KR101256112B1 - An alpha-Xylosidase mutants modified at their proton-donor/acceptor catalyst and high efficiency transglycosylation with the same - Google Patents

Abstract

The present invention relates to a quantum donor catalyst mutant enzyme of alpha-xylosidase, a method for preparing the enzyme, and an application of the enzyme.

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 quantum donor / receptor catalytically mutant enzyme of alpha-xyloidase, a method for preparing the enzyme, and an application of the enzyme.

Carbohydrates that perform a variety of physiological responses in vivo are recognized as disease prevention and treatment. Such carbohydrates have been made through organic synthesis so far, but the method of synthesizing oligosaccharides using enzymes has recently attracted a lot of attention due to advantages of reaction conditions and substrate specificities. The oligosaccharide synthesis technique using an enzyme is a method using a glycotransferase having a carbohydrate degrading enzyme or a glycosyltransferase using a nucleotide sugar as a substrate.

In general, sugar transfer enzymes are enzymes involved in the biosynthesis of sugar chains such as glycoproteins and glycolipids in a living body. Glycoproteins such as glycoproteins and glycolipids, which are reaction products, have been found to be important molecules that function as signal tacks between cells and extracellular matrices in differentiation and development, or as a tack of complex sugars. .

Among the carbohydrate degrading enzymes, the retaining glycosidase uses two amino acid residues having carboxyl groups as nucleophile catalysts and proton-donor / acceptor catalysts, respectively. To catalyze the sugar chain hydrolysis reaction. The persistent carbohydrate degrading enzymes decompose sugar chains in the substrate using nucleophilic catalysts of the respective enzymes, and the nucleophilic catalyst and the sugar-enzyme in which a part of the substrate is covalently bonded through anomer bond opposite to the original substrate. Generate intermediates. Subsequently, the anomeric product of the reaction product is reacted by a hydrolysis reaction that transfers the sugar in the sugar-enzyme intermediate into water by the action of a proton donor / receptor catalyst or a sugar transfer reaction that transfers to the hydroxyl group of a sugar receptor other than water. The reaction is complete with 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, since the generated sugar transition product is hydrolyzed by the enzyme again, the reaction must be controlled in time and the yield of the obtained sugar transfer product is also generally low. In addition, it is possible to transfer sugar to various hydroxyl groups of the sugar receptor, and the generated sugar transition products can be used as sugar receptors again, so that one or more sugar transition products are produced. 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, when the nucleophilic catalyst of the persistent carbohydrate degrading enzyme is inactivated to the inactive amino acid residue, it cannot form a sugar-enzyme intermediate and thus exhibit no hydrolytic activity. However, when analogs of sugar-enzyme intermediates are provided using fluoride sugars in the form of regioisomers opposite to the original substrate, the sugar transfer reaction proceeds successfully by the reaction of the proton donor catalyst remaining in the enzyme. do. At this time, the carbohydrate degrading enzyme variants in which the nucleophilic catalyst is mutated have lost hydrolytic activity and thus do not hydrolyze sugar transition products but accumulate products in the reaction solution. In this way, the nucleophilic catalyst of the persistent carbohydrate degrading enzyme is transformed by site-directed mutagenesis, and a processing enzyme that performs only a sugar transfer reaction by using a fluorine sugar in the form of a positional isomer opposite to the original substrate as a sugar donor It is named glycosynthase (J Am Chem Soc 1998, 120: 5583-5584). Since the first report of glycosyndase that transformed the 358th glutamate residue (Glu358), a nucleophilic catalyst of beta-glycosidase derived from Agrobacterium sp., Into alanine, The same strategy has been applied to various enzymes (Curr Opin Chem Biol. 2006, 10 (5): 509-519). However, most existing glycosidases are enzymes that synthesize beta-sugar chains, mostly derived from beta-glycosidase. In the case of alpha-glycosidase that can synthesize alpha-sugar chain, Schizosaccharomyces pombe alpha-gl Enzymes that mutated the nucleophilic catalyst of lycosidase are the only example (Biosci Biotechnol Biochem. 2002, 66 (4): 928-933). However, the alpha-glycosidase has a difficulty in using beta-anomeric fluorine sugar, which is difficult to synthesize and less stable than alpha-anomeric fluorine sugar, as a sugar donor.

The second method involves nucleophilic and quantum donor / receptor catalysis through directed evolution, but significantly reduces hydrolytic activity and maintains or improves translocation activity, thereby increasing the yield of sugar transfer products. A study has been reported (J Biol Chem. 2005, 280 (44): 37088-37097). However, this case also did not completely remove the hydrolytic activity of the sugar transition products, there has been no report on the application of alpha-glycosidase.

Alpha-xylosidase belongs to the surviving carbohydrate degrading enzyme which breaks down the reactive a-glycosidic linkage to produce a product having an alpha-anomer. Therefore, the production of various glycotransferases has been reported by using the sugar-transferase 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 in low yield.

The present invention solves the above problems, and the object of the present invention was devised by the necessity of the above, it is an object of the present invention mutating the alpha-xylidase quantum donor / receptor catalyst group, alpha-xyl to perform the sugar transfer reaction in high yield It is to provide a rosidase mutase.

It is another object of the present invention to provide a method for producing alpha-xylosidase mutase.

Another object of the present invention is to amplify non-reducing ends of analogues in which a-xylosyl fluoride and glucose, mannose, and their 2-hydroxyl groups are replaced by other reactors. The eggplant is to provide a method for preparing a sugar transfer product in which xylose is transferred by reacting the alpha-xylosidase mutase to a substrate including oligosaccharide and glycosides.

In order to achieve the above object, the present invention provides an alpha-xylosidase mutant enzyme polypeptide mutated with a natural alpha-xyloidase quantum donor / receptor catalyst group with an amino acid residue other than aspartate.

The term "polypeptide" described in the specification of the present invention refers to the entire length of the polypeptide according to the present invention. In a preferred embodiment, the term “polypeptide” includes polypeptides produced by isolated polypeptides and recombinant methods, such as by separating and purifying from a sample, by screening libraries and by protein synthesis by conventional methods. All of the above methods are known to those skilled in the art. Preferably, the entire polypeptide, or a portion thereof, can be synthesized by conventional synthetic methods, such as the Merrifield technique.

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

A “functional variant of a native alpha-xyloxidase or alpha-xyloxidase mutant enzyme” of a polypeptide described herein is a polypeptide having an amino acid sequence according to SEQ ID NO: 1 or 2 and / or SEQ ID NO: 3 or 4. Refers to a polypeptide having sequence homology with respect to, in particular, about 70%, preferably about 80%, particularly about 90%, especially about 95%, most preferably about 98%. Such functional variants are polypeptides which are homologous to the polypeptides according to the invention, for example, originating in organisms other than humans, preferably from non-human mammals such as mice, rats, monkeys and pigs. Another example of a functional variant is a polypeptide encoded by different alleles of genes in different individuals, different organs of one organism or at different stages of development. Functional variants preferably also include naturally occurring or synthetic mutations, especially those which significantly alter the activity of peptides encoded by these sequences. Such variants may also result from different conjugation of genes that encode, preferably.

"Functional variant" refers to a polypeptide having the same biological activity as the corresponding polypeptide according to the invention. This biological action can be analyzed by functional assays. To examine whether a candidate polypeptide is a functional variant of a polypeptide according to the present invention, the candidate polypeptide can be analyzed by functional assays generally known to those skilled in the art, which assays can be performed by the corresponding polypeptides according to the present invention. Suitable for analyzing the biological function of

The term “functional variant” also includes polypeptides expressed from mutated or differentially conjugated genes so long as the candidate functional variant polypeptides meet the criteria for functional variants for% sequence identity level. Such expression analysis can be carried out by methods generally known to those skilled in the art.

"Functional variants" of polypeptides range from 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. Preferably at least 50, such as at least 100, such as at least 200, such as at least 300, such as at least 400, such as at least 500, such as at least 600 amino acids, may be part of a polypeptide according to the invention. Also included are deletions of polypeptides according to the invention in the range of about 1 to 30, preferably about 1 to 15, especially about 1 to 5 amino acids, so long as they have substantially the same biological function as the corresponding polypeptides 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 anchor or phosphoryl groups may or may not be present in the variant.

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

The present invention also provides a gene encoding the alpha-xylosidase mutant enzyme of the present invention.

As used herein, the 'gene encoding alpha-xylosidase mutant enzyme' refers to the gene according to SEQ ID NO: 5 or 6 and / or variants thereof.

The term “coding gene” relates to a DNA sequence or a precursor thereof that encodes a separable bioactive polypeptide according to the invention. A polypeptide may be encoded by a sequence of the entire length or portion thereof, as long as the biological function, such as for example the sugar transfer activity of the present invention, is substantially maintained.

In the sequences of genes described above, for example, due to the degeneracy of the genetic code, there may be little change or an untranslated sequence may be attached to the 5 'and / or 3' ends of the nucleic acid without significantly affecting the activity of the encoded polypeptide. It is known that there is. The present invention therefore also encompasses 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 invention may be an RNA molecule, preferably a single or double chain RNA molecule. The sequence of nucleic acids may further comprise one or more introns and / or one polyA sequence.

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

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

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

As a preferred embodiment of the invention, the gene may comprise a gene having a sequence complementary to the gene according to the invention or a variant thereof. Preferably said gene comprises non-functional mutagenic variants of the genes according to the invention, or variants thereof.

In the present invention, the 'proton donor / receptor catalyst of alpha-xyloidase' refers to a function of supplying and receiving protons required when an enzyme hydrolyzes sugar chains among amino acid residues of alpha-xyloidase. Amino acid residues.

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

In one embodiment of the present invention, it is preferable that the quantum donor / receptor catalyst amino acid residue of the alpha-xylosidase is aspartate, and the aspartate in the PVHWGGDC amino acid sequence of the alpha-xylosidase. More preferably, most preferably, the 482th aspartate in SEQ ID NO: 1 or the 481th aspartate in SEQ ID NO: 2 is not limited thereto.

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

The contents of Table 4, which is one embodiment of the present invention, showed a 1 / 10000-fold decrease in variant activity. It also shows that pNP xyloside activity was lost.

In addition, the present invention is a mutated alpha-xyloidase quantum donor / receptor catalyst group in the amino acid residues other than aspartate through a method selected from specific positional variation, random variation, or variation of the natural microbial genome It provides a method for the production of alpha-xylosides mutant enzyme.

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

The present invention also provides a sugar transfer method using the alpha-xylosidase mutant enzyme of the present invention using a sugar transfer reaction catalyst, and preferably the alpha-xylyl fluoride as a sugar donor.

In one embodiment of the invention, the method preferably further comprises a sugar receptor, the sugar receptor is glucose, mannose, and analogs in which their 2-hydroxyl group is substituted by another reactor Are oligosaccharides and glycosides having non-reducing ends, 4-nitrophenyl beta glucoside, 4-nitrophenyl beta mannocoside, 4-nitrophenyl 2-deoxy 2-azido-beta glucoside, 4-nitrophenyl More preferably, it is a compound selected from the group consisting of beta cellobioside, and 4-nitrophenyl 2-deoxy 2-fluoro-beta cellobioside.

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

Hereinafter, the present invention will be described.

Alpha- Xylosidase Mutant  production

Many methods are known in the art for cloning alpha-xylosidase and introducing substitutions, deletions or insertions into genes (eg, alpha-xylosidase genes). Standard procedures for introducing (randomized and / or site specific) mutations can be used to obtain alpha-xylosidase variants of the invention. For further explanation of suitable techniques, see 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

Recombinant expression vectors comprising DNA structures encoding enzymes of the invention can be any vector that can readily be placed in a recombinant DNA process. The choice of vector will often depend on the host cell being introduced. Thus, the vector may be a spontaneously replicating vector, ie the vector is present as an extrachromosomal entity, the replication of which is independent of the replication of a chromosome such as, for example, a plasmid. Alternatively, the vector is introduced into a host cell. May be partially or wholly integrated into the host cell genome and replicated with the integrated chromosome. The vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to additional segments required for transcription of the DNA. to be. In general, expression vectors may be derived from plasmid or viral DNA, or contain both elements. The term “operably linked” refers to a segment that is arranged to function for its intended purpose, such as for example, where transcription is initiated in a promoter and continued in a DNA sequence encoding an 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 nonhomologous to the host cell. Examples of promoters suitable for use in a host cell of bacteria are Bacillus licheniformis maltogenic amylase gene, Bacillus subtilis alpha-amylase gene, Bacillus amyloliquefaciens alpha-amylase gene, Bacillus subtilis alkaline protease gene, or Bacillus pumilus xylosidase gene, or phage Lambda PR or PL promoter, phage T7. Includes lac, trp or tac promoters. DNA sequences encoding the enzymes of the invention can also be operably linked to suitable terminators, if desired.

Recombinant vectors of the invention may also include DNA sequences that allow the vector to replicate in the host cell in question. The vector may also include selectable markers, for example to compensate for defects in the host cell. 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.

To direct the enzyme of the invention into the secretory pathway of the host cell, secretory signal sequences (also known as leader sequences, prepro sequences or free sequences) can be provided in the recombinant vector. The secretory signal sequence is linked to the DNA sequence encoding the enzyme in the correct read frame. The secretory signal sequence is usually located 5 'in the DNA sequence encoding the enzyme. The secretory signal sequence may be normally associated with an enzyme or may come from a gene encoding another secreted protein.

Containing the present enzymes, promoters and optionally terminator and / or secretory signal sequences, the DNA sequences encoding for each, or collecting these sequences by appropriate PCR amplification schemes and containing the information necessary for replication or integration As that

Procedures used to insert them are well known to those skilled in the art (see, eg, Sambrook et al., Cited above).

Host cell

DNA sequences encoding the present enzymes introduced into host cells may be homologous or nonhomologous to the host in question. If homologous to a host cell, ie produced naturally by the host cell, it will generally be operably linked to other promoter sequences or, if applicable, to other secretory signal sequences and / or terminator sequences rather than to its natural environment. will be. The term "homologous" is intended to include DNA sequences encoding native enzymes in the host organism in question. The term “nonhomologous” is intended to include DNA sequences that are not naturally expressed by the host cell. Thus, the DNA sequence may be of other biological origin, 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, yeasts, fungi and plants.

In culture, examples of microbial host cells capable of producing the enzymes of the invention include 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 B. thuringiensis, or Streptomyces such as S. lividans or S. murinus, or Gram-negative bacteria such as Escherichia coli.

Bacterial transformation can be performed by protoplast transformation, electroporation, conjugation, or using reactive cells in a manner known in the art (see Sambrook et al., Supra).

When expressing enzymes in bacteria such as E. coli, 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 so that the granules are recovered and denatured and the enzyme is then refolded by dilution of the denaturant. In the latter case, the enzymes can be recovered from the periplasmic space by destroying the cells, for example by sonication or osmotic shock, to release the contents of the periplasmic space and recover the enzyme.

When expressing enzymes in Gram-positive bacteria, such as Bacillus or Streptomyces strains, the enzymes can be retained in the cytoplasm or induced into 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 with a DNA sequence encoding the enzyme is incubated under conditions allowing the production of the enzyme and the resulting enzyme is intracellular. Produced.

When an expression vector comprising a DNA sequence encoding an enzyme is transformed into a nonhomologous host cell, it may enable the production of nonhomologous recombinants of the enzymes of the invention. This allows the production of highly purified alpha-xylosidases characterized by the absence of homologous impurities.

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

General Molecular Biology Methods

DNA manipulations and transformations are performed using standard methods of molecular biology unless otherwise stated in the present specification (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". John Wiley and Sons, 1995; Harwood, CR, and Cutting, SM (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990).

DNA  Enzyme for Manipulation

Unless stated otherwise in the context of the present invention all enzymes for DNA manipulation, such as restriction endonucleases, ligase and the like, are obtained from Fermentas and DNA polymerase 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 shown that a protease in which the quantum donor / receptor catalyst group of the alpha-xylosidase belonging to the persistent carbohydrate degrading enzyme significantly degrades the hydrolytic activity, but the alpha-xylyl fluoride having fluorine which is a good leaving group When used as a sugar donor, the mutant enzymes have successfully undergone sugar transfer reactions, and thus oligosaccharides and glycosides having analogs at the non-reducing end with glucose, maltose and their 2-hydroxyl groups substituted with other reactors It was found that one molecule of xylose was regioselectively and stereospecifically translocated by alpha-sugar chain linkage to the non-reducing terminal 6-hydroxyl position of the sugar receptor. In particular, the non-reducing end of the sugar transition product produced through the enzymatic reaction is xylose having no 6-hydroxyl group, so it is not used as a sugar receptor for the secondary sugar transfer reaction, and the only sugar transition in the reaction solution is accumulated as a product so that the sugar transition The present invention has been completed by the discovery that the product can be easily produced in high purity.

As described above, the present invention is a glucose, by using a mutase that maintains the sugar transfer activity, but the hydrolysis activity is removed through a mutation on the proton / receptor catalyst group of alpha-xylosidase belonging to the persistent carbohydrate degrading enzyme Oligosaccharides having analogs substituted at the non-reducing end with mannose and their 2-hydroxyl groups substituted by other reactors and sugars by transferring axylose molecules via alpha-sugar chain bonds to the non-reducing terminal 6-hydroxyl positions of glycosides The transition product can be produced in high yield.

1 is a sugar or glycoside having alpha-xylyl fluoride as a sugar donor at a glucose non-reducing end after mutating aspartate, a proton / receptor catalyst of alpha-xylosidase belonging to a persistent carbohydrate degrading enzyme, to alanine This is a schematic diagram showing the sugar transition reaction occurs when using as a sugar receptor.
Figure 2 is an electrophoresis picture of the PCR product amplified E. coli K-12 alpha-xyloidase gene.
Figure 3 shows a restriction map of pETYicI6xH comprising the E. coli K-12 alpha-xyloxidase gene.
4 is a SDS-PAGE photograph showing a sample obtained during the purification of the recombinant E. coli K-12 alpha-xylosidase produced in E. coli by Ni-NTA chromatography.
Figure 5 is a diagram showing the change in activity of E. coli K-12 alpha-xylosidase with pH.
Figure 6 is a diagram showing the change in activity of E. coli K-12 alpha-xyloxidase with temperature.
Figure 7 shows a map of the pETYici-D482A6xH restriction enzyme containing a mutant gene in which aspartate, a quantum donor / receptor catalyst of E. coli K-12 alpha-xyloxidase, is mutated to alanine.
FIG. 8 is a thin layer chromatography photograph of a sugar transfer reaction product using E. coli K-12 alpha-xylosidase variant using alpha-xylyl fluoride and 4-nitrophenyl beta glucoside as a substrate.
FIG. 9 shows alpha-xylyl fluoride using mutant coenzyme solution selected from a mutant coenzyme solution substituted with an amino acid residue of ASp482, a proton donor / receptor catalyst of E. coli K-12 alpha-xylosidase. The result of analyzing the compound having 4-nitrophenyl under ultraviolet light after thin layer chromatography of the reaction product after the sugar transfer reaction using 4-nitrophenyl beta glucoside as a substrate.
10 is a sugar transfer reaction using alpha-xylyl fluoride and 4-nitrophenyl beta glucoside as substrates using a mutant enzyme substituted with alanine for ASp481, a quantum donor / receptor catalyst of Bacillus halodurans C-125. Figure shows the mass spectrometry of the 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 limited by the following examples.

Example  1: E. coli  Alpha from K-12 Xylosidase  gene( yicI ) Cloning  And alpha-xylosidase ( YicI ) production

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), incubated at 37 ° C and centrifuged to recover the cells and isolated chromosomal DNA (Dubnau, D. and RD Abenson, 1971, J. Mol. Biol., 56, 209-221). In other words, the recovered cells were treated with lysozyme to form protoplast cells, and then SDS (sodium dodecyl sulfate) was added to completely destroy the cells. Sodium chloride was added to the total reaction solution to precipitate the protein. After centrifugation, only the supernatant was taken, and a mixture of phenol: chloroform: isoamyl alcohol (ratio of the volume of each solution = 25: 24: 1) was added in the same volume as the supernatant to remove impurities such as protein in the solution. This process was repeated several times. A double volume of 99% ethanol was added to precipitate, which was wound up with a glass rod to separate and dissolved in 1 / 10xSSC buffer (3M sodium chloride, 0.3M sodium citrate).

The gene encoding the E. coli alpha-xylosidase (hereinafter referred to as YicI) was isolated from the YicI gene by PCR by the method of Table 2 using the primers of Table 1 below.

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-) to isolate the YicI gene. 3 ', SEQ ID NO: 8) were produced, respectively, and the primers are shown in Table 1 below.

order SEQ ID NO: 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

PCR product was obtained by PCR using E. coli K-12 strain chromosomal DNA using the forward and reverse primers. PCR products were obtained by electrophoresis, and the size of the PCR product was 2.2 kb. (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 ° C 30 sec Extension 72 ℃ 1min 30sec Cycle go to denaturation 33 cycles Final extension 72 ℃ 7 min

1-2. Cloning of E. coli alpha-xylosidase gene

For transformation, 5 ml LB liquid medium was inoculated with host cell E. coli MC1016 (New England Biolab (NEB, USA), incubated for 12 hours at 37 ° C, and 1.0 ml of the culture was inoculated in 50 ml of fresh LB liquid medium. Incubated at 600 nm until the absorbance became 0.5.

The cells were recovered by centrifuging 1.5 ml of the culture medium for which the absorbance was confirmed at 4 ° C. under 7000 × g for 5 minutes, and then suspended with 0.75 ml of transformation solution I (50 mM CaCl ₂ ) and left for 30 minutes in ice. . Thereafter, the cells were recovered by centrifugation at 4 ° C. under 6000 × g for 2 minutes, and the recovered cells were suspended in 0.15 ml of Transfection Solution II (100 mM CaCl ₂ ) and left in ice for 30 minutes.

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

The first selected strains were inoculated in 5 ml of LB liquid medium containing kanamycin, incubated at 37 ° C. for 12 hours, and centrifuged to obtain cells. The plasmid was separated from the cells recovered by the plasmid separation kit (Qiagen, USA) and cleaved under the same conditions using the same NdeI and XhoI restriction enzymes as above to confirm that the YicI gene of about 2.2 kb was included. It was.

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 comprises an E. coli alpha-xylosidase (YicI) gene and contains eight additional amino acid residues (Leu-Glu-6His) at the 3 'end of the alpha-xylosidase gene. have.

1-3 Culture of Transformants and Production of YicI

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. Selected transformants were inoculated in 2.5 L of LB liquid medium containing kanamycin and incubated at 37 ° C., when the absorbance was 0.6 at 600 nm, IPTG was added to a final concentration of 0.5 mM and incubated at 37 ° C. for 5 hours.

1-4. Enzyme purification

After culturing the transformants selected above, the cells were recovered by centrifugation for 30 minutes under conditions of 7,000 x g at 4 ℃. The recovered cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 300 mM NaCl and 10 mM imidazole, and the suspended cells were ultrasonically ground. The supernatant was collected by centrifugation for 30 minutes under conditions of 10,000 x g, and the obtained supernatant was purified by injection into Ni-NTA affinity chromatography.

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

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

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

2-1 Enzyme Titer

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

2-2 pH Characteristics

In order to determine the optimal reaction pH of the YicI of the present invention, each of various pH range buffers (pH 4, 5, 6 sodium acetate buffer; pH 6, 7, 8 sodium phosphate buffer; pH 8, 9 sodium borate buffer) Nitrophenyl alpha-xyloxide was added to prepare a 5 mM paranitrophenyl alpha-xyloxide substrate solution, and the enzyme was purified by the method of Example 2-1 using the purified YicI enzyme solution of Example 1-4. Titer was measured and relative activity was determined based on the highest absorbance values.

Figure 5 shows the enzyme activity according to the pH of the YicI enzyme, having 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

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

Figure 6 shows the enzyme activity according to the temperature of the YicI enzyme, having an optimum titer at 50 ℃ and maintained a titer of about 50% or more at 40 to 65 ℃.

Example 3: Preparation and Production of E. coli Derived Alpha-Xylosidase Mutase

3-1. Construction of YicI Quantum Donor Mutant (YicI-D482A)

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

order SEQ ID NO: 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 polymerase chain reaction was carried out using a reverse primer, Y7ID482A-F as a forward primer, and a T7 terminator primer (5-TATGCTAGTTATTGCTCAG-3; SEQ ID NO: 10) which binds to the recombinant vector. PCR product (YicID482A-rear) was obtained. In addition, to the recombinant vector pETYicI6xH, the T7 promoter primer (5-TAATACGACTCACTATAGGG-3; SEQ ID NO: 11) is used as a forward primer and the YicID482A-rear is subjected to a polymerase chain reaction using a reverse primer to provide an alpha provided by the present invention. A PCR product containing a gene encoding a YylI-D482A substituted with Alanine, a quantum donor catalyst of the YicI enzyme, ie, a YylI mutant enzyme, was obtained, and the size of the PCR product was about 2.2 kb. 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. A cleavage map of the vector of the recombinant vector pETYicI-D482A6xH is shown in FIG. 7. The pETYicI-D482A6xH vector is a gene comprising alpha-xylosidase mutase in which the quantum donor catalyst 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  Origin Alpha Xylosidase  Mutant enzyme ( YicI - D482A Measurement of hydrolysis activity

Enzyme activity against 1 mM 4-nitrophenyl alpha-xylose and alpha-xylyl fluoride was measured to determine the hydrolytic activity of YicI-D482A. Using 4-nitrophenyl alpha-xyl as a substrate, the reaction rate for each substrate concentration was measured according to Example 2-1. In the case of alpha-xylyl fluoride, the substrate is dissolved in 50 mM sodium phosphate (pH 7.0) buffer and reacted according to Example 2-1. 96-09 combination fluoride ion electrode) was used to measure the enzyme reaction rate. The reaction rate obtained according to the above method is summarized in Table 4 below in comparison with the reaction rate of the YicI enzyme.

YicI YicI-D482A 4-nitrophenyl alpha-xylose 100 0 Alpha-Xylosil Floride 100 3.7 x 10 ^-2

As shown in Table 4, 4-nitrophenyl alpha-xyl was not degraded at all, but the alpha-xylyl fluoride was used as a substrate, 3.7 × 10 ⁻⁴ times lower valence compared to wild type YicI enzyme. Degradation activity was shown.

Example  5: alpha Xylosyl Floridwa  4- Nitrophenyl -beta- Glucosides  Used YicI - D482A Transfer reaction using

5-1. Alpha- Xylosyl Floridwa  4- Nitrophenyl -Beta-glucoside (4-nitrophenyl-β-D- glucoside ) A former transition  Reaction product generation

To carry out the sugar transfer reaction, 0.2 mmole alpha-xylyl fluoride and 0.125 mmole of 4-nitrophenyl-beta-glucoside were dissolved in 5 mL of 50 mM sodium phosphate (pH 7.0) buffer, followed by the above example. YicI-D482A purified in 3-2 was added thereto and the reaction proceeded at 37 ° C., and the reaction solution was analyzed by thin layer chromatography. TLC results of the reaction are shown in FIG. 8. As can be seen in FIG. 8, all of 4-nitrophenyl-beta-glucoside used as sugar receptors were consumed, and only one sugar transition product was produced.

5-2. Alpha- Xylosyl Floridwa  4- Nitrophenyl -Beta-mannoside (4- nitrophenyl -D-D- mannoside ) A former transition Reaction products  produce

In order to carry out the sugar transfer reaction, 0.07 mmole alpha-xylyl fluoride and 0.05 mmole of 4-nitrophenyl-beta-glucoside were dissolved in 3 mL of 50 mM sodium phosphate (pH 7.0) buffer, followed by 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 Floridwa  4- Nitrophenyl -2- Deoxy -2- Azaido Beta-glucoside (4- nitrophenyl  2- deoxy -2- azido -D-D- glucoside ) A former transition  reaction

To carry out the sugar transfer reaction, 0.12 mmole alpha-xylyl 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 buffer, 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 Floridwa  4- Nitrophenyl Beta-cellobioside (4- nitrophenyl -β-D-cellobioside) A former transition  reaction

To perform the sugar transfer reaction, 0.27 mmole alpha-xylyl fluoride and 0.12 mmole of 4-nitrophenyl-beta-cellobioside were dissolved in 5 mL of 50 mM sodium phosphate (pH 7.0) buffer. 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 Floridwa  2,4- Dynatrophenyl -2- Deoxy -2- Floro Beta-cellobiosides (2,4- dinitrophenyl  2- deoxy -2- fluoro -D-D- cellobioside ) A former transition  reaction

To perform the sugar transfer reaction, 5 mL of 50 mM of 8.68 μmole alpha-xylyl fluoride and 8.68 μmole of 2,4-dytrophenyl-2-deoxy-2-fluoro-beta-cellobioside were added. After dissolving in sodium phosphate (pH 7.0) buffer, 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: A former transition Reaction products  refine

In Example 5-1, the reaction solution of 5-5 was injected into a C18 SEP PAK cartridge (Waters), washed with water to remove non-aryl sugars, and the sugar transition product was eluted with ethanol. Ethanol was removed from the eluted reaction product solution under a reduced pressure concentrator. The residue was added with 2 mL of pyridine and the same amount of acetic anhydride to acetylate all hydroxyl groups, then the solvent was removed and the sugar transfer product was analyzed by silica gel chromatography. After the solvent was completely removed through concentration under reduced pressure, the yield of sugar transfer product was calculated based on the weight of the sugar transfer product. The sugar transfer yield of the resulting product is shown in Table 5 below.

Example Party donor Sugar receptor Yield (%) 5-1 Alpha-Xylosil Floride 4-nitrophenyl beta glucoside 96 5-2 4-nitrophenyl beta mannoside 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: A former transition  Product structure analysis

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

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. ( Example 5-1 reaction product )

1 H NMR (δ 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 9.2 Hz, 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 (δ ppm, CDCl ₃ , 100 MHz): 170.16, 170.06, 170.03, 169.70, 169.40, 169.19 (CH ₃ CO); 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 C 35 H 43 NO 22 + 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. Example  5-2 Reaction products )

¹ H NMR (CDCl ₃ , 400 MHz): d 8.32 (m, 2H, Ar-H), 7.16 (m, 2H, Ar-H), 5.71 (dd, 1H, 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 , 100 MHz): 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  (remind Example  5-3 Reaction products )

¹ H NMR (δ ppm, CDCl ₃ , 400 MHz): d 8.35 (m, 2H, Ar-H), 7.21 (m, 2H, Ar-H), 5.51 (t, 1H, 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 -D-D- glucopyranoside  (remind Example  5-4 Reaction products )

¹ H NMR (δ 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 to 3.82 (m, 3H, H-5, H-5 '& H-5 "), 3.74 to 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 , 100 MHz): 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 -D-D- glucopyranoside  (remind Example  5-5 Reaction products )

¹ H NMR (δ ppm, 600 MHz, CDCl ₃ ): 2.01, 2.04, 2.05, 2.06, 2.07, 2.08, 2.10, 2.17 (s, 24H, 8 CH 3 CO); 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, 1H, J _{5 , 6} 4.8, J _{6a , 6b} 12.6 Hz, H6), 4.59 (dd, 1H, 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, 1H, 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 CH ₃ CO); 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  Quantum donor mutant library ( YicI - D482X Production

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

order SEQ ID NO: YicID482X-F 5-GTA CAC TGG GGT GGC NNN TGT TAC GC TAA CTA C-3 12

YicI mutants in which the quantum donor / receptor catalyst of the YicI enzyme is substituted with any amino acid by the method of Example 3-1 using the YicID482X-F as a forward primer to the recombinant vector pETYicI6xH of Examples 1-2 (YicI-D482X, X means any amino acid). The conditions of the PCR are as described in Table 2 above.

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

8-2. A former transition  Active YicI  Selection of quantum donors / receptors

After transforming the prepared pETYicI-D482X6xH vector library by the method of Example 1-2, 98 transformants were inoculated in 0.2 mL LB medium containing 20 μg / mL kanamycin, respectively. After producing YicI variants 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-xylyl fluoride and 0.1 mmole of 4-nitrophenyl-beta-glucoside substrate solution dissolved in 0.1 mL of 50 mM sodium phosphate (pH 7.0) buffer. The reaction was carried out at 37 ° C. for 12 hours, and the reaction solution was analyzed by thin layer chromatography. Figure 9 shows TLC data showing 18 of the 98 crude enzyme solutions. The QUAGEN plasmid separation kit was used to isolate the plasmid vector containing the gene encoding the YicI mutase from 16 samples exhibiting sugar transfer activity, and the nucleotide sequence was determined. As a result, transformants A4, A7, C3, C6, C10, E10, E12, and F1 were aspartate, YicI's quantum donor / receptor catalyst, alanine, and transformants C9, E2, E3, G1, H1 were serine. , Transformants E4, F6, F8 was confirmed to be 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 catalyst site amino acid residues 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 mutated to alanine, serine, or glycine at the 482th aspartate residue, which is a YicI proton donor / receptor catalyst, used a glycosylated product using alpha-xylyl fluoride as a sugar donor. It can be seen that the synthesis.

Example  9: Bacillus halodurans  Alpha- Xilocity days Mutant  Manufacturing and A former transition  Activity detection

9-1. Construction of Bacillus halodurans alpha-xyloxides variant (BHaX-D481A)

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

order SEQ ID NO: Forward primer
BHaXTOP 5'-TAGGGAACAACATATGAAATTTTCAGATGG-3 ' 13 Reverse primer
BHaXEND 5'-AACCCTCGAGGACGCCTAGGTGGACAACCA-3 ' 14

PCR products were obtained by B. halodurans C-125 strain chromosomal DNA using the forward primer and the reverse primer as in Example 1 above.

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

order SEQ ID NO: Forward Primer BHaXD481A-F 5'-CCTGTTCACTGGGGTGGAGCTTGTGCCGCAACT-3 ' 15

In Example 3-1, the YicID482A-F primer was replaced with BHaXD481A-F, and in the same manner as in Example 3-1, a gene encoding a mutant in which aspartate, a quantum donor / receptor of BHaX, was substituted with alanine pETBHaXD481A6xH was produced.

9-2. Production and Transfer of BHaX-D481A

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 carried out using purified BHaXD481A as in Example 5-1, and the reaction product was analyzed using a PE-Sciex API 300 triple quadrupole mass spectrometer (Sciex, Canada). Is the spectrum. As a result of analysis, the product having the same molecular weight (molecular weight = 433) as the sugar transfer product synthesized by YicI-D482A in Example 5-1 is a sodium salt (M + Na ⁺ , molecular weight = 433 + 23), potassium salt (M + K ⁺ , molecular weight = 433 + 39), and a dimeric sodium salt (2M + Na ⁺ , molecular weight = 433 x 2 + 23). Therefore, it was confirmed that BHaXD481A mutase also had the same glycotransfer activity as YicI-D482A through mutation of the BHaX quantum donor / receptor catalyst.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> An alpha-Xylosidase mutants modified at their          proton-donor / acceptor catalyst and high efficiency          transglycosylation with the same <160> 15 <170> Kopatentin 1.71 <210> 1 <211> 772 <212> PRT <213> YicI <400> 1 Met Lys Ile Ser Asp Gly Asn Trp Leu Ile Gln Pro Gly Leu Asn Leu   1 5 10 15 Ile His Pro Leu Gln Val Phe Glu Val Glu Gln Gln Asp Asn Glu Met              20 25 30 Val Val Tyr Ala Ala Pro Arg Asp Val Arg Glu Arg Thr Trp Gln Leu          35 40 45 Asp Thr Pro Leu Phe Thr Leu Arg Phe Phe Ser Pro Gln Glu Gly Ile      50 55 60 Val Gly Val Arg Ile Glu His Phe Gln Gly Ala Leu Asn Asn Gly Pro  65 70 75 80 His Tyr Pro Leu Asn Ile Leu Gln Asp Val Lys Val Thr Ile Glu Asn                  85 90 95 Thr Glu Arg Tyr Ala Glu Phe Lys Ser Gly Asn Leu Ser Ala Arg Val             100 105 110 Ser Lys Gly Glu Phe Trp Ser Leu Asp Phe Leu Arg Asn Gly Glu Arg         115 120 125 Ile Thr Gly Ser Gln Val Lys Asn Asn Gly Tyr Val Gln Asp Thr Asn     130 135 140 Asn Gln Arg Asn Tyr Met Phe Glu Arg Leu Asp Leu Gly Val Gly Glu 145 150 155 160 Thr Val Tyr Gly Leu Gly Glu Arg Phe Thr Ala Leu Val Arg Asn Gly                 165 170 175 Gln Thr Val Glu Thr Trp Asn Arg Asp Gly Gly Thr Ser Thr Glu Gln             180 185 190 Ala Tyr Lys Asn Ile Pro Phe Tyr Met Thr Asn Arg Gly Tyr Gly Val         195 200 205 Leu Val Asn His Pro Gln Cys Val Ser Phe Glu Val Gly Ser Glu Lys     210 215 220 Val Ser Lys Val Gln Phe Ser Val Glu Ser Glu Tyr Leu Glu Tyr Phe 225 230 235 240 Val Ile Asp Gly Pro Thr Pro Lys Ala Val Leu Asp Arg Tyr Thr Arg                 245 250 255 Phe Thr Gly Arg Pro Ala Leu Pro Pro Ala Trp Ser Phe Gly Leu Trp             260 265 270 Leu Thr Thr Ser Phe Thr Thr Asn Tyr Asp Glu Ala Thr Val Asn Ser         275 280 285 Phe Ile Asp Gly Met Ala Glu Arg Asn Leu Pro Leu His Val Phe His     290 295 300 Phe Asp Cys Phe Trp Met Lys Ala Phe Gln Trp Cys Asp Phe Glu Trp 305 310 315 320 Asp Pro Leu Thr Phe Pro Asp Pro Glu Gly Met Ile Arg Arg Leu Lys                 325 330 335 Ala Lys Gly Leu Lys Ile Cys Val Trp Ile Asn Pro Tyr Ile Gly Gln             340 345 350 Lys Ser Pro Val Phe Lys Glu Leu Gln Glu Lys Gly Tyr Leu Leu Lys         355 360 365 Arg Pro Asp Gly Ser Leu Trp Gln Trp Asp Lys Trp Gln Pro Gly Leu     370 375 380 Ala Ile Tyr Asp Phe Thr Asn Pro Asp Ala Cys Lys Trp Tyr Ala Asp 385 390 395 400 Lys Leu Lys Gly Leu Val Ala Met Gly Val Asp Cys Phe Lys Thr Asp                 405 410 415 Phe Gly Glu Arg Ile Pro Thr Asp Val Gln Trp Phe Asp Gly Ser Asp             420 425 430 Pro Gln Lys Met His Asn His Tyr Ala Tyr Ile Tyr Asn Glu Leu Val         435 440 445 Trp Asn Val Leu Lys Asp Thr Val Gly Glu Glu Glu Ala Val Leu Phe     450 455 460 Ala Arg Ser Ala Ser Val Gly Ala Gln Lys Phe Pro Val His Trp Gly 465 470 475 480 Gly Asp Cys Tyr Ala Asn Tyr Glu Ser Met Ala Glu Ser Leu Arg Gly                 485 490 495 Gly Leu Ser Ile Gly Leu Ser Gly Phe Gly Phe Trp Ser His Asp Ile             500 505 510 Gly Gly Phe Glu Asn Thr Ala Pro Ala His Val Tyr Lys Arg Trp Cys         515 520 525 Ala Phe Gly Leu Leu Ser Ser His Ser Arg Leu His Gly Ser Lys Ser     530 535 540 Tyr Arg Val Pro Trp Ala Tyr Asp Asp Glu Ser Cys Asp Val Val Arg 545 550 555 560 Phe Phe Thr Gln Leu Lys Cys Arg Met Met Pro Tyr Leu Tyr Arg Glu                 565 570 575 Ala Ala Arg Ala Asn Ala Arg Gly Thr Pro Met Met Arg Ala Met Met             580 585 590 Met Glu Phe Pro Asp Asp Pro Ala Cys Asp Tyr Leu Asp Arg Gln Tyr         595 600 605 Met Leu Gly Asp Asn Val Met Val Ala Pro Val Phe Thr Glu Ala Gly     610 615 620 Asp Val Gln Phe Tyr Leu Pro Glu Gly Arg Trp Thr His Leu Trp His 625 630 635 640 Asn Asp Glu Leu Asp Gly Ser Arg Trp His Lys Gln Gln His Gly Phe                 645 650 655 Leu Ser Leu Pro Val Tyr Val Arg Asp Asn Thr Leu Leu Ala Leu Gly             660 665 670 Asn Asn Asp Gln Arg Pro Asp Tyr Val Trp His Glu Gly Thr Ala Phe         675 680 685 His Leu Phe Asn Leu Gln Asp Gly His Glu Ala Val Cys Glu Val Pro     690 695 700 Ala Ala Asp Gly Ser Val Ile Phe Thr Leu Lys Ala Ala Arg Thr Gly 705 710 715 720 Asn Thr Ile Thr Val Thr Gly Ala Gly Glu Ala Lys Asn Trp Thr Leu                 725 730 735 Cys Leu Arg Asn Val Val Lys Val Asn Gly Leu Gln Asp Gly Ser Gln             740 745 750 Ala Glu Ser Glu Gln Gly Leu Val Val Lys Pro Gln Gly Asn Ala Leu         755 760 765 Thr Ile Thr Leu     770 <210> 2 <211> 773 <212> PRT <213> BHaX <400> 2 Met Lys Phe Ser Asp Gly Gln Trp Leu Thr Arg Gln Glu Tyr Thr Ile   1 5 10 15 Ile Gly Ala Val Gln Val His Glu Ile Ile Gln Gly Glu Ala Ser Met              20 25 30 Thr Val Leu Ala Ser Pro Lys Pro Ile Val Asp Arg Tyr Gly Gln Leu          35 40 45 Asp Thr Pro Leu Leu Glu Ile Thr Phe Ser Ala Pro Arg Arg Glu Met      50 55 60 Ile Arg Val Gln Ile Asp His His Lys Gly Gly Arg Lys Arg Gly Pro  65 70 75 80 Val Phe Thr Thr Lys His Asp His Lys His Gln Ala Ser Phe Thr Glu                  85 90 95 Thr Glu Thr Thr Ala Ile Leu Thr Ser Gly Asp Leu Ser Val Thr Val             100 105 110 His Lys Glu Gly Asp Trp Leu Ile Thr Phe Ser Tyr Gln Gly Lys Thr         115 120 125 Ile Thr Ser Ser Gly Ser Lys Ser Ile Ala His Ile Leu Ala Gln Asp     130 135 140 Gly Lys Ala Tyr Met Arg Glu Gln Leu Ser Leu Gln Pro Asp Glu Met 145 150 155 160 Ile Tyr Ala Leu Gly Glu Arg Phe Thr Pro Leu Ile Arg Asn Gly Gln                 165 170 175 Val Val Asp Ile Trp Asn Lys Asp Gly Gly Thr Asn Thr Glu Gln Ser             180 185 190 Tyr Lys Asn Val Pro Phe Tyr Leu Ser Asn Lys Gly Tyr Gly Val Phe         195 200 205 Val Asn His Pro Glu Trp Val Ser Phe Glu Val Gly Ser Glu Ser Val     210 215 220 Ser Lys Ser Gln Phe Ser Val Glu Gly His Arg Leu Asp Tyr Tyr Val 225 230 235 240 Met Ala Gly Pro Ser Met Lys Lys Val Ile Glu Ala Tyr Thr Asp Leu                 245 250 255 Thr Gly Lys Pro Ala Leu Pro Pro Ala Trp Ser Phe Gly Leu Trp Leu             260 265 270 Ser Thr Ser Phe Thr Thr As As Tyr Asp Glu Ala Thr Val Thr Gln Phe         275 280 285 Ile Asp Gly Met Asn Glu Arg Asp Leu Pro Val His Val Phe His Phe     290 295 300 Asp Cys Phe Trp Met Lys Glu Phe Glu Trp Cys Asn Phe Glu Trp Tyr 305 310 315 320 Arg Arg Val Phe Pro Glu Pro Glu Lys Met Leu Gln Arg Leu Lys Glu                 325 330 335 Lys Gly Leu Lys Leu Ser Val Trp Ile Asn Pro Tyr Ile Ala Gln Arg             340 345 350 Ser Pro Leu Phe Gln Glu Ala Ala Ala Asn Gly Tyr Leu Leu Lys Lys         355 360 365 Glu Asn Gly Asp Val Trp Gln Trp Asp Leu Trp Gln Pro Gly Met Gly     370 375 380 Val Val Asp Phe Thr Asn Pro Asp Ala Arg Ile Trp Tyr Gln Asp His 385 390 395 400 Leu Arg Arg Leu Leu Glu Met Gly Val Asp Cys Phe Lys Thr Asp Phe                 405 410 415 Gly Glu Arg Ile Pro Thr Asp Val Val Tyr His Asp Gly Ser Asp Pro             420 425 430 Glu Lys Met His Asn Tyr Tyr Thr Phe Leu Tyr Asn Gln Thr Val Phe         435 440 445 Asp Val Leu Lys Gln Val Lys Gly Asn His Glu Ala Val Leu Phe Ala     450 455 460 Arg Ser Ala Thr Ala Gly Ser Gln Gln Phe Pro Val His Trp Gly Gly 465 470 475 480 Asp Cys Ala Ala Thr Tyr Ser Ser Met Ala Glu Ser Leu Arg Gly Gly                 485 490 495 Leu Ser Leu Gly Met Ser Gly Phe Gly Tyr Trp Ser His Asp Ile Gly             500 505 510 Gly Phe Glu Ser Gln Ser Thr Ala Asp Leu Tyr Lys Arg Trp Thr Ala         515 520 525 Phe Gly Leu Leu Ser Ser His Ser Arg Leu His Gly Asn Lys Ser Tyr     530 535 540 Arg Val Pro Trp Val Tyr Asp Glu Glu Ala Thr Asp Val Leu Arg Gln 545 550 555 560 Phe Thr Lys Trp Lys Cys Arg Leu Met Pro Tyr Leu Tyr Ala Lys Ala                 565 570 575 Cys Glu Ala Arg Thr Thr Gly Leu Pro Leu Met Arg Ala Met Val Leu             580 585 590 Glu Phe Gln Asp Asp Pro Thr Cys Ala Phe Leu Asp Arg Gln Tyr Met         595 600 605 Leu Gly Asp Gln Leu Leu Val Ala Pro Ile Phe Asn Glu Glu Gly Leu     610 615 620 Ala His Tyr Tyr Val Pro Asp Gly Arg Trp Thr Asn Leu Leu Thr Gly 625 630 635 640 Lys Thr Val Glu Gly Gly Ser Trp Lys Lys Glu His His Asp His Leu                 645 650 655 Ser Ile Pro Leu Leu Val Arg Pro Asn Ser Ile Val Pro Ile Gly Ser             660 665 670 Val Asp Asp Arg Pro Asp Tyr Asp Tyr Thr Asp Asn Val Ala Phe His         675 680 685 Val Phe Ala Leu Glu Asn Tyr Ala Thr Thr Ser Ile Tyr Thr Val Glu     690 695 700 Gly Glu Glu Ala Leu Thr Leu Ser Ala Thr Arg Ser Thr Thr Thr Val 705 710 715 720 Thr Phe Asp Ile Ser Asp Gly Ser Lys Pro Trp Thr Val His Leu His                 725 730 735 Asp Val Thr Glu Val Ser Ser Val Glu Gly Ala Asp Phe Glu Ile Ser             740 745 750 Asp Gly Ser Val Ile Leu His Pro Ile Leu Asp Val Lys Gln Val Val         755 760 765 Val His Leu Gly Val     770 <210> 3 <211> 772 <212> PRT <213> Artificial Sequence <220> <223> YicI-D482A <400> 3 Met Lys Ile Ser Asp Gly Asn Trp Leu Ile Gln Pro Gly Leu Asn Leu   1 5 10 15 Ile His Pro Leu Gln Val Phe Glu Val Glu Gln Gln Asp Asn Glu Met              20 25 30 Val Val Tyr Ala Ala Pro Arg Asp Val Arg Glu Arg Thr Trp Gln Leu          35 40 45 Asp Thr Pro Leu Phe Thr Leu Arg Phe Phe Ser Pro Gln Glu Gly Ile      50 55 60 Val Gly Val Arg Ile Glu His Phe Gln Gly Ala Leu Asn Asn Gly Pro  65 70 75 80 His Tyr Pro Leu Asn Ile Leu Gln Asp Val Lys Val Thr Ile Glu Asn                  85 90 95 Thr Glu Arg Tyr Ala Glu Phe Lys Ser Gly Asn Leu Ser Ala Arg Val             100 105 110 Ser Lys Gly Glu Phe Trp Ser Leu Asp Phe Leu Arg Asn Gly Glu Arg         115 120 125 Ile Thr Gly Ser Gln Val Lys Asn Asn Gly Tyr Val Gln Asp Thr Asn     130 135 140 Asn Gln Arg Asn Tyr Met Phe Glu Arg Leu Asp Leu Gly Val Gly Glu 145 150 155 160 Thr Val Tyr Gly Leu Gly Glu Arg Phe Thr Ala Leu Val Arg Asn Gly                 165 170 175 Gln Thr Val Glu Thr Trp Asn Arg Asp Gly Gly Thr Ser Thr Glu Gln             180 185 190 Ala Tyr Lys Asn Ile Pro Phe Tyr Met Thr Asn Arg Gly Tyr Gly Val         195 200 205 Leu Val Asn His Pro Gln Cys Val Ser Phe Glu Val Gly Ser Glu Lys     210 215 220 Val Ser Lys Val Gln Phe Ser Val Glu Ser Glu Tyr Leu Glu Tyr Phe 225 230 235 240 Val Ile Asp Gly Pro Thr Pro Lys Ala Val Leu Asp Arg Tyr Thr Arg                 245 250 255 Phe Thr Gly Arg Pro Ala Leu Pro Pro Ala Trp Ser Phe Gly Leu Trp             260 265 270 Leu Thr Thr Ser Phe Thr Thr Asn Tyr Asp Glu Ala Thr Val Asn Ser         275 280 285 Phe Ile Asp Gly Met Ala Glu Arg Asn Leu Pro Leu His Val Phe His     290 295 300 Phe Asp Cys Phe Trp Met Lys Ala Phe Gln Trp Cys Asp Phe Glu Trp 305 310 315 320 Asp Pro Leu Thr Phe Pro Asp Pro Glu Gly Met Ile Arg Arg Leu Lys                 325 330 335 Ala Lys Gly Leu Lys Ile Cys Val Trp Ile Asn Pro Tyr Ile Gly Gln             340 345 350 Lys Ser Pro Val Phe Lys Glu Leu Gln Glu Lys Gly Tyr Leu Leu Lys         355 360 365 Arg Pro Asp Gly Ser Leu Trp Gln Trp Asp Lys Trp Gln Pro Gly Leu     370 375 380 Ala Ile Tyr Asp Phe Thr Asn Pro Asp Ala Cys Lys Trp Tyr Ala Asp 385 390 395 400 Lys Leu Lys Gly Leu Val Ala Met Gly Val Asp Cys Phe Lys Thr Asp                 405 410 415 Phe Gly Glu Arg Ile Pro Thr Asp Val Gln Trp Phe Asp Gly Ser Asp             420 425 430 Pro Gln Lys Met His Asn His Tyr Ala Tyr Ile Tyr Asn Glu Leu Val         435 440 445 Trp Asn Val Leu Lys Asp Thr Val Gly Glu Glu Glu Ala Val Leu Phe     450 455 460 Ala Arg Ser Ala Ser Val Gly Ala Gln Lys Phe Pro Val His Trp Gly 465 470 475 480 Gly Ala Cys Tyr Ala Asn Tyr Glu Ser Met Ala Glu Ser Leu Arg Gly                 485 490 495 Gly Leu Ser Ile Gly Leu Ser Gly Phe Gly Phe Trp Ser His Asp Ile             500 505 510 Gly Gly Phe Glu Asn Thr Ala Pro Ala His Val Tyr Lys Arg Trp Cys         515 520 525 Ala Phe Gly Leu Leu Ser Ser His Ser Arg Leu His Gly Ser Lys Ser     530 535 540 Tyr Arg Val Pro Trp Ala Tyr Asp Asp Glu Ser Cys Asp Val Val Arg 545 550 555 560 Phe Phe Thr Gln Leu Lys Cys Arg Met Met Pro Tyr Leu Tyr Arg Glu                 565 570 575 Ala Ala Arg Ala Asn Ala Arg Gly Thr Pro Met Met Arg Ala Met Met             580 585 590 Met Glu Phe Pro Asp Asp Pro Ala Cys Asp Tyr Leu Asp Arg Gln Tyr         595 600 605 Met Leu Gly Asp Asn Val Met Val Ala Pro Val Phe Thr Glu Ala Gly     610 615 620 Asp Val Gln Phe Tyr Leu Pro Glu Gly Arg Trp Thr His Leu Trp His 625 630 635 640 Asn Asp Glu Leu Asp Gly Ser Arg Trp His Lys Gln Gln His Gly Phe                 645 650 655 Leu Ser Leu Pro Val Tyr Val Arg Asp Asn Thr Leu Leu Ala Leu Gly             660 665 670 Asn Asn Asp Gln Arg Pro Asp Tyr Val Trp His Glu Gly Thr Ala Phe         675 680 685 His Leu Phe Asn Leu Gln Asp Gly His Glu Ala Val Cys Glu Val Pro     690 695 700 Ala Ala Asp Gly Ser Val Ile Phe Thr Leu Lys Ala Ala Arg Thr Gly 705 710 715 720 Asn Thr Ile Thr Val Thr Gly Ala Gly Glu Ala Lys Asn Trp Thr Leu                 725 730 735 Cys Leu Arg Asn Val Val Lys Val Asn Gly Leu Gln Asp Gly Ser Gln             740 745 750 Ala Glu Ser Glu Gln Gly Leu Val Val Lys Pro Gln Gly Asn Ala Leu         755 760 765 Thr Ile Thr Leu     770 <210> 4 <211> 773 <212> PRT <213> Artificial Sequence <220> <223> BHaX-D481A <400> 4 Met Lys Phe Ser Asp Gly Gln Trp Leu Thr Arg Gln Glu Tyr Thr Ile   1 5 10 15 Ile Gly Ala Val Gln Val His Glu Ile Ile Gln Gly Glu Ala Ser Met              20 25 30 Thr Val Leu Ala Ser Pro Lys Pro Ile Val Asp Arg Tyr Gly Gln Leu          35 40 45 Asp Thr Pro Leu Leu Glu Ile Thr Phe Ser Ala Pro Arg Arg Glu Met      50 55 60 Ile Arg Val Gln Ile Asp His His Lys Gly Gly Arg Lys Arg Gly Pro  65 70 75 80 Val Phe Thr Thr Lys His Asp His Lys His Gln Ala Ser Phe Thr Glu                  85 90 95 Thr Glu Thr Thr Ala Ile Leu Thr Ser Gly Asp Leu Ser Val Thr Val             100 105 110 His Lys Glu Gly Asp Trp Leu Ile Thr Phe Ser Tyr Gln Gly Lys Thr         115 120 125 Ile Thr Ser Ser Gly Ser Lys Ser Ile Ala His Ile Leu Ala Gln Asp     130 135 140 Gly Lys Ala Tyr Met Arg Glu Gln Leu Ser Leu Gln Pro Asp Glu Met 145 150 155 160 Ile Tyr Ala Leu Gly Glu Arg Phe Thr Pro Leu Ile Arg Asn Gly Gln                 165 170 175 Val Val Asp Ile Trp Asn Lys Asp Gly Gly Thr Asn Thr Glu Gln Ser             180 185 190 Tyr Lys Asn Val Pro Phe Tyr Leu Ser Asn Lys Gly Tyr Gly Val Phe         195 200 205 Val Asn His Pro Glu Trp Val Ser Phe Glu Val Gly Ser Glu Ser Val     210 215 220 Ser Lys Ser Gln Phe Ser Val Glu Gly His Arg Leu Asp Tyr Tyr Val 225 230 235 240 Met Ala Gly Pro Ser Met Lys Lys Val Ile Glu Ala Tyr Thr Asp Leu                 245 250 255 Thr Gly Lys Pro Ala Leu Pro Pro Ala Trp Ser Phe Gly Leu Trp Leu             260 265 270 Ser Thr Ser Phe Thr Thr As As Tyr Asp Glu Ala Thr Val Thr Gln Phe         275 280 285 Ile Asp Gly Met Asn Glu Arg Asp Leu Pro Val His Val Phe His Phe     290 295 300 Asp Cys Phe Trp Met Lys Glu Phe Glu Trp Cys Asn Phe Glu Trp Tyr 305 310 315 320 Arg Arg Val Phe Pro Glu Pro Glu Lys Met Leu Gln Arg Leu Lys Glu                 325 330 335 Lys Gly Leu Lys Leu Ser Val Trp Ile Asn Pro Tyr Ile Ala Gln Arg             340 345 350 Ser Pro Leu Phe Gln Glu Ala Ala Ala Asn Gly Tyr Leu Leu Lys Lys         355 360 365 Glu Asn Gly Asp Val Trp Gln Trp Asp Leu Trp Gln Pro Gly Met Gly     370 375 380 Val Val Asp Phe Thr Asn Pro Asp Ala Arg Ile Trp Tyr Gln Asp His 385 390 395 400 Leu Arg Arg Leu Leu Glu Met Gly Val Asp Cys Phe Lys Thr Asp Phe                 405 410 415 Gly Glu Arg Ile Pro Thr Asp Val Val Tyr His Asp Gly Ser Asp Pro             420 425 430 Glu Lys Met His Asn Tyr Tyr Thr Phe Leu Tyr Asn Gln Thr Val Phe         435 440 445 Asp Val Leu Lys Gln Val Lys Gly Asn His Glu Ala Val Leu Phe Ala     450 455 460 Arg Ser Ala Thr Ala Gly Ser Gln Gln Phe Pro Val His Trp Gly Gly 465 470 475 480 Ala Cys Ala Ala Thr Tyr Ser Ser Met Ala Glu Ser Leu Arg Gly Gly                 485 490 495 Leu Ser Leu Gly Met Ser Gly Phe Gly Tyr Trp Ser His Asp Ile Gly             500 505 510 Gly Phe Glu Ser Gln Ser Thr Ala Asp Leu Tyr Lys Arg Trp Thr Ala         515 520 525 Phe Gly Leu Leu Ser Ser His Ser Arg Leu His Gly Asn Lys Ser Tyr     530 535 540 Arg Val Pro Trp Val Tyr Asp Glu Glu Ala Thr Asp Val Leu Arg Gln 545 550 555 560 Phe Thr Lys Trp Lys Cys Arg Leu Met Pro Tyr Leu Tyr Ala Lys Ala                 565 570 575 Cys Glu Ala Arg Thr Thr Gly Leu Pro Leu Met Arg Ala Met Val Leu             580 585 590 Glu Phe Gln Asp Asp Pro Thr Cys Ala Phe Leu Asp Arg Gln Tyr Met         595 600 605 Leu Gly Asp Gln Leu Leu Val Ala Pro Ile Phe Asn Glu Glu Gly Leu     610 615 620 Ala His Tyr Tyr Val Pro Asp Gly Arg Trp Thr Asn Leu Leu Thr Gly 625 630 635 640 Lys Thr Val Glu Gly Gly Ser Trp Lys Lys Glu His His Asp His Leu                 645 650 655 Ser Ile Pro Leu Leu Val Arg Pro Asn Ser Ile Val Pro Ile Gly Ser             660 665 670 Val Asp Asp Arg Pro Asp Tyr Asp Tyr Thr Asp Asn Val Ala Phe His         675 680 685 Val Phe Ala Leu Glu Asn Tyr Ala Thr Thr Ser Ile Tyr Thr Val Glu     690 695 700 Gly Glu Glu Ala Leu Thr Leu Ser Ala Thr Arg Ser Thr Thr Thr Val 705 710 715 720 Thr Phe Asp Ile Ser Asp Gly Ser Lys Pro Trp Thr Val His Leu His                 725 730 735 Asp Val Thr Glu Val Ser Ser Val Glu Gly Ala Asp Phe Glu Ile Ser             740 745 750 Asp Gly Ser Val Ile Leu His Pro Ile Leu Asp Val Lys Gln Val Val         755 760 765 Val His Leu Gly Val     770 <210> 5 <211> 2319 <212> DNA <213> Artificial Sequence <220> <223> YicI-D482A <400> 5 atgaaaatta gcgatggaaa ctggttgatt caacctggcc tcaatttgat tcacccgctt 60 caggtgttcg aggttgaaca gcaggataat gaaatggtgg tctatgctgc cccccgtgat 120 gtgcgtgaac gtacctggca gcttgatacg cctttattta cgttgcgctt tttctcccca 180 caggaaggta ttgtcggtgt gcggattgag cattttcagg gggcgctgaa taacggtcct 240 cattatccgc tcaatatttt gcaggacgtg aaggtcacaa tcgaaaacac agaacgttat 300 gctgagttta aaagtggcaa cttaagcgcg cgtgtcagca aaggtgagtt ctggtcactg 360 gattttctgc gcaacggcga acgtattacc ggtagtcagg tgaaaaataa tggctacgtg 420 caggacacga ataatcaacg caattatatg tttgagcggc ttgatcttgg cgttggcgaa 480 acagtttacg gtctgggaga gcgctttact gccctggtgc gcaatggcca gacggtagag 540 acctggaacc gggacggcgg cacaagtact gaacaggcgt ataaaaatat cccgttctac 600 atgactaacc gtggttatgg ggtactggtc aatcatcccc agtgtgtctc ttttgaagtg 660 ggatcggaga aagtctccaa agtgcagttc agcgttgaga gtgaatatct cgaatacttt 720 gttatcgacg gcccgacgcc gaaagcggta cttgatcgtt atacccgctt tactggtcgt 780 ccggcgctgc cgcccgcgtg gtccttcggc ctgtggctaa ccacttcatt taccaccaac 840 tacgacgaag cgacggtaaa cagctttatc gatggtatgg cggaacgcaa tctgccgctg 900 catgttttcc actttgactg tttctggatg aaagccttcc agtggtgcga ttttgagtgg 960 gacccgctga ctttccctga cccggaaggg atgatccgcc gcctgaaagc gaaaggactg 1020 aaaatctgcg tctggattaa cccctatatc ggtcaaaaat cccccgtctt taaagagtta 1080 caagagaaag gctatttact caaacgcccg gacggttcgc tatggcagtg ggataaatgg 1140 cagccaggtc tggcgattta tgactttacc aatccggatg cctgcaaatg gtacgccgac 1200 aaactgaaag gtctggtcgc gatgggcgtt gattgcttta agaccgactt tggcgaacgt 1260 atcccaactg atgttcagtg gtttgacggt tccgatccgc agaaaatgca taaccattat 1320 gcgtacatct acaacgaact ggtgtggaac gtgctcaagg acaccgttgg tgaggaagaa 1380 gctgtcttgt ttgcccgctc ggcctccgtc ggtgcgcaga aattcccggt acactggggt 1440 ggcgcgtgtt acgctaacta cgaatcaatg gcggaaagcc tgcgcggtgg tttgtctatt 1500 ggcctttcag gttttggctt ctggagccac gatatcggcg gctttgaaaa taccgctccg 1560 gcgcacgttt acaaacgctg gtgcgcgttt ggtttgctct ccagccatag ccgtttacac 1620 ggtagcaaat cttatcgtgt gccgtgggcc tacgatgatg agtcctgtga tgtggtgcgc 1680 ttcttcacgc aactgaaatg ccgcatgatg ccgtatctgt atcgtgaagc tgcgcgtgcg 1740 aacgcgcggg gtacgccgat gatgcgggcc atgatgatgg agttcccgga cgatccggct 1800 tgtgattacc ttgaccgtca atacatgtta ggcgacaacg tgatggttgc gccggtgttc 1860 actgaagcgg gcgatgtgca gttctacctg ccggaaggtc gctggacaca cctgtggcac 1920 aacgatgaac tcgacggtag tcgctggcat aaacagcagc acggcttcct gagtctgccc 1980 gtttatgtgc gtgataacac tctactggcg ctgggcaaca acgatcaacg tcccgattac 2040 gtgtggcacg aaggcacggc attccacctc ttcaatctgc aagacgggca tgaagccgtc 2100 tgtgaagtgc ccgctgctga cggatcggtg atctttactt taaaagcagc acgtactggc 2160 aacacgatta ctgtgactgg tgcgggcgag gcgaagaact ggacactgtg cctgcgcaat 2220 gttgtgaaag taaatggtct gcaagacggt tcgcaggctg aaagtgagca ggggctggtg 2280 gtgaagcctc aagggaatgc gctgacaatt acgttgtaa 2319 <210> 6 <211> 2322 <212> DNA <213> Artificial Sequence <220> <223> BHaX-D481A <400> 6 atgaaatttt cagatggtca gtggctgaca cgacaagagt atacaattat aggagcggtt 60 caagttcatg agatcataca aggggaagca tccatgacgg ttctagcctc gccgaaaccg 120 attgtggatc ggtatggaca gcttgatacc ccgttactgg agatcacttt ttccgcccct 180 aggcgagaga tgatccgcgt gcaaatcgat catcataaag gagggagaaa acgaggtccg 240 gtttttacta ccaaacatga tcacaagcat caagcaagtt ttacagaaac ggagacgacg 300 gcaatcctca caagtggcga cctatccgtc accgtccata aagaaggtga ttggctcatt 360 accttttcct atcaaggaaa aacgatcaca tcgagtggtt caaaaagcat cgcccacatt 420 ctcgctcagg acggaaaggc ttatatgcgt gagcagctta gtctccagcc agatgaaatg 480 atttatgctt taggcgaacg gtttacacct ttgattcgaa acggtcaagt cgtcgacatt 540 tggaataaag atggcggtac gaacacggag caatcgtata aaaatgttcc cttttatctt 600 tctaacaaag ggtacggcgt gttcgtgaac catccggagt gggtgtcgtt cgaagtagga 660 tctgaaagtg tgtcgaagtc tcagtttagc gtcgaaggtc atcgccttga ttactatgtg 720 atggctgggc cgtcgatgaa aaaagtcatt gaagcctaca ccgatttaac gggaaaacca 780 gcattgccgc cggcctggtc gttcggtctt tggctgtcca catcgtttac aacgaactat 840 gatgaagcga ctgtcaccca atttattgat gggatgaatg aacgcgactt gcctgttcac 900 gtgtttcatt ttgattgctt ctggatgaag gaatttgaat ggtgtaattt tgaatggtat 960 cggcgggtat ttccagagcc agaaaaaatg ctacagcgct taaaagaaaa aggcttgaag 1020 ctatctgtct ggatcaaccc ttatattgcg caacgctctc cgctgtttca agaagcggct 1080 gcaaatggat acttactgaa aaaagaaaac ggtgatgtat ggcaatggga tttatggcaa 1140 ccagggatgg gcgtggttga ctttacgaac cctgacgctc ggatttggta tcaggaccac 1200 cttcgcaggc ttctagaaat gggcgttgat tgttttaaaa ccgatttcgg tgaacggata 1260 ccgaccgatg tcgtgtatca cgatggctct gacccagaaa aaatgcataa ttactacacg 1320 ttcctctata atcaaacggt gtttgacgtc ttgaaacaag taaagggtaa tcatgaagcc 1380 gtcttatttg ctcgatcggc cacggctggc agtcaacaat tccctgttca ctggggtgga 1440 gcttgtgccg caacttactc gtctatggcg gaaagcttac gagggggatt atctctcggt 1500 atgtctggtt ttggctactg gagtcacgac attggcggct ttgaaagcca atccactgct 1560 gacctttata agcgctggac cgcctttggt ttattatcga gccatagtcg tctgcatgga 1620 aataaatcgt atcgagtccc gtgggtttat gatgaggaag ccaccgatgt tcttcgtcag 1680 tttacaaaat ggaaatgtcg cctcatgcca tacctttatg cgaaagcatg tgaagcaaga 1740 acgaccgggc tcccactcat gcgtgcgatg gtgttagaat tccaagacga tccgacgtgt 1800 gcgtttttag atcgccaata catgctcggg gatcagttgc ttgtggctcc gatcttcaat 1860 gaagaagggc tcgctcatta ctacgttcct gatggtcgtt ggacaaatct cttaacaggc 1920 aaaactgtgg aaggtggtag ctggaagaaa gagcatcacg accatctgag catcccttta 1980 ctcgttcgcc caaattcgat cgttccgatc ggctcggtgg atgaccgtcc agactatgac 2040 tatacagaca atgtggcatt ccacgtgttt gcccttgaaa attacgcgac aacatccatc 2100 tatacagtag agggagagga agcactcact ttatctgcga ctcgctccac cactacagtc 2160 acatttgata tatctgatgg aagcaagcct tggaccgtac atttacacga tgtcacagag 2220 gtgtctagcg ttgagggagc tgactttgaa attagcgatg gctctgtcat cctccaccct 2280 atccttgatg tgaaacaggt ggttgtccac ctaggcgtct aa 2322 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cagaactaag gaacgcatat gaaaattagc 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 atcaagctcg agcaacgtaa ttgtcagcgc 30 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gtacactggg gtggcgcgtg ttacgctaac tac 33 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tatgctagtt attgctcag 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 taatacgact cactataggg 20 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gtacactggg gtggcnnntg ttacgctaac tac 33 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tagggaacaa catatgaaat tttcagatgg 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aaccctcgag gacgcctagg tggacaacca 30 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cctgttcact ggggtggagc ttgtgccgca act 33

Claims (17)

delete delete delete delete delete delete delete delete delete delete delete The 482th amino acid residue of SEQ ID NO: 1 was mutated from aspartate to alanine to remove alpha-xyloxidase mutagenase, which was hydrolyzed and retained glycosylated activity against the alpha-xylyl fluoride substrate. The sugar transfer method used as this reaction catalyst and containing a sugar donor. The method of claim 12, wherein the sugar donor is alpha-xyloxy fluoride. 13. The method of claim 12, wherein the method comprises glucose, mannose, 4-nitrophenyl beta glucoside, 4-nitrophenyl beta mannokoside, 4-nitrophenyl 2-deoxy 2-azido-beta glucoside as sugar acceptors. , 4-nitrophenyl beta cellobioside, and 4-nitrophenyl 2-deoxy 2-fluoro-beta cellobioside further comprises a compound selected from the group consisting of. delete delete 13. The method of claim 12, wherein said alpha-xyloxidase mutant enzyme is obtained after immobilization on a carrier or expression on a cell surface.

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