KR101768748B1 - Mutated sucrose isomerase and process for preparing the same - Google Patents

Abstract

The present invention relates to a mutant sucrose isomerase derived from a sucrose isomerase capable of producing a large amount of trehalulose from sucrose, a polynucleotide encoding the mutated sucrose isomerase, an expression comprising the polynucleotide A vector, a transformant into which the expression vector has been introduced into a host cell, and a method for producing the mutated sucrose isomerase using the transformant. The mutated sucrose isomerase provided by the present invention newly represents sugar substitution activity which is not exhibited by the conventional sucrose isomerase, and thus can produce isomaltos which is another peanut sugar by using sucrose. Therefore, Which can be widely used.

Description

Disclosed is a mutated sucrose isomerase and a process for preparing the same.

More particularly, the present invention relates to a mutant sucrose isomerase derived from a sucrose isomerase capable of producing a large amount of trehalulose from sucrose, A polynucleotide encoding the mutated sucrose isomerase, an expression vector containing the polynucleotide, a transformant into which the expression vector has been introduced into the host cell, and the mutant sucrose isomerase using the transformant And a method for manufacturing the same.

BACKGROUND ART [0002] Sugars are used as a main source for supplying energy used in living organisms, and are used not only as a component constituting various active ingredients in living bodies, but also as a substance for enhancing palatability of various foods. In recent years, an excessive amount of sugar has been introduced into the body for the purpose of enhancing the palatability of food, and there is a concern about the risk due to excessive introduction of such sugar. For example, an increase in the incidence of cardiovascular diseases, Increased incidence, and increased incidence of immune system metabolic disturbances. To reduce these risks, research is under way to curb excessive influx of sugar, to reduce the influx of sugars, to effectively consume imported sugars, and to develop low-risk sugars.

Among the above measures, a method for developing low-risk sugars has the effect of improving the palatability of foods, but also exhibits an effect of reducing the risk due to excessive introduction of sugars, and active research is under way to develop various substitute products . It is known that isomaltulose, stevia, tagatose, taumarine, erythritol, maltitol, xylitol and the like are known as the natural compounds, and as the artificial compound, Acesulfame potassium, aspartame, neotham, saccharin, sucralose, and the like are known. Among the above-mentioned substitutes, artificial compounds are advantageous because they are easy to produce and have a low production cost. However, studies on the effects on living organisms are still insufficient and their use is limited, while natural compounds have been proved to be safe for living organisms While there are advantages, there is a drawback that the production cost is relatively high. At present, research for lowering the production cost of a natural compound is considered to be more efficient than studying the effect of an artificial compound on a living body, and researches for reducing the production cost of a natural compound are actively conducted.

Among the above natural compounds, isomaltulose (α-D-glucopyranosyl-1,6-D-fructofuranos) is produced by reacting sucrose (α-D-glucopyranosyl-1,2-D-fructofuranos ), Which has the same caloric value as sucrose but has a low glycemic index (GI), and glucose monomer produced from its decomposition is slowly introduced into the blood, so that it can be used as a sweetener for foods for diabetic patients And it is known that when administered to patients with hyperlipidemia, they increase resistance to vascular diseases. In addition, physical and chemical properties such as excellent acid stability and low hygroscopicity have been clarified, and the use range of isomaltulose has been expanded.

The isomaltulose can be produced by enzymatic treatment of sucrose. Trehalulose or? -D-glucopyranosyl-1,1-d-fructofuranos is produced as a by-product. The activity may be selected from the group consisting of Enterobacter sp., Erwinia sp., Klebsiella sp., Pantoea sp., Protaminobacter sp. It has been confirmed in various microorganisms such as microorganisms that sucrose isomerase or isomaltulose synthase (EC 5.4.99.11), which is expressed from these microorganisms, ≪ / RTI > and trehalulose. Most sucrose isomerases produce isomaltulose more than trehalulose, although some strains ( Pseudomonas mesoacidophila MX-45 and Agrobacterium The sucrose isomerase expressed in radiobacter MX-232 is known to produce trehalulose more than isomaltulose. It is known that the cause of the expression of sucrose isomerase in the microorganism is related to the characteristics of the microorganism. That is, most of the microorganisms are microorganisms parasitic to plants, and most plants use sucrose as a carbon source. Therefore, it is possible to convert sucrose obtained from plants into isomaltulose or trehalulose, It is predicted that the sucrose isomerase is expressed.

On the other hand, despite the advantages mentioned above, isomaltulose is lower in water solubility than sucrose and the use of food is limited. For example, when isomaltulose is used instead of sucrose in jams, beverages and the like, recrystallization of isomaltulose occurs in the product after storage for a certain period of time, thereby deteriorating the quality of the product. As a solution to this problem, a method of using trehalulose produced with isomaltulose has been studied. Trehalulose was expected to replace isomaltulose because it exhibits similar properties to isomaltulose but exhibits the same level of water solubility as sucrose. However, since it is produced by sucrose isomerase in a relatively smaller amount than isomaltulose, its production unit price is relatively high, and the industrial applicability is low.

Various studies have been conducted to solve these problems. For example, Korean Patent Publication No. 1992-0008192 discloses a method for producing trehalulose from sucrose using a trehalulose-producing enzyme expressed in a microorganism belonging to the genus Pseudomonas sp. Or Agrobacterium sp. a and discloses technology to produce in large quantities, the US Patent Publication No. 2013-0295617 discloses a number derived from a microorganism of the genus Solarium Peck tobak Te (Pectobacterium carotovorum subsp. brasiliensis PBR1692) rule trehalose from sucrose using a cross-isomerase A method of producing a Ross is disclosed. However, since trehalulose has low productivity, it is difficult to use trehalulose in the industry. In order to solve such difficulties, it is required to develop a technology capable of producing trehalulose with higher productivity .

Under these circumstances, the present inventors have made extensive efforts to develop a technique capable of producing trehalulose with higher productivity, and as a result, found that a strain producing trehalulose in a large amount as compared with isomaltulose was discovered, It was confirmed that the sucrose isomerase can produce a large amount of trehalulose from sucrose and that the mutant of sucrose isomerase exhibits an enzyme activity not exhibited by the conventional sucrose isomerase, .

One object of the present invention is to provide a mutated sucrose isomerase derived from a sucrose isomerase derived from a microorganism of the genus Pektobacterium ( Pectobacterium carotovorum KKH 3-1).

It is another object of the present invention to provide a polynucleotide encoding said mutated sucrose isomerase.

It is still another object of the present invention to provide an expression vector comprising the polynucleotide.

It is still another object of the present invention to provide a transformant into which the above expression vector has been introduced.

It is still another object of the present invention to provide a method for producing the mutated sucrose isomerase using the transformant.

In order to develop a technique capable of producing trehalulose with higher productivity, the present inventors cultured various strains expressing conventional sucrose isomerase in a medium containing sucrose, and found that trehalulose and isomaltulose Were selected first. The one among the selected strains selected for the highest productivity of strains rules trehalose, and identified it results peck tobak Te Karo Solarium Sat borum KKH 3-1 strain (Pectobacterium carotovorum KKH 3-1). The polynucleotide encoding the sucrose isomerase was amplified in the above-identified strains and its sequence was determined. As a result, it was confirmed that the polynucleotide was a gene encoding a novel sucrose isomerase not known in the prior art.

As a result of identifying the sucrose isomerase from the gene, the enzyme was found to have a size of about 69 kDa, activity at pH 5.0 to 10.0, maximum activity at pH 6.0, glycine NaOH buffer (pH 9) Maintaining a half-life of 25.7 minutes at 55 ° C, a melting temperature of 55.5 ± 0.6 ° C, exhibiting enzyme activity in the temperature range of 30 to 50 ° C, and exhibiting enzyme activity at 40 ° C And the productivity of trehalulose increased as the reaction temperature was lower.

As a result of analyzing the three-dimensional structure of the sucrose isomerase, the arginine at position 325 plays an important role, and when the arginine is substituted with lysine, the structure of the active site of the enzyme is not changed The enzyme activity was predicted to change. In order to prove this, the arginine at position 325 was substituted with lysine to produce a mutated sucrose isomerase, and its activity was compared with the wild-type sucrose isomerase. As a result, the enzyme exhibited completely different activity from the wild type sucrose isomerase, , Which was not observed in the sucrose isomerase of the present invention. Finally, it has been confirmed that when the sugar chain is used as a substrate and the enzyme reaction of the mutated sucrose isomerase is carried out, the isomaltose is produced as a new reaction product.

As one embodiment for achieving the above-mentioned object, the present invention relates to a microorganism belonging to the genus Pectobacterium carotovorum KKH 3-1) and provides a novel sucrose isomerase capable of producing a large amount of trehalulose from sucrose.

The term " sucrose isomerase " of the present invention means an enzyme which acts on sucrose among isomerases which act on a specific substance and change its molecular stereostructure or the medium of a specific atomic group. Generally refers to an enzyme that converts sucrose to isomaltulose or trehalulose.

In the present invention, the sucrose isomerase enzyme is Peck tobak Te tobak the peck type of microorganism of the genus Solarium Te Solarium Caro sat borum KKH 3-1 (Pectobacterium can be interpreted as a novel sucrose isomerase capable of producing a large amount of trehalulose from sucrose containing the amino acid sequence of SEQ ID NO: 1 derived from a carotovorum KKH 3-1 strain, As long as it can exhibit an enzyme activity that mass-produces trehalulose rather than los, it is possible to include all peptides to which various amino acid sequences are added at the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 1. In addition, it may further comprise a targeting sequence, a tag, an amino acid sequence designed for specific purposes to increase the stability of the labeled residue, half-life or peptide.

In addition, if the amino acid sequence of the amino acid sequence of SEQ ID NO: 1 is mutated by a method such as addition, substitution, deletion, or the like, as long as it can exhibit an enzyme activity that massively produces trehalulose from sucrose rather than isomaltulose May be included in the category of sucrose isomerase provided by the present invention.

For example, the sucrose isomerase provided in the present invention may include a polypeptide having a sequence that differs from the amino acid sequence of SEQ ID NO: 1 by one or more amino acid residues. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, such as heat, pH and the like.

Finally, the sucrose isomerase of the present invention may be prepared by a chemical peptide synthesis method known in the art, or by amplifying the gene encoding the sucrose isomerase by PCR or by a known method, And then expressing the gene.

The sucrose isomerase provided by the present invention is similar to the conventional sucrose isomerase in that it produces isomaltulose and trehalulose from sucrose but can produce more trehalulose than isomaltulose And is distinguished from conventional sucrose isomerase. In addition, depending on the enzyme reaction temperature, the production ratio of trehalulose and isomaltulose produced from sucrose is changed. The lower the reaction temperature, the greater the amount of trehalulose produced. As the reaction temperature increases The amount of isomaltulose produced increases. Specifically, the modification temperature (Tm) of the sucrose isomerase provided by the present invention is 55.5 ° C, exhibits enzyme activity at 25 to 55 ° C, and can exhibit 60% or more of the maximum activity at 30 to 50 ° C . According to the present inventors's findings, when trehalulose and isomaltulose are produced from sucrose by using the sucrose isomerase of the present invention at 30 to 50 ° C, trehalulose and isomaltulose Losses can be produced at a ratio of 1: 1 (w / w), but at 30 ° C, trehalulose and isomaltulose can be produced at a ratio of about 4: 1 (w / w) Has not been known from any strain so far and has been discovered for the first time by the present inventors.

In fact, that is expressed from a peck tobak Te Solarium Caro sat borum KKH 3-1 strain and similar strains of Peg tobak Te Solarium Caro sat borum Brazilian cis PBR1692 (Pectobacterium carotovorum subsp. Brasiliensis PBR1692 ) strains expressing a sucrose isomerase of the present invention It is known that sucrose isomerase exhibits properties similar to those provided by the present invention (US Patent Publication No. 2013-0295617). The sucrose isomerase expressed in the known strains can produce a reaction product containing trehalulose and isomaltulose in a ratio of 95: 5 to 63: 37 (w / w) at 1 to 30 DEG C , And at the same reaction temperature, the productivity of trehalulose is relatively lower than that of the sucrose isomerase provided in the present invention. For example, at a reaction temperature of 30 DEG C, the sucrose isomerase of the present invention can produce trehalulose and isomaltulose at a ratio of about 4: 1 (w / w), but the known sucrose isomerization Since the enzyme can produce trehalulose and isomaltulose at a ratio of about 1.7: 1 (w / w), the sucrose isomerase of the present invention can be produced from known sucrose in terms of productivity of trehalulose Which is 2.3 times higher than that of the isomerization enzyme. In view of the fact that the sucrose isomerase provided in the present invention is not artificially mutated in order to enhance enzyme activity but is naturally expressed from microorganisms existing in nature, it is considered that enzyme activity which is 2.3 times higher than that of known enzyme It is extremely unusual to newly discover the enzyme that represents

Accordingly, the present inventors deposited the nucleotide sequence of the gene encoding the novel sucrose isomerase and the amino acid sequence of the enzyme provided in the present invention on GenBank on Aug. 13, 2013, and the accession number KF317210. 1.

As described above, the sucrose isomerase provided by the present invention is excellent not only in the productivity of trehalulose, but also in controlling the reaction temperature, the ratio of the produced trehalulose to isomaltulose is very large . The conventional sucrose isomerase cited above produces trehalulose at a ratio of at least 1.7 times that of isomaltulose, whereas the sucrose isomerase of the present invention is a trehalulose It is possible to produce Ross and isomaltulose in the same ratio.

These characteristics are very important in industry. That is, even when the production demand of trehalulose and isomaltulose required for industrially producing trehalulose and isomaltulose using the sucrose isomerase provided by the present invention is greatly changed, It is possible to meet the changed production requirement by merely adjusting the reaction temperature without any change. Considering that isomaltulose is used in a wider range than industrial trehalylulose and tremendous demand of isomaltulose rather than trehalulose, the sucrose isomerase provided in the present invention is a conventional It can be said that the industrial utility is very high compared with the cross isomerization enzyme. That is, the sucrose isomerase of the present invention can also be used for the production of isomaltulose.

In addition, the pH range capable of exhibiting the optimum activity of the sucrose isomerase provided by the present invention is a neutral range of pH 5.0 to 7.0, and an extremely high dangerous pH level (for example, strong acidity or strong alkalinity) is not required Therefore, when the sucrose isomerase of the present invention is used, safety can be maximally ensured in the production step.

As another embodiment for achieving the above object, the present invention provides a polynucleotide comprising a nucleotide sequence encoding the sucrose isomerase.

In the present invention, the nucleotide sequence constituting the polynucleotide may be a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 1 or a variety of nucleotides capable of being added to the N- or C-terminus of the amino acid sequence of SEQ ID NO: The nucleotide sequence encoding the amino acid sequence may be added to the 5'-terminal or 3'-terminal of the nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 1, preferably the amino acid sequence of SEQ ID NO: 1 The nucleotide sequence of SEQ ID NO: 2 capable of coding the sequence or the nucleotide sequence encoding various amino acid sequences which may be added to the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 1 is the nucleotide sequence of the nucleotide sequence of SEQ ID NO: Terminal < / RTI > or 3 ' -terminal.

Also, as long as it is capable of encoding a protein capable of exhibiting an enzyme activity that mass-produces trehalulose rather than isomaltulose from sucrose, a polynucleotide comprising a nucleotide sequence homologous to the nucleotide sequence described above May be included in the category of the polynucleotide provided by the present invention. Preferably, the polynucleotide may be a polynucleotide including a nucleotide sequence having 80% or more homology, more preferably a nucleotide sequence having 90% or more homology Polynucleotide, and most preferably a polynucleotide comprising a nucleotide sequence that exhibits at least 95% homology.

On the other hand, the polynucleotide may be mutated by substitution, deletion, insertion, or a combination of one or more bases. When the nucleotide sequence is prepared by chemically synthesizing, it is possible to use a method well known in the art, for example, a method described in Engels and Uhlmann, Angew Chem IntEd Engl., 37: 73-127, 1988 , Triesters, phosphites, phosphoramidites and H-phosphate methods, PCR and other auto primer methods, and oligonucleotide synthesis on solid supports.

In another embodiment for achieving the above object, the present invention provides an expression vector comprising the polynucleotide.

The term "expression vector" of the present invention means a gene construct comprising a gene insert that is capable of expressing a desired protein in an appropriate host cell, and an essential regulatory element operably linked to the expression of said gene insert. The expression vector includes expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc. The initiation codon and termination codon are generally regarded as part of the nucleotide sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

The term "operably linked" of the present invention means a state in which a nucleic acid sequence encoding a desired protein or RNA is functionally linked to a nucleic acid expression control sequence so as to perform a general function . For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with an expression vector can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.

In addition, the expression vector may include a signal sequence for the release of sucrose isomerase to facilitate the separation of the protein from the cell culture medium. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers. In addition, the expression vector may further comprise a protein tag which can be optionally removed using endopeptidase, in order to facilitate detection of the sucrose isomerase.

The term "tag " of the present invention means a molecule exhibiting quantifiable activity or property and includes a polypeptide fluorescent substance such as a fluorophore such as fluorescein, a fluorescent protein (GFP) Or may be a fluorescent molecule including; Myc tag, a Flag tag, a histidine tag, a Lucin tag, an IgG tag, and a strap tagidine tag. Particularly, in the case of using an epitope tag, a peptide tag composed of preferably 6 or more amino acid residues, more preferably 8 to 50 amino acid residues can be used.

In the present invention, the expression vector is not particularly limited as long as it is capable of producing the sucrose isomerase provided by the present invention by expressing the polynucleotide. The mammalian cell (for example, human, monkey, rabbit, rat, hamster , Or a vector capable of replicating and / or expressing the polynucleotide in an eukaryotic or prokaryotic cell comprising a plant cell, a yeast cell, an insect cell or a bacterial cell (e.g., Escherichia coli, etc.) , Preferably operably linked to a suitable promoter so that the polynucleotide can be expressed in the host cell, and can be a vector comprising at least one selectable marker, more preferably a commercially available plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119) Subtilis (Bacillus subtilis) - derived plasmid (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50), λ- phage (Charon4A, Charon21A, EMBL3, EMBL4 , λgt10, λgt11 and λZAP), retroviruses (retrovirus), adenoviruses adenovirus, vaccinia virus, baculovirus, and the like. Since the amount of expression of the protein and the expression of the expression vector are different depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.

As another embodiment for accomplishing the above object, the present invention provides a transformant wherein the expression vector is introduced into a host cell.

The transformant provided in the present invention is produced by introducing the expression vector provided in the present invention into a host cell, transforming the polynucleotide, and expressing the polynucleotide contained in the expression vector to produce the sucrose isomerase of the present invention .

The host cell to which the expression vector provided in the present invention can be introduced is not particularly limited as long as it is capable of producing the peptide by expressing the polynucleotide. However, the host cell may be any of E. coli, Streptomyces, Salmonella typhimurium Bacterial cells such as; Yeast cells such as Saccharomyces cerevisiae, and ski-inspected caromyces pombe; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, Bowmanella cells; Or plant cells.

Further, the transformant may be performed by various methods, though not one capable of producing a sucrose isomerase of the present invention showing the effect which can improve a variety of cellular activity at a high level, specifically limited to, CaCl ₂ precipitation, CaCl ₂ precipitation to Hanahan method with improved efficiency by using a reducing substance of DMSO (dimethyl sulfoxide), electroporation (electroporation), the calcium phosphate precipitation method, protoplast fusion method, a stirring method using a silicon carbide fiber, Agrobacterium Bacterial-mediated transformation, transformation with PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

As another embodiment for achieving the above object, the present invention provides a method for producing the sucrose isomerase of the present invention using the above transformant.

Specifically, the method for producing the sucrose isomerase of the present invention comprises the steps of: (a) culturing the transformant to obtain a culture; And (b) recovering the sucrose isomerase of the present invention from the culture.

As another method, a method for producing the sucrose isomerase of the present invention comprises the steps of: (a) obtaining a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1; (b) cloning the obtained polynucleotide to obtain an expression vector; (c) introducing the obtained expression vector into a host cell to obtain a transformant; And (d) culturing the transformant, and recovering mRFP comprising the amino acid sequence of SEQ ID NO: 1.

The term "cultivation" of the present invention means a method of growing the microorganism under an appropriately artificially controlled environmental condition. In the present invention, the method for culturing the transformant may be carried out by a method well known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing the sucrose isomerase of the present invention. However, it may be cultured continuously in a batch process or an injection batch or a repeated batch batch process .

The medium used for culturing can meet the requirements of a specific strain in an appropriate manner while controlling the temperature, pH and the like under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like. The carbon sources that can be used include glucose and xylose mixed sugar as main carbon sources, and sugar and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Oils and fats such as oils and the like, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used.

In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in the culture process in a batch manner, in an oil-feeding manner or in a continuous manner by an appropriate method, but it is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture.

In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. An oxygen or oxygen-containing gas (e.g., air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is usually 27 ° C to 37 ° C, preferably 30 ° C to 35 ° C. The culture is continued until the production amount of the sucrose isomerase is maximized. And usually for 10 to 100 hours for this purpose.

In addition, the step of recovering the sucrose isomerase from the culture can be carried out by a method known in the art. Specifically, the recovering method is not particularly limited as long as the recovered sucrose isomerase of the present invention can be recovered, but it is preferably centrifuged, filtered, extracted, sprayed, dried, (Eg ammonium sulfate precipitation), chromatography (for example, ion exchange, affinity, hydrophobicity and size exclusion) can be used.

In another aspect of the present invention, there is provided a mutant sucrose isomerase comprising the amino acid sequence of SEQ ID NO: 3, wherein the arginine at position 325 of the sucrose isomerase is substituted with lysine do.

In the present invention, the mutant was designed by the present inventors through analysis of three-dimensional structure of sucrose isomerase. The sucrose isomerase provided by the present invention exhibits properties that produce trehalulose and isomaltulose from sucrose and almost no glucose or fructose as a by-product. As a result, sucrose was degraded by the catalytic residues 223, 287 and 361 of the sucrose isomerase, and the resulting fructosyl moiety was cleaved with aromatic clamps 289 and 313 Phenylalanine) and formed a hydrogen bond with arginine at number 325, and hydrolysis products were not detected. In addition, oligocl-1, 6-glucosidase (BCOGL) derived from Bacillus cereus and the sucrose isomerase provided by the present invention exhibit different enzymatic activities, but when compared with their active sites, , Arginine (sucrose isomerase) or lysine (BCOGL) is present only in the amino acid at position 325, and almost the same structure is exhibited. Therefore, arginine at position 325 of the sucrose isomerase of the present invention is converted into lysine It was expected that the substitution would produce a sucrose isomerase having an altered enzyme activity without inducing a structural change of the enzyme active site, and a desired mutated sucrose isomerase was prepared using a conventional PCR method. As a result of evaluating the activity of the mutated sucrose isomerase, it was confirmed that, as expected, isomaltulose and trehalulose were produced from sucrose while producing a large amount of glucose and fructose, which are by-products thereof, It was confirmed that additional by-products were produced, and their physical properties were confirmed, and it was confirmed that isomaltose.

The mutated sucrose isomerase produces isomaltose and trehalulose from sucrose in accordance with its characteristics and, as expected, produces the by-products glucose and fructose, the conventional sucrose isomerase and But the effect of generating isomaltose is a characteristic that has not been reported from conventional sucrose isomerase and was first identified through mutated sucrose isomerase of the present invention.

Isomaltose is a disaccharide produced by combining two glucose molecules, and shows a completely different structure from sucrose, isomaltulose and trehalulose having a structure in which glucose and fructose are combined. Therefore, the isomaltose Was not produced by the activity of the sucrose isomerase. This is because sucrose is decomposed by 223 aspartic acid, 287 glutamic acid and 361 aspartic acid, which are the catalytic residues of sucrose isomerase, as expected through the above three-dimensional structure, and other glucose And it was expected that it would have been generated. To confirm this, it was confirmed that the amount of isomaltose produced was increased by reacting mutant sucrose isomerase with sucrose and glucose. Therefore, it was confirmed that the mutated sucrose isomerase has both activity as sucrose isomerase and activity as a glycosyltransferase.

In another aspect of the present invention for achieving the above object, the present invention provides a polynucleotide comprising a nucleotide sequence encoding the mutated sucrose isomerase, an expression vector comprising the polynucleotide, And a method for producing the sucrose isomerase of the present invention using the transformant and the transformant.

The polynucleotide comprising the nucleotide sequence encoding the mutated sucrose isomerase may be a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 3 or an N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 3 May be added to the 5'-terminal or 3'-terminal of the nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 3, preferably the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 or a base sequence encoding various amino acid sequences which may be added to the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 3 is the nucleotide sequence of SEQ ID NO: May be added to the 5'-terminal or 3'-terminal of the nucleotide sequence, and other characteristics are the same as described above. In addition, the method for producing the sucrose isomerase of the present invention using the polynucleotide-containing expression vector, the transformant into which the expression vector has been introduced into the host cell, and the transformant are also the same as described above.

The mutated sucrose isomerase provided by the present invention newly represents sugar substitution activity which is not exhibited by the conventional sucrose isomerase, and thus can produce isomaltos which is another peanut sugar by using sucrose. Therefore, Which can be widely used.

1 is a photograph showing the results of applying the culture supernatant (A) of 11 strains producing isomaltulose and trehalulose, or the reaction product (B) obtained by treating the above culture supernatant with an invertase to TLC analysis, The lane 1 represents a G-lane, the F-lane represents fructose, the S-lane represents sucrose, the P-lane represents isomaltulose, the T-lane represents trehalulose, No. 3 lane represents strain No. 15, No. 4 lane represents No. 16 strain, No. 5 lane represents No. 23 strain, No. 6 lane represents No. 14 strain, No. 2 lane represents No. 14 strain, No. 3 lane represents No. 15 strain, No. 4 lane represents No. 16 strain, No. 5 lane represents No. 23 strain, The lane 7 represents the strain No. 33, the lane No. 8 represents the strain No. 34, the lane No. 9 represents the strain No. 39, the lane No. 10 represents the strain No. 44, the lane No. 11 represents the strain No. 33, 45 < / RTI >
Figure 2 shows the results of culturing the Pectobacterium carotovorum KKH 3-1 strain in four different media and measuring the growth level (A) of the strain and the activity (B) of the sucrose isomerase expressed from the strain Fig.
FIG. 3 is a graph showing the activity of sucrose isomerase according to the concentration of sucrose contained in the medium. FIG.
FIG. 4 shows the result of comparing the amino acid sequence of the novel sucrose isomerase with the sequence of a known sucrose isomerase, wherein the sucrose isomerization motif (RLDRD) is represented by a red box, Represents an enzyme derived from Pectobacterium carotovorum KKH 3-1 of the present invention, and MutB represents an enzyme derived from Pseudomonas mesoacidophila MX-45 (Pseudomonas mesoacidophila MX-45) FMB-1 represents an enzyme derived from Enterobacter sp. FMB-1, ERSI represents an enzyme derived from Erwinia rhapontici NCPPB 1578, PalI represents an enzyme derived from Enterobacter sp. FMB-1, And KPSI represents an enzyme derived from Klebsiella planticola UQ 14S, and PDSI represents an enzyme derived from Pantoea disperse UQ68J. And SmuA represents an enzyme derived from protaminobacter rubrum CBS574 · 77 (Protaminobacter rubrum CBS574 · 77).
FIG. 5 is a photograph showing the result of SDS-PAGE analysis of a sample obtained in the course of purifying the sucrose isomerase of the present invention, wherein lane 1 represents a negative control (E. coli BL21), lane 2 represents cell lysate , And lane 3 represents a purified recombinant sucrose isomerase.
FIG. 6 shows the activity change of recombinant sucrose isomerase according to the reaction conditions. FIG. 6 (A) is a graph showing the activity change of recombinant isozymease according to the pH change, in which (●) represents sodium acetate buffer (?) Represents sodium citrate buffer (pH 5.0-6.0), (?) Represents sodium phosphate buffer (pH 6.0-8.0), and (Δ) represents sodium phosphate buffer 9.0), (1) represents glycine-NaOH buffer (pH 9.0-10.0); (B) is a graph showing the activity change of the recombinant sucrose isomerase according to the temperature change.
FIG. 7 shows the temperature characteristics of the recombinant sucrose isomerase, wherein (A) is a logarithm of the change in enzyme activity with temperature change, () denotes 50 ° C, and () denotes 55 ° C (?) Represents 60 占 폚; (B) is a graph showing the results of measuring the denaturation temperature of the recombinant sucrose isomerase.
Fig. 8 shows the results of three-dimensional structural analysis of recombinant sucrose isomerase, wherein 8a is a three-dimensional predicted structure of sucrose isomerase based on SmuA and 8b is an enlarged view of sucrose isomerase active site (crystal structure) , 8C is an enlarged view of the sucrose isomerase active site (crystal structure) associated with sucrose, the components are expressed in a light gray cartoon format: the catalytic residues D223, E287 and D361 are shown in pink; D94, H137, R231, R325, H360 and R448, which are reaction residues with the ligand, are shown in yellow; The aromatic clamps F289 and F313 are marked in red; RLDRD motifs R317, L318, D319, R320 and A321 are displayed in blue; The ligand was marked with a green bar.
9 shows the results of three-dimensional structural analysis of the respective active sites of oligo-1,6-glucosidase (BCOGL) and sucrose isomerase derived from Bacillus cereus and comparing them, wherein (A) represents BCOGL , And (B) represents sucrose isomerase.
Fig. 10 shows the result of displaying the active sites of the sucrose isomerase (A) and the mutated sucrose isomerase (B) in combination with sucrose in a light gray color comic form.
Fig. 11 shows the result of expressing the active site with a light gray color surface.
12 is an agarose gel electrophoresis of a 7,140-bp polynucleotide encoding an amino acid sequence obtained by substituting lysine for arginine at position 325 of the sucrose isomerase obtained by the PCR method, wherein the M-lane is a DNA marker, Lane is a PCR fragment amplified in pET-PCSI.
13 shows a vector map of pET-R325K, which is an expression vector containing a polynucleotide encoding an amino acid sequence in which arginine at position 325 of the sucrose isomerase is replaced with lysine.
14 shows the result of comparing the amino acid sequence of the sucrose isomerase with the amino acid sequence of the mutated sucrose isomerase derived from the DNA of the transformant into which the expression vector pET-R325K is introduced, wherein the substituted residue is a red box , PCSI represents a sucrose isomerase derived from Pectobacterium carotovorum KKH 3-1, and R325K represents a mutated sucrose isomerase.
FIG. 15 is a photograph showing the result of SDS-PAGE analysis of a product purified by using Ni-NTA affinity column chromatography to express a sucrose isomerase mutated from pET-R325K, and M lane is a standard marker Lane 1 represents a negative control (E. coli), lane 2 represents cell lysate, and lane 3 represents a purified mutant sucrose isomerase.
16 is a graph showing the results of analysis of reaction products of sucrose isomerase (black) and mutated sucrose isomerase (red) with HPAEC, wherein the first peak represents glucose and the second peak represents fructose , The third peak represents sucrose, the fourth peak represents trehalulose, and the fifth peak represents isomaltulose.
17A is a graph showing the results of HPAEC analysis of a sucrose isomerase (black) and a mutated sucrose isomerase (red) reaction product, wherein the first peak indicates glucose, the second peak indicates fructose The third peak indicates sucrose, the fourth peak indicates unknown substance, the fifth peak indicates trehalulose, and the sixth peak indicates isomaltulose.
FIG. 17 (B) is a graph showing the HPAEC analysis results of the reaction products produced by enzymatic reaction of sucrose isomerase (black) and mutated sucrose isomerase (red) performed using glucose, wherein 1 Wherein the first peak represents glucose, the second peak represents fructose, the third peak represents sucrose, the fourth peak represents unknown substance, the fifth peak represents trehalulose, the sixth peak represents iso Represents maltulose.
18 (A) is a graph showing the results of applying reaction products of mutated sucrose isomerase to a recycling preparative HPLC system.
FIG. 18 (B) is a graph showing the results of application of the fraction No. 3 to the HPAEC analysis among the nine fractions obtained through the recycling preparative HPLC system, wherein the black graph shows the mutated sucrose isomerase reaction product, The first peak represents glucose, the second peak represents fructose, the third peak represents sucrose, the fourth peak represents the unknown substance, and the fifth peak represents trehalulose And peak 6 indicates isomaltulose.
19 (A) is a graph showing the results of HPAEC analysis of a digestion product obtained by digesting the fraction No. 3 obtained in the recycling preparative HPLC system with -.alpha.-glucosidase, wherein the black fraction is the fraction 3 without α-glucosidase , Red represents fraction 3 treated with? -Glucosidase, the first peak represents glucose, and the second peak represents unknown substance.
FIG. 19 (B) is a graph showing the results of applying the fraction 3 and isomaltose obtained in the recycling preparative HPLC system to HPAEC analysis, wherein black indicates isomaltose, red indicates fraction 3, The peak indicates glucose and the second peak indicates unknown substance.
20 is a photograph showing the result of TLC analysis of the fraction No. 3 obtained in the recycling preparative HPLC system, wherein lane 1 represents a standard glucose, lane 2 represents a standard fructose, lane 3 represents a standard sucrose, Lane 4 represents the standard isomaltulose, lane 5 represents the fraction 3, and lane 6 represents 1% isomaltose.
FIG. 21 is a graph showing the results of analyzing the sugar transfer activity of mutated sucrose isomerase with respect to various hexoses by HPAEC analysis, wherein (A) shows the result of analysis of the reaction product using hexose as an aldehyde, (B) shows the result of analysis of the reaction product using galactose as hexadentate, and (C) shows the analysis result of the reaction product using mannose as hexadentate.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Trehalulose Production strain  Selection

53 plant-related microorganisms, including seven species of Enterobacteriaceae, three species of Pantoea subsp. Microorganisms and 43 species of Pectobacterium sp. Microorganisms, were isolated from various crops including vegetables and fruit trees and isolated Were cultured in LB medium (containing 1% (w / v) tryptone, 0.5% (w / v) yeast extract, and 1% (w / v) NaCl). The cultured strains were inoculated into 5 ml of SPY medium (4% sucrose, 1% peptone, and 0.4% yeast extract, pH 6.5) and cultured overnight at 30 ° C with vigorous stirring. 11 strains (13, 14, 15, 16, 23, 24, 33, 34, 39, 44 and 45 strains) producing isomaltulose and trehalulose were analyzed by the first selection Respectively.

A culture supernatant was obtained by culturing the selected strain (4 ° C., 12,000 × g), and this was applied to TLC analysis. Specifically, 3 μl of the culture supernatant was applied to activated silica gel K5F plates at 110 ° C. for 30 minutes and eluted with a developing solution (ethyl acetate: acetic acid: water = 4: 3: 0.8, v / v / v ) Was added to the plate at room temperature twice. The plate was then air-dried and immersed in a chromogenic reagent (methanol in which 0.5% (w / v) N- (1-naphthyl) -ethylenediamine and 5% (v / v) H ₂ SO ₄ had been dissolved) And developed in an oven at 110 ° C for 10 minutes to show a sugar-dotted region (Fig. 1 (A)). Also, the same method was applied to the reaction product obtained by adding invertase (Sigma-Aldrich Co.) to the culture supernatant (FIG. 1 (B)). At this time, glucose, fructose, sucrose, isomaltulose and trehalulose were used as standard substances.

1 is a photograph showing the results of applying the culture supernatant (A) of 11 strains producing isomaltulose and trehalulose, or the reaction product (B) obtained by treating the above culture supernatant with an invertase to TLC analysis, The lane 1 represents a G-lane, the F-lane represents fructose, the S-lane represents sucrose, the P-lane represents isomaltulose, the T-lane represents trehalulose, No. 3 lane represents strain No. 15, No. 4 lane represents No. 16 strain, No. 5 lane represents No. 23 strain, No. 6 lane represents No. 14 strain, No. 2 lane represents No. 14 strain, No. 3 lane represents No. 15 strain, No. 4 lane represents No. 16 strain, No. 5 lane represents No. 23 strain, The lane 7 represents the strain No. 33, the lane No. 8 represents the strain No. 34, the lane No. 9 represents the strain No. 39, the lane No. 10 represents the strain No. 44, the lane No. 11 represents the strain No. 33, 45 < / RTI > As shown in FIG. 1, it was confirmed that levels of trehalulose and isomaltulose were different depending on strains. That is, strains 14 (2 lanes), 16 strains (4 lanes), 44 strains (10 lanes) and 45 strains (11 lanes) (Lane 1), strain 15 (lane 3) and strain 39 (lane 9) were prepared from some sucrose at a level of isomaltulose and trehalulose And strain 23 (strain 5), strain 24 (strain 6) and strain 33 (strain 7) produced small amounts of isomaltulose and trehalulose from trace sucrose, Strain # 34 (lane 8) produced various products. When the sucrose present in the culture medium is decomposed into glucose and fructose by treatment of the invertase in the culture supernatant, the strains 13 (lane 1), 15 (strain 3) and 23 (Lane 7), Lane 34 (Lane 8), and Strain 39 (Lane 9), the sucrose remaining in the culture is degraded Glucose and fructose were formed. However, strains 14 (No. 2 lane), No. 16 strain (No. 4 lane), No. 44 strain (No. 10 lane) and No. 45 strain (No. 11 lane) It was confirmed that glucose and fructose were not formed due to no cross. Thus, four strains (strains 14, 16, 44 and 45), in which all sucrose was transformed into isomaltulose and trehalulose, were secondly selected.

The culture of the second selected strain was applied to the HPAEC analysis method to quantify the produced sugars. Specifically, the culture supernatant of the second selected strain was filtered to remove impurities, and each of the supernatants in which the impurities were removed was placed in an analytical CarboPac PA1 column (0.4) connected to a Dionex model DX-600 system equipped with an ED50 electrochemical detector cm × 25 cm; Dionex Co., Sunnyvale, Calif., USA), followed by isocratic separation by linear gradient for 10 to 40 minutes at a rate of 1 ml / min using 200 mM NaOH, Were quantitatively analyzed. As a result, it was confirmed that the production yield of isomaltulose and trehalulose was the best in the strain No. 45, and the strain No. 45 was finally selected.

As a result of the identification of the finally selected strain No. 45, it was confirmed that the strain No. 45 was Pectobacterium carotovorum KKH 3-1 (P. carotovorum KKH 3-1), which is a kind of microorganism of the genus Pektobacterium.

Example  2: Pectobacterium Carrotoborum KKH  3-1 Characterization of strains

0.5% peptone and 0.3% beef extract, 1.2% tryptone and 2.4% tryptone were added to the selected strain of Pectobacterium carotovorum KKH 3-1, which was finally selected in Example 1, yeast extract, and 0.4% glycerol) and peptone-yeast extract medium (PY; 1% peptone and 0.4% yeast extract) and incubated at 30 ° C for 30 hours. The activity of the sucrose isomerase was compared (Fig. 2).

Figure 2 shows the results of culturing the Pectobacterium carotovorum KKH 3-1 strain in four different media and measuring the growth level (A) of the strain and the activity (B) of the sucrose isomerase expressed from the strain Fig. As shown in FIG. 2, the Pctobacterium carotovorum KKH 3-1 strain cultured in the TB medium showed the highest proliferation level and the lowest sucrose isomerase activity, whereas the Pctobacterium luciferum cultured in the PY medium The tobomu KKH 3-1 strain showed low proliferation level and the highest sucrose isomerase activity.

Subsequently, 0 to 20% (w / v) sucrose was added to the PY medium to prepare a medium containing different amounts of sucrose. To each of the prepared medium, Pectobacterium carotovorum KKH 3 -1 strain, and then cultured at 30 ° C for 30 hours to measure the activity of sucrose isomerase (FIG. 3). The activity of the sucrose isomerase was determined by DNS (3,5-dinitrosalicylic acid) method. The activity of 1 unit of sucrose isomerase was determined by the amount of enzyme capable of producing 1 μM sucrose isomer Respectively.

FIG. 3 is a graph showing the activity of sucrose isomerase according to the concentration of sucrose contained in the medium. FIG. As shown in Fig. 3, in the medium containing sucrose up to 14% (w / v), the activity of sucrose isomerase was increased as the sucrose content was increased. However, in the medium containing sucrose, The activity of sucrose isomerase decreased as the content of cross increased.

Example  3: Pectobacterium Carrotoborum KKH  From strain 3-1 Derived Sucross Of the truncated enzyme gene Cloning

Example  3-1: Sucrose isomerase  Identification of genes

First, the following primers were designed based on conservative amino acid sequences in various sucrose isomerases.

Forward primer: 5'-CAT ATG TAC GAA CAA GTC TCC CAC TCG-3 '(SEQ ID NO: 5)

Reverse primer: 5'-CTC GAG AAG CTG TAA CCA ATA AAA GCC-3 '(SEQ ID NO: 6)

Next, PCR was performed using the genomic DNA extracted from the culture of the above-mentioned Pectobacterium carotovorum KKH 3-1 strain as a template and the primer designed above to obtain a DNA fragment. The PCR was carried out at 95 ° C for 5 minutes, 25 cycles (95 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 2 minutes) and 72 ° C for 5 minutes. The nucleotide sequence of the obtained DNA fragment was analyzed (SEQ ID NO: 1), and the analyzed nucleotide sequence was applied to BLASTN and BLASTP (http://www.ncbi.nlm.nih.gov/) to identify the nucleotide sequence and its analog (SEQ ID NO: 2) was searched (Fig. 4).

FIG. 4 shows the result of comparing the amino acid sequence of the novel sucrose isomerase with the sequence of a known sucrose isomerase, wherein the sucrose isomerization motif (RLDRD) is represented by a red box, Represents an enzyme derived from Pectobacterium carotovorum KKH 3-1 of the present invention, and MutB represents an enzyme derived from Pseudomonas mesoacidophila MX-45 (Pseudomonas mesoacidophila MX-45) FMB-1 represents an enzyme derived from Enterobacter sp. FMB-1, ERSI represents an enzyme derived from Erwinia rhapontici NCPPB 1578, PalI represents an enzyme derived from Enterobacter sp. FMB-1, And KPSI represents an enzyme derived from Klebsiella planticola UQ 14S, and PDSI represents an enzyme derived from Pantoea disperse UQ68J. And SmuA represents an enzyme derived from protaminobacter rubrum CBS574 · 77 (Protaminobacter rubrum CBS574 · 77). As shown in FIG. 4, the sucrose isomerase derived from the above-mentioned Pfactobacterium carotovorum KKH 3-1 strain has an amino acid sequence which is not identical to the sucrose isomerase derived from other conventional microorganisms in terms of amino acid sequence Respectively. In addition, about 95% identity with the sucrose isomerase derived from Pectobacterium carotovorum subsp. Brasiliensis PBR1692 belonging to the same genus as the Pectobacterium carotovorum KKH 3-1 strain of the present invention But it is composed of a new amino acid sequence which is not completely identical.

The present inventors deposited the nucleotide sequence of the gene encoding the novel sucrose isomerase and the amino acid sequence of the enzyme on GenBank on Aug. 13, 2013 and received accession number KF317210.1 .

Example  3-2: Sucrose isomerase  Gene Cloning , Expression and purification

The DNA fragment obtained in Example 3-1 was purified by applying to a purification kit (QIA quick gel extraction kit, Qiagen, Valencia, CA, USA) and cloned into a pGEMT-easy vector using a complementary restriction enzyme cleavage site Respectively. Then, the sucrose isomerase gene was extracted from the cloned vector, and the extracted gene was introduced into the pET-21a (+) vector to prepare an expression vector pET-PCSI. At this time, an expression vector was designed so that the sucrose isomerase expressed in the expression vector is expressed in the form that 6 histidine is bound to the C-terminal of the protein.

The prepared expression vector pET-PCSI was introduced into Escherichia coli BL21 to obtain a transformant. The obtained transformant was inoculated into 1 L of LB medium containing 0.1 mg / ml of ampicillin, . When the absorbance of the culture reached 0.5 at 600 nm, 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to induce expression of sucrose isomerase at 37 ° C. for 3 hours. The culture was then centrifuged at 6000 x g for 20 minutes at 4 DEG C to obtain cells and washed twice with 50 mM sodium phosphate buffer (pH 7.0). Wherein the washed cells were suspended in cell lysis buffer _{10㎖ (50 mM NaH 2 PO 4} , 300 mM NaCl and 10 mM imidazole, pH 7.0), treated in an ultrasonic hot water tank (4 min × 5 bun, output control 4, 50% duty cycle; VC-600, Sonics & Materials, Inc., Newtown, CT, USA) and centrifuged at 12,000 x g for 20 minutes at 4 占 폚.

The supernatant was applied to nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography equilibrated with a cell lysis buffer and washed with washing buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl and 20 mM imidazole, pH 7.0) It washed three times and then elution buffer _{(50 mM NaH 2 PO 4,} 300 mM NaCl and 250 mM imidazole, pH 7.0) to be eluted using recombinant cross-isomerase. Finally, the fraction containing the eluted recombinant sucrose isomerase was dialyzed to remove excess imidazole to thereby purify the recombinant sucrose isomerase (FIG. 5).

FIG. 5 is a photograph showing the result of SDS-PAGE analysis of a sample obtained in the course of purifying the sucrose isomerase of the present invention, wherein lane 1 represents a negative control (E. coli BL21), lane 2 represents cell lysate , And lane 3 represents a purified recombinant sucrose isomerase.

Example  4: Pectobacterium Carrotoborum KKH  From strain 3-1 Derived Sucross Characterization of the enrichment enzyme

The characteristics of the recombinant sucrose isomerase purified in Example 3-2 were analyzed.

Example  4-1: pH  Analysis of Enzyme Activities by Changes

Sodium acetate buffer (pH 5.0-6.0), sodium phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 7.0-9.0) and glycine-NaOH buffer (pH 9.0-5.0) 10.0), the change in the activity of the recombinant sucrose isomerase was measured by the DNS method and compared (Fig. 6 (A)).

FIG. 6A is a graph showing changes in the activity of the recombinant sucrose isomerase according to pH change. FIG. 6A shows sodium acetate buffer (pH 4.0-5.0), and FIG. 6C shows changes in sodium citrate buffer (?) Represents sodium phosphate buffer (pH 6.0-8.0), (?) Represents Tris-HCl buffer (pH 7.0-9.0) and (?) Represents glycine-NaOH buffer 9.0-10.0). As shown in FIG. 6 (A), the recombinant sucrose isomerase exhibited high activity at pH 5.0 to 7.0, and showed optimal activity at pH 6.0.

Example  4-2: Enzyme activity analysis by temperature change

The activity change of the recombinant sucrose isomerase was measured by a DNS method under the temperature condition of 25 to 60 ° C and then compared (FIG. 6 (B)).

6 (B) is a graph showing changes in the activity of the recombinant sucrose isomerase according to the temperature change. As shown in FIG. 6 (B), the recombinant sucrose isomerase exhibited high activity under temperature conditions of 30 to 50 ° C and showed optimal activity at 40 ° C.

Example  4-3: half-life temperature ( half - life 온도 ) Measure

The half-life temperature was determined by measuring the activity change of the recombinant sucrose isomerase in sodium citrate buffer (pH 6.0) for 30 minutes under temperature conditions of 50, 55 and 60 캜 (Fig. 7 (A)).

7 (A) is a graph showing the change in enzyme activity with temperature change as a log value, in which () indicates 50 ° C, () indicates 55 ° C, and () indicates 60 ° C . As shown in FIG. 7 (A), the activity of sucrose isomerase was maintained at 50 ° C., but the activity of sucrose isomerase decreased to 50% at 25 ° C. at 55 ° C., It was confirmed that the activity of the sucrose isomerase was almost not exhibited.

Example  4-4: Denaturation temperature ( melting 온도 ) Measure

DSF (differential scanning fluorimetry) method was used to measure the melting temperature of the recombinant sucrose isomerase.

Specifically, a sample was prepared by mixing 10 μl of an enzyme solution (0.1 mg / ml) and 10 μl of SYPRO orange (10 ×), applying the sample to a real-time PCR system and incubating the mixture at 30 ° C to 99 ° C Respectively. All fluorescence values were measured using an Applied Biosystems 7500 Fast RT-PCR system (Applied Biosystems, Grand Island, NY, USA) and run on a computer program (Sigma Plot 12.0 software, Systat Software Inc., Chicago, IL, USA and Accel program) were used to perform graph analysis and data statistical analysis (Fig. 7 (B)).

7 (B) is a graph showing the results of measurement of the denaturation temperature of the recombinant sucrose isomerase. As shown in FIG. 7 (B), the denaturation temperature of the recombinant sucrose isomerase was found to be 55.5 ± 0.6 ° C.

Example  4-5: Sucrose  Production of isomers

In order to confirm whether or not the reaction product was changed according to the reaction temperature of the recombinant sucrose isomerase, enzyme reaction was performed using sucrose as a substrate in a temperature range of 30 to 50 ° C. The reaction products were applied to the HPAEC analysis to quantitate the resulting glucose, fructose, trehalulose and isomaltulose (Table 1).

Changes in activity of recombinant sucrose isomerase according to reaction temperature Reaction temperature
(° C) Glucose and
Fructose (%) Trehalulose
(%) Isomaltulose
(%) ratio
(Trehalulose / isomaltulose) 30
35
40
45
50 0.06
0.35
0.89
1.64
2.56 79.16
70.92
63.26
54.61
47.54 20.72
28.38
34.96
42.10
47.33 79/21
71/29
64/36
56/44
50/50

As shown in Table 1, it was confirmed that the amount of trehalulose increased as the reaction temperature was lower, while the amount of isomaltulose increased as the reaction temperature increased.

Example  4-6: Various Sucrose isomerase  Character rating

The characteristics of the sucrose isomerase provided in the present invention were evaluated by comparing the main characteristics of the sucrose isomerase expressed from various strains known in the art with the recombinant sucrose isomerase provided in the present invention (Table 2).

Information and enzymatic characteristics of sucrose isomerase from various strains Strain
number Registration Number
Homology
(%) Molecular Weight
(kDa) pI Optimum temperature
(° C) Optimum pH Product Ratio (%) Trehalulose Isomaltulose #One
#2
# 3
#4
# 5
# 6
# 7
#8 KF317210
ACF42098.1
AAP57084.1
AAP57085.1
AAK82938.1
AAP57083.1
EKF64560
ABC33903.1 100
66
67
70
70
66
68
67 68.8
70.2
73.3
70.0
69.9
69.4
69.6
66.8 5.96
6.02
6.62
6.62
6.58
6.99
7.11
5.30 40
50
30
35
35
35
30
40 6.0
5.0-6.0
7.0
6.0
6.0
5.0
5.5
5.8 64
22
15
15
21
3
9
91 36
78
85
66
83
91
86
8

Strain # 1 represents Pectobacterium carotovorum KKH 3-1, and # 2 represents Enterobacter sp. FMB-1, # 3 represents Erwinia rhapontici NCPPB 1578, # 4 represents Klebsiella planticola UQ 14S, and # 5 represents Klebsiella sp. LX3, # 6 represents Pantoea disperse UQ68J, # 7 represents Protaminobacter rubrum CBS574-77, and # 8 represents Pseudomonas mesoacidophila MX-45.

Example  5: Sucrose isomerase  3D structure analysis and Mutant  making

The three-dimensional structure of the recombinant sucrose isomerase was analyzed and based on this, a mutant sucrose isomerase exhibiting mutated enzyme activity was produced.

Example  5-1: 3D structure analysis

Analysis of the three-dimensional structure of the recombinant sucrose isomerase was carried out according to a known method using SWISSMODEL (http://swissmodel.expasy.org), an automated comparative protein modeling server (Arnold K, et al., Bioinformatics. 22: 195-201, 2006; Kiefer F, et al., Nucleic Acids Research., 37: D387-D92, 2009; Peitsch M., Bio / technology., 13: 658-660, 1995). At this time, the crystal structure of SmuA (pdb code = 3GBD), which is a sucrose isomerase having a crystal structure already identified, was obtained from Brookhaven Protein Data Bank (PDB, http://www.rcsb.org) The structure was obtained from the PubChem database, and based on this, the three-dimensional structure of the recombinant sucrose isomerase was predicted.

In addition, using the AutoDock 4.2 software, the sucrose was bound to the active site of the sucrose isomerase. For this purpose, a grid box was used. The binding analysis result was changed to a file that can be used in AutoDock4 using AutoDock Tools 1.5.6. , And the analysis results were visualized using PyMOL (http://www.pymol.org) (FIGS. 8A to 8C).

Figure 8a shows the three-dimensional predicted structure of sucrose isomerase based on SmuA and Figure 8b shows an enlarged view of the sucrose isomerase active site (crystal structure), the components of which are expressed in a light gray cartoon format: D223, E287 and D361 are indicated in pink; D94, H137, R231, R325, H360 and R448, which are reaction residues with the ligand, are shown in yellow; The aromatic clamps F289 and F313 are marked in red; The RLDRD motifs R317, L318, D319, R320 and A321 are shown in blue. Each residue was labeled according to the protein number of the sucrose isomerase. Figure 8c shows an enlarged view of the sucrose isomerase active site (crystal structure) associated with sucrose, the components being expressed in a light gray cartoon format and the ligand being indicated by a green bar.

As shown in Figs. 8A to 8C, sucrose isomerase exhibited the structure of the enzyme belonging to the family of the typical glycoside hydrolase family 13, and the active site exhibited the property of the GH13 enzyme. As a result of molecular binding studies to identify the optimal mode of conjugation with sucrose, sucrose was degraded by the catalytic residues 223 aspartic acid, 287 glutamic acid and 361 aspartic acid, The tosyl moiety was found to bind to aromatic clamps (phenylalanine at 289 and 313) and form a hydrogen bond with arginine at number 325, indicating that hydrolysis products would not be generated.

Example  5-2: Mutated Sucrose isomerase  design

The arginine at position 325 is a part of the fructose binding site and is known to bind to the ligand sucrose and is expected to form a hydrogen bond with the fructosyl moiety after binding to sucrose, By-product, such as isomers, is not produced, and the isomerization reaction is expected to be carried out. Therefore, it was expected that the substitution of arginine at position 325 with another amino acid would cause a change in the activity of sucrose isomerase.

Although oligo-1,6-glucosidase (BCOGL) derived from Bacillus cereus and sucrose isomerase exhibit different enzymatic activities, the active sites thereof are compared (Fig. 9) (BCOGL, FIG. 9 (A)) or arginine (sucrose isomerase, FIG. 9 (B)). Therefore, it was expected that the substitution of arginine 325 of sucrose isomerase with lysine would change the activity of sucrose isomerase.

Thus, a simulated computer program was used to simulate the substitution of lysine for arginine at position 325 of the sucrose isomerase (FIGS. 10 and 11).

FIG. 10 shows the result of displaying the active sites of the sucrose isomerase (A) and the mutated sucrose isomerase (B) in combination with sucrose in a light gray color comic form, and FIG. 11 shows the result of displaying the active site as a light gray color surface . As shown in FIGS. 10 and 11, it was confirmed that even when the arginine at position 325 of the sucrose isomerase was replaced with lysine, the active site was opened and sucrose could be bound. However, since arginine is not present, it can not form a hydrogen bond with the fructosyl moiety, resulting in the formation of by-products such as glucose and fructose, and the reaction products can be predicted to change.

Example  5-3: Mutated Sucrose isomerase  making

Example  5-3-1: Mutated Sucrose isomerase  Obtaining the polynucleotide encoding

PCR was carried out using QuikChange site-directed mutagenesis kit (Stratagene) using pET-PCSI as a template and the primers shown below to obtain 7,140 bp coding amino acid sequence in which arginine at position 325 of the sucrose isomerase was substituted with lysine Of the polynucleotide was obtained (Fig. 12). At this time, the PCR conditions were 25 cycles (94 ° C for 1 minute, 57 ° C for 1 minute, and 72 ° C for 8 minutes).

R325K-F: 5'-cgatcgcgccgtagaagaaaaatggcgacgga-3 '(SEQ ID NO: 7)

R325K-R: 5'-tccgtcgccatttttcttctacggcgcgatcg-3 '(SEQ ID NO: 8)

12 is an agarose gel electrophoresis of a 7,140-bp polynucleotide encoding an amino acid sequence obtained by substituting lysine for arginine at position 325 of the sucrose isomerase obtained by the PCR method, wherein the M-lane is a DNA marker, Lane is a PCR fragment amplified in pET-PCSI. As shown in FIG. 12, it was confirmed that the desired polynucleotide was amplified.

Example  5-3-2: Mutated Sucrose isomerase  Production of Expression Vectors Containing Polynucleotides to be Coded

The polynucleotide obtained in Example 5-3-1 was treated with a restriction enzyme DpnI and introduced into the pET21a vector to obtain a polynucleotide encoding an amino acid sequence in which arginine at position 325 of the sucrose isomerase was substituted with lysine To construct an expression vector pET-R325K (Fig. 13).

13 shows a vector map of pET-R325K, which is an expression vector containing a polynucleotide encoding an amino acid sequence in which arginine at position 325 of the sucrose isomerase is replaced with lysine.

Example  5-3-3: Mutated Sucrose isomerase  Production of transgenic plants expressing

The expression vector pET-R325K prepared in Example 5-3-2 was introduced into E. coli BL21, which is a host cell, to obtain a transformant, and the nucleotide sequence of the DNA thus obtained was analyzed (Fig. 14).

14 shows the result of comparing the amino acid sequence of the sucrose isomerase with the amino acid sequence of the mutated sucrose isomerase derived from the DNA of the transformant into which the expression vector pET-R325K is introduced, wherein the substituted residue is a red box , PCSI represents a sucrose isomerase derived from Pectobacterium carotovorum KKH 3-1, and R325K represents a mutated sucrose isomerase. As shown in FIG. 14, the transformant was confirmed to contain a gene coding for a sucrose isomerase enzyme in which arginine at position 325 was substituted with lysine.

Example  5-3-4: Mutated from the transformant Sucrose isomerase  production

The transformant was cultured in the same manner as in Example 3-2, except that the transformant prepared in Example 5-3-3 was used, and the cultured transformant was pulverized Next, the sucrose isomerase mutated from the application to Ni-NTA affinity column chromatography was purified. Samples obtained during the purification process were then analyzed by SDS-PAGE (Fig. 15).

FIG. 15 is a photograph showing the result of SDS-PAGE analysis of a product purified by using Ni-NTA affinity column chromatography to express a sucrose isomerase mutated from pET-R325K, and M lane is a standard marker Lane 1 represents a negative control (E. coli), lane 2 represents cell lysate, and lane 3 represents a purified mutant sucrose isomerase. As shown in FIG. 15, the mutant sucrose isomerase having a size of about 69 kDa was purified, which was confirmed to be at the same level as the theoretical predicted level (68,723.91 Da).

Example  6: Mutated Sucrose isomerase  Character rating

Example  6-1: Mutated Sucrose isomerase  Enzyme activity

In order to evaluate the characteristics of the mutated sucrose isomerase produced in Example 5-3-4, a sucrose isomerase and a mutant sucrose isomerase using 5% (w / v) sucrose as a substrate After performing the enzyme reaction, the reaction product was applied to the HPAEC analysis (Fig. 16).

16 is a graph showing the results of analysis of reaction products of sucrose isomerase (black) and mutated sucrose isomerase (red) with HPAEC, wherein the first peak represents glucose and the second peak represents fructose , The third peak represents sucrose, the fourth peak represents trehalulose, and the fifth peak represents isomaltulose. As shown in Figure 16, unlike sucrose isomerase, which consumes almost all sucrose and produces trehalulose and isomaltulose as the main reaction products, the mutated sucrose isomerase does not consume sucrose , And glucose and fructose in addition to trehalulose and isomaltulose.

Example  6-2: Variation of reaction temperature Sucrose isomerase  Enzyme activity

As shown in Table 1 of Example 4-4, the sucrose isomerase changes the reaction product depending on the reaction temperature, and therefore, it was examined whether the mutated sucrose isomerase exhibited similar characteristics.

Specifically, each reaction product was obtained in the same manner as in Example 6-1, except that the enzyme reaction was carried out at 30 to 50 ° C, and this was applied to the HPAEC analysis (Table 3).

HPAEC analysis of sucrose isomerase (wild type) and mutated sucrose isomerase (mutant) reaction products at various reaction temperatures Reaction temperature (캜) Glucose / fructose content The content ratio of trehalulose and isomaltulose Wild type Mutant Wild type Mutant 30
35
40
45
50 0.06
0.35
0.89
1.64
2.56 2.40
3.20
4.00
5.60
5.60 79/21
71/29
64/36
56/44
50/50 51/49
50/50
39/61
25/75
23/77

As shown in Table 3, it was confirmed that the sucrose isomerase mutated with sucrose isomerase showed a difference in the content of glucose and fructose contained in the reaction product and the content ratio of trehalulose and isomaltulose in the reaction product Respectively. Specifically, the mutated sucrose isomerase produced a large amount of glucose and fructose, which are by-products of the reaction, as compared with the wild-type sucrose isomerase, and produced more isomaltulose than trehalulose.

However, as the reaction temperature increases, the production amount of glucose and fructose, which are byproducts of the reaction, increases and the amount of isomaltulose produced is increased rather than trehalulose, which is shared by the sucrose isomerase and the mutant sucrose isomerase .

Example  6-3: Variation of response time Sucrose isomerase  Enzyme activity

An enzyme reaction was carried out in the same manner as in Example 6-1, except that the enzyme reaction was carried out overnight, and the resulting reaction product was applied to the HPAEC analysis (Fig. 17 (A)).

17A is a graph showing the results of HPAEC analysis of a sucrose isomerase (black) and a mutated sucrose isomerase (red) reaction product, wherein the first peak indicates glucose, the second peak indicates fructose The third peak indicates sucrose, the fourth peak indicates unknown substance, the fifth peak indicates trehalulose, and the sixth peak indicates isomaltulose. As shown in FIG. 17 (A), if an enzymatic reaction is performed for a long time using a mutated sucrose isomerase, an unknown substance (peak 4) which is not contained in the reaction product of the wild-type sucrose isomerase is produced Respectively.

To confirm whether or not the monosaccharide affects the formation of the unknown substance, the same method as in Example 6-1, except that 5% (w / v) glucose was added and the enzyme reaction was performed overnight , And the reaction products obtained therefrom were applied to HPAEC analysis (Fig. 17 (B)).

FIG. 17 (B) is a graph showing the HPAEC analysis results of the reaction products produced by enzymatic reaction of sucrose isomerase (black) and mutated sucrose isomerase (red) performed using glucose, wherein 1 Wherein the first peak represents glucose, the second peak represents fructose, the third peak represents sucrose, the fourth peak represents unknown substance, the fifth peak represents trehalulose, the sixth peak represents iso Represents maltulose. As shown in FIG. 17 (B), when glucose is added and the enzyme reaction of the sucrose isomerase and the mutated sucrose isomerase is performed for a long time, an unknown substance is produced. As compared with the case of using sucrose isomerase, When the isomerization enzyme was used, it was confirmed that the unknown substance was produced in a larger amount.

In Example 5-2, since arginine is not present in the active site of the mutated sucrose isomerase, it is predicted that byproducts such as glucose and fructose will be formed due to the failure to form a hydrogen bond with the fructosyl moiety Therefore, even when glucose is not treated, the mutated sucrose isomerase produces glucose, and thus, unknown substances are produced.

Example  6-4: Identification of unknown substances

Example  6-4-1: recycling preparative HPLC  System to obtain active fractions

The reaction product of 3 ml of mutated sucrose isomerase obtained in Example 6-3 was applied to a recycling preparative HPLC system (LC-9104, JAI, Tokyo, Japan) to purify the unknown substance. At this time, a column of JAIGEL-W251 (JAI, Tokyo, Japan), a column of JAIGEL-W252 (JAI, Tokyo, Japan) and an RI detector RI-50 (JAI, Tokyo, Japan) , And the speed was set at 3 ml / min (Fig. 18 (A)).

18 (A) is a graph showing the results of applying reaction products of mutated sucrose isomerase to a recycling preparative HPLC system. As shown in Figure 18 (A), a total of 9 fractions were obtained through a recycling preparative HPLC system and the nine fractions obtained were applied to the HPAEC analysis (Figure 18 (B)).

FIG. 18 (B) is a graph showing the results of application of the fraction No. 3 to the HPAEC analysis among the nine fractions obtained through the recycling preparative HPLC system, wherein the black graph shows the mutated sucrose isomerase reaction product, The first peak represents glucose, the second peak represents fructose, the third peak represents sucrose, the fourth peak represents the unknown substance, and the fifth peak represents trehalulose And peak 6 indicates isomaltulose. As shown in FIG. 18 (B), the nine fractions were applied to the HPAEC analysis. As a result, it was confirmed that the fraction No. 3 contained a large amount of unknown substances.

Example  6-4-2: α- Glucosidase  Hydrolysis Reaction product  analysis

5 占 퐇 of? -Glucosidase enzyme solution was added to 20 占 퐇 of the fraction No. 3 obtained in Example 6-4-1, followed by reaction at 37 占 폚, and then the reaction product obtained was applied to the HPAEC analysis (A)).

19 (A) is a graph showing the results of HPAEC analysis of a digestion product obtained by digesting the fraction No. 3 obtained in the recycling preparative HPLC system with -.alpha.-glucosidase, wherein the black fraction is the fraction 3 without α-glucosidase , Red represents fraction 3 treated with? -Glucosidase, the first peak represents glucose, and the second peak represents unknown substance. As shown in FIG. 19 (A), it was confirmed that all of the unknown substances were decomposed into glucose by? -Glucosidase. Therefore, it was found that the unknown substance is a saccharide composed only of glucose. In light of the molecular weight of the unknown substance, the unknown substance is a substance composed of two glucose, and the two glucose are decomposed by? -Glucosidase Since it is bound by the? -1,6 bond, the unknown substance is expected to be isomaltose in which two glucose are bonded by? -1,6 bonds.

Thus, fraction 3 and isomaltose were applied to HPAEC analysis (FIG. 19 (B)).

FIG. 19 (B) is a graph showing the results of applying the fraction 3 and isomaltose obtained in the recycling preparative HPLC system to HPAEC analysis, wherein black indicates isomaltose, red indicates fraction 3, The peak indicates glucose and the second peak indicates unknown substance. As shown in FIG. 19 (B), it was confirmed that the unknown substance and isomaltose show a peak at the same position as the result of HPAEC analysis. As expected, the unknown substance was found to be isomaltose.

From the above results, it can be seen that the mutated sucrose isomerase not only decomposes sucrose to produce glucose, but also transfers glucose to the glucose, thereby showing a sugar transfer activity for producing a disaccharide. The above sugar chain activity was analyzed to be an enzyme activity which was not exhibited at all in conventional sucrose isomerase and newly generated enzyme activity as arginine 325 of sucrose isomerase was substituted with lysine.

Example  6-4-3: Fraction of the third fraction TLC  analysis

Through the example 6-4-2, it was confirmed that the unknown substance contained in the fraction No. 3 obtained in the recycling preparative HPLC system was isomaltose, and it was confirmed by TLC analysis (FIG. 20).

20 is a photograph showing the result of TLC analysis of the fraction No. 3 obtained in the recycling preparative HPLC system, wherein lane 1 represents a standard glucose, lane 2 represents a standard fructose, lane 3 represents a standard sucrose, Lane 4 represents the standard isomaltulose, lane 5 represents the fraction 3, and lane 6 represents 1% isomaltose. As shown in FIG. 20, it was confirmed that the unknown substance contained in fraction No. 3 exhibited the same TLC analysis as isomaltose.

Example  6-4-4: Mutated Sucrose isomerase Sugar transfer activity  Verification

In order to evaluate the sugar transfer activity of the mutated sucrose isomerase, an enzyme reaction using various hexoses (sucrose, glucose, fructose, alose, galactose or mannose) alone as a substrate was carried out. The final concentration of the sucrose and each hexane was 5% (w / v). The concentration of the mutant sucrose isomerase was set at 0.01 mg / ), And the reaction conditions were set at 40 DEG C for 1 hour (Table 4).

Measurement of sugar transfer activity of sucrose isomerase (wild type and variant) using various saccharides as substrate temperament Whether the sugar transfer product (isomaltose) is produced Wild type Mutant Glucose
Fructose
Sucrose
Glucose + Fructose
Sucrose + glucose
Sucrose + Fructose
Sucross + Al Ross
Sucrose + galactose
Sucrose + Mannos ×
×
×
×
×
×
×
×
× ×
×
○
×
○
×
○
○
○

As shown in Table 4, the isomaltose produced by the sugar transfer activity was not detected in the reaction product using the wild type sucrose isomerase, but was detected only in the reaction product using the mutated sucrose isomerase. Among the reaction products using mutated sucrose isomerase, isomaltose was detected only in a reaction product containing glucose, which is a sugar receptor, or its corresponding hexose (alos, galactose and mannose).

On the other hand, since the reaction product containing sucrose and fructose contains sucrose capable of producing glucose, it is theoretically possible to produce isomaltose, but glucose produced from sucrose due to excessive fructose Was reacted with an excessive amount of fructose to form sucrose, and a trace amount of glucose was not bound to each other to form isomaltose.

From the above results, it was confirmed that isomaltose was produced when sucrose and hexose (alos, galactose and mannose) were used. Therefore, the reaction product was applied to HPAEC analysis (FIG. 21).

FIG. 21 is a graph showing the results of analyzing the sugar transfer activity of mutated sucrose isomerase with respect to various hexoses by HPAEC analysis, wherein (A) shows the result of analysis of the reaction product using hexose as an aldehyde, (B) shows the result of analysis of a reaction product using galactose as a hexose, (C) shows the result of analysis of a reaction product using mannose as hexose, the first peak indicates glucose, the second peak indicates the fructose , The third peak represents isomaltose, the fourth peak represents trehalulose, the fifth peak represents isomaltulose, and the blue point represents the hexasaccharide (alos, galactose and mannose) used And the red dot represents the product that has not yet been identified. As shown in FIG. 21, the production level of isomaltose was changed according to the type of hexose used even when 6-valent sugar was used, and it was confirmed that the level of isomaltose production was the highest when mannose was used.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Mutated sucrose isomerase and process for preparing same <130> PA131372 / KR <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 591 <212> PRT <213> Artificial Sequence <220> <223> Pectobacterium carotovorum KKH 3-1, sucrose isomerase <400> 1 Met Leu Ala Ala Gly Leu Ser Phe Ser Ala Val Asn Tyr Ala Ala Thr   1 5 10 15 Asn His Asn Glu Gln Asp Thr Lys Thr Val Ile Ala Val Asn Asp Gly              20 25 30 Val Ser Ala His Pro Val Trp Trp Lys Glu Ala Val Phe Tyr Gln Val          35 40 45 Tyr Pro Arg Ser Phe Lys Asp Ser Asn Gly Asp Gly Ile Gly Asp Leu      50 55 60 Lys Gly Leu Thr Glu Lys Leu Asp Tyr Leu Lys Thr Leu Gly Ile Asn  65 70 75 80 Ala Ile Trp Ile Asn Pro His Tyr Asp Ser Pro Asn Thr Asp Asn Gly                  85 90 95 Tyr Asp Ile Arg Asp Tyr Arg Lys Ile Met Lys Glu Tyr Gly Thr Met             100 105 110 Asp Asp Phe Asp Asn Leu Ile Ala Glu Met Lys Lys Arg Asp Met Arg         115 120 125 Leu Met Ile Asp Val Val Val Asn His Thr Ser Asn Glu His Lys Trp     130 135 140 Phe Val Glu Ser Lys Lys Ser Lys Asp Asn Pro Tyr Arg Asp Tyr Tyr 145 150 155 160 Ile Trp Arg Asp Gly Lys Asp Gly Thr Pro Pro Asn Asn Tyr Pro Ser                 165 170 175 Phe Phe Gly Gly Ser Ala Trp Gln Lys Asp Asn Val Thr Gln Gln Tyr             180 185 190 Tyr Leu His Tyr Phe Gly Val Gln Gln Pro Asp Leu Asn Trp Asp Asn         195 200 205 Pro Lys Val Arg Glu Glu Val Tyr Asp Met Leu Arg Phe Trp Ile Asp     210 215 220 Lys Gly Val Ser Gly Leu Arg Met Asp Thr Val Ala Thr Phe Ser Lys 225 230 235 240 Asn Pro Ala Phe Pro Asp Leu Thr Pro Glu Gln Leu Lys Asn Phe Ala                 245 250 255 Tyr Thr Tyr Thr Gln Gly Pro Asn Leu His Arg Tyr Ile Gln Glu Met             260 265 270 His Gln Lys Val Leu Ala Lys Tyr Asp Val Val Ser Ala Gly Glu Ile         275 280 285 Phe Gly Val Pro Leu Glu Glu Ala Ala Pro Phe Ile Asp Gln Arg Arg     290 295 300 Lys Glu Leu Asp Met Ala Phe Ser Phe Asp Leu Ile Arg Leu Asp Arg 305 310 315 320 Ala Val Glu Glu Arg Trp Arg Arg Asn Asp Trp Thr Leu Ser Gln Phe                 325 330 335 Arg Gln Ile Asn Asn Arg Leu Val Asp Met Ala Gly Gln Tyr Gly Trp             340 345 350 Asn Thr Phe Leu Ser Asn His Asp Asn Pro Arg Ala Val Ser His         355 360 365 Phe Gly Asp Asp Arg Pro Glu Trp Arg Ile Arg Ser Ala Lys Ala Leu     370 375 380 Ala Thr Leu Ala Leu Thr Gln Arg Ala Thr Pro Phe Ile Tyr Gln Gly 385 390 395 400 Asp Glu Leu Gly Met Thr Asn Tyr Pro Phe Thr Ser Leu Ser Glu Phe                 405 410 415 Asp Asp Ile Glu Val Lys Gly Phe Trp Gln Asp Phe Val Glu Thr Gly             420 425 430 Lys Val Lys Pro Asp Val Phe Leu Glu Asn Val Lys Gln Thr Ser Arg         435 440 445 Asp Asn Ser Arg Thr Pro Phe Gln Trp Ser Asn Ala Glu Gln Ala Gly     450 455 460 Phe Thr Thr Gly Thr Pro Trp Phe Arg Ile Asn Pro Asn Tyr Lys Asn 465 470 475 480 Ile Asn Ala Glu Asp Gln Thr Gln Asn Pro Asp Ser Ile Phe His Phe                 485 490 495 Tyr Arg Gln Leu Ile Ala Leu Arg His Ala Thr Pro Ala Phe Thr Tyr             500 505 510 Gly Ala Tyr Gln Asp Leu Asp Pro Asn Asn Asn Glu Val Leu Ala Tyr         515 520 525 Thr Arg Glu Leu Asn Gln Gln Arg Tyr Leu Val Val Val Asn Phe Lys     530 535 540 Glu Lys Pro Val His Tyr Ala Leu Pro Lys Thr Leu Ser Ile Lys Gln 545 550 555 560 Thr Leu Leu Glu Ser Gly Gln Lys Asp Lys Val Ala Pro Asn Ala Thr                 565 570 575 Ser Leu Glu Leu Gln Pro Trp Gln Ser Gly Ile Tyr Gln Leu Asn             580 585 590 <210> 2 <211> 1776 <212> DNA <213> Artificial Sequence <220> <223> Pectobacterium carotovorum KKH 3-1, sucrose isomerase <400> 2 atgctggcgg caggattatc attctcagcc gttaattatg cggcaacaaa tcataatgag 60 caggatacaa agacggtaat agccgtcaat gatggcgttt ctgcgcatcc cgtgtggtgg 120 aaagaagctg tgttctatca ggtttatcca cgttcattta aagacagtaa cggtgatggg 180 attggtgatc ttaaaggcct gaccgaaaag ctcgattatt taaaaacact cggtattaat 240 gccatctgga ttaaccccca ttatgattca ccgaataccg ataacggata cgatattcgt 300 gattatcgaa agataatgaa agaatatggc acaatggatg acttcgacaa cctcattgca 360 gaaatgaaaa agcgtgatat gcgactaatg atagatgttg tcgttaatca caccagcaat 420 gaacacaaat ggtttgttga aagtaagaaa tcaaaagata atccttatcg cgactattac 480 atttggcgcg atggtaaaga tggcacaccg cctaataatt acccctcctt cttcggcggt 540 tccgcctggc agaaagataa cgtaacacag caatattatt tacactattt tggcgtacag 600 cagcccgatc tgaattggga taatcccaaa gtacgtgaag aagtgtacga tatgctgcgt 660 ttctggattg ataaaggcgt ttccgggcta cgtatggata ccgtagcaac cttctctaag 720 aatccggcat ttcccgatct cacgccagaa caattgaaaa acttcgctta tacctacaca 780 caaggtccaa atctgcaccg ttacattcag gaaatgcacc agaaggtgct ggcaaaatac 840 gacgtcgttt ccgcaggtga aattttcggt gtaccgcttg aagaagcggc cccgtttatc 900 gatcaacgtc gtaaagagct cgatatggcc ttctcattcg atcttatccg tctcgatcgc 960 gccgtagaag aaagatggcg acggaatgat tggacgctat cccagttccg tcagatcaac 1020 aatcgactgg tcgatatggc cgggcaatat ggctggaata ctttcttcct gagcaaccat 1080 gataacccgc gtgctgtatc acacttcggt gacgatcgcc ctgagtggcg tatccgttct 1140 gctaaagcgt tggcaaccct agcgttaaca cagcgagcga ccccgtttat ttatcaaggg 1200 gacgaattgg gcatgaccaa ctatccgttt acctccttgt ctgaatttga tgacattgaa 1260 gttaaaggtt tctggcagga ctttgtagag accggaaaag tgaaacctga tgtctttttg 1320 gaaaacgtga aacaaacgag tcgcgataac agccgaaccc cattccaatg gagcaatgcg 1380 gaacaggcgg gctttactac aggtacgccc tggttccgta ttaaccccaa ctacaagaac 1440 atcaatgcag aggatcaaac gcaaaaccca gattccatct tccatttcta ccgtcagctg 1500 atcgcactgc gtcacgccac gccggcgttc acctacggag cctatcagga tctcgatccg 1560 aataataacg aggtacttgc ttatactcgt gaactcaacc agcaacgtta tctggttgtg 1620 gtgaacttta aagagaaacc ggtgcattac gcgctgccga aaacgctttc catcaagcaa 1680 acactgttgg aaagtgggca gaaagataag gtagcaccga atgcaacctc acttgagctc 1740 cagccgtggc aatctggtat ttatcagctg aactag 1776 <210> 3 <211> 591 <212> PRT <213> Artificial Sequence <220> <223> mutated sucrose isomerase <400> 3 Met Leu Ala Ala Gly Leu Ser Phe Ser Ala Val Asn Tyr Ala Ala Thr   1 5 10 15 Asn His Asn Glu Gln Asp Thr Lys Thr Val Ile Ala Val Asn Asp Gly              20 25 30 Val Ser Ala His Pro Val Trp Trp Lys Glu Ala Val Phe Tyr Gln Val          35 40 45 Tyr Pro Arg Ser Phe Lys Asp Ser Asn Gly Asp Gly Ile Gly Asp Leu      50 55 60 Lys Gly Leu Thr Glu Lys Leu Asp Tyr Leu Lys Thr Leu Gly Ile Asn  65 70 75 80 Ala Ile Trp Ile Asn Pro His Tyr Asp Ser Pro Asn Thr Asp Asn Gly                  85 90 95 Tyr Asp Ile Arg Asp Tyr Arg Lys Ile Met Lys Glu Tyr Gly Thr Met             100 105 110 Asp Asp Phe Asp Asn Leu Ile Ala Glu Met Lys Lys Arg Asp Met Arg         115 120 125 Leu Met Ile Asp Val Val Val Asn His Thr Ser Asn Glu His Lys Trp     130 135 140 Phe Val Glu Ser Lys Lys Ser Lys Asp Asn Pro Tyr Arg Asp Tyr Tyr 145 150 155 160 Ile Trp Arg Asp Gly Lys Asp Gly Thr Pro Pro Asn Asn Tyr Pro Ser                 165 170 175 Phe Phe Gly Gly Ser Ala Trp Gln Lys Asp Asn Val Thr Gln Gln Tyr             180 185 190 Tyr Leu His Tyr Phe Gly Val Gln Gln Pro Asp Leu Asn Trp Asp Asn         195 200 205 Pro Lys Val Arg Glu Glu Val Tyr Asp Met Leu Arg Phe Trp Ile Asp     210 215 220 Lys Gly Val Ser Gly Leu Arg Met Asp Thr Val Ala Thr Phe Ser Lys 225 230 235 240 Asn Pro Ala Phe Pro Asp Leu Thr Pro Glu Gln Leu Lys Asn Phe Ala                 245 250 255 Tyr Thr Tyr Thr Gln Gly Pro Asn Leu His Arg Tyr Ile Gln Glu Met             260 265 270 His Gln Lys Val Leu Ala Lys Tyr Asp Val Val Ser Ala Gly Glu Ile         275 280 285 Phe Gly Val Pro Leu Glu Glu Ala Ala Pro Phe Ile Asp Gln Arg Arg     290 295 300 Lys Glu Leu Asp Met Ala Phe Ser Phe Asp Leu Ile Arg Leu Asp Arg 305 310 315 320 Ala Val Glu Glu Lys Trp Arg Arg Asn Asp Trp Thr Leu Ser Gln Phe                 325 330 335 Arg Gln Ile Asn Asn Arg Leu Val Asp Met Ala Gly Gln Tyr Gly Trp             340 345 350 Asn Thr Phe Leu Ser Asn His Asp Asn Pro Arg Ala Val Ser His         355 360 365 Phe Gly Asp Asp Arg Pro Glu Trp Arg Ile Arg Ser Ala Lys Ala Leu     370 375 380 Ala Thr Leu Ala Leu Thr Gln Arg Ala Thr Pro Phe Ile Tyr Gln Gly 385 390 395 400 Asp Glu Leu Gly Met Thr Asn Tyr Pro Phe Thr Ser Leu Ser Glu Phe                 405 410 415 Asp Asp Ile Glu Val Lys Gly Phe Trp Gln Asp Phe Val Glu Thr Gly             420 425 430 Lys Val Lys Pro Asp Val Phe Leu Glu Asn Val Lys Gln Thr Ser Arg         435 440 445 Asp Asn Ser Arg Thr Pro Phe Gln Trp Ser Asn Ala Glu Gln Ala Gly     450 455 460 Phe Thr Thr Gly Thr Pro Trp Phe Arg Ile Asn Pro Asn Tyr Lys Asn 465 470 475 480 Ile Asn Ala Glu Asp Gln Thr Gln Asn Pro Asp Ser Ile Phe His Phe                 485 490 495 Tyr Arg Gln Leu Ile Ala Leu Arg His Ala Thr Pro Ala Phe Thr Tyr             500 505 510 Gly Ala Tyr Gln Asp Leu Asp Pro Asn Asn Asn Glu Val Leu Ala Tyr         515 520 525 Thr Arg Glu Leu Asn Gln Gln Arg Tyr Leu Val Val Val Asn Phe Lys     530 535 540 Glu Lys Pro Val His Tyr Ala Leu Pro Lys Thr Leu Ser Ile Lys Gln 545 550 555 560 Thr Leu Leu Glu Ser Gly Gln Lys Asp Lys Val Ala Pro Asn Ala Thr                 565 570 575 Ser Leu Glu Leu Gln Pro Trp Gln Ser Gly Ile Tyr Gln Leu Asn             580 585 590 <210> 4 <211> 1776 <212> DNA <213> Artificial Sequence <220> <223> mutated sucrose isomerase <400> 4 atgctggcgg caggattatc attctcagcc gttaattatg cggcaacaaa tcataatgag 60 caggatacaa agacggtaat agccgtcaat gatggcgttt ctgcgcatcc cgtgtggtgg 120 aaagaagctg tgttctatca ggtttatcca cgttcattta aagacagtaa cggtgatggg 180 attggtgatc ttaaaggcct gaccgaaaag ctcgattatt taaaaacact cggtattaat 240 gccatctgga ttaaccccca ttatgattca ccgaataccg ataacggata cgatattcgt 300 gattatcgaa agataatgaa agaatatggc acaatggatg acttcgacaa cctcattgca 360 gaaatgaaaa agcgtgatat gcgactaatg atagatgttg tcgttaatca caccagcaat 420 gaacacaaat ggtttgttga aagtaagaaa tcaaaagata atccttatcg cgactattac 480 atttggcgcg atggtaaaga tggcacaccg cctaataatt acccctcctt cttcggcggt 540 tccgcctggc agaaagataa cgtaacacag caatattatt tacactattt tggcgtacag 600 cagcccgatc tgaattggga taatcccaaa gtacgtgaag aagtgtacga tatgctgcgt 660 ttctggattg ataaaggcgt ttccgggcta cgtatggata ccgtagcaac cttctctaag 720 aatccggcat ttcccgatct cacgccagaa caattgaaaa acttcgctta tacctacaca 780 caaggtccaa atctgcaccg ttacattcag gaaatgcacc agaaggtgct ggcaaaatac 840 gacgtcgttt ccgcaggtga aattttcggt gtaccgcttg aagaagcggc cccgtttatc 900 gatcaacgtc gtaaagagct cgatatggcc ttctcattcg atcttatccg tctcgatcgc 960 gccgtagaag aaaaatggcg acggaatgat tggacgctat cccagttccg tcagatcaac 1020 aatcgactgg tcgatatggc cgggcaatat ggctggaata ctttcttcct gagcaaccat 1080 gataacccgc gtgctgtatc acacttcggt gacgatcgcc ctgagtggcg tatccgttct 1140 gctaaagcgt tggcaaccct agcgttaaca cagcgagcga ccccgtttat ttatcaaggg 1200 gacgaattgg gcatgaccaa ctatccgttt acctccttgt ctgaatttga tgacattgaa 1260 gttaaaggtt tctggcagga ctttgtagag accggaaaag tgaaacctga tgtctttttg 1320 gaaaacgtga aacaaacgag tcgcgataac agccgaaccc cattccaatg gagcaatgcg 1380 gaacaggcgg gctttactac aggtacgccc tggttccgta ttaaccccaa ctacaagaac 1440 atcaatgcag aggatcaaac gcaaaaccca gattccatct tccatttcta ccgtcagctg 1500 atcgcactgc gtcacgccac gccggcgttc acctacggag cctatcagga tctcgatccg 1560 aataataacg aggtacttgc ttatactcgt gaactcaacc agcaacgtta tctggttgtg 1620 gtgaacttta aagagaaacc ggtgcattac gcgctgccga aaacgctttc catcaagcaa 1680 acactgttgg aaagtgggca gaaagataag gtagcaccga atgcaacctc acttgagctc 1740 cagccgtggc aatctggtat ttatcagctg aactag 1776 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 catatgtacg aacaagtctc ccactcg 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 ctcgagaagc tgtaaccaat aaaagcc 27 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R325K-F <400> 7 cgatcgcgcc gtagaagaaa aatggcgacg ga 32 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R325K-R <400> 8 tccgtcgcca tttttcttct acggcgcgat cg 32

Claims (7)

Arginine, which is the amino acid 325 of sucrose isomerase derived from Pectobacterium carotovorum KKH 3-1, is replaced by lysine, and a mutant sucrose isomerized with the amino acid sequence of SEQ ID NO: 3 enzyme.
The method according to claim 1,
Wherein said mutated sucrose isomerase exhibits sucrose isomerase activity and glycosyltransferase activity.
A polynucleotide consisting of a nucleotide sequence encoding the mutated sucrose isomerase of claim 1.
The method of claim 3,
A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4.
An expression vector comprising the polynucleotide of claim 3.
A transformant wherein the expression vector of claim 5 is introduced into a host cell.
(a) culturing the transformant of claim 6 to obtain a culture; And
(b) recovering the mutated sucrose isomerase of claim 1 from said culture.

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