KR101560311B1 - METHOD FOR SCREENING α-2,3 AND α-2,6 SIALYLTRANSFERASE VARIANTS AND THEIR APPLICATION FOR SYNTHESIS OF SIALYLOLIGOSACCHARIDES - Google Patents

Abstract

The present invention relates to a method for searching for a sialic acid transferase mutant, a characteristic of the variant, a gene encoding the sialic acid transferase mutant enzyme, and the synthesis of sialyl oligosaccharides using the gene. The present invention provides an α-2,3-sialyltransferase derived from Pasteurella and an α-2,6-sialyltransferase derived from a photobacterium, which are improved through protein engineering mutations. In addition, the sialic acid transferase variants of the present invention reduce side reactions in the 3'- and 6'-sialyl oligosaccharide production reactions, increase productivity and thereby reduce production costs.

Description

Methods for searching for variants of α-2,3 and α-2,6 sialic acid transferases and their application to the production of sialyl oligosaccharides {METHOD FOR SCREENING α-2,3 AND α-2,6 SIALYLTRANSFERASE VARIANTS AND THEIR APPLICATION FOR SYNTHESIS OF SIALYLOLIGOSACCHARIDES}

The present invention relates to a method for searching for variants of sialic acid transferase through protein engineering enzyme mutations, characterization of the produced variants, and synthesizing sialyl oligosaccharides using these sialic acid transferase variants.

Breast milk not only provides essential nutrients to infants, but also provides a variety of health benefits beyond the concept of simple nutrients. Human milk oligosaccharides are composed of functional ingredients, and contain 5-10 g of oligosaccharides per 1 L, which is 100-200 times more than milk, and more than 130 types of human milk oligosaccharides have been identified so far. The content and structural diversity of oligosaccharides is very specific in breast milk, unlike milk. Among human milk oligosaccharides, 3'- and 6'-sialyl lactose of sialyl oligosaccharide is contained 5-10 times more than milk, and specifically, 0.1-0.3 g/L of 3'-sialyl lactose and 0.3- 0.5 g/L of 6'-sialyllactose.

Looking at the functions of sialyl oligosaccharides in the human body, first, gangliosides, GM1 (Gal-GalNAc-3'-sialyllactose-ceramide), GM2 (GalNAc-3'-sialyllactose-ceramide), and GM3 (3' -sialyllactose-ceramide) acts as a receptor for in vivo signal transduction in nervous tissue, and is involved in brain development and memory formation. Second, it serves to promote treatment by inducing leukocytes to the inflamed area in the form of sialyl Lewis X and/or sialyl Lewis A (Sialyl-LewisX, sialyl-LewisA) from leukocytes or bone marrow cells. Third, studies that act as inhibitors to prevent adhesion to the epithelial surface of host cells during the initial infection process of bacteria or viruses have been conducted. Among sialyl oligosaccharides, sialyllactose, in which sialic acid is linked to α-2,3 and α-2,6, can be a recognition material for hemagglutinin of avian and human influenza viruses, respectively, so that the virus is a host It can competitively inhibit invasion by binding to sugars on the cell surface. Currently, studies on various functions of sialyl lactose are active, and the synthesis of sialyl oligosaccharides present in breast milk can be applied to various industrial applications such as food, medicine, or filters, and bioconversion processes using these enzymes are being actively studied. .

However, the search for a sialic acid transferase capable of producing sialyl oligosaccharides including sialylactose and studies on the reaction characteristics of the enzyme have been conducted, but studies on the improvement of the discovered enzymes have been insignificant.

Leloir glycosyltransferase uses a nucleotide-type sugar as a donor. Since sugars in the form of nucleotides are usually very expensive, many studies have been conducted on biotransformation processes to produce these substances. The sialic acid sugar transfer enzyme of the present invention uses cytidine monophosphate- N -acetylneuraminic acid as a donor substrate, and a bioconversion process for synthesizing this substance has been previously studied.

Cytidine one phosphate-N-acetylneuraminic acid was synthesized in the N-acetylglucosamine (N -acetyl-D-glucosamine) , blood from a substrate of Rubin acid (pyruvate) and cytidine yl phosphate (CMP) N-acetylglucosamine- The process of synthesis using five enzymes corresponding to 2-epimerase, acetate kinase, cytidine monophosphate kinase, N -acetylneuraminic acid aldolase and cytidine monophosphate N-acetylneuraminic acid synthase Was built. Finally, to produce 3'-, 6'-sialyl oligosaccharide, α-2,3, α-2,6 sialic acid transferase is used with the production of cytidine monophosphate-N-acetylneuraminic acid. Thus, a system to produce the target substance was established.

In this regard, Jinchem Co., Ltd. established a process for separating and purifying the target substance in order to mass-produce sialyl lactose among sialyl oligosaccharides, and in particular, attempted mass production of 3'-sialyl lactose. However, in order to economically mass-produce 3'- and 6'-sialyl lactose, enzyme mutations aimed at improving the function of α-2,3 and α-2,6 transferases to increase the productivity of the target substance. Need

Sialic acid transferase is one cytidine phosphate-N-acetylneuraminic acid (Cytidine monophosphate N -acetylneuraminic acid, CMP -Neu5Ac) N from the - part of the galactose receptors per acetylneuraminic acid α-2,3 or α- It is delivered by the bond of 2,6 to produce oligosaccharides. Among them, the sialic acid transferase corresponding to GT-B folding is two domains that can bind CMP-Neu5Ac, a donor substrate that transfers sialic acid, and lactose (Galβ1,4Glc), a sialic acid receptor. It has a substrate-binding pocket and can perform protein engineering mutations on amino acid residues located in the pocket when the sialic acid transferase is improved.

The α-2,3 sialic acid transferase used in the present invention was first explored by the Xi chen group, is derived from Pasteurella multocida strain, and has four enzyme activities according to pH. It is an enzyme. This enzyme has a function of α-2,3 sialic acid transfer to galactose, the main reaction in a wide pH range (pH 6.0-10.0), and between pH 4.5-7.0, α-2 is lower than that of α-2,3 transition. , Has the activity of 6 sialic acid transfer. In addition, it has the activity of sialidase to decompose α-2,3 bonded sialic acid at pH 5.0-5.5, and another galactoside from sialyl galactoside having α-2,3 bonds at pH 5.5-6.5. It has the activity of trans sialidase to Rho. This sialic acid transferase is easily expressed as a soluble protein in E. coli, has high activity of α-2,3 sialic acid transfer, has a wide substrate specificity, and is easy to produce various sialyl oligosaccharides.

On the other hand, the α-2,6 sialic acid transferase used in the present invention is derived from a strain of Photobacterium damselae , and in terms of the structure folding and sequence of the glycotransferase, α-2,6 sialic acid transferase is derived from Pasteurella. It belongs to the same GT family 80 as -2,3 sialic acid transferase. In the case of α-2,6 sialic acid transferase derived from photobacterium, the activities of α-2,6 sialidase and transsialidase were recently revealed. Since the activity is very insignificant (more than 150 times), the activity of sialic acid transfer can be considered to be most. In the case of α-2,6 sialic acid transferase, there are few side reactions, most of the activities of α-2,6 sialic acid transfer are active, and the substrate specificity is diverse, whereas the difference in enzyme activity is derived from Pasteurella. It has a disadvantage that is 5-6 times lower than that of α-2,3 sialic acid transferase.

Therefore, in order to increase the production efficiency of various sialyl oligosaccharides having α-2,3 and α-2,6 bonds, the functions of α-2,3 and α-2,6 sialic acid transferases having various substrate specificities as described above. There is a need to prepare these improved variants and apply them to siallyloligosaccharide production.

Republic of Korea Patent Publication No. 10-2008-0093087 (2008.10.20) Korean Registered Patent Publication No. 10-0886650 (2009.02.25)

The present invention is to prepare a variant through protein engineering mutations of α-2,3 and α-2,6 sialic acid transferase, and to effectively produce sialyl oligosaccharide using the variant.

In addition, the variant of the α-2,3 sialic acid transferase of the present invention inhibits the generation of side reactions, thereby enabling a batch-type integrated reaction, speeding up the catalytic reaction, and thus various 3'-sialic acids including 3'-sialyllactose. It is aimed at improving the productivity and efficiency of riloligosaccharide.

In addition, the variant of the α-2,6 sialic acid transferase of the present invention has an improved catalytic activity, thereby improving the productivity of various 6'-sialyl oligosaccharides including 6'-sialyl lactose.

The present invention provides a method for searching for variants through protein engineering mutations of α-2,3 and α-2,6 sialic acid transferases, and gene sequence information encoding the variants.

It also provides the analysis of the properties of these variants and the use of them to produce sialyl oligosaccharides. That is, the present invention uses α-2,3 sialic acid transferase derived from Pasteurella and α-2,6 sialic acid transferase derived from photobacterium improved through protein engineering mutations, and 3′- and 6′ -It is applied to the production reaction of siallyl oligosaccharide.

In addition, the present invention provides a hybrid, that is, a semi-rational method for searching for a variant of a sialic acid transferase. This method combines directed evolution and rational design, and is intended to secure only a small number of high-quality variant libraries. This method refers to performing mutation by selecting the desired part of the protein and selecting the residue of a specific amino acid using the sequence, structure and function of the protein, and a computer program.

Specifically, the present invention provides the following.

(1) A method of searching for a sialic acid transferase variant comprising the following steps:

Analyzing functional residues to perform saturation mutation through multiple sequence alignment and alanine scanning among residues selected within 5 to 20 Å from the two substrate binding sites of the sialic acid transferase crystal structure or model structure.

(2) A method for searching for a variant of sialic acid transferase according to (1), characterized in that alanine scanning and saturation mutant search are performed using an indicator by pH change at a temperature of 20 to 70°C and a pH of 6 to 10. .

(3) α-2,3 sialic acid transferase variant represented by any one sequence selected from the following (a) to (g):

(a) the amino acid sequence of SEQ ID NO: 1;

(b) the amino acid sequence of SEQ ID NO: 2;

(c) the amino acid sequence of SEQ ID NO: 3;

(d) the amino acid sequence of SEQ ID NO: 4;

(e) the amino acid sequence of SEQ ID NO: 5;

(f) a sequence in which the 313th or 265th amino acid in any one of SEQ ID NOs: 1 to 5 is substituted with a hydrophilic amino acid;

(g) A sequence in which the 313th or 265th amino acid in any one of SEQ ID NOs: 1 to 5 is substituted with a hydrophilic amino acid, and an amino acid sequence having 97% or more homology with SEQ ID NOs: 1 to 5.

(4) DNA encoding the α-2,3 sialic acid transferase variant according to (3).

(5) DNA composed of any one of the sequences of SEQ ID NOs: 6 to 10 according to (4).

(6) A recombinant DNA vector comprising the DNA according to (4) or (5).

(7) A host cell transformed with the recombinant DNA vector according to (6).

(8) Extract of host cells transformed with the recombinant DNA vector according to (6).

*(9) A method for producing a 3'-sialyl oligosaccharide using any one selected from the group consisting of a host cell transformed with the recombinant DNA vector according to (6) and an extract of the host cell as a biocatalyst.

(10) α-2,6 sialic acid transferase variant represented by any one sequence selected from the following (a) to (g):

(a) the amino acid sequence of SEQ ID NO: 11;

(b) the amino acid sequence of SEQ ID NO: 12;

(c) the amino acid sequence of SEQ ID NO: 13;

(d) the amino acid sequence of SEQ ID NO: 14;

(e) the amino acid sequence of SEQ ID NO: 15;

(f) a sequence in which the 411th amino acid in any one of SEQ ID NOs: 11 to 15 is substituted with a small size or a hydrophilic amino acid, or the 433th amino acid is substituted with a hydrophilic amino acid;

(g) a sequence in which the 411 th amino acid in any one of SEQ ID NOs: 11 to 15 is substituted with a small size or a hydrophilic amino acid, or the 433 th amino acid is substituted with a hydrophilic amino acid, as in SEQ ID NOs: 11 to 15 and 55 Amino acid sequences with% or more homology.

(11) DNA encoding the α-2,6 sialic acid transferase variant according to (10).

(12) The DNA composed of any one of the sequences of SEQ ID NOs: 16 to 20 according to (11).

(13) A recombinant DNA vector comprising the DNA according to (11) or (12).

(14) A host cell transformed with the recombinant DNA vector according to (13).

(15) Extract of host cells transformed with the recombinant DNA vector according to (13).

(16) A method for producing 6'-sialyl oligosaccharide using any one selected from the group consisting of a host cell transformed with the recombinant DNA vector according to (13) and an extract of the host cell as a biocatalyst.

The method for producing a variant of the present invention can be applied to other glycotransferases, particularly glycotransferases having a GT-B folded structure. In addition, it has the effect of increasing the productivity of 3'-sialyl oligosaccharide and increasing production efficiency, and of increasing the productivity of 6'-sialyl oligosaccharide.

Specifically, by using a variant of α-2,3 sialic acid transferase with improved catalytic function, it is possible to increase the productivity of 3'-sialyl oligosaccharide including 3'-sialyl lactose and thereby reduce production cost. In addition, it enables a batch-type integrated reaction with the production of cytidine monophosphate-N -acetylneuraminic acid due to a decrease in the side reaction of the α-2,6 sialic acid transition at pH 4.5 to 7.0.

In addition, through the use of a variant of α-2,6 sialic acid transferase with improved catalytic function, the rate of α-2,6 sialic acid transfer, which is the rate determining step in the batch-type integral reaction with monophosphate -N-acetylneuraminic acid, is improved. By improving, it is possible to increase the productivity of 6'-sialyl oligosaccharide and thereby reduce the production cost.

Accordingly, through mass production of the 3'- and 6'-sialyl oligosaccharides, various applications such as food, pharmaceuticals, and cosmetics can be used.

1 is a 3 using α-2,3 and α-2,6 sialic acid transferases using a receptor containing cytidine monophosphate -N -acetylneuraminic acid (CMP-Neu5Ac) and galactose as substrates in the present invention. A schematic diagram of the synthesis of'-, 6'-sialyl oligosaccharide is shown.
Figure 2 shows the relative specific activity (specific activity) compared to the wild-type mutant in which a single amino acid of each of the α-2,3 and α-2,6 sialic acid transfer enzymes was changed in the present invention.
3 is a table showing the kinetic parameters of the single amino acid substitution variants with high intrinsic activity and their combinatorial variants, together with the wild-type values, which have been searched for in the present invention. Figure 3a shows the kinetic parameters of a single amino acid substitution variant of α-2,3 sialic acid transferase and a combination variant thereof, and Figure 3b is a single amino acid substitution variant of α-2,6 sialic acid transferase and combinations thereof Represents the kinetic variable of the enemy variant.
FIG. 4 is a graph showing the amount of 6'-sialyl lactose produced when the yield of 3'-sialyl lactose is saturated at pH 4.5 to 7.0 in a reaction using a purified α-2,3 sialic acid transferase. , Wild-type and 313-th arginine were transformed into asparagine, aspartic acid, tyrosine, threonine, and histidine, respectively, and the result of quantification of 6'-sialyllactose using combination variants R313N/T265S and R313H/T265S. Figure 4a shows the amount of 6'-sialyl lactose produced at pH 4.5 to pH 6.0, Figure 4b shows the amount of 6'-sialyl lactose produced at pH 6.5 to pH 7.0, and the unit is? to be.
Figure 5 shows the amino acid sequence of the α-2,3 sialic acid transferase variant derived from Pasteurella multocida explored in the present invention, Figure 5a is the α-2,3 sialic acid transferase The amino acid sequence of R313N in which arginine at the 313th is substituted with asparagine (SEQ ID NO: 1), FIG. 5B is an R313H amino acid sequence in which arginine at the 313th of α-2,3 sialic acid transferase is substituted with histidine (SEQ ID NO: 2), Figure 5c is the amino acid sequence of T265S in which the 265 th threonine of α-2,3 sialic acid transferase is substituted with serine (SEQ ID NO: 3), Figure 5D is a combination amino acid sequence of R313N and T265S (SEQ ID NO: 4), 5e is the combinatorial amino acid sequence of R313H and T265S (SEQ ID NO: 5). Here, since the α-2,3 sialic acid transferase of the present invention has 24 amino acids removed from the N-terminus, when counting from the first methionine sequence, methionine is numbered 25.
Figure 6 shows the DNA sequence of the α-2,3 sialic acid transferase variant explored in the present invention, Figure 6a is the DNA sequence of R313N (SEQ ID NO: 6), Figure 6b is the DNA sequence of R313H (SEQ ID NO: 7) , Figure 6c is a DNA sequence of T265S (SEQ ID NO: 8), Figure 6d is a combinatorial DNA sequence of R313N and T265S (SEQ ID NO: 9), Figure 6e is a combinatorial DNA sequence of R313H and T265S (SEQ ID NO: 10).
Figure 7 shows the amino acid sequence of the α-2,6 sialic acid transferase variant derived from Photobacterium damselae explored in the present invention, Figure 7a is 411 of the α-2,6 sialic acid transferase The amino acid sequence of I411T in which th isoleucine was substituted with threonine (SEQ ID NO: 11), FIG. 7B is the amino acid sequence of L433S in which the 433 th leucine of α-2,6 sialic acid transferase was substituted with serine (SEQ ID NO: 12), FIG. 7c is an amino acid sequence of L433T in which the 433 th leucine of α-2,6 sialic acid transferase is substituted with threonine (SEQ ID NO: 13), Figure 7d is a combination amino acid sequence of I411T and L433S (SEQ ID NO: 14), Figure 7e Is the combined amino acid sequence of I411T and L433T (SEQ ID NO: 15).
Figure 8 shows the DNA sequence of the α-2,6 sialic acid transferase mutant explored in the present invention, Figure 8a is the DNA sequence of I411T (SEQ ID NO: 16), Figure 8b is the DNA sequence of L433S (SEQ ID NO: 17) , Figure 8c is a DNA sequence of L433T (SEQ ID NO: 18), Figure 8d is a combinatorial DNA sequence of I411T and L433S (SEQ ID NO: 19), Figure 8e is a combinatorial DNA sequence of I411T and L433T (SEQ ID NO: 20).

Terms used in the present invention are commonly used in the art, and anyone skilled in the art will understand their meaning, but briefly described in the present specification is as follows:

(1) Sialic acid transferase refers to an enzyme that transfers N -acetylneuraminic acid from cytidine monophosphate-N -acetylneuraminic acid to receptor saccharide substances, as a loire sugar transferase. In addition, the variants of α-2,3 and α-2,6 sialic acid transferases produced through the present invention include cytidine monophosphate- N -acetylneuraminic acid donor substrate as well as cytidine monophosphate diaminoneuraminic acid. It can be applied to various derivative substrates including (CMP-KDN, CMP-deaminoneuraminic acid), cytidine monophosphate- N -glycolylneuraminic acid (CMP-Neu5Gc, CMP- N-glycolylneuraminic acid).

(2) α-2,3 sialic acid transferase was first explored by the Xi chen group , derived from Pasteurella multocida strain, and is a multifunctional enzyme with four enzyme activities depending on pH. . This enzyme has a function of α-2,3 sialic acid transfer to galactose, the main reaction in a wide pH range (pH 6.0-10.0), and between pH 4.5-7.0, α-2 is lower than that of α-2,3 transition. , Has the activity of 6 sialic acid transfer. In addition, it has the activity of sialidase to decompose α-2,3 bonded sialic acid at pH 5.0-5.5, and another galactoside from sialyl galactoside having α-2,3 bonds at pH 5.5-6.5. It has the activity of trans sialidase to Rho. The α-2,3 sialic acid transferase used in the present invention is a form in which 24 amino acids have been removed from the N-terminus, and the number of the first amino acid is 25. This shows the amino acid sequence of SEQ ID NO: 21 and the DNA sequence of SEQ ID NO: 22.

(3) α-2,6 sialic acid transferase is derived from the strain of Photobacterium damselae, and in terms of the structure folding and sequence of the glycotransferase, α-2,3 derived from Pasteurella It belongs to the same GT family 80 as sialic acid transferase. When the α-2,6 sialic acid transferase used in the present invention is counted from the first methionine sequence, methionine is number 1. This shows the amino acid sequence of SEQ ID NO: 23 and the DNA sequence of SEQ ID NO: 24.

(4) One of the receptor substrates, lactose, is an oligosaccharide composed of Galβ1,4Glc (galactose and glucose are linked by β1,4 bonds). In addition, variants of α-2,3 and α-2,6 sialic acid transferases produced through the present invention are not only lactose receptor substrates, but also various lactose derivative substrates, N -acetyllactosamine (LacNAc), and azaido β -D-galactopyranosyl-(1-4)-β-D-glucopyranoside (LacβN3), 3-azidopropyl β-D-galactopyranosyl-(1-4)-β-D- Glucopyranoside (LacβProN3), methyl β-D-galactopyranosyl-(1-4)-β-D-glucopyranoside (LacβOMe), and LewisX, Galβ1,4(Fucα1,3)GlcNAc ), Lewis A (LewisA, Galβ1,3(Fucα1,4)GlcNAc) and Lacto- N -tetraose (LNT, Galβ1,3GlcNAcβ1,3Galβ1,4Glc). .

(5) 3'- and 6'-sialyl oligosaccharides refer to oligosaccharides in which N -acetylneuraminic acid (sialic acid) is linked to the galactose portion by α-2,3 or α-2,6 bonds, as shown in FIG. , Galactose or other sugars may further bind to glucose.

In addition, variants of α-2,3 and α-2,6 sialic acid transferases produced through the present invention include 3'-sialyllactose, 3'-sialyllactosamine, and Sialyl LewisX (Sialyl-LewisX). , Sialyl Lewis A (Sialyl-LewisA), 6'-sialyllactose, 6'-sialyllactosamine and sialyl LNT a, b, c (Sialyl-LNT, Neu5Acα2,3Galβ1,3 GlcNAcβ1,3Galβ1,4Glc/ Galβ1,3(Neu5Acα2,6)GlcNAcβ1,3Galβ1,4Glc/ Neu5Acα2,6Galβ1,3GlcNAcβ1,3Galβ1,4Glc) It can be applied to the production of various sialyl oligosaccharides, including.

(6) 3'- and 6'-sialylactose is a triose consisting of Neu5Acα2,3/2,6Galβ1,4Glc in which sialic acid is linked to galactose of lactose by α-2,3 or α-2,6 bonds. Means substance.

(7) Cell extract refers to the microbial extract of the present invention in which sialic acid transferase is expressed.

(8) Whole-cell reaction refers to a reaction using cell contents by disrupting a cell containing a specific enzyme, or using whole cells without separating and purifying the enzyme.

(9) Hydrophilic amino acid refers to an amino acid containing an atom (oxygen, nitrogen, sulfur) with a large electronegativity capable of forming hydrogen bonds with water in a functional group, serine, Threonine, cysteine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, or arginine ( arginine).

(10) Small-sized amino acids refer to amino acids with small functional groups, and include glycine, alanine, serine, threonine, or cysteine.

(11) PCR is a polymerase chain reaction, which refers to a method of specifically amplifying a certain region of DNA.

(12) Site directed mutagenesis refers to introducing a change in a designated nucleotide sequence at a designated position in a gene.

(13) Saturation mutagenesis refers to introducing changes in various nucleotide sequences at designated positions in a gene. Saturation mutation refers to inserting a mutation through PCR by inserting an NNK codon instead of a sequence to be mutated on a primer of a complementary sequence that binds to the template strand. At this time, in the NNK codon, N means A, T, G, C of the nucleotide, and K means T, G.

(14) A vector refers to a polynucleotide consisting of single-stranded, double-stranded, circular or super-stranded DNA or RNA, and may include components that are operatively linked at an appropriate distance to produce a recombinant protein.

These components may include an origin of replication, a promoter, an enhancer, a 5'mRNA leader sequence, a ribosome binding site, a nucleic acid cassette, a termination and polyadenylation site, or a selectable label template. One or more may be missing depending on the situation. The nucleic acid cassette may contain a restriction enzyme site for insertion of a recombinant protein to be expressed. In the functional vector, the nucleic acid cassette contains a nucleic acid sequence to be expressed including a translation initiation and termination site, and if necessary, a vector capable of inserting two types of cassettes into the vector is used. Can additionally be sequenced.

The gene inserted into the recombinant vector may be E. coli strain BW25113 (DE3) for expression, or BL21 (DE3), but may vary depending on the type of the inserted vector. Such vectors and expression strains can be easily selected by those skilled in the art.

(15) The pH indicator is mainly used to know the neutralization point during titration or to know the concentration of hydrogen ions. The indicator is in an acid type and a base type according to the hydrogen ion index, and the color tone is different, and this area is called a discoloration area. The concentration of hydrogen ions according to the absorbance can be measured by a spectrophotometric method. Types of pH indicators include cresol red and phenol red used in the present invention, depending on the pH range, thymol blue, bromophenol blue, and methyl red. ), bromothymol blue, etc.

(16) Specific activity refers to the activity per unit amount of pure protein from which impurities and other proteins are removed through enzyme purification, and the amount of enzyme that catalyzes the substrate change of 1 μmol per minute is usually 1 mg. It is expressed in the number of units per unit.

(17) Enzyme kinetics deals with the rate at which an enzyme reacts with a substrate per unit time and converts it, and the rate equation of v=V _max [S]/( K _m +[S]) is established and the reaction rate (v ) substrate concentration ([S]) to one of V _max by examining the dependence dynamics variables (kinetic parameter) represents the maximum reaction velocity limit V _max by the molar concentration [E] ₀ of the enzyme k _cat (turnover number of: It is called the amount of substrate a molecule of enzyme converts in a given time. K _m is a Michaelis constant and represents the apparent dissociation constant for enzymes and substrates. k _cat / K _m means that k _cat and K _m are expressed together, and the higher the value, the faster the conversion rate from the substrate to the product.

Meanwhile, in the present invention, the single amino acid substitution variant R313N of R313 of α-2,3 sialic acid transferase represents the amino acid sequence of SEQ ID NO: 1, and includes a protein having a hydrophilic amino acid sequence at the 313th amino acid position and this variant sequence. All enzymes that have more than 97% homology and have sialic acid transfer activity are also possible. DNA encoding the protein of SEQ ID NO: 1 is SEQ ID NO: 6 and may also include all DNA sequences encoding the amino acids. Here, since the α-2,3 sialic acid transferase of the present invention has 24 amino acids removed from the N-terminus, when counting from the first methionine sequence, methionine is numbered 25.

In addition, the single amino acid substitution variant R313H of R313 of α-2,3 sialic acid transferase represents the amino acid sequence of SEQ ID NO: 2, a protein having a hydrophilic amino acid sequence at the 313th amino acid position, and 97% containing this variant sequence Any enzyme having the above homology and having sialic acid transfer activity is also possible. DNA encoding the protein of SEQ ID NO: 2 is SEQ ID NO: 7 and all DNA sequences encoding the amino acids may also be included.

In addition, the single amino acid substitution variant T265S of T265 of α-2,3 sialic acid transferase represents the amino acid sequence of SEQ ID NO: 3, a protein having a hydrophilic amino acid sequence at the 265th amino acid position, and 97% containing this variant sequence Any enzyme having the above homology and having sialic acid transfer activity is also possible. DNA encoding the protein of SEQ ID NO: 3 is SEQ ID NO: 8, and all DNA sequences encoding the amino acids may also be included.

In addition, the combinatorial variant of R313N and T265S of α-2,3 sialic acid transferase represents the amino acid sequence of SEQ ID NO: 4, and the combinatorial variant of R313H and T265S represents the amino acid sequence of SEQ ID NO: 5. In addition, a protein having a hydrophilic amino acid sequence at the 313th amino acid position or the 265th amino acid position, and an enzyme having 97% or more homology containing this variant sequence and having sialic acid transfer activity are all possible. The DNA encoding the protein of SEQ ID NO: 4 is SEQ ID NO: 9, the DNA encoding the protein of SEQ ID NO: 5 is SEQ ID NO: 10, and all DNA sequences encoding the above amino acids may also be included.

As an example having more than 97% homology with the above-specified variant of α-2,3 sialic acid transferase, it contains the mutated sequence of the above variant, and is named as having the activity of α-2,3 sialic acid transferase, Or, as a predicted sequence, a sequence derived from the genus of Pasteurella , in particular, multocida species may be included.

In the present invention, the single amino acid substitution variant I411T of I411 of α-2,6 sialic acid transferase represents the amino acid sequence of SEQ ID NO: 11, and a protein having a small size or hydrophilic amino acid sequence at the 411th amino acid position, and this variant sequence All enzymes that have a homology of 55% or more contained and have sialic acid transfer activity are also possible. The DNA encoding the protein of SEQ ID NO: 11 is SEQ ID NO: 16, and may also include all DNA sequences encoding the above amino acids.

In addition, the single amino acid substitution variant L433S of L433 of α-2,6 sialic acid transferase represents the amino acid sequence of SEQ ID NO: 12, L433T represents the amino acid sequence of SEQ ID NO: 13, and a hydrophilic amino acid sequence at the 433th amino acid position. Enzymes having a sialic acid transfer activity having a homology of 55% or more containing a protein having and this mutant sequence are also possible. DNA encoding the protein of SEQ ID NO: 12 and 13 is SEQ ID NO: 17 and 18, and all DNA sequences encoding the above amino acids may also be included.

In addition, the combinatorial variant of I411T and L433S of α-2,6 sialic acid transferase represents the amino acid sequence of SEQ ID NO: 14, and the combinatorial variant of I411T and L433T represents the amino acid sequence of SEQ ID NO: 15. In addition, a protein having a small size or a hydrophilic amino acid sequence at the 411th amino acid position or the 433th amino acid position, and an enzyme having 55% or more homology containing this variant sequence and having sialic acid transfer activity are also possible. The DNA encoding the protein of SEQ ID NO: 14 is SEQ ID NO: 19, the DNA encoding the protein of SEQ ID NO: 15 is SEQ ID NO: 20, and all DNA sequences encoding the above amino acids may also be included.

As an example having 55% or more homology with the above-specified variant of α-2,6 sialic acid transferase, it is named as containing the mutated sequence of the above variant and has the activity of α-2,6 sialic acid transferase, or As a predicted sequence, a sequence derived from the genus Photobacterium, particularly damseale and leiognathi species may be included. In addition, photobacterium Jt-Ish-224 α-2,6 sial, which is a template protein forming the protein model structure of the present invention, because it has 55% homology with the α-2,6 sialic acid transferase of the present invention. The sequence of acid transferase may be included.

Selection and mutation of sialic acid transferase mutation target part

The α-2,3 sialic acid transferase of the present invention confirmed the substrate-binding pocket portion from the crystal structure, and the α-2,6 sialic acid transferase of the other photobacterium-derived α-2,6 sialic acid transferase The substrate-binding portion was confirmed from the model structure using the crystal structure as a template. Cytidine monophosphate- N -acetylneuraminic acid and residues located within 5 to 20 Å from the receptor substrate binding site were selected from α-2,3 sialic acid transferase and α-2,6 sialic acid transferase, respectively. In the present invention, multiple sequence alignment was performed using sequence information of bioinformatics, and the preservation of amino acid residues at specific positions in the protein structure despite evolutionary pressures plays a very important role in the structure and function of the protein. They are excluded as mutant moieties because they are likely to play a direct role in the catalytic process in particular.

Among the residues selected above among the substrate-binding residues, position-directed mutation to alanine was performed to select functional residues to perform saturation mutation. Substitution with alanine is the same as the removal of a moiety, which can be interpreted as to whether a particular moiety contributes to an important catalytic activity due to its interaction with the substrate. After substitution with alanine, the enzyme activity was measured by a colorimetric method to compare the difference in activity with the wild strain. It was confirmed that the substituted moieties that lost the activity of sialic acid transfer are moieties that critically control the catalytic activity of the substrate. In the present invention, the alanine-substituted mutants that have lost the activity of sialic acid transfer were recognized as “non-replaceable” residues due to the strong possibility that their original residues play an important role in catalytic activity.

In addition, in the present invention, an alanine-substituted variant that maintains the degree of folding of the expressed protein compared to the wild strain was selected. In conclusion, among the alanine-substituted variants of each α-2,3 and α-2,6 sialic acid transferase, it exhibits at least 30%, preferably at least 50%, and more preferably at least 60% of the activity compared to the wild strain. Residues that retain folding were selected as functional residues to perform the next step, saturation mutation. By performing saturation mutations on the residues of the alanine-substituted mutant retaining the original activity and folding of the enzyme, leaving the residues that essentially contribute to the interaction with the substrate in the catalytic reaction, that is, the main activity of the sialic acid transfer. It can induce'neutral drift' of the enzyme. This means that through saturation mutation, an enzyme-substrate complex of an active type that fits more appropriately to the substrate than the wild strain is produced.

In the present invention, through the method for searching for a variant of sialic acid transferase described above, the 313th or 265th amino acid in any one of the amino acid sequences of SEQ ID NOs: 1 to 5 and SEQ ID NOs: 1 to 5 is substituted with a hydrophilic amino acid. Any one selected from an amino acid sequence having at least 97% homology with SEQ ID NOs: 1 to 5 as a sequence in which the 313 th or 265 th amino acid is substituted with a hydrophilic amino acid in the sequence and any one of SEQ ID NOs: 1 to 5 The α-2,3 sialic acid transferase variant represented by the sequence of was selected, and the 411th amino acid in any one of the amino acid sequences of SEQ ID NOs: 11 to 15 and SEQ ID NOs: 11 to 15 is a small size or hydrophilic amino acid Or a sequence in which the 433 th amino acid is substituted with a hydrophilic amino acid and the 411 th amino acid in any one of SEQ ID NOs: 11 to 15 is substituted with a small size or a hydrophilic amino acid, or the 433 th amino acid is hydrophilic As a sequence substituted with an amino acid, an α-2,6 sialic acid transferase variant represented by any one sequence selected from amino acid sequences having 55% or more homology with SEQ ID NOs: 11 to 15 was selected.

Intrinsic activity of sialic acid transferase variants

The intrinsic activity of the single amino acid substitutional variants of α-2,3 and α-2,6 sialic acid transferase can be analyzed by using the same amount of protein, respectively, through the enzyme activity assay method using the pH indicator.

In the case of the α-2,3 sialic acid transferase variant, the arginine of R313 is located on a loop near the glucose of lactose. The variant in which arginine of R313 is substituted with small amino acids such as alanine and glycine exhibits neutral activity compared to wild strains, and variants substituted with hydrophilic amino acids such as serine, threonine, tyrosine, aspartic acid, asparagine or histidine are wild. It exhibits about 1.5 times more activity than the main In addition, a variant in which threonine of T265 located within 20 Å of cytidine monophosphate-N -acetylneuraminic acid is substituted with a small amino acid such as glycine exhibits neutral activity compared to wild strains and is hydrophilic such as serine or asparagine. Variants substituted with amino acids show higher activity compared to wild strains. In addition, the variant in which the substitution of R313 and the substitution of T265 are combined shows higher activity than that of a single variant.

On the other hand, in the case of α-2,6 sialic acid transferase, I411 and L433 are located within a distance of 5 to 20 Å from cytidine monophosphate-N-acetylneuraminic acid. The variants in which leucine of L433 is substituted with a small amino acid or a hydrophilic amino acid exhibit increased activity compared to the wild strain, and in particular, the variant substituted with serine or threonine exhibits an increased activity of about 3 times compared to the wild strain. In addition, variants in which the isoleucine of I411 is substituted with a small-sized amino acid or a hydrophilic amino acid exhibit an increased activity compared to the wild strain, and in particular, the variant substituted with threonine exhibits an increased activity of about 2 times compared to the wild strain. In addition, the variant in which the substitution of I411 and the substitution of L433 are combined shows a higher activity than that of the single variant, and the I411T/L433T combination variant exhibits an increased activity of about 5 times compared to the wild strain.

Kinetic parameters of sialic acid transferase variants

To determine the effect of each mutation on the substrate donor and acceptor, the kinetic parameters were measured for single amino acid substitution and combinatorial variants. The measurement of the kinetic variable can be analyzed through the method of searching for variants using the colorimetric method.

α-2,3 sialic acid R313 and / or T265 have been replaced by a hydrophilic amino acid variant of the transferase are cytidine phosphate yl - N - k _cat for both the substrate increases the k _cat values, and for acetylneuraminic acid lactose / K _m increases. In particular, R313N and R313H, single amino acid substitution variants of α-2,3 sialic acid transferase, increased k _cat values for cytidine monophosphate-N -acetylneuraminic acid and lactose, and k _cat / K for both substrates. _{In m} , R313N increases by about 1.4 times and R313H by about 1.2 times compared to wild strains. Combination variants R313N/T265S and R313H/T265S increase k _cat values for both substrates, and both combination variants increase k _cat / K _m for cytidine monophosphate-N -acetylneuraminic acid by about 1.6 times. . In addition, R313N/T265S and R313H/T265S increase k _cat / K _m for lactose by about 1.5 and 1.8 times, respectively, compared to wild strains.

Variants in which I411 and/or L433 of α-2,6 sialic acid transferase is substituted with a small amino acid or hydrophilic amino acid have an increased k _cat value for cytidine monophosphate-N -acetylneuraminic acid and lactose. In particular, increases with respect to the acceptor substrate lactose, and all variants are the k _cat value is increased, while the reduction in the bonding strength between the substrate and k _cat values from the six-fold compared to the wild state up to 27 times. In contrast, I411T, a single amino acid substitution variant, increased k _cat / K _m for cytidine monophosphate-N -acetylneuraminic acid and lactose, respectively, by about 2.4 times and about 1.8 times compared to wild strains. In addition, k _cat / K _m of L433S and L433T increases by about 2.6 times and about 6.7 times for cytidine monophosphate- N -acetylneuraminic acid, respectively, and about 3 times and about 2.6 times for lactose. Among the combination variants, I411T/L433T increased k _cat / K _m for cytidine monophosphate-N -acetylneuraminic acid and lactose, respectively, about 8-fold and about 3.9-fold compared to wild strains.

α-2,6 sialic acid transfer side reaction activity of the α-2,3 sialic acid transferase mutant strain

In the present invention, when arginine is replaced at the 313th amino acid position of α-2,3 sialic acid transferase, and converted to other hydrophilic amino acids (N, D, Y, T, H), the intrinsic activity of the enzyme is increased. In addition to confirming that, a side reaction of α-2,6 sialic acid transfer can be confirmed for these mutants.

The side-reactivity of the α-2,6 sialic acid transfer of the α-2,3 sialic acid transferase mutant was reduced by 4 to 30 times compared to the wild strain in the amount of 6'-sialylactose at pH 4.5 to 6.0, pH 6.5. To pH 7.0, there is no or significant decrease in the production of 6'-sialyl lactose.

[ Example ]

Specific methods of the present invention will be described in detail as examples, but the technical scope of the present invention is not limited to these examples.

Enzyme activity analysis method using pH indicator

The measurement of the activity of the sugar transfer enzyme through the colorimetric method is a method of measuring the change in pH due to the proton that occurs when the glycosidic bond between the sugar donor and the acceptor is formed. This is proportional to productivity.

In the present invention, cresol red according to the optimum active pH of α-2,3 sialic acid transferase and phenol red according to the optimum active pH of α-2,6 sialic acid transferase were used. Enzyme activity analysis was carried out by mixing 1 to 5 mM cytidine monophosphate- N -acetylneuraminic acid and lactose, and 0.1 to 1 mM pH indicator in 1 to 10 mM Tris-HCl buffer solution to a total volume of 100 uL 96-tablets. Proceed in a (well) plate. The reaction was carried out for 5 to 10 minutes, and in the case of cresol red, the increase in absorbance at 405 nm, which turns yellow, and in the case of phenol red, the decrease in absorbance at 560 nm, where the red color decreases, was performed by using a spectrum machine at intervals of 30 seconds. Analyzed using. The concentration of generated hydrogen ions was calculated by a calibration curve according to the concentration of hydrochloric acid (HCl). The enzymatic reaction was performed with 10% of the total reaction volume of the enzyme, and the substrate mixture was added to the enzyme. Enzyme activity (Unit) was defined as the amount of enzyme required to generate 1 umole of hydrogen ions generated during the formation of glycosidic bonds per minute at room temperature.

A method for searching for variants using saturation mutation and colorimetric method

The AGA and ACC sequences corresponding to amino acid positions 313 and 265 of α-2,3 sialic acid transferase are randomly substituted NNK sequences (N is A, C, G or T, and K is G or T). Using the introduced primers, the entire vector was PCR to construct a library. Since the α-2,3 sialic acid transferase of the present invention has 24 amino acids removed from the N-terminus, when counting from the first methionine sequence, methionine is numbered 25.

The ATT and CTG sequences corresponding to amino acid positions 411 and 433 of α-2,6 sialic acid transferase are randomly substituted NNK sequences (N is A, C, G or T, and K is G or T). Using the introduced primers, the entire vector was PCR to construct a library. When the α-2,6 sialic acid transferase of the present invention is counted from the first methionine sequence, methionine is number 1.

The amplified gene of sialic acid transferase including the vector sequence was transformed into E. coli DH5α after treatment with DpnI enzyme to remove the original plasmid. Mutant genes were extracted from all colonies generated, and this was transformed into E. coli BW25113 (DE3). Each transformed colony was inoculated into 500 uL of ampicillin-containing LB medium on 96-tablets, and cultured with shaking at 30 to 37°C for 18 to 24 hours, and then part of the culture medium was added to 100 ug mL ^-1 ampicillin and IPTG (Isopropyl β). -D-1-thiogalactopyranoside) was inoculated into 500 uL of a new LB medium containing, and incubated for 18 to 40 hours at 18 to 30°C. After centrifugation, the cultured cells were resuspended in 100 uL of 1-10 mM Tris buffer, and then 10 uL of whole cells were used for the mutant screening reaction, or 50 uL of the cells were used for the BugBuster protein. After resuspension with an extraction reagent and centrifugation, 10 uL of the cell extract was obtained, and 10 uL of this was used in the mutant screening reaction. The reaction solution of 90 uL contains 1 to 10 mM Tris buffer solution, 1 to 5 mM cytidine monophosphate-N-acetylneuraminic acid and lactose, 0.1 to 1 mM pH indicator, and whole cells or cell extracts of sialic acid transferase At the same time as 10 uL was added, the reaction proceeded, and the reaction rate of 10 to 30 minutes was observed at a time interval of 1 minute compared to that of wild strains.

Purification of sialic acid transfer enzyme

The wild strain and mutant of the sialic acid transferase transformed in E. coli BW25113 (DE3) were expressed using the inducer IPTG in a culture volume of 50 mL, and only pure protein was purified using a Ni-NTA column. First, after protein expression, the cells were disrupted with a sonic disruptor, and then centrifuged to obtain a cell extract. The cell extract was added to a column equilibrated with a 50 mM Tris buffer solution (pH 8.0) to which 5 mM imidazole and 300 mM sodium chloride were added, and bound with nickel resin for 1 hour at 0°C. Subsequently, proteins that could not be bound to the resin were spilled, and other non-specifically bound proteins were removed with a Tris buffer solution containing 50 mM imidazole. Finally, only the desired protein was eluted with a Tris buffer solution containing 250 mM imidazole. The eluted protein was desalted using a filtration column to remove imidazole, and only the active protein was finally obtained, and the protein amount was measured using the Bradford protein quantification kit, and the same amount of protein was used. After the reaction, intrinsic activity, kinetic parameters, and α-2,6 sialic acid transfer side reaction activity of the α-2,3 sialic acid transferase mutant were measured.

Measurement of intrinsic activity of sialic acid transferase variants

The intrinsic activity of the single amino acid substitution mutants of α-2,3 and α-2,6 sialic acid transferase was analyzed by the enzyme activity assay method using the pH indicator using the same amount of protein, respectively, and the reaction was from 5 to It was calculated as the activity per mg of enzyme (unit) when the conversion yield of 10 to 25% compared to the initial receptor substrate concentration was displayed by proceeding for 10 minutes, which is shown in FIG. 2.

In the case of the α-2,3 sialic acid transferase variant, the arginine of R313 is located on a loop near the glucose of lactose. For the variants of R313, the variants substituted with small sized amino acids such as alanine and glycine showed neutral activity compared to wild strains and were substituted with hydrophilic amino acids such as serine, threonine, tyrosine, aspartic acid, asparagine or histidine. The mutants showed more than 1.5 times the activity compared to the wild strain.

For the T265 variant located within 20 Å of cytidine monophosphate- N -acetylneuraminic acid, the variant substituted with glycine, serine or asparagine was found to have similar or high activity to wild strains.

As a result of comparing the relative intrinsic activity of the R313 mutant compared to the wild type, it is as shown in Fig.2, and as a result, R313 was able to accept relatively various mutants, of which the intrinsic activity of R313N was 231% compared to the wild type, among the single mutants. It was the highest. In addition, the relative intrinsic activity relative to the wild type for the T265 mutant is shown in FIG. 2.

On the other hand, in the case of α-2,6 sialic acid transferase, I411 and L433 are located within a distance of 5 to 20 Å from cytidine monophosphate-N-acetylneuraminic acid. Among the variants of L433, L433S and L433T showed three-fold increased activity compared to wild-type. Among the mutants of I411, I411T was found to exhibit twice the activity compared to the wild type. In the case of wild-type α-2,6 sialic acid transferase, the expression in the pET28a vector was increased compared to the pET15b vector, and the found mutants were cloned into the pET28a vector to confirm their intrinsic activity. The intrinsic activity of the variant is shown in Fig. 2(b).

In the present invention, a combination of each of the single amino acid substitution variants of R313 and the single amino acid substitution variants of T265 of α-2,3 sialic acid transferase having high intrinsic activity was made. Among the combination variants, R313H/T265S And R313N/T265S showed the highest intrinsic activity of 237% and 216% compared to wild strains.

In the present invention, a single amino acid substitution variant of I411T of α-2,6 sialic acid transferase having a high intrinsic activity and a single amino acid substitution variant of L433S and L433T each were made in combination. Among the combination variants, I411T/ L433S and I411T/L433T showed high intrinsic activity of 194% and 510% compared to wild strains.

Measurement of kinetic parameters of sialic acid transferase variants

The measurement of the kinetic variable was analyzed through the method of searching for variants using the colorimetric method, and the reaction was performed at room temperature for 5 to 10 minutes, and the conversion yield was 10 to 25% compared to the initial receptor substrate concentration at intervals of every 30 seconds. The initial reaction rate of was measured. The kinetic parameters were measured for both the donor substrate, cytidine monophosphate- N -acetylneuraminic acid and the acceptor substrate, lactose, and the substrate concentration ranged from 0.1 to 30 mM. The kinematic variables of k _cat and K _m were obtained through nonlinear regression analysis of the Michaelis-Menten equation using the SigmaPlot program. The kinetic variables for the wild strains and variants of α-2,3 and α-2,6 sialic acid transferase are shown in FIG. 3.

Single amino acid substitution variants of α-2,3 sialic acid transferase, R313N and R313H , increased k _cat values for cytidine monophosphate-N -acetylneuraminic acid and lactose, and k _cat / K _m for both substrates. R313N increased by 1.4 times and R313H by 1.2 times compared to wild strains. Combination variants R313N/T265S and R313H/T265S increased k _cat values for both substrates, and both combination variants increased k _cat / K _m 1.6 times for cytidine monophosphate-N -acetylneuraminic acid. In addition, for lactose, R313N/T265S and R313H/T265S increased k _cat / K _m 1.5 and 1.8 times, respectively, compared to wild strains.

In the case of the α-2,6 sialic acid transferase variant, k _cat values were increased for both substrates. In particular, the receptor for the substrate, lactose, sikimyeo all variants are reduced whereas the bonding strength between the substrate increases the value of k _cat, k _cat values were increased in a six-fold compared to the wild state up to 27 times. I411T, a single amino acid substitution variant, increased k _cat / K _m for cytidine monophosphate-N -acetylneuraminic acid and lactose, respectively, by 2.4 and 1.8 times compared to wild strains. In addition, k _cat / K _m of L433S and L433T increased 2.6 times and 6.7 times for cytidine monophosphate- N -acetylneuraminic acid, respectively, and 3 times and 2.6 times for lactose. Among the combination variants, I411T/L433T had a greater effect of increasing activity than I411T/L433S, and k _cat / K _m for cytidine monophosphate-N -acetylneuraminic acid and lactose, respectively, increased 8-fold and 3.9-fold compared to wild strains. .

α-2,6 sialic acid transfer side reaction activity of the α-2,3 sialic acid transferase mutant strain

The α-2,6 sialic acid transfer side reaction activity was measured for R313 variants R313N, R313D, R313Y, R313T, R313H, R313N/T265S, and R313H/T265S of α-2,3 sialic acid transferase after protein purification. Enzymatic reaction was carried out in a buffer solution of 50 mM MES containing 10 mM cytidine monophosphate-N -acetylneuraminic acid and 5 mM lactose in a buffer solution of pH 4.5 to pH 7, and the same amount of purified enzyme was added thereto, followed by reaction at room temperature for 30 minutes. After the reaction, heat was applied for 1 minute to denature the protein, and then removed after centrifugation. The 3'-sialyl lactose and 6'-sialyl lactose generated on the supernatant were quantitatively analyzed through Bio-LC. As a result of measuring the side reactivity of the α-2,6 sialic acid transfer of the α-2,3 sialic acid transferase mutant, the production of 6'-sialylactose at pH 4.5 to 6.0 decreased by 4-30 times compared to wild strains. And, it was confirmed that all of the production amount of 6'-sialyl lactose disappeared except for R313Y (15-fold decrease) at pH 6.5 to pH 7.0, which is shown in FIG. 4.

<110> Seoul National University R&DB Foundation GeneChem Inc. <120> METHOD FOR SCREENING a-2,3 AND a-2,6 SIALYLTRANSFERASE VARIANTS AND THEIR APPLICATION FOR SYNTHESIS OF SIALYLOLIGOSACCHARIDES <130> Y13KP-021 <160> 24 <170> KopatentIn 2.0 <210> 1 <211> 391 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 1 Met Lys Thr Ile Thr Leu Tyr Leu Asp Pro Ala Ser Leu Pro Ala Leu 1 5 10 15 Asn Gln Leu Met Asp Phe Thr Gln Asn Asn Glu Asp Lys Thr His Pro 20 25 30 Arg Ile Phe Gly Leu Ser Arg Phe Lys Ile Pro Asp Asn Ile Ile Thr 35 40 45 Gln Tyr Gln Asn Ile His Phe Val Glu Leu Lys Asp Asn Arg Pro Thr 50 55 60 Glu Ala Leu Phe Thr Ile Leu Asp Gln Tyr Pro Gly Asn Ile Glu Leu 65 70 75 80 Asn Ile His Leu Asn Ile Ala His Ser Val Gln Leu Ile Arg Pro Ile 85 90 95 Leu Ala Tyr Arg Phe Lys His Leu Asp Arg Val Ser Ile Gln Gln Leu 100 105 110 Asn Leu Tyr Asp Asp Gly Ser Met Glu Tyr Val Asp Leu Glu Lys Glu 115 120 125 Glu Asn Lys Asp Ile Ser Ala Glu Ile Lys Gln Ala Glu Lys Gln Leu 130 135 140 Ser His Tyr Leu Leu Thr Gly Lys Ile Lys Phe Asp Asn Pro Thr Ile 145 150 155 160 Ala Arg Tyr Val Trp Gln Ser Ala Phe Pro Val Lys Tyr His Phe Leu 165 170 175 Ser Thr Asp Tyr Phe Glu Lys Ala Glu Phe Leu Gln Pro Leu Lys Glu 180 185 190 Tyr Leu Ala Glu Asn Tyr Gln Lys Met Asp Trp Thr Ala Tyr Gln Gln 195 200 205 Leu Thr Pro Glu Gln Gln Ala Phe Tyr Leu Thr Leu Val Gly Phe Asn 210 215 220 Asp Glu Val Lys Gln Ser Leu Glu Val Gln Gln Ala Lys Phe Ile Phe 225 230 235 240 Thr Gly Thr Thr Thr Trp Glu Gly Asn Thr Asp Val Arg Glu Tyr Tyr 245 250 255 Ala Gln Gln Gln Leu Asn Leu Leu Asn His Phe Thr Gln Ala Glu Gly 260 265 270 Asp Leu Phe Ile Gly Asp His Tyr Lys Ile Tyr Phe Lys Gly His Pro 275 280 285 Asn Gly Gly Glu Ile Asn Asp Tyr Ile Leu Asn Asn Ala Lys Asn Ile 290 295 300 Thr Asn Ile Pro Ala Asn Ile Ser Phe Glu Val Leu Met Met Thr Gly 305 310 315 320 Leu Leu Pro Asp Lys Val Gly Gly Val Ala Ser Ser Leu Tyr Phe Ser 325 330 335 Leu Pro Lys Glu Lys Ile Ser His Ile Ile Phe Thr Ser Asn Lys Gln 340 345 350 Val Lys Ser Lys Glu Asp Ala Leu Asn Asn Pro Tyr Val Lys Val Met 355 360 365 Arg Arg Leu Gly Ile Ile Asp Glu Ser Gln Val Ile Phe Trp Asp Ser 370 375 380 Leu Lys Gln Leu Gly Gly Gly 385 390 <210> 2 <211> 391 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 2 Met Lys Thr Ile Thr Leu Tyr Leu Asp Pro Ala Ser Leu Pro Ala Leu 1 5 10 15 Asn Gln Leu Met Asp Phe Thr Gln Asn Asn Glu Asp Lys Thr His Pro 20 25 30 Arg Ile Phe Gly Leu Ser Arg Phe Lys Ile Pro Asp Asn Ile Ile Thr 35 40 45 Gln Tyr Gln Asn Ile His Phe Val Glu Leu Lys Asp Asn Arg Pro Thr 50 55 60 Glu Ala Leu Phe Thr Ile Leu Asp Gln Tyr Pro Gly Asn Ile Glu Leu 65 70 75 80 Asn Ile His Leu Asn Ile Ala His Ser Val Gln Leu Ile Arg Pro Ile 85 90 95 Leu Ala Tyr Arg Phe Lys His Leu Asp Arg Val Ser Ile Gln Gln Leu 100 105 110 Asn Leu Tyr Asp Asp Gly Ser Met Glu Tyr Val Asp Leu Glu Lys Glu 115 120 125 Glu Asn Lys Asp Ile Ser Ala Glu Ile Lys Gln Ala Glu Lys Gln Leu 130 135 140 Ser His Tyr Leu Leu Thr Gly Lys Ile Lys Phe Asp Asn Pro Thr Ile 145 150 155 160 Ala Arg Tyr Val Trp Gln Ser Ala Phe Pro Val Lys Tyr His Phe Leu 165 170 175 Ser Thr Asp Tyr Phe Glu Lys Ala Glu Phe Leu Gln Pro Leu Lys Glu 180 185 190 Tyr Leu Ala Glu Asn Tyr Gln Lys Met Asp Trp Thr Ala Tyr Gln Gln 195 200 205 Leu Thr Pro Glu Gln Gln Ala Phe Tyr Leu Thr Leu Val Gly Phe Asn 210 215 220 Asp Glu Val Lys Gln Ser Leu Glu Val Gln Gln Ala Lys Phe Ile Phe 225 230 235 240 Thr Gly Thr Thr Thr Trp Glu Gly Asn Thr Asp Val Arg Glu Tyr Tyr 245 250 255 Ala Gln Gln Gln Leu Asn Leu Leu Asn His Phe Thr Gln Ala Glu Gly 260 265 270 Asp Leu Phe Ile Gly Asp His Tyr Lys Ile Tyr Phe Lys Gly His Pro 275 280 285 His Gly Gly Glu Ile Asn Asp Tyr Ile Leu Asn Asn Ala Lys Asn Ile 290 295 300 Thr Asn Ile Pro Ala Asn Ile Ser Phe Glu Val Leu Met Met Thr Gly 305 310 315 320 Leu Leu Pro Asp Lys Val Gly Gly Val Ala Ser Ser Leu Tyr Phe Ser 325 330 335 Leu Pro Lys Glu Lys Ile Ser His Ile Ile Phe Thr Ser Asn Lys Gln 340 345 350 Val Lys Ser Lys Glu Asp Ala Leu Asn Asn Pro Tyr Val Lys Val Met 355 360 365 Arg Arg Leu Gly Ile Ile Asp Glu Ser Gln Val Ile Phe Trp Asp Ser 370 375 380 Leu Lys Gln Leu Gly Gly Gly 385 390 <210> 3 <211> 391 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 3 Met Lys Thr Ile Thr Leu Tyr Leu Asp Pro Ala Ser Leu Pro Ala Leu 1 5 10 15 Asn Gln Leu Met Asp Phe Thr Gln Asn Asn Glu Asp Lys Thr His Pro 20 25 30 Arg Ile Phe Gly Leu Ser Arg Phe Lys Ile Pro Asp Asn Ile Ile Thr 35 40 45 Gln Tyr Gln Asn Ile His Phe Val Glu Leu Lys Asp Asn Arg Pro Thr 50 55 60 Glu Ala Leu Phe Thr Ile Leu Asp Gln Tyr Pro Gly Asn Ile Glu Leu 65 70 75 80 Asn Ile His Leu Asn Ile Ala His Ser Val Gln Leu Ile Arg Pro Ile 85 90 95 Leu Ala Tyr Arg Phe Lys His Leu Asp Arg Val Ser Ile Gln Gln Leu 100 105 110 Asn Leu Tyr Asp Asp Gly Ser Met Glu Tyr Val Asp Leu Glu Lys Glu 115 120 125 Glu Asn Lys Asp Ile Ser Ala Glu Ile Lys Gln Ala Glu Lys Gln Leu 130 135 140 Ser His Tyr Leu Leu Thr Gly Lys Ile Lys Phe Asp Asn Pro Thr Ile 145 150 155 160 Ala Arg Tyr Val Trp Gln Ser Ala Phe Pro Val Lys Tyr His Phe Leu 165 170 175 Ser Thr Asp Tyr Phe Glu Lys Ala Glu Phe Leu Gln Pro Leu Lys Glu 180 185 190 Tyr Leu Ala Glu Asn Tyr Gln Lys Met Asp Trp Thr Ala Tyr Gln Gln 195 200 205 Leu Thr Pro Glu Gln Gln Ala Phe Tyr Leu Thr Leu Val Gly Phe Asn 210 215 220 Asp Glu Val Lys Gln Ser Leu Glu Val Gln Gln Ala Lys Phe Ile Phe 225 230 235 240 Ser Gly Thr Thr Thr Trp Glu Gly Asn Thr Asp Val Arg Glu Tyr Tyr 245 250 255 Ala Gln Gln Gln Leu Asn Leu Leu Asn His Phe Thr Gln Ala Glu Gly 260 265 270 Asp Leu Phe Ile Gly Asp His Tyr Lys Ile Tyr Phe Lys Gly His Pro 275 280 285 Arg Gly Gly Glu Ile Asn Asp Tyr Ile Leu Asn Asn Ala Lys Asn Ile 290 295 300 Thr Asn Ile Pro Ala Asn Ile Ser Phe Glu Val Leu Met Met Thr Gly 305 310 315 320 Leu Leu Pro Asp Lys Val Gly Gly Val Ala Ser Ser Leu Tyr Phe Ser 325 330 335 Leu Pro Lys Glu Lys Ile Ser His Ile Ile Phe Thr Ser Asn Lys Gln 340 345 350 Val Lys Ser Lys Glu Asp Ala Leu Asn Asn Pro Tyr Val Lys Val Met 355 360 365 Arg Arg Leu Gly Ile Ile Asp Glu Ser Gln Val Ile Phe Trp Asp Ser 370 375 380 Leu Lys Gln Leu Gly Gly Gly 385 390 <210> 4 <211> 391 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 4 Met Lys Thr Ile Thr Leu Tyr Leu Asp Pro Ala Ser Leu Pro Ala Leu 1 5 10 15 Asn Gln Leu Met Asp Phe Thr Gln Asn Asn Glu Asp Lys Thr His Pro 20 25 30 Arg Ile Phe Gly Leu Ser Arg Phe Lys Ile Pro Asp Asn Ile Ile Thr 35 40 45 Gln Tyr Gln Asn Ile His Phe Val Glu Leu Lys Asp Asn Arg Pro Thr 50 55 60 Glu Ala Leu Phe Thr Ile Leu Asp Gln Tyr Pro Gly Asn Ile Glu Leu 65 70 75 80 Asn Ile His Leu Asn Ile Ala His Ser Val Gln Leu Ile Arg Pro Ile 85 90 95 Leu Ala Tyr Arg Phe Lys His Leu Asp Arg Val Ser Ile Gln Gln Leu 100 105 110 Asn Leu Tyr Asp Asp Gly Ser Met Glu Tyr Val Asp Leu Glu Lys Glu 115 120 125 Glu Asn Lys Asp Ile Ser Ala Glu Ile Lys Gln Ala Glu Lys Gln Leu 130 135 140 Ser His Tyr Leu Leu Thr Gly Lys Ile Lys Phe Asp Asn Pro Thr Ile 145 150 155 160 Ala Arg Tyr Val Trp Gln Ser Ala Phe Pro Val Lys Tyr His Phe Leu 165 170 175 Ser Thr Asp Tyr Phe Glu Lys Ala Glu Phe Leu Gln Pro Leu Lys Glu 180 185 190 Tyr Leu Ala Glu Asn Tyr Gln Lys Met Asp Trp Thr Ala Tyr Gln Gln 195 200 205 Leu Thr Pro Glu Gln Gln Ala Phe Tyr Leu Thr Leu Val Gly Phe Asn 210 215 220 Asp Glu Val Lys Gln Ser Leu Glu Val Gln Gln Ala Lys Phe Ile Phe 225 230 235 240 Ser Gly Thr Thr Thr Trp Glu Gly Asn Thr Asp Val Arg Glu Tyr Tyr 245 250 255 Ala Gln Gln Gln Leu Asn Leu Leu Asn His Phe Thr Gln Ala Glu Gly 260 265 270 Asp Leu Phe Ile Gly Asp His Tyr Lys Ile Tyr Phe Lys Gly His Pro 275 280 285 Asn Gly Gly Glu Ile Asn Asp Tyr Ile Leu Asn Asn Ala Lys Asn Ile 290 295 300 Thr Asn Ile Pro Ala Asn Ile Ser Phe Glu Val Leu Met Met Thr Gly 305 310 315 320 Leu Leu Pro Asp Lys Val Gly Gly Val Ala Ser Ser Leu Tyr Phe Ser 325 330 335 Leu Pro Lys Glu Lys Ile Ser His Ile Ile Phe Thr Ser Asn Lys Gln 340 345 350 Val Lys Ser Lys Glu Asp Ala Leu Asn Asn Pro Tyr Val Lys Val Met 355 360 365 Arg Arg Leu Gly Ile Ile Asp Glu Ser Gln Val Ile Phe Trp Asp Ser 370 375 380 Leu Lys Gln Leu Gly Gly Gly 385 390 <210> 5 <211> 391 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 5 Met Lys Thr Ile Thr Leu Tyr Leu Asp Pro Ala Ser Leu Pro Ala Leu 1 5 10 15 Asn Gln Leu Met Asp Phe Thr Gln Asn Asn Glu Asp Lys Thr His Pro 20 25 30 Arg Ile Phe Gly Leu Ser Arg Phe Lys Ile Pro Asp Asn Ile Ile Thr 35 40 45 Gln Tyr Gln Asn Ile His Phe Val Glu Leu Lys Asp Asn Arg Pro Thr 50 55 60 Glu Ala Leu Phe Thr Ile Leu Asp Gln Tyr Pro Gly Asn Ile Glu Leu 65 70 75 80 Asn Ile His Leu Asn Ile Ala His Ser Val Gln Leu Ile Arg Pro Ile 85 90 95 Leu Ala Tyr Arg Phe Lys His Leu Asp Arg Val Ser Ile Gln Gln Leu 100 105 110 Asn Leu Tyr Asp Asp Gly Ser Met Glu Tyr Val Asp Leu Glu Lys Glu 115 120 125 Glu Asn Lys Asp Ile Ser Ala Glu Ile Lys Gln Ala Glu Lys Gln Leu 130 135 140 Ser His Tyr Leu Leu Thr Gly Lys Ile Lys Phe Asp Asn Pro Thr Ile 145 150 155 160 Ala Arg Tyr Val Trp Gln Ser Ala Phe Pro Val Lys Tyr His Phe Leu 165 170 175 Ser Thr Asp Tyr Phe Glu Lys Ala Glu Phe Leu Gln Pro Leu Lys Glu 180 185 190 Tyr Leu Ala Glu Asn Tyr Gln Lys Met Asp Trp Thr Ala Tyr Gln Gln 195 200 205 Leu Thr Pro Glu Gln Gln Ala Phe Tyr Leu Thr Leu Val Gly Phe Asn 210 215 220 Asp Glu Val Lys Gln Ser Leu Glu Val Gln Gln Ala Lys Phe Ile Phe 225 230 235 240 Ser Gly Thr Thr Thr Trp Glu Gly Asn Thr Asp Val Arg Glu Tyr Tyr 245 250 255 Ala Gln Gln Gln Leu Asn Leu Leu Asn His Phe Thr Gln Ala Glu Gly 260 265 270 Asp Leu Phe Ile Gly Asp His Tyr Lys Ile Tyr Phe Lys Gly His Pro 275 280 285 His Gly Gly Glu Ile Asn Asp Tyr Ile Leu Asn Asn Ala Lys Asn Ile 290 295 300 Thr Asn Ile Pro Ala Asn Ile Ser Phe Glu Val Leu Met Met Thr Gly 305 310 315 320 Leu Leu Pro Asp Lys Val Gly Gly Val Ala Ser Ser Leu Tyr Phe Ser 325 330 335 Leu Pro Lys Glu Lys Ile Ser His Ile Ile Phe Thr Ser Asn Lys Gln 340 345 350 Val Lys Ser Lys Glu Asp Ala Leu Asn Asn Pro Tyr Val Lys Val Met 355 360 365 Arg Arg Leu Gly Ile Ile Asp Glu Ser Gln Val Ile Phe Trp Asp Ser 370 375 380 Leu Lys Gln Leu Gly Gly Gly 385 390 <210> 6 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 6 atgaaaacaa tcacgctgta tttagatcct gcctccttac cggcattaaa tcagctgatg 60 gactttacgc aaaataatga agataaaaca catccacgta tttttggtct ttctcgcttt 120 aaaatccctg acaacattat tacacagtat caaaatatcc atttcgtcga actcaaagat 180 aatcgtccca ctgaagcact ttttacgatt ttagatcaat accctggtaa cattgagtta 240 aatatacact taaatattgc tcattccgtt caattaattc gtccgatttt ggcatatcgt 300 tttaaacatt tagatcgtgt atcaattcag cagttaaatc tttatgacga tggctcaatg 360 gaatatgttg atttagaaaa agaagaaaat aaagatattt ccgcagaaat taagcaagca 420 gaaaaacaac tttctcacta tttgcttact ggcaaaataa aatttgataa cccaactatt 480 gctcgttatg tctggcaatc cgcgttccca gtaaaatatc attttttaag tacagactat 540 tttgaaaaag ccgaattttt acaaccacta aaagaatatt tagcagaaaa ttatcaaaaa 600 atggactgga ctgcttacca acagctgact ccagaacagc aagcattcta cttaacattg 660 gtaggcttca atgacgaagt caagcagtcg ctagaagtgc aacaagctaa atttatcttt 720 accggcacga caacttggga aggaaatacc gatgtgcgag aatactacgc acagcaacaa 780 cttaatttac ttaatcactt tacccaagct gagggcgatt tatttattgg tgatcattat 840 aaaatctact ttaaagggca tcctaatggt ggtgaaatta atgactacat tctgaacaat 900 gctaaaaata tcaccaatat ccctgccaat atttcctttg aagtattgat gatgacaggc 960 ttattacctg ataaagtggg tggtgttgca agttcactgt atttctcctt accaaaagaa 1020 aaaattagcc atattatttt cacatcgaat aaacaagtga aaagcaaaga agatgcgcta 1080 aataatccgt atgttaaggt catgcgtcgt ttaggtataa ttgacgaatc acaagtcatc 1140 ttttgggaca gtttaaaaca gttgggtgga ggt 1173 <210> 7 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 7 atgaaaacaa tcacgctgta tttagatcct gcctccttac cggcattaaa tcagctgatg 60 gactttacgc aaaataatga agataaaaca catccacgta tttttggtct ttctcgcttt 120 aaaatccctg acaacattat tacacagtat caaaatatcc atttcgtcga actcaaagat 180 aatcgtccca ctgaagcact ttttacgatt ttagatcaat accctggtaa cattgagtta 240 aatatacact taaatattgc tcattccgtt caattaattc gtccgatttt ggcatatcgt 300 tttaaacatt tagatcgtgt atcaattcag cagttaaatc tttatgacga tggctcaatg 360 gaatatgttg atttagaaaa agaagaaaat aaagatattt ccgcagaaat taagcaagca 420 gaaaaacaac tttctcacta tttgcttact ggcaaaataa aatttgataa cccaactatt 480 gctcgttatg tctggcaatc cgcgttccca gtaaaatatc attttttaag tacagactat 540 tttgaaaaag ccgaattttt acaaccacta aaagaatatt tagcagaaaa ttatcaaaaa 600 atggactgga ctgcttacca acagctgact ccagaacagc aagcattcta cttaacattg 660 gtaggcttca atgacgaagt caagcagtcg ctagaagtgc aacaagctaa atttatcttt 720 accggcacga caacttggga aggaaatacc gatgtgcgag aatactacgc acagcaacaa 780 cttaatttac ttaatcactt tacccaagct gagggcgatt tatttattgg tgatcattat 840 aaaatctact ttaaagggca tcctcatggt ggtgaaatta atgactacat tctgaacaat 900 gctaaaaata tcaccaatat ccctgccaat atttcctttg aagtattgat gatgacaggc 960 ttattacctg ataaagtggg tggtgttgca agttcactgt atttctcctt accaaaagaa 1020 aaaattagcc atattatttt cacatcgaat aaacaagtga aaagcaaaga agatgcgcta 1080 aataatccgt atgttaaggt catgcgtcgt ttaggtataa ttgacgaatc acaagtcatc 1140 ttttgggaca gtttaaaaca gttgggtgga ggt 1173 <210> 8 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 8 atgaaaacaa tcacgctgta tttagatcct gcctccttac cggcattaaa tcagctgatg 60 gactttacgc aaaataatga agataaaaca catccacgta tttttggtct ttctcgcttt 120 aaaatccctg acaacattat tacacagtat caaaatatcc atttcgtcga actcaaagat 180 aatcgtccca ctgaagcact ttttacgatt ttagatcaat accctggtaa cattgagtta 240 aatatacact taaatattgc tcattccgtt caattaattc gtccgatttt ggcatatcgt 300 tttaaacatt tagatcgtgt atcaattcag cagttaaatc tttatgacga tggctcaatg 360 gaatatgttg atttagaaaa agaagaaaat aaagatattt ccgcagaaat taagcaagca 420 gaaaaacaac tttctcacta tttgcttact ggcaaaataa aatttgataa cccaactatt 480 gctcgttatg tctggcaatc cgcgttccca gtaaaatatc attttttaag tacagactat 540 tttgaaaaag ccgaattttt acaaccacta aaagaatatt tagcagaaaa ttatcaaaaa 600 atggactgga ctgcttacca acagctgact ccagaacagc aagcattcta cttaacattg 660 gtaggcttca atgacgaagt caagcagtcg ctagaagtgc aacaagctaa atttatcttt 720 agtggcacga caacttggga aggaaatacc gatgtgcgag aatactacgc acagcaacaa 780 cttaatttac ttaatcactt tacccaagct gagggcgatt tatttattgg tgatcattat 840 aaaatctact ttaaagggca tcctagaggt ggtgaaatta atgactacat tctgaacaat 900 gctaaaaata tcaccaatat ccctgccaat atttcctttg aagtattgat gatgacaggc 960 ttattacctg ataaagtggg tggtgttgca agttcactgt atttctcctt accaaaagaa 1020 aaaattagcc atattatttt cacatcgaat aaacaagtga aaagcaaaga agatgcgcta 1080 aataatccgt atgttaaggt catgcgtcgt ttaggtataa ttgacgaatc acaagtcatc 1140 ttttgggaca gtttaaaaca gttgggtgga ggt 1173 <210> 9 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 9 atgaaaacaa tcacgctgta tttagatcct gcctccttac cggcattaaa tcagctgatg 60 gactttacgc aaaataatga agataaaaca catccacgta tttttggtct ttctcgcttt 120 aaaatccctg acaacattat tacacagtat caaaatatcc atttcgtcga actcaaagat 180 aatcgtccca ctgaagcact ttttacgatt ttagatcaat accctggtaa cattgagtta 240 aatatacact taaatattgc tcattccgtt caattaattc gtccgatttt ggcatatcgt 300 tttaaacatt tagatcgtgt atcaattcag cagttaaatc tttatgacga tggctcaatg 360 gaatatgttg atttagaaaa agaagaaaat aaagatattt ccgcagaaat taagcaagca 420 gaaaaacaac tttctcacta tttgcttact ggcaaaataa aatttgataa cccaactatt 480 gctcgttatg tctggcaatc cgcgttccca gtaaaatatc attttttaag tacagactat 540 tttgaaaaag ccgaattttt acaaccacta aaagaatatt tagcagaaaa ttatcaaaaa 600 atggactgga ctgcttacca acagctgact ccagaacagc aagcattcta cttaacattg 660 gtaggcttca atgacgaagt caagcagtcg ctagaagtgc aacaagctaa atttatcttt 720 agcggcacga caacttggga aggaaatacc gatgtgcgag aatactacgc acagcaacaa 780 cttaatttac ttaatcactt tacccaagct gagggcgatt tatttattgg tgatcattat 840 aaaatctact ttaaagggca tcctaatggt ggtgaaatta atgactacat tctgaacaat 900 gctaaaaata tcaccaatat ccctgccaat atttcctttg aagtattgat gatgacaggc 960 ttattacctg ataaagtggg tggtgttgca agttcactgt atttctcctt accaaaagaa 1020 aaaattagcc atattatttt cacatcgaat aaacaagtga aaagcaaaga agatgcgcta 1080 aataatccgt atgttaaggt catgcgtcgt ttaggtataa ttgacgaatc acaagtcatc 1140 ttttgggaca gtttaaaaca gttgggtgga ggt 1173 <210> 10 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 10 atgaaaacaa tcacgctgta tttagatcct gcctccttac cggcattaaa tcagctgatg 60 gactttacgc aaaataatga agataaaaca catccacgta tttttggtct ttctcgcttt 120 aaaatccctg acaacattat tacacagtat caaaatatcc atttcgtcga actcaaagat 180 aatcgtccca ctgaagcact ttttacgatt ttagatcaat accctggtaa cattgagtta 240 aatatacact taaatattgc tcattccgtt caattaattc gtccgatttt ggcatatcgt 300 tttaaacatt tagatcgtgt atcaattcag cagttaaatc tttatgacga tggctcaatg 360 gaatatgttg atttagaaaa agaagaaaat aaagatattt ccgcagaaat taagcaagca 420 gaaaaacaac tttctcacta tttgcttact ggcaaaataa aatttgataa cccaactatt 480 gctcgttatg tctggcaatc cgcgttccca gtaaaatatc attttttaag tacagactat 540 tttgaaaaag ccgaattttt acaaccacta aaagaatatt tagcagaaaa ttatcaaaaa 600 atggactgga ctgcttacca acagctgact ccagaacagc aagcattcta cttaacattg 660 gtaggcttca atgacgaagt caagcagtcg ctagaagtgc aacaagctaa atttatcttt 720 agcggcacga caacttggga aggaaatacc gatgtgcgag aatactacgc acagcaacaa 780 cttaatttac ttaatcactt tacccaagct gagggcgatt tatttattgg tgatcattat 840 aaaatctact ttaaagggca tcctcatggt ggtgaaatta atgactacat tctgaacaat 900 gctaaaaata tcaccaatat ccctgccaat atttcctttg aagtattgat gatgacaggc 960 ttattacctg ataaagtggg tggtgttgca agttcactgt atttctcctt accaaaagaa 1020 aaaattagcc atattatttt cacatcgaat aaacaagtga aaagcaaaga agatgcgcta 1080 aataatccgt atgttaaggt catgcgtcgt ttaggtataa ttgacgaatc acaagtcatc 1140 ttttgggaca gtttaaaaca gttgggtgga ggt 1173 <210> 11 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 11 Met Cys Asn Ser Asp Asn Thr Ser Leu Lys Glu Thr Val Ser Ser Asn 1 5 10 15 Ser Ala Asp Val Val Glu Thr Glu Thr Tyr Gln Leu Thr Pro Ile Asp 20 25 30 Ala Pro Ser Ser Phe Leu Ser His Ser Trp Glu Gln Thr Cys Gly Thr 35 40 45 Pro Ile Leu Asn Glu Ser Asp Lys Gln Ala Ile Ser Phe Asp Phe Val 50 55 60 Ala Pro Glu Leu Lys Gln Asp Glu Lys Tyr Cys Phe Thr Phe Lys Gly 65 70 75 80 Ile Thr Gly Asp His Arg Tyr Ile Thr Asn Thr Thr Leu Thr Val Val 85 90 95 Ala Pro Thr Leu Glu Val Tyr Ile Asp His Ala Ser Leu Pro Ser Leu 100 105 110 Gln Gln Leu Ile His Ile Ile Gln Ala Lys Asp Glu Tyr Pro Ser Asn 115 120 125 Gln Arg Phe Val Ser Trp Lys Arg Val Thr Val Asp Ala Asp Asn Ala 130 135 140 Asn Lys Leu Asn Ile His Thr Tyr Pro Leu Lys Gly Asn Asn Thr Ser 145 150 155 160 Pro Glu Met Val Ala Ala Ile Asp Glu Tyr Ala Gln Ser Lys Asn Arg 165 170 175 Leu Asn Ile Glu Phe Tyr Thr Asn Thr Ala His Val Phe Asn Asn Leu 180 185 190 Pro Pro Ile Ile Gln Pro Leu Tyr Asn Asn Glu Lys Val Lys Ile Ser 195 200 205 His Ile Ser Leu Tyr Asp Asp Gly Ser Ser Glu Tyr Val Ser Leu Tyr 210 215 220 Gln Trp Lys Asp Thr Pro Asn Lys Ile Glu Thr Leu Glu Gly Glu Val 225 230 235 240 Ser Leu Leu Ala Asn Tyr Leu Ala Gly Thr Ser Pro Asp Ala Pro Lys 245 250 255 Gly Met Gly Asn Arg Tyr Asn Trp His Lys Leu Tyr Asp Thr Asp Tyr 260 265 270 Tyr Phe Leu Arg Glu Asp Tyr Leu Asp Val Glu Ala Asn Leu His Asp 275 280 285 Leu Arg Asp Tyr Leu Gly Ser Ser Ala Lys Gln Met Pro Trp Asp Glu 290 295 300 Phe Ala Lys Leu Ser Asp Ser Gln Gln Thr Leu Phe Leu Asp Ile Val 305 310 315 320 Gly Phe Asp Lys Glu Gln Leu Gln Gln Gln Tyr Ser Gln Ser Pro Leu 325 330 335 Pro Asn Phe Ile Phe Thr Gly Thr Thr Thr Trp Ala Gly Gly Glu Thr 340 345 350 Lys Glu Tyr Tyr Ala Gln Gln Gln Val Asn Val Ile Asn Asn Ala Ile 355 360 365 Asn Glu Thr Ser Pro Tyr Tyr Leu Gly Lys Asp Tyr Asp Leu Phe Phe 370 375 380 Lys Gly His Pro Ala Gly Gly Val Ile Asn Asp Ile Ile Leu Gly Ser 385 390 395 400 Phe Pro Asp Met Ile Asn Ile Pro Ala Lys Thr Ser Phe Glu Val Leu 405 410 415 Met Met Thr Asp Met Leu Pro Asp Thr Val Ala Gly Ile Ala Ser Ser 420 425 430 Leu Tyr Phe Thr Ile Pro Ala Asp Lys Val Asn Phe Ile Val Phe Thr 435 440 445 Ser Ser Asp Thr Ile Thr Asp Arg Glu Glu Ala Leu Lys Ser Pro Leu 450 455 460 Val Gln Val Met Leu Thr Leu Gly Ile Val Lys Glu Lys Asp Val Leu 465 470 475 480 Phe Trp Ala <210> 12 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 12 Met Cys Asn Ser Asp Asn Thr Ser Leu Lys Glu Thr Val Ser Ser Asn 1 5 10 15 Ser Ala Asp Val Val Glu Thr Glu Thr Tyr Gln Leu Thr Pro Ile Asp 20 25 30 Ala Pro Ser Ser Phe Leu Ser His Ser Trp Glu Gln Thr Cys Gly Thr 35 40 45 Pro Ile Leu Asn Glu Ser Asp Lys Gln Ala Ile Ser Phe Asp Phe Val 50 55 60 Ala Pro Glu Leu Lys Gln Asp Glu Lys Tyr Cys Phe Thr Phe Lys Gly 65 70 75 80 Ile Thr Gly Asp His Arg Tyr Ile Thr Asn Thr Thr Leu Thr Val Val 85 90 95 Ala Pro Thr Leu Glu Val Tyr Ile Asp His Ala Ser Leu Pro Ser Leu 100 105 110 Gln Gln Leu Ile His Ile Ile Gln Ala Lys Asp Glu Tyr Pro Ser Asn 115 120 125 Gln Arg Phe Val Ser Trp Lys Arg Val Thr Val Asp Ala Asp Asn Ala 130 135 140 Asn Lys Leu Asn Ile His Thr Tyr Pro Leu Lys Gly Asn Asn Thr Ser 145 150 155 160 Pro Glu Met Val Ala Ala Ile Asp Glu Tyr Ala Gln Ser Lys Asn Arg 165 170 175 Leu Asn Ile Glu Phe Tyr Thr Asn Thr Ala His Val Phe Asn Asn Leu 180 185 190 Pro Pro Ile Ile Gln Pro Leu Tyr Asn Asn Glu Lys Val Lys Ile Ser 195 200 205 His Ile Ser Leu Tyr Asp Asp Gly Ser Ser Glu Tyr Val Ser Leu Tyr 210 215 220 Gln Trp Lys Asp Thr Pro Asn Lys Ile Glu Thr Leu Glu Gly Glu Val 225 230 235 240 Ser Leu Leu Ala Asn Tyr Leu Ala Gly Thr Ser Pro Asp Ala Pro Lys 245 250 255 Gly Met Gly Asn Arg Tyr Asn Trp His Lys Leu Tyr Asp Thr Asp Tyr 260 265 270 Tyr Phe Leu Arg Glu Asp Tyr Leu Asp Val Glu Ala Asn Leu His Asp 275 280 285 Leu Arg Asp Tyr Leu Gly Ser Ser Ala Lys Gln Met Pro Trp Asp Glu 290 295 300 Phe Ala Lys Leu Ser Asp Ser Gln Gln Thr Leu Phe Leu Asp Ile Val 305 310 315 320 Gly Phe Asp Lys Glu Gln Leu Gln Gln Gln Tyr Ser Gln Ser Pro Leu 325 330 335 Pro Asn Phe Ile Phe Thr Gly Thr Thr Thr Trp Ala Gly Gly Glu Thr 340 345 350 Lys Glu Tyr Tyr Ala Gln Gln Gln Val Asn Val Ile Asn Asn Ala Ile 355 360 365 Asn Glu Thr Ser Pro Tyr Tyr Leu Gly Lys Asp Tyr Asp Leu Phe Phe 370 375 380 Lys Gly His Pro Ala Gly Gly Val Ile Asn Asp Ile Ile Leu Gly Ser 385 390 395 400 Phe Pro Asp Met Ile Asn Ile Pro Ala Lys Ile Ser Phe Glu Val Leu 405 410 415 Met Met Thr Asp Met Leu Pro Asp Thr Val Ala Gly Ile Ala Ser Ser 420 425 430 Ser Tyr Phe Thr Ile Pro Ala Asp Lys Val Asn Phe Ile Val Phe Thr 435 440 445 Ser Ser Asp Thr Ile Thr Asp Arg Glu Glu Ala Leu Lys Ser Pro Leu 450 455 460 Val Gln Val Met Leu Thr Leu Gly Ile Val Lys Glu Lys Asp Val Leu 465 470 475 480 Phe Trp Ala <210> 13 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 13 Met Cys Asn Ser Asp Asn Thr Ser Leu Lys Glu Thr Val Ser Ser Asn 1 5 10 15 Ser Ala Asp Val Val Glu Thr Glu Thr Tyr Gln Leu Thr Pro Ile Asp 20 25 30 Ala Pro Ser Ser Phe Leu Ser His Ser Trp Glu Gln Thr Cys Gly Thr 35 40 45 Pro Ile Leu Asn Glu Ser Asp Lys Gln Ala Ile Ser Phe Asp Phe Val 50 55 60 Ala Pro Glu Leu Lys Gln Asp Glu Lys Tyr Cys Phe Thr Phe Lys Gly 65 70 75 80 Ile Thr Gly Asp His Arg Tyr Ile Thr Asn Thr Thr Leu Thr Val Val 85 90 95 Ala Pro Thr Leu Glu Val Tyr Ile Asp His Ala Ser Leu Pro Ser Leu 100 105 110 Gln Gln Leu Ile His Ile Ile Gln Ala Lys Asp Glu Tyr Pro Ser Asn 115 120 125 Gln Arg Phe Val Ser Trp Lys Arg Val Thr Val Asp Ala Asp Asn Ala 130 135 140 Asn Lys Leu Asn Ile His Thr Tyr Pro Leu Lys Gly Asn Asn Thr Ser 145 150 155 160 Pro Glu Met Val Ala Ala Ile Asp Glu Tyr Ala Gln Ser Lys Asn Arg 165 170 175 Leu Asn Ile Glu Phe Tyr Thr Asn Thr Ala His Val Phe Asn Asn Leu 180 185 190 Pro Pro Ile Ile Gln Pro Leu Tyr Asn Asn Glu Lys Val Lys Ile Ser 195 200 205 His Ile Ser Leu Tyr Asp Asp Gly Ser Ser Glu Tyr Val Ser Leu Tyr 210 215 220 Gln Trp Lys Asp Thr Pro Asn Lys Ile Glu Thr Leu Glu Gly Glu Val 225 230 235 240 Ser Leu Leu Ala Asn Tyr Leu Ala Gly Thr Ser Pro Asp Ala Pro Lys 245 250 255 Gly Met Gly Asn Arg Tyr Asn Trp His Lys Leu Tyr Asp Thr Asp Tyr 260 265 270 Tyr Phe Leu Arg Glu Asp Tyr Leu Asp Val Glu Ala Asn Leu His Asp 275 280 285 Leu Arg Asp Tyr Leu Gly Ser Ser Ala Lys Gln Met Pro Trp Asp Glu 290 295 300 Phe Ala Lys Leu Ser Asp Ser Gln Gln Thr Leu Phe Leu Asp Ile Val 305 310 315 320 Gly Phe Asp Lys Glu Gln Leu Gln Gln Gln Tyr Ser Gln Ser Pro Leu 325 330 335 Pro Asn Phe Ile Phe Thr Gly Thr Thr Thr Trp Ala Gly Gly Glu Thr 340 345 350 Lys Glu Tyr Tyr Ala Gln Gln Gln Val Asn Val Ile Asn Asn Ala Ile 355 360 365 Asn Glu Thr Ser Pro Tyr Tyr Leu Gly Lys Asp Tyr Asp Leu Phe Phe 370 375 380 Lys Gly His Pro Ala Gly Gly Val Ile Asn Asp Ile Ile Leu Gly Ser 385 390 395 400 Phe Pro Asp Met Ile Asn Ile Pro Ala Lys Ile Ser Phe Glu Val Leu 405 410 415 Met Met Thr Asp Met Leu Pro Asp Thr Val Ala Gly Ile Ala Ser Ser 420 425 430 Thr Tyr Phe Thr Ile Pro Ala Asp Lys Val Asn Phe Ile Val Phe Thr 435 440 445 Ser Ser Asp Thr Ile Thr Asp Arg Glu Glu Ala Leu Lys Ser Pro Leu 450 455 460 Val Gln Val Met Leu Thr Leu Gly Ile Val Lys Glu Lys Asp Val Leu 465 470 475 480 Phe Trp Ala <210> 14 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 14 Met Cys Asn Ser Asp Asn Thr Ser Leu Lys Glu Thr Val Ser Ser Asn 1 5 10 15 Ser Ala Asp Val Val Glu Thr Glu Thr Tyr Gln Leu Thr Pro Ile Asp 20 25 30 Ala Pro Ser Ser Phe Leu Ser His Ser Trp Glu Gln Thr Cys Gly Thr 35 40 45 Pro Ile Leu Asn Glu Ser Asp Lys Gln Ala Ile Ser Phe Asp Phe Val 50 55 60 Ala Pro Glu Leu Lys Gln Asp Glu Lys Tyr Cys Phe Thr Phe Lys Gly 65 70 75 80 Ile Thr Gly Asp His Arg Tyr Ile Thr Asn Thr Thr Leu Thr Val Val 85 90 95 Ala Pro Thr Leu Glu Val Tyr Ile Asp His Ala Ser Leu Pro Ser Leu 100 105 110 Gln Gln Leu Ile His Ile Ile Gln Ala Lys Asp Glu Tyr Pro Ser Asn 115 120 125 Gln Arg Phe Val Ser Trp Lys Arg Val Thr Val Asp Ala Asp Asn Ala 130 135 140 Asn Lys Leu Asn Ile His Thr Tyr Pro Leu Lys Gly Asn Asn Thr Ser 145 150 155 160 Pro Glu Met Val Ala Ala Ile Asp Glu Tyr Ala Gln Ser Lys Asn Arg 165 170 175 Leu Asn Ile Glu Phe Tyr Thr Asn Thr Ala His Val Phe Asn Asn Leu 180 185 190 Pro Pro Ile Ile Gln Pro Leu Tyr Asn Asn Glu Lys Val Lys Ile Ser 195 200 205 His Ile Ser Leu Tyr Asp Asp Gly Ser Ser Glu Tyr Val Ser Leu Tyr 210 215 220 Gln Trp Lys Asp Thr Pro Asn Lys Ile Glu Thr Leu Glu Gly Glu Val 225 230 235 240 Ser Leu Leu Ala Asn Tyr Leu Ala Gly Thr Ser Pro Asp Ala Pro Lys 245 250 255 Gly Met Gly Asn Arg Tyr Asn Trp His Lys Leu Tyr Asp Thr Asp Tyr 260 265 270 Tyr Phe Leu Arg Glu Asp Tyr Leu Asp Val Glu Ala Asn Leu His Asp 275 280 285 Leu Arg Asp Tyr Leu Gly Ser Ser Ala Lys Gln Met Pro Trp Asp Glu 290 295 300 Phe Ala Lys Leu Ser Asp Ser Gln Gln Thr Leu Phe Leu Asp Ile Val 305 310 315 320 Gly Phe Asp Lys Glu Gln Leu Gln Gln Gln Tyr Ser Gln Ser Pro Leu 325 330 335 Pro Asn Phe Ile Phe Thr Gly Thr Thr Thr Trp Ala Gly Gly Glu Thr 340 345 350 Lys Glu Tyr Tyr Ala Gln Gln Gln Val Asn Val Ile Asn Asn Ala Ile 355 360 365 Asn Glu Thr Ser Pro Tyr Tyr Leu Gly Lys Asp Tyr Asp Leu Phe Phe 370 375 380 Lys Gly His Pro Ala Gly Gly Val Ile Asn Asp Ile Ile Leu Gly Ser 385 390 395 400 Phe Pro Asp Met Ile Asn Ile Pro Ala Lys Thr Ser Phe Glu Val Leu 405 410 415 Met Met Thr Asp Met Leu Pro Asp Thr Val Ala Gly Ile Ala Ser Ser 420 425 430 Ser Tyr Phe Thr Ile Pro Ala Asp Lys Val Asn Phe Ile Val Phe Thr 435 440 445 Ser Ser Asp Thr Ile Thr Asp Arg Glu Glu Ala Leu Lys Ser Pro Leu 450 455 460 Val Gln Val Met Leu Thr Leu Gly Ile Val Lys Glu Lys Asp Val Leu 465 470 475 480 Phe Trp Ala <210> 15 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Artificial <400> 15 Met Cys Asn Ser Asp Asn Thr Ser Leu Lys Glu Thr Val Ser Ser Asn 1 5 10 15 Ser Ala Asp Val Val Glu Thr Glu Thr Tyr Gln Leu Thr Pro Ile Asp 20 25 30 Ala Pro Ser Ser Phe Leu Ser His Ser Trp Glu Gln Thr Cys Gly Thr 35 40 45 Pro Ile Leu Asn Glu Ser Asp Lys Gln Ala Ile Ser Phe Asp Phe Val 50 55 60 Ala Pro Glu Leu Lys Gln Asp Glu Lys Tyr Cys Phe Thr Phe Lys Gly 65 70 75 80 Ile Thr Gly Asp His Arg Tyr Ile Thr Asn Thr Thr Leu Thr Val Val 85 90 95 Ala Pro Thr Leu Glu Val Tyr Ile Asp His Ala Ser Leu Pro Ser Leu 100 105 110 Gln Gln Leu Ile His Ile Ile Gln Ala Lys Asp Glu Tyr Pro Ser Asn 115 120 125 Gln Arg Phe Val Ser Trp Lys Arg Val Thr Val Asp Ala Asp Asn Ala 130 135 140 Asn Lys Leu Asn Ile His Thr Tyr Pro Leu Lys Gly Asn Asn Thr Ser 145 150 155 160 Pro Glu Met Val Ala Ala Ile Asp Glu Tyr Ala Gln Ser Lys Asn Arg 165 170 175 Leu Asn Ile Glu Phe Tyr Thr Asn Thr Ala His Val Phe Asn Asn Leu 180 185 190 Pro Pro Ile Ile Gln Pro Leu Tyr Asn Asn Glu Lys Val Lys Ile Ser 195 200 205 His Ile Ser Leu Tyr Asp Asp Gly Ser Ser Glu Tyr Val Ser Leu Tyr 210 215 220 Gln Trp Lys Asp Thr Pro Asn Lys Ile Glu Thr Leu Glu Gly Glu Val 225 230 235 240 Ser Leu Leu Ala Asn Tyr Leu Ala Gly Thr Ser Pro Asp Ala Pro Lys 245 250 255 Gly Met Gly Asn Arg Tyr Asn Trp His Lys Leu Tyr Asp Thr Asp Tyr 260 265 270 Tyr Phe Leu Arg Glu Asp Tyr Leu Asp Val Glu Ala Asn Leu His Asp 275 280 285 Leu Arg Asp Tyr Leu Gly Ser Ser Ala Lys Gln Met Pro Trp Asp Glu 290 295 300 Phe Ala Lys Leu Ser Asp Ser Gln Gln Thr Leu Phe Leu Asp Ile Val 305 310 315 320 Gly Phe Asp Lys Glu Gln Leu Gln Gln Gln Tyr Ser Gln Ser Pro Leu 325 330 335 Pro Asn Phe Ile Phe Thr Gly Thr Thr Thr Trp Ala Gly Gly Glu Thr 340 345 350 Lys Glu Tyr Tyr Ala Gln Gln Gln Val Asn Val Ile Asn Asn Ala Ile 355 360 365 Asn Glu Thr Ser Pro Tyr Tyr Leu Gly Lys Asp Tyr Asp Leu Phe Phe 370 375 380 Lys Gly His Pro Ala Gly Gly Val Ile Asn Asp Ile Ile Leu Gly Ser 385 390 395 400 Phe Pro Asp Met Ile Asn Ile Pro Ala Lys Thr Ser Phe Glu Val Leu 405 410 415 Met Met Thr Asp Met Leu Pro Asp Thr Val Ala Gly Ile Ala Ser Ser 420 425 430 Thr Tyr Phe Thr Ile Pro Ala Asp Lys Val Asn Phe Ile Val Phe Thr 435 440 445 Ser Ser Asp Thr Ile Thr Asp Arg Glu Glu Ala Leu Lys Ser Pro Leu 450 455 460 Val Gln Val Met Leu Thr Leu Gly Ile Val Lys Glu Lys Asp Val Leu 465 470 475 480 Phe Trp Ala <210> 16 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 16 atgtgtaata gtgacaatac cagcttgaaa gaaacggtaa gctctaattc tgcagatgta 60 gtagaaacag aaacttacca actgacaccg attgatgctc ctagctcttt tttatctcat 120 tcttgggagc aaacatgtgg cacacctatc ttgaatgaaa gtgacaagca agcgatatct 180 tttgattttg ttgctccaga gttaaagcaa gatgaaaagt attgttttac ttttaaaggt 240 attacaggcg atcataggta tatcacaaat acaacattaa ctgttgttgc acctacgcta 300 gaagtttaca tcgatcatgc atccttacca tcgctacagc agcttatcca cattattcaa 360 gcaaaagatg aatacccaag taatcaacgt tttgtctctt ggaagcgtgt aactgttgat 420 gctgataatg ccaataagtt aaacattcat acttatccat taaaaggcaa taatacctca 480 ccagaaatgg tggcagcgat tgatgagtat gctcagagca aaaatcgatt gaatatagag 540 ttctatacaa atacagctca tgtttttaat aatttaccac ctattattca acctttatat 600 aataacgaga aggtgaaaat ttctcatatt agtttgtatg atgatggttc ttctgaatat 660 gtaagtttat atcaatggaa agatacacca aataagatag aaacattaga aggtgaagta 720 tcgcttcttg ctaattattt agcaggaaca tctccggatg caccaaaagg aatgggaaat 780 cgttataact ggcataaatt atatgacact gattattact ttttgcgcga agattacctt 840 gacgttgaag caaacctaca tgatttacgt gattatttag gctcttccgc aaagcaaatg 900 ccatgggatg aatttgctaa attatctgat tctcagcaaa cactattttt agatattgtg 960 ggttttgata aagagcaatt gcaacaacaa tattcacaat ccccactacc aaactttatt 1020 tttaccggca caacaacttg ggctgggggg gaaacgaaag agtattatgc tcagcaacaa 1080 gtaaatgtga ttaataatgc gatcaatgaa actagccctt attatttagg taaagactac 1140 gatctatttt tcaaggggca tcctgctggt ggcgttatta acgacatcat tcttggaagc 1200 ttccctgata tgatcaatat tccagccaag acttcatttg aggtcttgat gatgacggat 1260 atgttgcctg atacagtagc tggtattgcg agctctctgt acttcacaat tcctgccgat 1320 aaagttaatt ttattgtatt tacttcatct gacactatta ctgatcgtga agaggctctt 1380 aaatcaccat tagtacaagt gatgctaacg ttgggtattg ttaaagaaaa agatgttctg 1440 ttctgggct 1449 <210> 17 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 17 atgtgtaata gtgacaatac cagcttgaaa gaaacggtaa gctctaattc tgcagatgta 60 gtagaaacag aaacttacca actgacaccg attgatgctc ctagctcttt tttatctcat 120 tcttgggagc aaacatgtgg cacacctatc ttgaatgaaa gtgacaagca agcgatatct 180 tttgattttg ttgctccaga gttaaagcaa gatgaaaagt attgttttac ttttaaaggt 240 attacaggcg atcataggta tatcacaaat acaacattaa ctgttgttgc acctacgcta 300 gaagtttaca tcgatcatgc atccttacca tcgctacagc agcttatcca cattattcaa 360 gcaaaagatg aatacccaag taatcaacgt tttgtctctt ggaagcgtgt aactgttgat 420 gctgataatg ccaataagtt aaacattcat acttatccat taaaaggcaa taatacctca 480 ccagaaatgg tggcagcgat tgatgagtat gctcagagca aaaatcgatt gaatatagag 540 ttctatacaa atacagctca tgtttttaat aatttaccac ctattattca acctttatat 600 aataacgaga aggtgaaaat ttctcatatt agtttgtatg atgatggttc ttctgaatat 660 gtaagtttat atcaatggaa agatacacca aataagatag aaacattaga aggtgaagta 720 tcgcttcttg ctaattattt agcaggaaca tctccggatg caccaaaagg aatgggaaat 780 cgttataact ggcataaatt atatgacact gattattact ttttgcgcga agattacctt 840 gacgttgaag caaacctaca tgatttacgt gattatttag gctcttccgc aaagcaaatg 900 ccatgggatg aatttgctaa attatctgat tctcagcaaa cactattttt agatattgtg 960 ggttttgata aagagcaatt gcaacaacaa tattcacaat ccccactacc aaactttatt 1020 tttaccggca caacaacttg ggctgggggg gaaacgaaag agtattatgc tcagcaacaa 1080 gtaaatgtga ttaataatgc gatcaatgaa actagccctt attatttagg taaagactac 1140 gatctatttt tcaaggggca tcctgctggt ggcgttatta acgacatcat tcttggaagc 1200 ttccctgata tgatcaatat tccagccaag atttcatttg aggtcttgat gatgacggat 1260 atgttgcctg atacagtagc tggtattgcg agctctagtt acttcacaat tcctgccgat 1320 aaagttaatt ttattgtatt tacttcatct gacactatta ctgatcgtga agaggctctt 1380 aaatcaccat tagtacaagt gatgctaacg ttgggtattg ttaaagaaaa agatgttctg 1440 ttctgggct 1449 <210> 18 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 18 atgtgtaata gtgacaatac cagcttgaaa gaaacggtaa gctctaattc tgcagatgta 60 gtagaaacag aaacttacca actgacaccg attgatgctc ctagctcttt tttatctcat 120 tcttgggagc aaacatgtgg cacacctatc ttgaatgaaa gtgacaagca agcgatatct 180 tttgattttg ttgctccaga gttaaagcaa gatgaaaagt attgttttac ttttaaaggt 240 attacaggcg atcataggta tatcacaaat acaacattaa ctgttgttgc acctacgcta 300 gaagtttaca tcgatcatgc atccttacca tcgctacagc agcttatcca cattattcaa 360 gcaaaagatg aatacccaag taatcaacgt tttgtctctt ggaagcgtgt aactgttgat 420 gctgataatg ccaataagtt aaacattcat acttatccat taaaaggcaa taatacctca 480 ccagaaatgg tggcagcgat tgatgagtat gctcagagca aaaatcgatt gaatatagag 540 ttctatacaa atacagctca tgtttttaat aatttaccac ctattattca acctttatat 600 aataacgaga aggtgaaaat ttctcatatt agtttgtatg atgatggttc ttctgaatat 660 gtaagtttat atcaatggaa agatacacca aataagatag aaacattaga aggtgaagta 720 tcgcttcttg ctaattattt agcaggaaca tctccggatg caccaaaagg aatgggaaat 780 cgttataact ggcataaatt atatgacact gattattact ttttgcgcga agattacctt 840 gacgttgaag caaacctaca tgatttacgt gattatttag gctcttccgc aaagcaaatg 900 ccatgggatg aatttgctaa attatctgat tctcagcaaa cactattttt agatattgtg 960 ggttttgata aagagcaatt gcaacaacaa tattcacaat ccccactacc aaactttatt 1020 tttaccggca caacaacttg ggctgggggg gaaacgaaag agtattatgc tcagcaacaa 1080 gtaaatgtga ttaataatgc gatcaatgaa actagccctt attatttagg taaagactac 1140 gatctatttt tcaaggggca tcctgctggt ggcgttatta acgacatcat tcttggaagc 1200 ttccctgata tgatcaatat tccagccaag atttcatttg aggtcttgat gatgacggat 1260 atgttgcctg atacagtagc tggtattgcg agctctacgt acttcacaat tcctgccgat 1320 aaagttaatt ttattgtatt tacttcatct gacactatta ctgatcgtga agaggctctt 1380 aaatcaccat tagtacaagt gatgctaacg ttgggtattg ttaaagaaaa agatgttctg 1440 ttctgggct 1449 <210> 19 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 19 atgtgtaata gtgacaatac cagcttgaaa gaaacggtaa gctctaattc tgcagatgta 60 gtagaaacag aaacttacca actgacaccg attgatgctc ctagctcttt tttatctcat 120 tcttgggagc aaacatgtgg cacacctatc ttgaatgaaa gtgacaagca agcgatatct 180 tttgattttg ttgctccaga gttaaagcaa gatgaaaagt attgttttac ttttaaaggt 240 attacaggcg atcataggta tatcacaaat acaacattaa ctgttgttgc acctacgcta 300 gaagtttaca tcgatcatgc atccttacca tcgctacagc agcttatcca cattattcaa 360 gcaaaagatg aatacccaag taatcaacgt tttgtctctt ggaagcgtgt aactgttgat 420 gctgataatg ccaataagtt aaacattcat acttatccat taaaaggcaa taatacctca 480 ccagaaatgg tggcagcgat tgatgagtat gctcagagca aaaatcgatt gaatatagag 540 ttctatacaa atacagctca tgtttttaat aatttaccac ctattattca acctttatat 600 aataacgaga aggtgaaaat ttctcatatt agtttgtatg atgatggttc ttctgaatat 660 gtaagtttat atcaatggaa agatacacca aataagatag aaacattaga aggtgaagta 720 tcgcttcttg ctaattattt agcaggaaca tctccggatg caccaaaagg aatgggaaat 780 cgttataact ggcataaatt atatgacact gattattact ttttgcgcga agattacctt 840 gacgttgaag caaacctaca tgatttacgt gattatttag gctcttccgc aaagcaaatg 900 ccatgggatg aatttgctaa attatctgat tctcagcaaa cactattttt agatattgtg 960 ggttttgata aagagcaatt gcaacaacaa tattcacaat ccccactacc aaactttatt 1020 tttaccggca caacaacttg ggctgggggg gaaacgaaag agtattatgc tcagcaacaa 1080 gtaaatgtga ttaataatgc gatcaatgaa actagccctt attatttagg taaagactac 1140 gatctatttt tcaaggggca tcctgctggt ggcgttatta acgacatcat tcttggaagc 1200 ttccctgata tgatcaatat tccagccaag acttcatttg aggtcttgat gatgacggat 1260 atgttgcctg atacagtagc tggtattgcg agctctagtt acttcacaat tcctgccgat 1320 aaagttaatt ttattgtatt tacttcatct gacactatta ctgatcgtga agaggctctt 1380 aaatcaccat tagtacaagt gatgctaacg ttgggtattg ttaaagaaaa agatgttctg 1440 ttctgggct 1449 <210> 20 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Artificial <400> 20 atgtgtaata gtgacaatac cagcttgaaa gaaacggtaa gctctaattc tgcagatgta 60 gtagaaacag aaacttacca actgacaccg attgatgctc ctagctcttt tttatctcat 120 tcttgggagc aaacatgtgg cacacctatc ttgaatgaaa gtgacaagca agcgatatct 180 tttgattttg ttgctccaga gttaaagcaa gatgaaaagt attgttttac ttttaaaggt 240 attacaggcg atcataggta tatcacaaat acaacattaa ctgttgttgc acctacgcta 300 gaagtttaca tcgatcatgc atccttacca tcgctacagc agcttatcca cattattcaa 360 gcaaaagatg aatacccaag taatcaacgt tttgtctctt ggaagcgtgt aactgttgat 420 gctgataatg ccaataagtt aaacattcat acttatccat taaaaggcaa taatacctca 480 ccagaaatgg tggcagcgat tgatgagtat gctcagagca aaaatcgatt gaatatagag 540 ttctatacaa atacagctca tgtttttaat aatttaccac ctattattca acctttatat 600 aataacgaga aggtgaaaat ttctcatatt agtttgtatg atgatggttc ttctgaatat 660 gtaagtttat atcaatggaa agatacacca aataagatag aaacattaga aggtgaagta 720 tcgcttcttg ctaattattt agcaggaaca tctccggatg caccaaaagg aatgggaaat 780 cgttataact ggcataaatt atatgacact gattattact ttttgcgcga agattacctt 840 gacgttgaag caaacctaca tgatttacgt gattatttag gctcttccgc aaagcaaatg 900 ccatgggatg aatttgctaa attatctgat tctcagcaaa cactattttt agatattgtg 960 ggttttgata aagagcaatt gcaacaacaa tattcacaat ccccactacc aaactttatt 1020 tttaccggca caacaacttg ggctgggggg gaaacgaaag agtattatgc tcagcaacaa 1080 gtaaatgtga ttaataatgc gatcaatgaa actagccctt attatttagg taaagactac 1140 gatctatttt tcaaggggca tcctgctggt ggcgttatta acgacatcat tcttggaagc 1200 ttccctgata tgatcaatat tccagccaag acttcatttg aggtcttgat gatgacggat 1260 atgttgcctg atacagtagc tggtattgcg agctctacct acttcacaat tcctgccgat 1320 aaagttaatt ttattgtatt tacttcatct gacactatta ctgatcgtga agaggctctt 1380 aaatcaccat tagtacaagt gatgctaacg ttgggtattg ttaaagaaaa agatgttctg 1440 ttctgggct 1449 <210> 21 <211> 391 <212> PRT <213> Pasteurella multocida <400> 21 Met Lys Thr Ile Thr Leu Tyr Leu Asp Pro Ala Ser Leu Pro Ala Leu 1 5 10 15 Asn Gln Leu Met Asp Phe Thr Gln Asn Asn Glu Asp Lys Thr His Pro 20 25 30 Arg Ile Phe Gly Leu Ser Arg Phe Lys Ile Pro Asp Asn Ile Ile Thr 35 40 45 Gln Tyr Gln Asn Ile His Phe Val Glu Leu Lys Asp Asn Arg Pro Thr 50 55 60 Glu Ala Leu Phe Thr Ile Leu Asp Gln Tyr Pro Gly Asn Ile Glu Leu 65 70 75 80 Asn Ile His Leu Asn Ile Ala His Ser Val Gln Leu Ile Arg Pro Ile 85 90 95 Leu Ala Tyr Arg Phe Lys His Leu Asp Arg Val Ser Ile Gln Gln Leu 100 105 110 Asn Leu Tyr Asp Asp Gly Ser Met Glu Tyr Val Asp Leu Glu Lys Glu 115 120 125 Glu Asn Lys Asp Ile Ser Ala Glu Ile Lys Gln Ala Glu Lys Gln Leu 130 135 140 Ser His Tyr Leu Leu Thr Gly Lys Ile Lys Phe Asp Asn Pro Thr Ile 145 150 155 160 Ala Arg Tyr Val Trp Gln Ser Ala Phe Pro Val Lys Tyr His Phe Leu 165 170 175 Ser Thr Asp Tyr Phe Glu Lys Ala Glu Phe Leu Gln Pro Leu Lys Glu 180 185 190 Tyr Leu Ala Glu Asn Tyr Gln Lys Met Asp Trp Thr Ala Tyr Gln Gln 195 200 205 Leu Thr Pro Glu Gln Gln Ala Phe Tyr Leu Thr Leu Val Gly Phe Asn 210 215 220 Asp Glu Val Lys Gln Ser Leu Glu Val Gln Gln Ala Lys Phe Ile Phe 225 230 235 240 Thr Gly Thr Thr Thr Trp Glu Gly Asn Thr Asp Val Arg Glu Tyr Tyr 245 250 255 Ala Gln Gln Gln Leu Asn Leu Leu Asn His Phe Thr Gln Ala Glu Gly 260 265 270 Asp Leu Phe Ile Gly Asp His Tyr Lys Ile Tyr Phe Lys Gly His Pro 275 280 285 Arg Gly Gly Glu Ile Asn Asp Tyr Ile Leu Asn Asn Ala Lys Asn Ile 290 295 300 Thr Asn Ile Pro Ala Asn Ile Ser Phe Glu Val Leu Met Met Thr Gly 305 310 315 320 Leu Leu Pro Asp Lys Val Gly Gly Val Ala Ser Ser Leu Tyr Phe Ser 325 330 335 Leu Pro Lys Glu Lys Ile Ser His Ile Ile Phe Thr Ser Asn Lys Gln 340 345 350 Val Lys Ser Lys Glu Asp Ala Leu Asn Asn Pro Tyr Val Lys Val Met 355 360 365 Arg Arg Leu Gly Ile Ile Asp Glu Ser Gln Val Ile Phe Trp Asp Ser 370 375 380 Leu Lys Gln Leu Gly Gly Gly 385 390 <210> 22 <211> 1173 <212> DNA <213> Pasteurella multocida <400> 22 atgaaaacaa tcacgctgta tttagatcct gcctccttac cggcattaaa tcagctgatg 60 gactttacgc aaaataatga agataaaaca catccacgta tttttggtct ttctcgcttt 120 aaaatccctg acaacattat tacacagtat caaaatatcc atttcgtcga actcaaagat 180 aatcgtccca ctgaagcact ttttacgatt ttagatcaat accctggtaa cattgagtta 240 aatatacact taaatattgc tcattccgtt caattaattc gtccgatttt ggcatatcgt 300 tttaaacatt tagatcgtgt atcaattcag cagttaaatc tttatgacga tggctcaatg 360 gaatatgttg atttagaaaa agaagaaaat aaagatattt ccgcagaaat taagcaagca 420 gaaaaacaac tttctcacta tttgcttact ggcaaaataa aatttgataa cccaactatt 480 gctcgttatg tctggcaatc cgcgttccca gtaaaatatc attttttaag tacagactat 540 tttgaaaaag ccgaattttt acaaccacta aaagaatatt tagcagaaaa ttatcaaaaa 600 atggactgga ctgcttacca acagctgact ccagaacagc aagcattcta cttaacattg 660 gtaggcttca atgacgaagt caagcagtcg ctagaagtgc aacaagctaa atttatcttt 720 accggcacga caacttggga aggaaatacc gatgtgcgag aatactacgc acagcaacaa 780 cttaatttac ttaatcactt tacccaagct gagggcgatt tatttattgg tgatcattat 840 aaaatctact ttaaagggca tcctagaggt ggtgaaatta atgactacat tctgaacaat 900 gctaaaaata tcaccaatat ccctgccaat atttcctttg aagtattgat gatgacaggc 960 ttattacctg ataaagtggg tggtgttgca agttcactgt atttctcctt accaaaagaa 1020 aaaattagcc atattatttt cacatcgaat aaacaagtga aaagcaaaga agatgcgcta 1080 aataatccgt atgttaaggt catgcgtcgt ttaggtataa ttgacgaatc acaagtcatc 1140 ttttgggaca gtttaaaaca gttgggtgga ggt 1173 <210> 23 <211> 483 <212> PRT <213> Photobacterium damselae <400> 23 Met Cys Asn Ser Asp Asn Thr Ser Leu Lys Glu Thr Val Ser Ser Asn 1 5 10 15 Ser Ala Asp Val Val Glu Thr Glu Thr Tyr Gln Leu Thr Pro Ile Asp 20 25 30 Ala Pro Ser Ser Phe Leu Ser His Ser Trp Glu Gln Thr Cys Gly Thr 35 40 45 Pro Ile Leu Asn Glu Ser Asp Lys Gln Ala Ile Ser Phe Asp Phe Val 50 55 60 Ala Pro Glu Leu Lys Gln Asp Glu Lys Tyr Cys Phe Thr Phe Lys Gly 65 70 75 80 Ile Thr Gly Asp His Arg Tyr Ile Thr Asn Thr Thr Leu Thr Val Val 85 90 95 Ala Pro Thr Leu Glu Val Tyr Ile Asp His Ala Ser Leu Pro Ser Leu 100 105 110 Gln Gln Leu Ile His Ile Ile Gln Ala Lys Asp Glu Tyr Pro Ser Asn 115 120 125 Gln Arg Phe Val Ser Trp Lys Arg Val Thr Val Asp Ala Asp Asn Ala 130 135 140 Asn Lys Leu Asn Ile His Thr Tyr Pro Leu Lys Gly Asn Asn Thr Ser 145 150 155 160 Pro Glu Met Val Ala Ala Ile Asp Glu Tyr Ala Gln Ser Lys Asn Arg 165 170 175 Leu Asn Ile Glu Phe Tyr Thr Asn Thr Ala His Val Phe Asn Asn Leu 180 185 190 Pro Pro Ile Ile Gln Pro Leu Tyr Asn Asn Glu Lys Val Lys Ile Ser 195 200 205 His Ile Ser Leu Tyr Asp Asp Gly Ser Ser Glu Tyr Val Ser Leu Tyr 210 215 220 Gln Trp Lys Asp Thr Pro Asn Lys Ile Glu Thr Leu Glu Gly Glu Val 225 230 235 240 Ser Leu Leu Ala Asn Tyr Leu Ala Gly Thr Ser Pro Asp Ala Pro Lys 245 250 255 Gly Met Gly Asn Arg Tyr Asn Trp His Lys Leu Tyr Asp Thr Asp Tyr 260 265 270 Tyr Phe Leu Arg Glu Asp Tyr Leu Asp Val Glu Ala Asn Leu His Asp 275 280 285 Leu Arg Asp Tyr Leu Gly Ser Ser Ala Lys Gln Met Pro Trp Asp Glu 290 295 300 Phe Ala Lys Leu Ser Asp Ser Gln Gln Thr Leu Phe Leu Asp Ile Val 305 310 315 320 Gly Phe Asp Lys Glu Gln Leu Gln Gln Gln Tyr Ser Gln Ser Pro Leu 325 330 335 Pro Asn Phe Ile Phe Thr Gly Thr Thr Thr Trp Ala Gly Gly Glu Thr 340 345 350 Lys Glu Tyr Tyr Ala Gln Gln Gln Val Asn Val Ile Asn Asn Ala Ile 355 360 365 Asn Glu Thr Ser Pro Tyr Tyr Leu Gly Lys Asp Tyr Asp Leu Phe Phe 370 375 380 Lys Gly His Pro Ala Gly Gly Val Ile Asn Asp Ile Ile Leu Gly Ser 385 390 395 400 Phe Pro Asp Met Ile Asn Ile Pro Ala Lys Ile Ser Phe Glu Val Leu 405 410 415 Met Met Thr Asp Met Leu Pro Asp Thr Val Ala Gly Ile Ala Ser Ser 420 425 430 Leu Tyr Phe Thr Ile Pro Ala Asp Lys Val Asn Phe Ile Val Phe Thr 435 440 445 Ser Ser Asp Thr Ile Thr Asp Arg Glu Glu Ala Leu Lys Ser Pro Leu 450 455 460 Val Gln Val Met Leu Thr Leu Gly Ile Val Lys Glu Lys Asp Val Leu 465 470 475 480 Phe Trp Ala <210> 24 <211> 1449 <212> DNA <213> Photobacterium damselae <400> 24 atgtgtaata gtgacaatac cagcttgaaa gaaacggtaa gctctaattc tgcagatgta 60 gtagaaacag aaacttacca actgacaccg attgatgctc ctagctcttt tttatctcat 120 tcttgggagc aaacatgtgg cacacctatc ttgaatgaaa gtgacaagca agcgatatct 180 tttgattttg ttgctccaga gttaaagcaa gatgaaaagt attgttttac ttttaaaggt 240 attacaggcg atcataggta tatcacaaat acaacattaa ctgttgttgc acctacgcta 300 gaagtttaca tcgatcatgc atccttacca tcgctacagc agcttatcca cattattcaa 360 gcaaaagatg aatacccaag taatcaacgt tttgtctctt ggaagcgtgt aactgttgat 420 gctgataatg ccaataagtt aaacattcat acttatccat taaaaggcaa taatacctca 480 ccagaaatgg tggcagcgat tgatgagtat gctcagagca aaaatcgatt gaatatagag 540 ttctatacaa atacagctca tgtttttaat aatttaccac ctattattca acctttatat 600 aataacgaga aggtgaaaat ttctcatatt agtttgtatg atgatggttc ttctgaatat 660 gtaagtttat atcaatggaa agatacacca aataagatag aaacattaga aggtgaagta 720 tcgcttcttg ctaattattt agcaggaaca tctccggatg caccaaaagg aatgggaaat 780 cgttataact ggcataaatt atatgacact gattattact ttttgcgcga agattacctt 840 gacgttgaag caaacctaca tgatttacgt gattatttag gctcttccgc aaagcaaatg 900 ccatgggatg aatttgctaa attatctgat tctcagcaaa cactattttt agatattgtg 960 ggttttgata aagagcaatt gcaacaacaa tattcacaat ccccactacc aaactttatt 1020 tttaccggca caacaacttg ggctgggggg gaaacgaaag agtattatgc tcagcaacaa 1080 gtaaatgtga ttaataatgc gatcaatgaa actagccctt attatttagg taaagactac 1140 gatctatttt tcaaggggca tcctgctggt ggcgttatta acgacatcat tcttggaagc 1200 ttccctgata tgatcaatat tccagccaag atttcatttg aggtcttgat gatgacggat 1260 atgttgcctg atacagtagc tggtattgcg agctctctgt acttcacaat tcctgccgat 1320 aaagttaatt ttattgtatt tacttcatct gacactatta ctgatcgtga agaggctctt 1380 aaatcaccat tagtacaagt gatgctaacg ttgggtattg ttaaagaaaa agatgttctg 1440 ttctgggct 1449

Claims (9)

An α-2,6-sialyltransferase mutant represented by any one of sequences selected from the following (a) to (f):
(a) the amino acid sequence of SEQ ID NO: 11;
(b) an amino acid sequence of SEQ ID NO: 12;
(c) the amino acid sequence of SEQ ID NO: 13;
(d) an amino acid sequence of SEQ ID NO: 14;
(e) the amino acid sequence of SEQ ID NO: 15; And
(f) a sequence in which the 411th amino acid in the multiple sequence alignment is substituted with threonine in the amino acid sequence of SEQ ID NO: 11 to 15, or the 433th amino acid in the multiple sequence alignment is substituted with serine or threonine. A DNA encoding an alpha-2,6-sialyltransferase mutant according to claim 1. 3. The method of claim 2,
A DNA consisting of any one of the sequences of SEQ ID NOS: 16 to 20; 4. A recombinant DNA vector comprising the DNA according to claim 2 or 3. A host cell transformed with a recombinant DNA vector according to claim 4. An extract of a host cell transformed with the recombinant DNA vector according to claim 4. A method for producing 6'-sialyl-oligosaccharides using a host cell transformed with the recombinant DNA vector according to claim 4 and an extract of said host cell as a biocatalyst. A method for searching for an? -2,6-sialyltransferase variant according to claim 1, comprising the steps of:
Analyzing a functional moiety that will perform a saturation mutation through multiple sequence alignment and alanine scanning, in a selected moiety within 5-20 Angstroms of the two substrate binding sites of the crystal structure or model structure of the sialyltransferase. 9. The method according to claim 8, wherein an alanine scanning and a saturation mutant search are performed using an indicator at a temperature of 20 to 70 DEG C and a pH change at a pH of 6 to 10.

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