KR20110128167A - An alpha-xylosidase mutants modified at their proton-donor/acceptor catalyst and high efficiency transglycosylation with the same - Google Patents

Abstract

PURPOSE: An alpha-xylosidase mutant of proton-donor/acceptor catalyst is provided to obtain transflycosylation products with high yield. CONSTITUTION: An alpha-xylosidase mutant is prepared by substituting 482th amino acid residue, aspartate with serine. The alpha-xylosidase mutant is prepared by mutating by site mutation, random mutation, and selection among natural microorganism genomes. The alpha-xylosidase mutant is prepared using a recombinant microorganism. A transglycosylation is performed using the alpha-xylosidase mutant as a translycosylation catalyst.

Description

{An alpha-Xylosidase mutants modified at their proton-donor/acceptor catalyst and high efficiency transglycosylation with the same}

The present invention relates to a proton donor/receptor catalyzed variant enzyme of alpha-xyloseidase, a method for preparing the enzyme, and an application of the enzyme.

Carbohydrates that mediate various physiological responses in vivo are recognized as disease prevention and treatment. These carbohydrates have been achieved through organic synthesis so far, but the method of synthesizing oligosaccharides using enzymes has recently attracted much attention because of the advantages of reaction conditions and substrate specificity. Oligosaccharide synthesis technology using enzymes includes a method of using a glycosyltransferase that uses a glycosyltransferase that uses a nucleotide sugar as a substrate or a glycosyltransferase of a carbohydrate-degrading enzyme.

In general, sugar transfer enzymes are enzymes involved in the biosynthesis of sugar chains such as glycoproteins and glycolipids in a living body. It has been found that sugar chains such as glycoproteins and glycolipids, which are reaction products, are important molecules that function as a tack of complex saccharides or signal transduction between cells and between cells and extracellular matrix during differentiation and development. .

Among the glycosidases, the retaining glycosidase uses two amino acid residues having a carboxyl group as a nucleophile catalyst and a proton-donor/acceptor catalyst, respectively. To catalyze the sugar chain hydrolysis reaction. The persistent carbohydrate degrading enzymes degrade sugar chain bonds in the substrate using the nucleophilic catalytic group of each enzyme, and sugar-enzymes in which a nucleophilic catalyst and a part of the substrate are covalently bonded through an anomer bond opposite to the original substrate. It produces an intermediate. After that, the anomer of the reaction product is originally through a hydrolysis reaction in which the sugar in the sugar-enzyme intermediate is transferred to water by the action of the proton donation/receptor catalytic group, or a sugar transfer reaction in which the sugar in the sugar acceptor other than water is transferred to the hydroxyl group. The reaction is completed as it becomes the same as that of the substrate (Acc Chem Res, 2000, 33(1):11-18).

* However, in the case of the sugar transfer reaction using the persistent carbohydrate degrading enzyme, the resulting sugar transfer product is hydrolyzed again by the enzyme, so the reaction must be controlled over time, and the yield of the obtained sugar transfer product is generally low. In addition, sugar transfer is possible with various hydroxyl groups possessed by the sugar receptor, and the generated sugar transfer product can be used as a sugar receptor again, so that more than one sugar transfer product is produced.Therefore, the separation and purification process of the desired substance is additionally required. There is a problem that increases. In order to solve this problem, protein engineering technology using genetic engineering technology has been applied.

As a first example, if the nucleophilic catalytic group of a persistent carbohydrate degrading enzyme is mutated into an inactive amino acid residue, a sugar-enzyme intermediate cannot be formed and thus hydrolytic activity is not exhibited. However, if an analog of a sugar-enzyme intermediate is provided using fluoride sugar in the form of regioisomer opposite to the original substrate, the sugar transfer reaction proceeds successfully due to the reaction of the proton donor catalytic group remaining in the enzyme. do. At this time, the carbohydrate degrading enzyme mutants having mutated nucleophilic catalytic groups have lost their hydrolytic activity, so they cannot hydrolyze the sugar transfer product and accumulate the product in the reaction solution. In this way, a processing enzyme that performs only sugar transfer reactions by mutating the nucleophilic catalytic group of the persistent carbohydrate degrading enzyme by site-directed mutagenesis and using the fluorine sugar in the form of the regioisomer opposite to the original substrate is used as a sugar donor. It is named glycosynthase (J Am Chem Soc 1998, 120:5583-5584). Since the first reported glycosynthase in which the 358 th glutamate residue (Glu358), which is the nucleophilic catalyst group of beta-glycosidase derived from Agrobacterium sp., is transformed into alanine was reported for the first time. The same strategy has been applied to various enzymes (Curr Opin Chem Biol. 2006, 10(5):509-519). However, most of the existing glycosidases are enzymes that synthesize only beta-sugar chains derived from beta-glycosidase, and in the case of alpha-glycosidase capable of synthesizing alpha-sugar chains, Schizosaccharomyces pombe alpha-glycerides An enzyme that mutated the nucleophilic catalytic group of lycosidase is the only example (Biosci Biotechnol Biochem. 2002, 66(4):928-933). However, the alpha-glycosidase has a difficulty in using a beta-anomeric fluorosaccharide as a sugar donor, which is difficult to synthesize and has poor stability compared to the alpha-anomeric fluorosaccharide.

The second method is to increase the yield of sugar transfer products by developing a mutant that retains nucleophilicity and proton donation/receptor catalytic groups through directed evolution, but the hydrolytic activity is significantly reduced and the sugar transfer activity is maintained or improved. Studies have been reported (J Biol Chem. 2005, 280(44):37088-37097). However, in this case, the hydrolysis activity of the sugar transfer product was not completely removed, and no application examples for alpha-glycosidase have been reported.

Alpha-xylosidase belongs to the persistent carbohydrate degrading enzyme that produces a product having an alpha-anomer by decomposing a-glycosidic linkage of reactive substances. Therefore, the production of various glycosyltransferases has been reported using the glycotransferase activity of alpha-xylosidase (FEBS J. 2007, 274(23):6074-6084). However, it does not overcome the disadvantages of the sugar transfer reaction using the persistent carbohydrate degrading enzyme and has a problem of generating two or more by-products with a low yield.

The present invention solves the above problems and is conceived by the necessity of the above, and an object of the present invention is to mutate the proton donation/receptor catalytic group of alpha-xylosidase, and to perform the sugar transfer reaction in high yield. It is to provide a rosidase mutant enzyme.

Another object of the present invention is to provide a method for producing the alpha-xylosidase mutant enzyme.

Another object of the present invention is to provide an analogue in which a-xylosyl fluoride and glucose, mannose, and their 2-hydroxyl groups are substituted with other reactive groups at the non-reducing end. Eggplant provides a method of preparing a glycotransfer product to which xylose has been transferred by reacting the alpha-xylosidase mutant enzyme with a substrate containing oligosaccharides and glycosides.

In order to achieve the above object, the present invention provides an alpha-xylosidase mutant enzyme polypeptide in which the proton donation/receptor catalyst of natural alpha-xyloseidase is mutated to an amino acid residue other than aspartate.

The term "polypeptide" as described herein refers to the full length of a polypeptide according to the invention. In a preferred embodiment, the term “polypeptide” includes isolated polypeptides and polypeptides prepared by recombinant methods, such as by isolating and purifying from a sample, by screening a library, and by protein synthesis by conventional methods. And, all of the above-described methods are known to those skilled in the art. Preferably, the entire polypeptide or a part thereof can be synthesized by a conventional synthetic method such as the Merrifield method.

The natural alpha-xylosidase polypeptide sequence of the present invention refers to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or 2.

"Functional variant of a native alpha-xyloseidase or alpha-xyloseidase mutant enzyme" of the polypeptide described in the specification of the present invention refers to a polypeptide having an amino acid sequence according to SEQ ID NO: 1 or 2 and/or SEQ ID NO: 3 or 4. It refers to a polypeptide having sequence homology with respect to, in particular about 70%, preferably about 80%, in particular about 90%, particularly about 95%, most preferably about 98%. Such functional variants are polypeptides that are homologous to the polypeptide according to the invention, for example originating in non-human organisms, preferably originating from non-human mammals such as mice, rats, monkeys and pigs. Other examples of functional variants are polypeptides encoded by different alleles of genes in different individuals, different organs of an organism, or at different stages of development. Functional variants preferably also comprise naturally occurring or synthetic mutations, in particular mutations that significantly alter the activity of the peptides encoded by these sequences. In addition, such variants may preferably arise from different junctions of the encoding gene.

“Functional variant” refers to a polypeptide having the same biological action as a corresponding polypeptide according to the invention. These biological actions can be analyzed by functional assays. In order to test whether the candidate polypeptide is a functional variant of the polypeptide according to the present invention, the candidate polypeptide can be analyzed by a functional assay method generally known to those skilled in the art, and such assay method is the corresponding polypeptide according to the present invention. It is suitable for analyzing the biological function of

In addition, the term “functional variant” includes a polypeptide expressed from a mutated gene or from a differentially conjugated gene as long as the candidate functional variant polypeptide meets the criteria for a functional variant for% sequence identity level. Such expression analysis can be carried out by methods generally known to those skilled in the art.

"Functional variants" of a polypeptide refer to about 7 to about 1000 amino acids, preferably 10 or more amino acids, more preferably 20 or more, most, so long as they have substantially the same biological function as the corresponding polypeptide according to the invention. It may preferably be part of a polypeptide according to the invention having a length of 50 or more, such as 100 or more, such as 200 or more, such as 300 or more, such as 400 or more, such as 500 or more, such as 600 or more amino acids in length. Also included are deletions of the polypeptides according to the invention in the range of about 1 to 30, preferably about 1 to 15, in particular about 1 to 5 amino acids, as long as they have substantially the same biological function as the corresponding polypeptide according to the invention. do. For example, the first amino acid, methionine, may not be present without significantly altering the function of the polypeptide. In addition, post-translational modifications, such as lipid anchors or phosphoryl groups, may or may not be present in the variant.

"Sequence identity" refers to when the polypeptide is determined by eg BLASTP 2.0.1 and when the nucleic acid is determined by eg by BLASTN 2.014 (where filter is installed and BLOSUM is 62; Altschul et al., 1997, Nucleic Acids Res. , 25: 3389-3402), refers to the degree of identity (% identity) of two sequences.

In addition, the present invention provides a gene encoding the alpha-xylosidase mutant enzyme of the present invention.

The'gene encoding the alpha-xylosidase mutant enzyme' described in the specification of the present invention refers to a gene according to SEQ ID NO: 5 or 6 and/or a variant thereof.

The term “coding gene” relates to a DNA sequence encoding a separable bioactive polypeptide according to the present invention or a precursor thereof. Polypeptides may be encoded by the entire length of the coding sequence or a part of the sequence as long as the biological function, such as the glycotransferase activity of the present invention, is substantially maintained.

There may be small changes in the sequence of the genes described above, such as due to degeneration of the genetic code, or the untranslated sequence may be attached to the 5'and/or 3'end of the nucleic acid without significantly affecting the activity of the encoded polypeptide. It is known that there is. Accordingly, the present invention also includes so-called naturally occurring nucleic acids and artificially generated "variants" of the nucleic acids described above.

Preferably, the gene according to the invention is DNA or RNA, preferably DNA, in particular double-stranded DNA. In particular, the gene according to the present invention may be an RNA molecule, preferably a single-stranded or double-stranded RNA molecule. The sequence of the nucleic acid may further comprise one or more introns and/or one polyA sequence.

Genes according to the present invention can be prepared by methods generally known to those skilled in the art and are described below.

A “variant” of a gene may be a homologue obtained from another species with sequence identity of preferably 80%, in particular 90%, most preferably 95%.

A “variant” of a gene is about 8 or more nucleotides in length, preferably about 16 or more nucleotides in length, particularly about 21 or more nucleotides in length, more preferably about 30 nucleotides in length, as long as it has similar activity to the corresponding polypeptide according to the invention. It may be part of a gene according to the invention having a length of at least 4 nucleotides, more preferably at least about 40 nucleotides in length, and most preferably at least about 50 nucleotides in length. This activity can be assayed using the functional assay method described above.

As a preferred embodiment of the present invention, the gene may include a gene or a variant thereof having a sequence complementary to the gene according to the present invention. Preferably, the gene comprises a non-functional mutant variant of the gene according to the present invention, or a variant thereof.

In the present invention, the term'proton donation/receptor catalytic group of alpha-xylosidase' refers to the function of supplying and receiving protons required when enzymes hydrolyze sugar chains among the amino acid residues of alpha-xylosidase. Refers to an amino acid residue.

In one embodiment of the present invention, the amino acid residue other than aspartate is preferably alanine, serine, or glycine, but is not limited thereto.

In one embodiment of the present invention, the proton donation/receptor catalyst catalytic amino acid residue of alpha-xyloseidase is preferably aspartate, and the aspartate in the PVHWGGDC amino acid sequence of the alpha-xyloseidase It is more preferred, and most preferably, the 482th aspartate in SEQ ID NO: 1 or the 481th aspartate in SEQ ID NO: 2, but is not limited thereto.

In one embodiment of the present invention, the mutant enzyme preferably has a hydrolytic activity reduced by 1/10 to 1/1000,000 times, but is not limited thereto.

* In the contents of Table 4, which is a specific example of the present invention, it was shown that the mutant activity was reduced by 1/10000 times. In addition, it was shown at the same time that the activity against pNP xyloside was lost.

In addition, the present invention is the present invention in which the proton donation/receptor catalyst of alpha-xylosidase is mutated to amino acid residues other than aspartate through a method selected from a specific positional mutation method, a random mutation method, or a mutation of a natural microbial genome. It provides a method of preparing an alpha-xyloseidase mutant enzyme of.

In one embodiment of the present invention, the alpha-xylosidase mutant enzyme is preferably produced using a recombinant microorganism, but is not limited thereto.

In addition, the present invention provides a sugar transfer method in which the alpha-xylose mutant enzyme of the present invention is used as a sugar transfer reaction catalyst, and preferably alpha-xylosyl fluoride is used as a sugar donor.

In one embodiment of the present invention, the method preferably further comprises a sugar receptor, and the sugar receptor is an analog in which glucose, mannose, and their 2-hydroxyl groups are substituted with other reactive groups. It is preferable that they are oligosaccharides and glycosides having at the non-reducing terminal, 4-nitrophenyl beta glucoside, 4-nitrophenyl beta mannokoside, 4-nitrophenyl 2-deoxy 2-azido-beta glucoside, 4-nitrophenyl It is more preferable that it is a compound selected from the group consisting of beta cellobioside, and 4-nitrophenyl 2-deoxy 2-fluoro-beta cellobioside, but is not limited thereto.

In one embodiment of the present invention, the alpha-xylosidase mutant enzyme is preferably obtained after being immobilized on a carrier or expressed on a cell surface, but is not limited thereto.

Hereinafter, the present invention will be described.

Alpha- Xylosidase Variant production

Many methods are well known in the art for cloning alpha-xylosidase and introducing substitutions, deletions or insertions into genes (e.g., alpha-xylosidase genes). In general, cloning of genes and into said genes. Standard procedures for introducing mutations (random and/site specific) can be used to obtain the alpha-xylosidase variants of the present invention. For further explanation of suitable techniques, Example 1 herein and (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al., (eds. ) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, CR, and Cutting, SM (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990), and WO 96/34946. Reference.

Expression vector

A recombinant expression vector comprising a DNA structure encoding an enzyme of the present invention can be any vector that can readily be subjected to the recombinant DNA process. The choice of vector will often depend on the host cell into which it is introduced. Thus, the vector may be a spontaneously replicating vector, i.e. the vector exists as an extrachromosomal entity, and its replication is independent of the replication of a chromosome, for example a plasmid. Alternatively, the vector is introduced into a host cell. The vector may be partially or wholly integrated into the host cell genome and replicated together with the integrated chromosome. The vector is preferably an expression vector in which a DNA sequence encoding an enzyme of the present invention is operably linked to an additional segment required for transcription of DNA. to be. In general, expression vectors can be derived from plasmid or viral DNA, or contain elements of both. The term “operably linked” refers to that a segment is arranged to act for its intended purpose, for example, transcription is initiated at a promoter and continues in the DNA sequence encoding the enzyme.

The promoter may be any DNA sequence that exhibits transcriptional activity in the host cell of choice and may be derived from a gene encoding a protein that is homologous or non-homologous to the host cell. Examples of suitable promoters for use in the host cell of bacteria are examples Bacillus licheniformis maltogenic amylase gene, Bacillus subtilis alpha-amylase gene, Bacillus amyloliquefaciens alpha-amylase gene, Bacillus subtilis alkali protease gene, or Bacillus pumilus xylosidase gene-derived promoter, or phage Lambda PR or E co promoter lac, trp or tac promoters. The DNA sequence encoding the enzyme of the invention can also be operatively linked to a suitable terminator, if necessary.

The recombinant vector of the invention may also contain a DNA sequence that allows the vector to replicate in the host cell in question. The vector may also contain selectable markers, e.g. to compensate for defects in the host cell. It is a gene product, or a gene encoding resistance to antibiotics such as kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycin, or the like, or resistance to heavy metals or herbicides.

* In order to induce the enzyme of the present invention into the secretory pathway of the host cell, a secretion signal sequence (also known as a leader sequence, prepro sequence, or free sequence) may be provided in a recombinant vector. The secretion signal sequence is linked to a DNA sequence encoding an enzyme in the correct reading frame. The secretory signal sequence is usually located 5'in the DNA sequence encoding the enzyme. The secretory signal sequence may be normally enzyme related or may come from a gene encoding another secreted protein.

The enzyme, promoter, and optionally terminator and/or secretion signal sequence, DNA sequence encoding for each of them is ligated, or these sequences are collected by an appropriate PCR amplification scheme, and in a suitable vector containing the information necessary for replication or integration. As that

The process used to insert them is well known to those skilled in the art (see, e.g., Sambrook et al., cited above).

Host cell

The DNA sequence encoding the enzyme introduced into the host cell may be homologous or non-homologous to the host in question. If homologous to the host cell, i.e. produced naturally by the host cell, it will generally be operably linked to a promoter sequence other than its natural environment, but to other secretory signal sequences and/or terminator sequences, if applicable. will be. The term “homology” is intended to include a DNA sequence encoding an enzyme native to the host organism in question. The term “unhomologous” is intended to include DNA sequences that are not naturally expressed by a host cell. Thus, the DNA sequence may be derived from another organism, or it may be a synthetic sequence.

The host cell into which the DNA structure or recombinant vector of the present invention is introduced may be any cell capable of producing the enzyme and includes higher eukaryotic cells including bacteria, yeast, fungi and plants.

In culture, examples of microbial host cells capable of producing the enzyme of the present invention are strains such as strain Corynebacterium glutamicum, B. subtilis, B. licheniformis, B. lentus, B.brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or Bacillus, such as B. thuringiensis, or Streptomyces such as S. lividans or S. murinus, or gram-negative bacteria such as Escherichia coli.

Transformation of bacteria can be carried out by protoplast transformation, electroporation, conjugation, or using reactive cells in a manner known per se (see, Sambrook et al., supra).

When expressing enzymes in bacteria such as E. coli, the enzymes are typically retained in the cytoplasm as insoluble granules (known as inclusion bodies) or can be induced into the periplasmic space by bacterial secretion sequences. In the former case, the cells are lysed, the granules are recovered and denatured, and then the enzyme is refolded by dilution of the denaturant. In the latter case, the enzyme can be recovered from the periplasmic space by destroying the cells by, for example, ultrasonic treatment or osmotic shock, releasing the contents of the periplasmic space and recovering the enzyme.

When expressing the enzyme in Gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme can be retained in the cytoplasm or induced into the extracellular medium by the secretory sequence of the bacteria. In the latter case, the enzyme can be recovered from the medium as described below.

Alpha- Xylosidase How to produce

The present invention provides a method for producing an isolated enzyme according to the present invention, wherein a suitable host cell transformed using a DNA sequence encoding the enzyme is cultured under conditions that allow the production of the enzyme, and the resulting enzyme is intracellularly Is produced.

When an expression vector containing a DNA sequence encoding an enzyme is transformed into a non-homologous host cell, it can enable the production of a non-homologous recombinant of the enzyme of the present invention. This makes it possible to produce highly purified alpha-xylosidase characterized in the absence of homologous impurities.

The medium used to cultivate the transformed host cell can be any conventional medium suitable for growing the host cell in question. The expressed alpha-xylosidase and mutant enzyme are present in the cytoplasm, and the cells are separated from the medium through centrifugation or filtration, and the coenzyme solution is obtained through lysozyme or ultrasonic disruption, and then the medium by salt such as ammonium sulfate. It can be recovered from it by a chromatographic procedure such as ion exchange chromatography, affinity chromatography and the like, following a well-known process including precipitating the protein component of.

General molecular biology method

Unless otherwise stated in the specification of the present invention, DNA manipulation and transformation are performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harborlab., Cold Spring Harbor, NY ; A usubel, FM et a1. (eds.) "Current protocols in Molecular Biology". JohnWiley and Sons, 1995; Harwood, CR, and Cutting, SM (eds.) "Molecular Biological Methods for Bacillus". JohnWiley and Sons, 1990).

DNA Enzymes for manipulation

Unless otherwise stated in the specification of the present invention, all enzymes for DNA manipulation, such as restriction endonucleases, ligases, etc., are obtained from Fermentas, and DNA polymerase is obtained from Roche. Enzymes for DNA manipulation are used according to the supplier's instructions.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention have produced alpha-xylosyl fluoride having a good leaving group, fluorine, although the proton donor/receptor catalytic group of alpha-xylosidase, which belongs to the long-acting carbohydrate degrading enzyme, is mutated significantly lowers the hydrolytic activity. When used as a sugar donor, oligosaccharides and glycosides having at the non-reducing end of which the mutant enzymes successfully carry out a sugar transfer reaction, and an analog in which glucose, maltose and their 2-hydroxyl groups are substituted with other reactive groups, are sugar acceptors. As used, it was found that one molecule of xylose was regioselectively and stereospecifically transferred to the non-reducing terminal 6-hydroxyl position of the sugar receptor by alpha-sugar chain bonds. In particular, the non-reducing end of the sugar transfer product generated through the above enzymatic reaction is xylose that does not have a 6-hydroxyl group, so it is not used as a sugar receptor for the secondary sugar transfer reaction, and is accumulated as the only sugar transfer product in the reaction solution. The present invention was completed by discovering that the product can be easily prepared with high purity.

As described above, the present invention uses a mutant enzyme that maintains the sugar transfer activity through the mutation of the proton donation/receptor catalytic group of alpha-xyloseidase, which belongs to the persistent carbohydrate degrading enzyme, to glucose, Sugars by transferring one molecule of xylose to the 6-hydroxyl position of the non-reducing terminal of oligosaccharides and glycosides having an analog substituted with a different reactive group of mannose and their 2-hydroxyl groups at the non-reducing terminal through an alpha-sugar chain bond. Transformation products can be produced in high yield.

1 is a sugar or glycoside having an alpha-xylosyl fluoride as a sugar donor at the non-reducing end of glucose after mutating aspartate, a proton donor/receptor catalytic group of alpha-xylosidase belonging to a persistent carbohydrate degrading enzyme, to alanine. It is a schematic diagram showing the sugar transfer reaction that occurs when is used as a sugar acceptor.
Fig. 2 is an electrophoresis picture of a PCR product amplifying the E. coli K-12 alpha-xylosidase gene.
Figure 3 shows the restriction enzyme map of pETYicI6xH containing the E. coli K-12 alpha-xylosidase gene.
4 is an SDS-PAGE photograph showing a sample obtained during the process of purifying recombinant E. coli K-12 alpha-xylosidase produced in E. coli through Ni-NTA chromatography.
5 is a diagram showing changes in the activity of E. coli K-12 alpha-xylosidase according to pH.
6 is a diagram showing changes in the activity of E. coli K-12 alpha-xylosidase according to temperature.
Figure 7 shows a pETYici-D482A6xH restriction enzyme map containing a mutant gene in which aspartate, which is a proton donation/receptor catalyst group of E. coli K-12 alpha-xylosidase, is mutated into alanine.
FIG. 8 is a photograph of a thin layer chromatography analysis of a sugar transfer reaction product using an E. coli K-12 alpha-xylosidase variant using alpha-xyloxyl fluoride and 4-nitrophenyl beta glucoside as substrates.
9 shows alpha-xylosyl fluoride using a mutant coenzyme solution selected from a variant library in which ASp482, which is a proton donation/receptor catalyst group of E. coli K-12 alpha-xylosidase, is substituted with an arbitrary amino acid residue. This is a photograph of a result of analyzing a compound having 4-nitrophenyl under ultraviolet light after a sugar transfer reaction using 4-nitrophenyl beta glucoside as a substrate, after thin layer chromatography of the reaction product.
FIG. 10 shows a glucose transfer reaction using alpha-xylosyl fluoride and 4-nitrophenyl beta glucoside as substrates using a mutant enzyme in which ASp481, which is a proton donor/acceptor catalyst group of Bacillus halodurans C-125, is substituted with allinine. It is a figure showing the mass spectrometry spectrum of one reaction solution and the structural formula of the product.

Hereinafter, the present invention will be described through non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1: E. coli K-12-derived alpha-xylene to let dehydratase gene (yicI) Cloning and alpha-xylene production to let dehydratase (YicI)

1-1 E. coli Alpha- Xylosidase Gene amplification

E. coli K-12 was inoculated in 50 ml of LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl), cultured at 37°C, centrifuged to recover cells, and chromosomal DNA was isolated (Dubnau, D. and RD Abenson, 1971, J. Mol. Biol., 56, 209-221). That is, the recovered cells were treated with lysozyme to make protoplast cells, and then SDS (sodium dodecyl sulfate) was added to completely destroy the cells, and then sodium chloride was added so that the concentration was 1 mol to the total reaction solution to precipitate proteins. This was centrifuged to take only the supernatant, and a mixture of phenol:chloroform:isoamyl alcohol (a volume ratio of each solution = 25:24:1) was added in the same volume as the supernatant to remove impurities such as proteins in the solution. This process was repeated several times. Twice the volume of 99% ethanol was added to precipitate, and this was separated by winding it with a glass rod and dissolved in 1/10xSSC buffer solution (3M sodium chloride, 0.3M sodium citrate).

For the gene encoding the E. coli alpha-xylosidase (hereinafter, YicI), the YicI gene was isolated by performing PCR by the method of Table 2 below using the primers shown in Table 1 below.

To isolate the YicI gene, forward primer YicITOP (5'-CAG AAC TAA GGA ACG CAT ATG AAA ATT AGC-3' SEQ ID NO: 7) and reverse primer YicIEND (5'-ATC AAG CTC GAG CAA CGT AAT TGT CAG CGC- 3'and SEQ ID NO: 8) were prepared, respectively, and the primers are shown in Table 1 below.

order Sequence number Forward Primer YicITOP 5'-CAG AAC TAA GGA ACG CAT ATG AAA ATT AGC-3' 7 Reverse primer YicIEND 5'-ATC AAG CTC GAG CAA CGT AAT TGT CAG CGC-3' 8

Polymerase chain reaction (PCR) was performed on the chromosomal DNA of E. coli K-12 strain using the forward and reverse primers to obtain a PCR product, and it was confirmed that the size of the PCR product was 2.2 kb through electrophoresis. Was done (Fig. 2). The conditions of the PCR are as described in Table 2 below.

STEP Temperature Reaction time First(1st) denaturation 95 ℃ 5 min Second(2st) denaturation 95 ℃ 1 min Annealing 55 ℃ 30 sec Extension 72 ℃ 1min 30sec Cycle go to denaturation 33 cycles Final extension 72 ℃ 7 min

1-2. E. coli Alpha- Xylosidase gene Cloning

For transformation, host cells, E. coli MC1016 (New England Biolab (NEB, USA)) were inoculated in 5 ml LB liquid medium and cultured at 37°C for 12 hours, and 1.0 ml of the culture medium was inoculated into 50 ml of new LB liquid medium. Then, it was incubated until the absorbance reached 0.5 at 600 nm.

1.5 ml of the culture solution having confirmed the absorbance was centrifuged for 5 minutes at 7000 xg at 4° C. to recover the cells, then _{suspended in 0.75 ml of Transformation Solution I (50 mM CaCl 2} ) and left in ice for 30 minutes. . Thereafter, the cells were recovered by centrifugation at 4° C. for 2 minutes under 6000 xg conditions, and the recovered cells _{were suspended in 0.15 ml of Transformation Solution II (100 mM CaCl 2} ) and left in ice for 30 minutes.

The YicI gene amplified in Example 1-1 was treated with restriction enzymes NdeI and XhoI at 37° C. for 6 hours, and a solution ligated with pET-29b expression vector (Novagen, Germany) treated with the same restriction enzyme The transformation was mixed with 0.2 ml of E. coli MC1061 (New England Biolab, USA), left to stand for 1 hour, and then subjected to heat shock at 42° C. for 2 minutes. 0.8 ml of LB liquid medium was mixed with the heat shocked mixed solution, and then incubated at 37° C. for 1 hour. The culture medium cultured for 1 hour was plate-plated on LB agar medium containing kanamycin (final concentration 20 μg/ml), and strains showing resistance were first selected.

The first selected strain was inoculated into 5 ml of LB liquid medium containing kanamycin, cultured at 37° C. for 12 hours, and centrifuged to obtain cells. Isolation of the plasmid from the cells recovered with a plasmid isolation kit (Qiagen, USA) and digestion under the same conditions as above using the same NdeI and XhoI restriction enzymes as above, confirming that the YicI gene of about 2.2 kb is included. I did.

PETYicI6xH was prepared through the ligation, and a cleavage map of the vector of the prepared recombinant vector pETYicI6xH is shown in FIG. 3. The pETYicI6xH vector contains an E. coli alpha-xylosidase (YicI) gene, and contains 8 additional amino acid residues (Leu-Glu-6His) at the 3'end of the alpha-xylosidase gene, have.

1-3 cultivation of transformants and YicI Production of

The pETYicI6xH vector prepared above was transformed into E. coli BL21 (DE3) in the same manner as in Example 1-2, and then kanamycin resistance was checked to select the strain transformed with the vector, that is, a transformant. The selected transformants were inoculated in 2.5 L of LB liquid medium containing kanamycin and cultured at 37°C. When the absorbance at 600 nm reached 0.6, IPTG was added to a final concentration of 0.5 mM, followed by incubation at 37°C for 5 hours.

1-4. Enzyme purification

After culturing the transformant strain selected above, the cells were recovered by centrifugation for 30 minutes at 7,000 x g at 4°C. The recovered cells were suspended in a 50 mM Tris-HCl buffer solution (pH 7.5) containing 300 mM NaCl and 10 mM imidazole, and the suspended cells were subjected to ultrasonic grinding. The culture solution subjected to ultrasonic pulverization was centrifuged at 10,000 x g for 30 minutes to obtain a supernatant, and the obtained supernatant was injected into Ni-NTA affinity chromatography and purified.

Fig. 4 shows an electrophoresis picture of a sample for each purification step of YicI produced in E. coli BL21 (DE3) transformed with the recombinant vector pETYicI6xH. M in FIG. 4 is a size marker, lane 1 is a supernatant obtained by ultrasonic grinding of the transformant, and lane 2 is YicI enzyme purified by Ni-NTA affinity chromatography of a sample of the supernatant obtained after ultrasonic grinding. to be.

As shown in Figure 4, it can be seen that the YicI enzyme (lane 2) was efficiently purified through a Ni-NTA affinity chromatography process, and the size of the alpha-xylosidase was about 88 Da on SDS-PAGE. By indicating the molecular weight of YicI, a value similar to the theoretical molecular weight inferred from the amino acid sequence of YicI was shown.

Example 2: E. coli Alpha- from K-12 Xylosidase ( YicI ) Of

2-1 enzyme Titer

450 μl of 5 mM paranitrophenyl alpha-xyloside solution dissolved in 50 mM sodium phosphate (pH 7.0) buffer solution was preheated at 37° C. for 5 minutes, 50 μl of enzyme was added, and reacted for 10 minutes. After stopping the reaction by adding 500 μl of 1M sodium bicarbonate solution, measure the absorbance at 405 nm to measure the amount of nitrophenol produced. The reaction without the enzyme is used as a control. 1 unit refers to the amount of enzyme required to form 1 μmol of nitrophenol for 1 minute under specific reaction conditions.

2-2 pH characteristic

In order to determine the optimal reaction pH of YicI of the present invention, the buffer solution (pH 4, 5, 6 sodium acetate buffer; pH 6, 7, 8 sodium phosphate buffer; pH 8, 9 sodium borate buffer) of various pH ranges is para. To prepare a 5 mM paranitrophenyl alpha-xyloside substrate solution by adding nitrophenyl alpha-xyloside, enzymes were prepared by the method of Example 2-1 using the purified YicI enzyme solution of Example 1-4. The titer was measured and the relative activity was measured based on the highest absorbance value.

Figure 5 shows the enzyme activity according to the pH of the YicI enzyme, has an optimum titer at pH 6.5 and maintained a titer of 50% or more at pH 5.0 to 7.5.

2-3. Temperature characteristics

In order to determine the optimum reaction temperature of the E. coli-derived alpha-xylosidase of the present invention, a 5 mM paranitrophenyl alpha-xyloside substrate solution was prepared using 50 mM sodium phosphate buffer (pH 7.0). , After heating the substrate solution in various temperature ranges of 20 to 60° C., enzyme activity was measured according to the method of Example 2-1, and then absorbance was measured to measure relative enzyme activity.

6 shows the enzyme activity according to the temperature of the YicI enzyme, and has an optimal titer at 50°C and maintained a titer of about 50% or more at 40 to 65°C.

Example 3: E. coli Derived alpha- Xylosidase Production and production of mutant enzymes

3-1. YicI Proton donor mutant ( YicI - D482A ) Production

YicID482A-F (SEQ ID NO: 9) was prepared to replace Asp482, which is a proton donor catalyst group of the YicI enzyme, with alanine, and the primers are shown in Table 3 below.

order Sequence number YicID482A-F 5-GTA CAC TGG GGT GGC GCG TGT TAC GC TAA CTA C-3 9

For the recombinant vector pETYicI6xH of Example 1-2, the YicID482A-F was used as a forward primer, and the T7 terminator primer (5-TATGCTAGTTATTGCTCAG-3; SEQ ID NO: 10) binding to the recombinant vector was used as a reverse primer. By carrying out a PCR product (YicID482A-rear) was obtained. In addition, for the recombinant vector pETYicI6xH, the T7 promoter primer (5-TAATACGACTCACTATAGGG-3; SEQ ID NO: 11) was used as a forward primer, and the YicID482A-rear was subjected to a polymerase chain reaction using a reverse primer. -A PCR product containing a gene encoding YicI-D482A in which the proton donor catalytic group of the xylosidase mutant enzyme, that is, the YicI enzyme, was substituted with Alanine was obtained, and the size of the PCR product was about 2.2 kb through electrophoresis. Confirmed. The conditions of the two PCRs are as described in Table 2 above.

The PCR product containing the gene encoding YicI-D482A was digested with NdeI and XhoI, and then ligated to pET29b as in Example 1-2 to prepare a recombinant vector pETYicI-D482A6xH, and Example 1- Transformants were prepared according to 3 and 1-4, and YicI-D482A was produced. The cleavage map of the vector of the recombinant vector pETYicI-D482A6xH is shown in FIG. 7. The pETYicI-D482A6xH vector is a gene containing an alpha-xylosidase mutase in which the proton donor catalytic group of the YicI enzyme is substituted with alanine and eight additional amino acid residues (Leu-Glu-6xHis) at the C-terminus of the enzyme Have.

3-2. YicI Proton donor mutant ( YicI - D482A ) production

The prepared pETYicI-D482A6xH vector was transformed by the method of Example 1-3, and then purified according to the method of Example 1-4.

Example 4. E. coli-derived alpha-let in xylene kinase mutant enzyme hydrolytic activity measured in (YicI D482A)

In order to measure the hydrolytic activity of YicI-D482A, enzyme activities against 1 mM 4-nitrophenyl alpha-xylose and alpha-xylosyl fluoride were measured. Using 4-nitrophenyl alpha-xylose as a substrate, the reaction rate per concentration of each substrate was measured according to Example 2-1. In the case of alpha-xyloxyl fluoride, the substrate was dissolved in a 50 mM sodium phosphate (pH 7.0) buffer solution and reacted according to Example 2-1, and the amount of fluorine released by the enzymatic reaction was measured with a fluorine ion analyzer (Orion 96-09 combination fluoride ion electrode) was used to measure the enzyme reaction rate. The reaction rate obtained according to the above method was compared with the reaction rate of YicI enzyme and summarized in Table 4 below.

YicI YicI-D482A 4-nitrophenyl alpha-xylose 100 0 Alpha-Xylosyl Fluoride 100 3.7 x 10 ^-2

As shown in Table 4 above, 4-nitrophenyl alpha-xylose was not degraded at all, but when alpha-xylosyl fluoride was used as a substrate, it was 3.7 x 10 ^-4 times lower than that of wild type YicI enzyme. Showed decomposition activity.

Example 5: Alpha- Xylosyl Fluoride and 4 -nitrophenyl -beta- glucoside Glucose transfer reaction using YicI-D482A

5-1. Alpha- Xylosyl Fluoride with 4-nitro-phenyl-beta-glucoside (4-nitrophenyl-β-D-glucoside) produced dangjeon the reaction product with

To carry out the sugar transfer reaction, 0.2 mmole alpha-xylosyl fluoride and 0.125 mmole of 4-nitrophenyl-beta-glucoside were dissolved in 5 mL of 50 mM sodium phosphate (pH 7.0) buffer solution, and the above Example YicI-D482A purified in 3-2 was added and the reaction was carried out at 37°C, and the reaction solution was analyzed through a thin layer chromatography method. Fig. 8 shows the TLC results of the reaction. As can be seen from FIG. 8, it can be seen that all of the 4-nitrophenyl-beta-glucoside used as a sugar receptor was consumed, and only one sugar transfer product was produced.

5-2. Alpha- Xylosyl Fluoride with 4-nitro-phenyl-beta-manno side (4-nitrophenyl-β-D-mannoside) generated dangjeon the reaction product with

To perform the sugar transfer reaction, 0.07 mmole alpha-xylosyl fluoride and 0.05 mmole of 4-nitrophenyl-beta-glucoside were dissolved in 3 mL of 50 mM sodium phosphate (pH 7.0) buffer solution, and the above Example The reaction was carried out in the same manner as in 5-1 and analyzed by TLC to confirm that a reaction product was formed.

5-3. Alpha- Xylosyl Fluoride with 4-nitro-phenyl-2-deoxy-2-ahjayi even-beta-glucoside (4-nitrophenyl 2-deoxy- 2-azido-β-D-glucoside) The reaction using the dangjeon

To carry out the glucose transfer reaction, 0.12 mmole alpha-xylosyl fluoride and 0.06 mmole of 4-nitrophenyl-2-deoxy-2-azido-beta-glucoside were added to 3 mL of 50 mM sodium phosphate (pH 7.0). ) After dissolving in a buffer solution, the reaction was carried out in the same manner as in Example 5-1 and analyzed by TLC. The reaction was carried out in the same manner as in Example 5-1 and analyzed by TLC to confirm that a reaction product was formed.

5-4. Alpha- Xylosyl Fluoride with 4-nitro-phenyl-beta-cell bio-side (4-nitrophenyl-β-D-cellobioside) a reaction using a dangjeon

To perform the sugar transfer reaction, 0.27 mmole alpha-xyloxyl fluoride and 0.12 mmole of 4-nitrophenyl-beta-cellobioside were dissolved in 5 mL of 50 mM sodium phosphate (pH 7.0) buffer solution, and the above The reaction was carried out in the same manner as in Example 5-1 and analyzed by TLC. The reaction was carried out in the same manner as in Example 5-1 and analyzed by TLC to confirm that a reaction product was formed.

5-5. Alpha- Xylosyl Fluoride and 2,4-phenyl-2-deoxy-2-flow-nitro-beta-cell bio-side (2,4-dinitrophenyl 2-deoxy- 2-fluoro-β-D-cellobioside) dangjeon using This reaction

To perform the glucose transfer reaction, 8.68 μmole alpha-xylosyl fluoride and 8.68 μmole of 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-cellobioside were added to 5 mL of 50 mM. After dissolving in sodium phosphate (pH 7.0) buffer solution, the reaction was carried out in the same manner as in Example 5-1 and analyzed by TLC. The reaction was carried out in the same manner as in Example 5-1 and analyzed by TLC to confirm that a reaction product was formed.

Example 6: Party transfer Reaction product refine

The reaction solution of Example 5-1 to 5-5 was injected into a C18 SEP PAK cartridge (Waters), washed with water to remove non-aryl sugar, and the sugar transfer product was eluted with ethanol. Ethanol was removed from the eluted reaction product solution with a vacuum concentrator. After adding 2 mL of pyridine and acetic anhydride to the residue to acetylate all hydroxyl groups, the solvent was removed and the sugar transfer product was analyzed through silica gel chromatography. Again, the yield of the sugar transfer product was calculated relative to the sugar receptor used in the reaction by using the weight of the sugar transfer product obtained after completely removing the solvent through vacuum concentration. The yield of sugar transfer of the resulting product is shown in Table 5 below.

Example Party donor Sugar receptor Yield (%) 5-1 Alpha-Xylosyl Fluoride 4-nitrophenyl beta glucoside 96 5-2 4-nitrophenyl beta mannocoside 93 5-3 4-nitrophenyl 2-deoxy 2-azido-beta glucoside 88 5-4 4-nitrophenyl beta cellobioside 80 5-5 4-nitrophenyl 2-deoxy 2-fluoro-beta cellobioside 90

Example 7: Party transfer Structural analysis of products

The reaction product purified in Example 6 was dissolved in methanol, all hydroxyl groups were acetylated using acetic anhydride, and then the solvent was removed through concentration under reduced pressure. Only the acetylated sugar transfer product was separated from the residue through Silica gel chromatography, and the structure was determined by analyzing the sugar transfer product by dissolving it in chloroform (CDCl3) and then analyzing it with 400 MHz using a Bruker AV-400 spectrometer.

7 -1. 4-Nitrophenyl[( 2,3,4- tri -O- acetyl -α-D- xylopyranosyl )-(1→6)-O- 2,3,4-tri-O-acetyl-β-D- glucopyranoside. (Reaction product of Example 5-1)

1 H NMR (? ppm, CDCl ₃ , 400 MHz): 8.30 (m,2H), 7.12 (m, 2H) (C ₆ H ₄ NO ₂ ); 5.47 (t, 1H, J _{3' ,4'} 10.0 Hz, H-3), 5.31 (t, 1H, J _{3 ,4} 8.8 Hz, H-3), 5.26 (t, 1H, J2,3 9.2Hz, H-2), 5.17 (d, 1H, J _{1 ,2} 7.6 Hz, H-1), 5.00 (t, 1H, J _{4 ,5} 10.0 Hz, H-4), 4.97 (d, 1H, J _{1' ,2'} 3.2 Hz, H-1'), 4.88 (dt, 1H, J _{4' ,5'a} = J _{4' ,5'b} 6.0 Hz, H-4'), 4.78 (dd, 1H, J _{2 ' ,3'} 10.0 Hz, H-2'), 3.97 (m, 1H, H-5), 3.78 (dd, 1H, J _{5 ,6a} 8.0 Hz, & J _{6a ,6b} 10.4 Hz, H-6a), 3.64(dd, 1H, J _{5'a ,5'b} 10.8 Hz, H-5'a), 3.45(dd, 1H, J _{4' ,5'b} 6.0 Hz, H-5'b), 3.43 (dd , 1H, J _{5 ,6b} 2.4 Hz, H-6b); 2.08-1.73 (6s, 18H, CH ₃ CO). 13 CNMR (?ppm, CDCl ₃ , 100 MHz): 170.16, 170.06, 170.03, 169.70, 169.40, 169.19 (CH3CO); 161.24, 143.32, 126.22 (2C), 116.50 (2C) (C ₆ H ₄ NO ₂ ); 98.14, 95.69, 73.36, 72.41, 70.97, 70.95, 68.99, 68.85, 68.72, 66.22, 58.41 (sugar ring); 20.81, 20.74, 20.64, 20.61, 20.56, 20.52, 20.16 (CH ₃ CO). EISMS: Calcd for C35H43NO22+Na ⁺ :852.7. Found:852.7.

7-2 4- Nitrophenyl [( 2,3,4- tri - O - acetyl -α-D- xylopyranosyl )-(1→6)- O - 2,3,4-tri- O -acetyl-β-D -mannopyranoside. (Reaction product of Example 5-2)

¹ H NMR (CDCl ₃ , 400 MHz): d 8.32 (m, 2 H, Ar-H), 7.16 (m, 2 H, Ar-H), 5.71 (dd, 1 H, J _{2 ,3} 2.0 Hz, H-3), 5.52 (t, 1H, J _{3' ,4'} 10.0 Hz, H-3'), 5.35 (d, 1H, J _{1 ,2} 0.8 Hz, H-1), 5.20 (t, 1H, J _{3 ,4} 9.0 Hz, H-4), 5.17 (dd, 1H, H-3), 5.02 (d, 1H, J _{1' ,2'} 3.6 Hz, H-1'), 4.90 (ddd, 1H, H-4'), 4.81 (dd, 1H, J _{2' ,3'} 10.4 Hz, H-2'), 3.96 (m, 1H, H-5), 3.91 (dd, 1H, J _{5 ,6a} 8.4 Hz , H-6a), 3.68 (dd, 1H, J _4',5'a 5.6 Hz & J _{5'a ,5'b} 10.8 Hz, H-5'a), 3.51 (t, 1H,J _{4' , 5'b} = J _{5'a ,5'b} 10.8 Hz, H-5'b), 3.46 (dd, 1H, J _{5 ,6b} 1.2 Hz, J _{6a ,6b} 10.0 Hz, H-6), 2.25 (s , 3H, C H ₃ CO), 2.11 (s, 3H, C H ₃ CO), 2.10 (s,3H, C H ₃ CO), 2.09 (s, 3H, C H ₃ CO), 2.03 (s, 3H , C H ₃ CO), 1.74 (s, 3H, C H ₃ CO). _{^{13 CNMR (CDCl 3, 100MHz)}} : d 170.42, 170.39, 170.23, 170.06, 169.95, 169.89 (CH 3 C O); 161.36, 143.48, 126.43 (2C), 116.63 (2C) (Ar-C); 96.29 (C-1), 95.84 (C-1'), 74.13 (C-5), 71.28 (C-2'), 70.85 (C-3), 69.16, 69.13 (C-3'&4'), 68.69 (C-2), 66.68 (C-6), 66.24 (C-4), 58.70 (C-5'); 21.00, 20.97 (2C), 20.87, 20.73, 20.37 ( C H ₃ CO). EISMS: Calcd for C ₃₅ H ₄₃ NO ₂₂ +Na ⁺ :852.7. Found:852.7.

7-3 4- Nitrophenyl [( 2,3,4- tri - O - acetyl -α-D- xylopyranosyl )-(1→6)-2- deoxy-2-azido- O -3,4-di- O -acetyl-β-D-glucopyranoside (reaction product of Example 5-3)

¹ H NMR (δppm, CDCl ₃ , 400 MHz): d 8.35 (m, 2 H, Ar-H), 7.21 (m, 2 H, Ar-H), 5.51 (t, 1 H, J _{2' ,3 '} = J _{3' ,4'} 10.0 Hz, H-3'), 5.13 (dd, 1H, J _{2 ,3} 9.6 Hz, J _{3 ,4} 10.0 Hz, H-3), 5.06 (d, 1H, J _{1 ,2} 8.4 Hz, H-1), 5.01 (d, 1H, J _{1' ,2'} 3.2 Hz, H-1'), 4.95 (t, 1H, J _{4 ,5} 9.6 Hz, H-4), 4.90 (dd, 1H, H-4'), 4.80 (dd, 1H, H-2'), 3.96 (m, 1H, H-5), 3.83 (dd, 1H, J _{2 ,3} 10.0 Hz, H-2 ), 3.80 (dd, 1H, J _{5 ,6a} 8.0 Hz, J _{6a ,6b} 10.4 Hz, H-6a), 3.69 (dd, 1H, J _{5'a ,5'b} 10.4 Hz, H-5'a) , 3.47 (t, 2H, J _{5 ,6b} 10.4 Hz, J _{4' ,5'b} 10.4 Hz, H-6b &H-5'b), 2.12 (s, 3H, C H ₃ CO), 2.09 (s +s, 6H, C H ₃ CO), 2.07 (s, 3H, C H ₃ CO), 1.77 (s, 3H, C H ₃ CO). ¹³ C NMR (CDCl ₃ , 100 MHz): d 170.43, 170.28, 169.98 (2 C), 169.83 (CH ₃ C O); 161.25, 143.78, 126.56 (2C), 116.94 (2C) (Ar-C); 99.81 (C-1), 95.90 (C-1'), 73.62 (C-5), 72.32 (C-3), 71.21 (C-2'), 69.19, 69.12, 68.95 (C-3', 4') & 4), 66.40 (C-6), 63.73 (C-2), 58.68 (C-5'); 20.99, 20.91, 20.86 (2C), 20.44 ( C H ₃ CO).

7-4 4- Nitrophenyl [( 2,3,4- tri -O- acetyl -α-D- xylopyranosyl )-(1→6)-O- 2,3,4-tri-O-acetyl-β-D -glucopyranosyl-(1→4)-O-2,3,6-tri-O-acetyl-β-D-glucopyranoside (reaction product of Example 5-4)

¹ H NMR (?ppm, CDCl ₃ , 400 MHz): 8.18 (m, 2H), 7.01 (m, 2H) (C ₆ H ₄ NO ₂ ); 5.43 (t, 1H, J _{3 ",4"} 9.6 Hz, H-3"), 5.29 (t, 1H, J _{3' ,4'} 9.2 Hz, H-4'), 5.17 (d, 1H, J _{1 ,2} 7.6 Hz, H-1), 5.14 (m, 2H, H-2 &H-3'), 5.03 (d, 1H, J _{1 ",2"} 4.0 Hz, H-1"), 4.98 (m , 2H, H-4"& H-3), 4.86 (dd, 1H, J _{2' ,3'} 9.6 Hz, H-2'), 4.78 (dd, 1H, J _{2 ",3"} 10.4 Hz, H -2"), 4.57 (d, 1H, J _{1' ,2'} 7.6 Hz, H-1'), 4.52 (dd, 1H, J _{5 ,6a} 2.0 Hz & J _{6a ,6b} 12.0 Hz, H-6a) , 4.11 (dd, 1H, J _{5 ,6b} 6.0 Hz, H-6b),3.93~3.82 (m, 3H, H-5, H-5'&H-5"), 3.74 ~ 3.58 (m, 4H, H-4, H-5", H-6'a &H-6'b), 2.10-1.97 (8s, 27H, C H ₃ CO). _{^{13 CNMR (d ppm, CDCl 3}} , 100MHz): 170.25, 170.16 (2C), 169.93, 169.89, 169.52, 169.40 (2C), 169.09 (CH 3 C O); 161.13, 143.24, 125.78 (2C), 116.55 (2C) ( C ₆ H ₄ NO ₂ ); 99.97, 97.80, 96.22, 77.20, 75.25, 73.17, 73.11, 72.71, 72.33, 71.68, 71.12, 70.93 (2C), 69.27, 69.03, 68.93, 67.29 (sugar ring); 20.79, 20.73, 20.69 (2C), 20.65 (2C), 20.57, 20.52 (2C) ( C H ₃ CO).

7-5 2,4- Dinitrophenyl [( 2,3,4- tri -O- acetyl -α-D- xylopyranosyl )-(1→6)- O-2,3,4-tri-O-acetyl-β -D-glucopyranosyl-(1→4)-2-deoxy-2-fluoro-O-3,6-di-O-acetyl-β-D-glucopyranoside (reaction product of Example 5-5)

¹ H NMR (?ppm, 600 MHz, CDCl ₃ ): 2.01, 2.04, 2.05, 2.06, 2.07, 2.08, 2.10, 2.17 (s, 24 H, 8 CH3CO); 3.56 (dd, 1 H, J _{5 ,6} 2.7,J _{6'a ,6'b} 11.1 Hz, H6'), 3.69 (ddd, 1 H, J _{5' ,6'} 2.4, J _{5' ,6'} 5.4 , J _{4' ,5'} 10.2 Hz, H5), 3.74 (dd, 1 H, J _{5' ,6'} 5.5, J _{6'a ,6'b} 10.8 Hz, H6'), 3.77 (dd, 1 H, J _{4 ",5"} 6.0, J _{5 "a,5"b} 11.1 Hz, H5"), 3.86 (dd, 1 H, J _{4 ",5"} 6.0, J _{5 "a,5"b} 10.2 Hz, H5 "), 3.95 (ddd, 1 H, J _{5 ,6} 2.4, J _{5 ,6} 5.4, J _{4 ,5} 9.6 Hz, H5), 4.03 (t, 1 H, J _{3 ,4} = J _{4 ,5} 9.6 Hz , H4), 4.12 (dd, 1 H, J _{5 ,6} 4.8, J _{6a ,6b} 12.6 Hz, H6), 4.59 (dd, 1 H, J _{5 ,6} 2.1, J _{6a ,6b} 12.3 Hz, H6), 4.62 (d, 1 H, J _{1' ,2'} 7.8 Hz, H1'), 4.69 (dt, 1 H, J _{2 ,F} 49.2, J _{1 ,2} = J _{2 ,3} 7.5 Hz, H2), 4.82 ( dd, 1 H, J _1",2" 3.6, J _{2 ",3"} 10.2 Hz, H2"), 4.89 (dd, 1 H, J _{1' ,2'} 7.8, J _{2' ,3'} 9.6 Hz, H2'), 5.00 (t, 1 H, J _{3' ,4'} = J _{4' ,5'} 10.2 Hz, H4'), 5.00 (sixt, 1 H, J _{4 ",5"} = J _{4 ",5 "} 6.3, J _{3 ",4"} 9.9 Hz, H4"), 5.07 (d, 1 H, J _{1 ",2"} 3.0 Hz, H1"), 5.20 (t, 1 H, J _{2' ,3'} = J _{3' ,4'} 9.6 Hz, H3'), 5.44 (t, 1 H, J _{2 ,3} = J _{3 ,4} 9.6 Hz, H3), 5.46 (dd, 1 H, J _{1 ,2} 7.8, J _{1 ,F} 2.4 Hz, H1), 5.48 (t, 1 H, J _{2 ",3"} = J _{3 ",4"} 9.9 Hz, H3"), 7.39 (d, 1 H, J 9.6 Hz, DNP), 8.44 (dd, 1H, J 2.4, J 9.0 Hz, DNP), 8.75 (d, 1H, J 3.0 Hz, DNP). ¹³ CNMR (150 MHz, CDCl ₃ ): 20.78, 20.86, 20.89, 20.95, 21.11, (8 CH3CO); 59.09, 61.60, (C6, C6'); 67.15 (C5"), 69.09, 69.21, 69.38, 71.14, 71.90, 72.91, 73.47, 73.52, (C4, C5, C2', C3', C4', C5', C2", C3", C4"); 71.58 (C3, J _{3 ,F} 20.9 Hz), 88.62 (C2, J _{2 ,F} 191 Hz), 96.39 (C1"), 98.11 (C1, J _{1 ,F} 25.8 Hz), 99.97 (C1'), 117.69, 121.86, 128.87, 140.21, 142.25, 153.61 (DNP); 169.29, 169.49, 169.67, 170.16, 170.24, 170.43 (8 CH3CO)

Example 8: YicI Quantum donor/receptor Catalytic Random mutation

* 8-1. YicI proton donor mutant library ( YicI - D482X ) construction

YicID482X-F (5-GTA CAC TGG GGT GGC NNN TGT TAC GC TAA CTA C-3, N is an arbitrary nucleotide sequence, SEQ ID NO: 12) to replace Asp482, the proton donor catalyst group of the YicI enzyme, with an arbitrary amino acid And, the primers are shown in Table 6 below.

order Sequence number YicID482X-F 5-GTA CAC TGG GGT GGC NNN TGT TAC GC TAA CTA C-3 12

* YicI mutant in which the proton donation/receptor catalyst group of the YicI enzyme was substituted with an arbitrary amino acid by the method of Example 3-1 using the YicID482X-F for the recombinant vector pETYicI6xH of Example 1-2 as a forward primer. (YicI-D482X, X means any amino acid) of the genes were obtained. The conditions of the PCR are as described in Table 2 above.

The PCR product containing the genes encoding YicI-D482X was prepared in the same manner as in Example 3-1 to prepare a recombinant vector library pETYicI-D482X6xH, and a transformant was prepared according to Example 1-2.

8-2. Party transfer Active YicI Quantum donor/receptor selection

After transforming the prepared pETYicI-D482X6xH vector library by the method of Example 1-2, 98 transformants were inoculated into 0.2 mL LB medium containing 20 μg/mL kanamycin, respectively. After producing YicI mutants by the method of Example 1-3, lysozyme was added to a final concentration of 1 mg/mL to destroy cells to prepare a crude enzyme solution. Each crude enzyme solution was mixed 1:1 with 0.2 mmole alpha-xylosyl fluoride dissolved in 0.1 mL of 50 mM sodium phosphate (pH 7.0) buffer solution and 0.1 mmole of 4-nitrophenyl-beta-glucoside substrate solution. , The reaction was performed at 37°C for 12 hours, and the reaction solution was analyzed through thin layer chromatography. Figure 9 is TLC data showing the results of 18 out of 98 crude enzyme solutions. A plasmid vector containing a gene encoding a YicI mutase was isolated from 16 samples showing glucose transfer activity, and the nucleotide sequence was determined. As a result, transformants A4, A7, C3, C6, C10, E10, E12, and F1 are aspartate, which is the proton donor/receptor catalytic group of YicI, as alanine, and transformants C9, E2, E3, G1, and H1 as serine. , It was confirmed that transformants E4, F6, and F8 were substituted with glycine. The mutant amino acids of the 18 mutants are summarized in Table 7.

Mutant enzyme A4 A5 A7 C3 C6 C9 C10 D2 E2 E3 E4 E10 E12 F1 F6 F8 G1 H4 Proton donor/receptor catalytic group position amino acid residue A D A A A S A D S S G A A A G G S S

As shown in Table 7, the enzymes in which the 482th aspartide residue, which is the YicI proton donation/receptor catalyst group, is mutated to alanine, serine, or glycine, uses alpha-xylosyl fluoride as a sugar donor to obtain a sugar transfer product. It can be seen that it is synthesized.

Example 9: Bacillus Days when a xylene - halodurans Alpha Preparation of variants and detection of glucose transfer activity

9-1. Bacillus halodurans Alpha- Xylosids Variant ( BHaX - D481A ) Production

After culturing B. halodurans C-125 by the method of Example 1-1, chromosomal DNA was isolated. To isolate the gene encoding the B. halodurans alpha-xylosidase (hereinafter BHaX), the forward primer BHaXTOP (5'-TAG GGA ACA ACA TAT GAA ATT TTC AGA TGG-3' SEQ ID NO: 13) and reverse primer BHaXEND (5'-AAC CCT CGA GGA CGC CTA GGT GGA CAA CCA-3', SEQ ID NO: 14) was prepared, respectively, and PCR was performed according to the method of Table 2 to isolate the BHaX gene. The primer sequence is shown in Table 8 below.

order Sequence number Forward primer
BHaXTOP 5'-TAGGGAACAACATATGAAATTTTCAGATGG-3' 13 Reverse primer
BHaXEND 5'-AACCCTCGAGGACGCCTAGGTGGACAACCA-3' 14

B. halodurans C-125 strain chromosomal DNA was subjected to PCR as in Example 1 using the forward and reverse primers to obtain a PCR product.

BHaXD481A-F (5'-CCTGTTCACTGGGGTGGAGCTTGTGCCGCAACT-3' SEQ ID NO: 15) was prepared to replace Asp481, which is a proton donation/receptor catalyst group of the BHaX enzyme, with alanine, and the primers are shown in Table 9 below.

order Sequence number Forward primer BHaXD481A-F 5'-CCTGTTCACTGGGGTGGAGCTTGTGCCGCAACT-3' 15

In Example 3-1, YicID482A-F primer was replaced with BHaXD481A-F, and in the same manner as in Example 3-1, aspartate, which is a proton donor/acceptor of BHaX, contains a gene encoding a mutant enzyme substituted with alanine. pETBHaXD481A6xH was prepared.

9-2. BHaX - D481A Production of and Party transfer reaction

The prepared pETBHaXD481A6xH vector was transformed by the method of Example 1-3, and then purified according to the method of Example 1-4. The sugar transfer reaction was performed as in Example 5-1 using the purified BHaXD481A, and the reaction product was analyzed using a PE-Sciex API 300 triple quadrupole mass spectrometer (Sciex, Canada), and Figure 10 shows the reaction product. Is the spectrum of. As a result of the analysis, the product having the same molecular weight (molecular weight = 433) as the glycotransfer product synthesized by YicI-D482A in Example 5-1 was sodium salt (M + Na ⁺ , molecular weight = 433 + 23), potassium salt (M + K ⁺ , molecular weight = 433 + 39), and the sodium salt of the dimer (2M + Na ⁺ , molecular weight = 433 x 2 + 23). Therefore, it was confirmed that the BHaXD481A mutant enzyme also has the same glycotransfer activity as the YicI-D482A through the mutation of the proton donor/receptor catalytic group of BHaX.

<110> KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> A Chimeric protein, A Preparation method thereof, A Nanosenser fixed with the same and An application thereof <150> KR10-2009-0019355 <151> 2009-03-06 <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 catatgcatc accatcacca tcacgacatt gacccgtata aagaa 45 <210> 2 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cccactccct ccgccaccgt cttccaaatt acttcccacc ca 42 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ctcgagagta ccgcctcccc cactccctcc gccacc 36 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggatccggat ccggtggcgg agggtctggg ggaggcggt 39 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggcggagggt ctgggggagg cggttccagg ggattagtag tcagctat 48 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aagcttttaa acaacagtag tttccggaag 30 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ctcgaggcac cgaaagctga taac 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gaattcgtca gcttttggtg cttg 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gaattcgcac cgaaagctga taac 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ggatccgtca gcttttggtg cttg 24 <210> 11 <211> 189 <212> DNA <213> SPAB <400> 11 gcaccgaaag ctgataacaa attcaacaaa gaacaacaaa atgctttcta tgaaatctta 60 catttaccta acttaaatga agaacaacgc aatggtttca tccaaagctt aaaagatgac 120 ccaagccaaa gcgctaacct tttagcagaa gctaaaaagc taaatgatgc acaagcacca 180 aaagctgac 189 <210> 12 <211> 234 <212> DNA <213> HBV capsid protein <400> 12 atggacattg acccgtataa agaatttgga gcttctgtgg agttactctc ttttttgcct 60 tctgacttct ttccttctat tcgagatctc ctcgacaccg cctctgctct gtatcgggag 120 gccttagagt ctccggaaca ttgttcacct caccatacag cactcaggca agctattctg 180 tgttggggtg agttgatgaa tctggccacc tgggtgggaa gtaatttgga agac 234 <210> 13 <211> 78 <212> PRT <213> HBV capsid protein <400> 13 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp 65 70 75 <210> 14 <211> 207 <212> DNA <213> HBV capsid protein <400> 14 tccagggaat tagtagtcag ctatgtcaac gttaatatgg gcctaaaaat cagacaacta 60 ttgtggtttc acatttcctg tcttactttt ggaagagaaa ctgttcttga gtatttggtg 120 tcttttggag tgtggattcg cactcctccc gcttacagac caccaaatgc ccctatctta 180 tcaacacttc cggaaactac tgttgtt 207 <210> 15 <211> 69 <212> PRT <213> HBV capsid protein <400> 15 Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 1 5 10 15 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 20 25 30 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 35 40 45 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 50 55 60 Glu Thr Thr Val Val 65 <210> 16 <211> 63 <212> PRT <213> Protein A <400> 16 Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe 1 5 10 15 Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly 20 25 30 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu 35 40 45 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp 50 55 60

Claims (7)

An alpha-xylosidase mutant enzyme in which the hydrolysis activity of 482 amino acid residues of SEQ ID NO: 1 is mutated from aspartate to serine is eliminated and the sugar transfer activity is maintained. A method for preparing the alpha-xyloxidase mutant enzyme of claim 1, wherein the quantum donor-catalyzed amino acid residues of alpha-xyloxidase are mutated by a method selected from specific positional variation, random variation, or mutation of a natural microbial genome. . 3. The method of claim 2, wherein the alpha-xyloidase mutant enzyme is produced using recombinant microorganisms. The method of claim 1, wherein the alpha-xylosidase mutant enzyme of claim 1 is used as a sugar transfer reaction catalyst and comprises a sugar donor. 5. The method according to claim 4, wherein said sugar donor is alpha-xylyl fluoride. The method of claim 4, wherein the sugar receptor is glucose, mannose, 4-nitrophenyl beta glucoside, 4-nitrophenyl beta mannocoside, 4-nitrophenyl 2-deoxy 2-azido-beta The method of claim 1 further comprising a compound selected from the group consisting of glucoside, 4-nitrophenyl beta cellobioside, and 4-nitrophenyl 2-deoxy 2-fluoro-beta cellobioside. 5. The method of claim 4, wherein said alpha-xylosidase mutant enzyme is obtained after immobilization on a carrier or expression on a cell surface.

Images