a crude enzyme solution containing the BFFKP and its variants M01 to M10;

[0131] 2.1 Screening of L-Fucose kinase activity

[0132] Prepare 20 mL of 2× reaction solution (pH = 8.5~9.0, temperature 38 degrees) as shown in Table 3.

[0133] Table 3. L-Fucose kinase screening reaction solution

[0134]

[0135] Weigh the above reagents and completely dissolve them in 15 mL of water. Add 4 M NaOH to adjust the pH to 9.0, and then make up to 20 mL.

[0136] The steps for screening L-Fucose kinase activity are as follows (deep well plate reaction):

[0137] Add 200 μL of 2× reaction solution and 100 μL of H2O to each well, mix by pipetting, then add 100 μL of the wild-type BFFKP (WT) or mutant M01~M10 crude enzyme solution of FKP, place on a shaker at 38°C, 800 rpm, and react for 2 h.

[0138] 100 μL of supernatant was taken from each well into a deep-well plate, and 900 μL of 60% acetonitrile was added to dilute the plate 10 times to quench the reaction, and the mixture was shaken at 1000 rpm for 10 min at 37°C and centrifuged at 4000 rpm for 10 min. The supernatant was filtered with a 0.45 μm filter head and then subjected to liquid phase detection.

[0139] After comparing the results (such as Figure 1 As shown in the figure, after truncation, the C-terminal mutant M03 (497-949aa) has L-Fucose kinase activity, but the enzyme activity is relatively low. Among the mutants, M07 has the highest L-Fucose kinase reaction activity.

[0140] 2.2 Screening of GDP-Fucose pyrophosphorylase activity

[0141] First, use M07 to prepare a fucose-1-phosphate (Fuc-1-P) reaction solution 1. The preparation process of the reaction solution 1 is as follows:

[0142] Take 0.3283g of L-Fucose, 1.1023g of ATP-2Na, 0.2517g of MnCl2, and 0.2423g of Tris, and completely dissolve them in 15mL of water. Add 4M NaOH to adjust the pH to 9.0, and then adjust the volume to 20mL. Add 10mL of water, mix well, and add 10mL of M07 bacterial solution. Place in a water bath shaker at 38℃ and shake for 4 hours.

[0143] Then adjust to acidic pH = 6.5~7.0 to obtain reaction solution 1, which serves as the starting substrate for the second step reaction.

[0144] Take 20 mL of reaction solution 1 (containing Fuc-1-P, concentration is about 50 mM), adjust the pH to 6.5-7.0 with HCl, then add 0.952 g anhydrous magnesium chloride (50 mM), 0.68 g GTP-2Na (Fuc-1-P and GTP-2Na are added at a ratio of 1:1.2, 60 mM), and then adjust the pH to 7.0 with NaOH to obtain step 2 reaction solution 2.

[0145] Take a 96-well plate, add 300 μL of reaction solution 2 to each well, add 100 μL of wild-type (WT) or mutant M01~M10 bacterial solution of FKP, pipette to mix, place on a shaker at 38°C, 800 rpm, and react for 4 hours.

[0146] 100 μL of supernatant was taken from each well into a deep-well plate, and 900 μL of 60% acetonitrile was added to dilute the plate 10 times to quench the reaction, and the mixture was shaken at 1000 rpm for 10 min at 37°C and centrifuged at 4000 rpm for 10 min. The supernatant was filtered with a 0.45 μm filter head and then subjected to liquid phase detection.

[0147] Comparison results of GDP-Fucose pyrophosphorylase activity Figure 2 As shown in the figure, after truncation, the N-terminal mutant M01 (1~496aa) still has GDP-Fucose pyrophosphorylase activity, but it is reduced. The activity of M02 (1~564aa) is improved, and the pyrophosphorylase activity of mutant M10 is the highest.

[0148] Example 2 One-pot enzymatic preparation of GDP-L-Fucose using natural enzyme BFFKP

[0149] GDP-L-Fucose was prepared by one-pot enzymatic method using natural enzyme BFFKP. The reaction formula is as follows: Figure 3 As shown;

[0150] 1. Reagent preparation

[0151] Substrate solution: accurately weigh 1662 mg ATP-2Na (molecular weight 551.145 g / mol), 490 mg L-fucose (molecular weight 164.16 g / mol), and 2041 mg GTP-2Na (molecular weight 567.2 g / mol), dissolve in 10 mL deionized water, adjust the pH to 7.5, and finally make up to 15 mL; final concentration: 200 mM ATP, 200 mM L-Fucose, 240 mM GTP).

[0152] Tris-HCl solution (1M, molecular weight 121.1 g / mol, pH 7.5): Weigh 12.11 g Tris into a 250 mL beaker, add about 80 mL of deionized water, stir thoroughly to dissolve, add concentrated hydrochloric acid to adjust the pH to 7.5, and dilute the solution to 100 mL.

[0153] MgCI2 solution (1M, molecular weight 95.211 g / mol): Accurately weigh 0.952 g of anhydrous magnesium chloride, dissolve it in deionized water and make up to 10 mL.

[0154] 2. Preparation of crude enzyme solution

[0155] Dilute 1M Tris-HCl (pH 7.5) solution to 50mM. Mix BFFKP bacteria and 50mMTris-HCl (pH 7.5) solution at a mass ratio of 1:8. Break the cell wall at 40% power for 15 minutes in a cell disruptor (homogeneous bacteria disruption) to dissolve the intracellular proteins in the buffer, then centrifuge and take the supernatant for use to obtain BFFKP crude enzyme solution. Mix PPA bacteria and 50mM Tris-HCl (pH 7.5) solution at a mass ratio of 1:10. Break the cell wall at 40% power for 15 minutes in a cell disruptor to dissolve the intracellular proteins in the buffer, then centrifuge and take the supernatant for use to obtain PPA crude enzyme solution.

[0156] 3. Reaction solution

[0157] Take 800μL substrate solution, add 50μL Tris-HCl solution (1M, pH 7.5), 50μL MgCI2 solution (1M) and 100μL BFFKP crude enzyme solution in sequence, and finally add 2U PPA crude enzyme solution, mix well and react at 38℃ for 6h; after the reaction, take 100μL supernatant into a test tube, add 900μL 60% acetonitrile according to the dilution 10 times to quench the reaction, shake at 37℃, 1000rpm for 10min, and centrifuge at 4000rpm for 10 min. Take the supernatant and filter it with a 0.45μm filter head, send it to HPLC for analysis, and the results are as follows: Figure 4 As shown, there is a large amount of ATP and GTP remaining. Calculated yield: 8.5 g / L of GDP-L-Fucose is generated.

[0158] Example 3 A two-step enzymatic method (WT+WT, M07+M07, M10+M10, M07+M10) was used to prepare GDP-L-Fucose by enzyme-catalyzed cascade reaction

[0159] The two-step enzymatic method (WT+WT, M07+M07, M10+M10, M07+M10) was used to perform enzyme-catalyzed cascade reactions to prepare GDP-L-Fucose. Figure 5 As shown;

[0160] 1. Solvent preparation

[0161] Tris-HCl solution (1M, pH 8.5): Weigh 12.11 g Tris into a 250 mL beaker, add about 80 mL of deionized water, and stir thoroughly to dissolve. Adjust the pH to 8.5 and dilute the solution to 100 mL.

[0162] Substrate solution 1: Accurately weigh 1662 mg ATP-2Na and 490 mg L-fucose, dissolve in 10 mL deionized water, adjust the pH to 8.5-9.0, and finally make up to 15 mL; final concentration: 200 mM ATP, 200 mM L-Fucose;

[0163] Substrate solution 2: MnCl2 solution (1M, molecular weight 125.844 g / mol): Accurately weigh 1.258 g of anhydrous manganese chloride, dissolve it in deionized water and make up to 10 mL;

[0164] Substrate solution 3: MgCl2 solution (1M, molecular weight 95.211 g / mol): Accurately weigh 0.952 g of anhydrous magnesium chloride, dissolve it in deionized water and make up to 10 mL;

[0165] 2. Reaction steps

[0166] Step 1: Take 800 μL of substrate solution 1, add 50 μL of Tris-HCl solution (1M, pH 8.5), 50 μL of substrate solution 2 (1M MnCl2 solution) and 100 μL of crude enzyme solution (WT, M07, M10, M07) in sequence, mix well and react at 38°C for 2 hours.

[0167] Step 2: After the first step, add 50 μL of substrate solution 3 (1M MgCl2 solution), slowly add 0.136 g of GTP-2Na (240 mM) and adjust the pH to 6.5-7.0 with ammonia water, add 2 U of PPA crude enzyme solution, and finally add 100 μL of crude enzyme solution (WT, M07, M10 or M10 crude enzyme solution), mix well and react at 38°C for 4 hours.

[0168] For each reaction, take 100 μL of the reaction solution, dilute it 10 times, add 900 μL of 60% acetonitrile to quench the reaction, shake at 37°C, 1000 rpm for 10 min, and centrifuge at 4000 rpm for 10 min. Take the supernatant and filter it with a 0.45 μm filter head for liquid phase detection. Calculate the product GDP-fucose yield (g / L).

[0169] The results of the two-step enzymatic method (WT+WT, M07+M07, M10+M10, M07+M10) for preparing GDP-L-Fucose by enzyme-catalyzed cascade reaction are shown in Figure 2. Figure 6As shown in Table 4. The GDP-L-Fucose yields prepared by the two-step enzyme-catalyzed cascade reaction of WT+WT, M07+M07, M10+M10, and M07+M10 were 10.8 g / L, 40.3 g / L, 30.5 g / L, and 64.1 g / L, respectively; among them, the GDP-L-Fucose yield prepared by the two-step enzyme-catalyzed cascade reaction of M07+M10 was significantly better than that of the other three groups. The table also compares the GDP-L-Fucose yields of the one-pot method used in Example 1 and the two-step method of this example. The results show that the GDP-L-Fucose yield obtained by the two-step method is higher.

[0170] Table 4. Single enzyme (WT, M07 or M10) and double enzyme (M07+M10)

[0171] Two-step reaction results

[0172]

[0173] Example 4 10mL system, using two mutants of FKP enzyme (M07+M10), two-step enzymatic method and PPA (pyrophosphate hydrolase) for enzyme-catalyzed cascade reaction to prepare GDP-L-Fucose

[0174] Step 1: Take 8 mL of substrate solution 1 (200 mM ATP and 200 mM L-Fucose), add 500 μL Tris-HCl solution (1 M, pH 8.5), 500 μL of substrate solution 2 (1 M MnCl2 solution) and 1 mL of M07 crude enzyme solution in sequence, mix well and react at 38 °C for 2 h. The L-fucose reaction was complete by HPLC. The conversion rate of substrate L-fucose to the product Fuc-1-P obtained after the first step reaction was 80% (see Figure 7 ).

[0175] Step 2: After the reaction, add 500 μL of substrate solution 3 (1M MgCl2 solution), slowly add 1.36 g of GTP-2Na and adjust the pH to 6.5-7.0 with ammonia water, add 20 U of PPA enzyme, and finally add 1 mL of M10 crude enzyme solution, mix well and react at 38°C for 4 h. HPLC liquid phase detection reaction shows that the product of the first step is not completely converted into GDP-L-fucose (such as Figure 8 As shown in Figure 3, the GDP-L-fucose yield was 136 mM, and the conversion rate of Fuc-1-P to GDP-L-fucose was 85%.

[0176] Example 5 1L system - using FKP enzyme (M07+M10) - step-by-step enzymatic method, using ATP regeneration system and PPA to hydrolyze pyrophosphate to prepare GDP-L-Fucose

[0177] 1L system - using FKP enzyme (M07+M10) - step-by-step enzymatic method - using ATP regeneration system and PPA to hydrolyze pyrophosphate, the reaction formula for preparing GDP-L-Fucose is as follows Fig. 9 As shown;

[0178] Step 1: Add 32.8 g L-fucose (200 mM), 3.0 g adenosine triphosphate monosodium salt (ATP-2Na, 5.4 mM), and 6.3 g anhydrous manganese chloride (50 mM) to 1 L 50 mM Tris-HCl solution (pH 8.5), adjust the pH value of the solution to 8.5-9.0, then add 2000 U of PPK crude enzyme solution and 100 mL of M07 crude enzyme solution (the amount added is 10% of the total reaction system in step 1), and incubate at 38 o C was gently stirred and the pH of the reaction system was maintained between 8.5 and 9.0. After 2 hours, HPLC detected that the L-fucose reaction was basically complete, and the conversion rate of the substrate L-fucose to the Fuc-1-P product obtained after the first step reaction was nearly 100%.

[0179] Step 2: After adjusting the pH of the reaction solution to 7.0 with HCl, add 113 g of guanosine triphosphate monosodium salt (GTP, 200 mM), 4.8 g of anhydrous magnesium chloride (50 mM), and then add 100 mL of M10 crude enzyme solution (the addition amount is 9% to 11% of the reaction system, which is 9.09% at this time), PPA enzyme 2000 U, and incubate at 38 o C and gently stir the reaction system to maintain the pH between 6.5 and 7.0. After 4 hours, GDP-L-fucose was detected by HPLC to be almost completely generated (such as Fig.10 As shown), the yield of GDP-L-fucose is 170mM, and the conversion rate of the product after the first step reaction to GDP-L-fucose is 85%.

[0180] The reaction solution containing GDP-L-Fucose was adjusted to pH 2.0, the enzyme in the reaction system was denatured and precipitated, and the protein solid was removed by centrifugation. The crude product was obtained by two steps of isopropanol purification: first, at -20 oC, add 40% (v / v) by volume, let stand for 0.5h. Centrifuge to separate the supernatant and precipitate. Under -20 degrees, add isopropanol to the supernatant again to 80% (v / v), let stand for 0.5h, and centrifuge to separate the precipitate. Continue to purify, dissolve the solid in water, and directly load it onto the D201 anion exchange resin purification column to remove adenosine diphosphate and free phosphate impurities. Finally, the product is desalted, concentrated and crystallized using a reverse osmosis membrane (ethanol: water, 2:1, v:v) to obtain 60.1 grams of white powder. The product was confirmed to be GDP-L-fucose by mass spectrometry. HPLC purity is 95.6% (see spectrum Fig.11 ), mass spectrometry results are shown in Fig.12 , GDP-L-fucose, molecular weight: 587.334 g / mol.

[0181] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A L-fucokinase / GDP-L-fucose pyrophosphorylase variant, characterized in that including L-fucokinase variants and / or GDP-L-fucose pyrophosphorylase variants; The L-fucokinase variant includes an L-fucokinase / GDP-L-fucose pyrophosphorylase with an amino acid sequence as shown in SEQ ID NO: 1 having the following mutations: Glutamine at position 623 is mutated to serine; and / or Glutamine at position 627 is mutated to glycine; and / or The tyrosine at position 629 is mutated to serine; and / or Lysine at position 631 is mutated to arginine; and / or Aspartic acid at position 644 was mutated to alanine; The GDP-L-fucose pyrophosphorylase variant includes an L-fucokinase / GDP-L-fucose pyrophosphorylase with an amino acid sequence as shown in SEQ ID NO: 1 and having the following mutations: Arginine at position 592 is mutated to lysine; and / or Proline at position 711 is mutated to alanine; and / or Glycine at position 713 is mutated to alanine; and / or The serine at position 714 is mutated to threonine; and / or The leucine at position 722 was mutated to alanine.

2. The L-fucokinase / GDP-L-fucose pyrophosphorylase variant according to claim 1, characterized in that The L-fucokinase variants include: The glutamine at position 623 of the L-fucokinase / GDP-L-fucose pyrophosphorylase as described in SEQ ID NO: 1 is mutated to serine and the glutamine at position 627 is mutated to glycine; and / or The tyrosine at position 629 of the L-fucokinase / GDP-L-fucose pyrophosphorylase described in SEQ ID NO: 1 is mutated to serine and the lysine at position 631 is mutated to arginine; and / or The L-fucokinase / GDP-L-fucose pyrophosphorylase of SEQ ID NO: 1 has tyrosine at position 629 mutated to serine, lysine at position 631 mutated to arginine, and aspartic acid at position 644 mutated to alanine; and / or The L-fucokinase / GDP-L-fucose pyrophosphorylase of SEQ ID NO: 1, wherein the glutamine at position 623 is mutated to serine, the glutamine at position 627 is mutated to glycine, the tyrosine at position 629 is mutated to serine, and the lysine at position 631 is mutated to arginine; The GDP-L-fucose pyrophosphorylase variants include: The L-fucokinase / GDP-L-fucose pyrophosphorylase of SEQ ID NO: 1 has arginine at position 592 mutated to lysine and proline at position 711 mutated to alanine; and / or The L-fucokinase / GDP-L-fucose pyrophosphorylase of SEQ ID NO: 1 has proline at position 711 mutated to alanine, glycine at position 713 mutated to alanine, and serine at position 714 mutated to threonine; and / or In the L-fucokinase / GDP-L-fucose pyrophosphorylase described in SEQ ID NO: 1, the arginine at position 592 is mutated to lysine, the proline at position 711 is mutated to alanine, the serine at position 714 is mutated to threonine and / or the leucine at position 722 is mutated to alanine.

3. A composition, characterized in that Comprising Composition 1 and / or Composition 2: The composition 1 comprises the L-fucokinase variant and polyphosphate kinase in the L-fucokinase / GDP-L-fucose pyrophosphorylase variant according to claim 1 or 2; The composition 2 comprises the GDP-L-fucose pyrophosphorylase variant in the L-fucokinase / GDP-L-fucose pyrophosphorylase variant according to claim 1 or 2 and pyrophosphatase.

4. Biomaterial, characterized in that Including at least one of the following A) to D): A), a nucleic acid encoding the L-fucokinase / GDP-L-fucose pyrophosphorylase variant according to any one of claims 1 to 3 or the composition according to claim 3; B), a recombinant vector containing the nucleic acid described in A); C), transforming or transfecting host cells with the recombinant vector described in B); D) culturing the mixture obtained by culturing the host cells described in C).

5. The biomaterial according to claim 4, characterized in that The backbone of the recombinant vector is pET28a.

6. The biomaterial according to claim 4, characterized in that The host cell is Escherichia coli.

7. Application of at least one of the following i) to iii) in the synthesis of GDP-L-Fucose: i), the L-fucokinase / GDP-L-fucose pyrophosphorylase variant according to claim 1 or 2; ii), the composition according to claim 3; iii) The biomaterial according to any one of claims 4 to 6.

8. A method for preparing GDP-L-Fucose, characterized in that: The method comprises synthesizing GDP-L-Fucose by using at least one of the following methods I) to IV): I), the L-fucokinase / GDP-L-fucose pyrophosphorylase variant according to claim 1 or 2; II), the composition according to claim 3; III), the biomaterial according to any one of claims 4 to 6.

9. The preparation method according to claim 8, characterized in that: The steps include: Step 1, L-fucose kinase variant catalyzes L-fucose to generate Fuc-1-P in the presence of auxiliary 1; Step 2, the GDP-L-fucose pyrophosphorylase variant catalyzes Fuc-1-P to generate GDP-L-fucose in the presence of auxiliary 2; The auxiliary material 1 includes an auxiliary material 1a and / or an auxiliary material 1b; The auxiliary substance 1a includes Tris-HCl, ATP, manganese chloride and polyphosphate kinase; The auxiliary substance 1b includes Tris-HCl, ATP, and manganese chloride; The auxiliary substance 2 includes GTP, magnesium chloride and pyrophosphatase.

10. The preparation method according to claim 9, characterized in that: In step 1, the pH for catalyzing L-fucose to generate Fuc-1-P is 8.5-9.0, and the reaction time is 1.5-3h; In step 2, the pH for catalyzing Fuc-1-P to generate GDP-L-fucose is 6.5-7.0, and the reaction time is 3-5 h.

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