KR100921980B1 - Nostoc sp-derived amylopullulanase and preparation method of maltooligosaccharide with the same - Google Patents

Abstract

The present invention relates to a amylopullulanase derived from Nostak strain strain PCC73102 ( Nostoc sp PCC73102) and a method for producing maltooligosaccharides from starch using the same, and more particularly, the Nostak strain strain PCC73102 ( Nostoc sp) PCC73102) and a novel amylopullulanase (amylopullulanase) isolated from the starch-containing substrate to a high-purity malto oligosaccharide having 7 to 11 glucose molecules of the present invention.

Nostak, amylofluranease, starch, maltooctaos

Description

Nostril-derived amylofluranease and manufacturing method of high purity maltooligosaccharide using the same {NOSTOC SP-DERIVED AMYLOPULLULANASE AND PREPARATION METHOD OF MALTOOLIGOSACCHARIDE WITH THE SAME}

The present invention relates to amylopullulanase derived from Nostoc sp PCC73102 ( Nostoc sp PCC73102) and a method for producing maltooligosaccharides from starch using the same.

An acid hydrolysis method and an enzyme method are mainly used to produce malto oligosaccharides having a long length of maltohexaose (G6) or more from starch.

Enzyme-based methods include a method of preparing maltohexaose and maltoheptaose by applying alpha-amylase derived from microorganisms to starch (US Pat. No. 5,739,024; E. Ben Messaoud et al. Enzyme Microb.Technol 34: 662-666. However, in the manufacturing method, maltohexaose and maltoheptaose constitute the main products, and malto oligosaccharides having short chain lengths such as glucose, maltose, maltotriose, maltotetraose, and maltopentaose are generated in large quantities to remove them. As a separation and purification process is required, there is a problem that excessive production cost is required.

In addition, there has been reported a method of enzymatically synthesizing by applying cyclomaltodextrinase (Cyclomaltodextrinase, CDase) to gamma-cyclodextrin as a method for producing maltooctaose using an enzyme (Uchida et al. Carbohydr. Res . 287: 271-274. 1996). However, since maltose, maltotriose, maltotetraose and maltopentaose are produced in a large amount in the reaction process, there is a problem in that even in the above method, a production yield is low and a purification process for removing by-products is required. Because of the high price of gamma-cyclodextrin used in the production process, an increase in production costs is inevitable in industrial production.

An object of the present invention is to provide an amylofluranease derived from Nostak strains in order to solve the problems of the prior art.

In addition, an object of the present invention is to provide a production method and a transformant for producing the amylofluranease.

In addition, an object of the present invention is to provide a method for producing malto oligosaccharides containing maltooctaose by reacting amylopullulanase (amylopullulanase) derived from Nostak strains to a substrate containing starch.

The inventors have found that the hydrolysis rate of alpha-1,4 glucoside bonds of amylopullulanase derived from Nostoc sp. PCC73102 ( Nostoc sp. PCC73102) is selective for chain length, so that starch is used as a substrate. In order to proceed with the hydrolysis reaction, maltoheptaose (maltoheptaose (G7), maltooctatase (G8), maltooctaose (maltooctaose (G8) and maltononaose (G9)) having 7 to 9 glocose molecules (maltogosaccharides as main products) maltooligosaccharide mixtures, in particular maltooligosaccharide mixtures containing maltooctaose as the main product, can be prepared, by controlling the reaction time between the enzyme and the substrate, the reaction conditions and the amount of enzyme relative to the substrate malto oligosaccharides containing maltooctaose from starch The present invention was completed by discovering that it can be easily prepared in high purity.

In order to achieve the above object, the present invention provides an amylofluranease derived from Nostoc sp.

The present invention also provides a production method for producing the amylofluranease and a transformant for producing the same.

The present invention also provides a method for producing maltooligosaccharides containing maltooctaose by reacting amylopullulanase derived from Nostak strains to a substrate containing starch.

Hereinafter, the present invention will be described in more detail.

The amylofluranease provided in the present invention is derived from Nostoc sp., Preferably from Nostoc sp. PCC73102 ( Nostoc sp PCC73102). The amylofluranease is an enzyme that exhibits optimal activity at 25 ° C and exhibits 60% or more of optimal activity at 10 ° C to 40 ° C. In addition, the amylofluranease is an enzyme that exhibits optimal activity at pH 7.5 and exhibits at least 50% of the optimal activity at pH 7.0 to pH 8.0. Preferably the amylofluranease is an enzyme that exhibits high activity at 20 ° C to 30 ° C and at pH 7.0 to pH 7.5.

Amylofluranease of the present invention may be a polypeptide having a molecular weight of 55.2 kDa to 55.8 kDa, preferably about 55.5 kDa, and preferably having an amino acid sequence of SEQ ID NO: 4.

Amylofluranease of the present invention has alpha-1,4-glucoside linkage and alpha-1,6-glucoside linkage hydrolytic activity, and in detail, pullulan is an optimal substrate, It decomposes the alpha-1,6-glucoside bond of to produce maltotriose, and has hydrolyzing ability against the alpha-1,4-glucoside bond of maltooligosaccharide. In addition, the amylofluranease of the present invention can produce malto oligosaccharides by decomposing alpha-1,6 glucoside bonds of starch including starch, and decomposing the starch or pretreated starch to produce malto oligosaccharides. Can hydrolyze maltooligosaccharides, but cannot hydrolyze cyclodextrins.

Maltooligosaccharide in the present invention refers to a sugar in which three or more glucose molecules are bound by alpha-1,4 bonds, and specifically, malto oligosaccharides having 3 to 50 glucose molecules, preferably malto oligosaccharides having 3 to 30 glucose molecules. And, more preferably, may be malto oligosaccharide having 3 to 20 glucose molecules, for example, maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), maltoheptaose (G7), malto It may be octaose (G8), maltononaose (G9), malto decaos (G10), malto de decause (G11) or maldoto de chaos (G12), most preferably maltooctaose.

In particular, the hydrolysis capacity and hydrolysis rate for the alpha-1,4 glucoside bonds of the amylofluranease of the present invention are selective for the chain length, that is, the number of glucose molecules.

Specifically, when maltooligosaccharide is used as the substrate, the substrate below the maltotriose cannot hydrolyze, but has hydrolysis ability against alpha-1,4-glucoside bonds of glucose 4 or more maltooligosaccharides.

In addition, the amylofluranease is characterized by selective hydrolytic ability to the number of glucose molecules. Specifically, the amyl as pullulan tyrosinase are 25 ℃ and pH was reacted under the conditions of 7, glow was measured to quantify the amount of coarse, malto-octahydro OSU of K _{m /} Kcat value of malto cyclohepta agarose K _{m /} Kcat than the value 2.5 times to 3.5 times, preferably 3 times, say 55 times to 65 times the K _{m /} Kcat value of the buttocks or this K _{m /} Kcat value of agarose malto octa agarose, preferably 59 times to 63 times , More preferably 60 to 62 times, most preferably 61 times higher. According to the above characteristics, when the starch is hydrolyzed by using the enzyme to prepare a maltooligosaccharide mixture, the main component of the maltooligosaccharide mixture is malto oligosaccharides having a molecular weight of 7 to 11 of the glucose, preferably maltoheptaose, maltoocta Os and maltononaose, most preferably maltooctaose.

Further, when the amylofluranease of the present invention is reacted with starch as a substrate, malto-oligosaccharides containing maltooctaose from starch, preferably maltose, are controlled by adjusting the amount of enzyme, reaction time and reaction conditions. Malto oligosaccharide further comprising octaose as a main component, and further comprising maltoheptaose, maltononase or a mixture thereof, more preferably maltooctaose as a main component, glucose number 7, and number of glucose molecules 9 to 11 Maltooligosaccharides, more preferably maltooctaose, further comprising one or more selected from the group consisting of maltooligosaccharides can be easily obtained in high purity.

The gene of amylofluranease of the present invention is composed of a polynucleotide comprising a nucleic acid sequence encoding a protein of SEQ ID NO: 4. An example of a nucleic acid sequence encoding the protein of SEQ ID NO: 4 is a nucleic acid sequence set forth in SEQ ID NO: 3. The gene sequence of SEQ ID NO: 3 is shown in FIG. 2.

Amylofluranease of the present invention can be isolated from Nostak strains or prepared by genetic engineering methods. The genetic engineering method may be a conventionally known method, and may be a method for producing a protein using eukaryotic cells derived from prokaryote, eukaryote or eukaryote. Preferably, the method for producing amylofluranease of the present invention may be produced through a microbial expression production system, but is not limited thereto.

For example, the production method of amylofluranease comprises the steps of: (a) preparing a recombinant vector by inserting a DNA sequence encoding amylofluranease isolated from a Nostagus strain into a vector; (b) introducing the recombinant vector into a host cell to prepare a transformant; (c) culturing the transformant may include producing amylofluranease.

In the step (a), the recombinant vector may be a recombinant vector inserted into a known vector so that the gene encoding the amylofluranease can be expressed. The known vector may be appropriately selected and used according to the type of host cell, and may be any type of vector proposed for expressing a heterologous protein in a microorganism, more preferably in yeast, E. coli or fungus. In addition, the vector may further comprise means for facilitating the separation of conventional selection markers, reporter genes or recombinant proteins. The selection marker may be an antibiotic resistance gene or a gene related to oxotropy.

The vector may be selected and used according to the type of host cell. For example, it may be a pET-29b (+) vector having a Kanamycin resistance gene as a T7 promoter, a T7 transcription terminator and a selection marker, but is not limited thereto.

The recombinant vector may be preferably pETNPul6xH prepared by combining pET-29b (+) with DNA encoding amylofluranease of SEQ ID NO: 4. A cleavage map of the recombinant vector pETNPul6xH is shown in FIG. 3.

Step (b) is to prepare a transformant, the host cell may be a prokaryote, eukaryote or eukaryote-derived cells, preferably E. coli, lactic acid bacteria, yeast or fungi. The method for preparing a transformant may be a conventional method, and preferably, may be a method for preparing a transformant using CaCl ₂ and heat-shock.

Step (c) is a step of culturing the transformant to produce amylofluranease. The medium for culturing the transformant may select a suitable medium according to the host cell, and the culture conditions may also be suitable for the host cell. Can be selected.

Escherichia coli ( E. coli ) BL21 (DE3) / pETNPul6xH, a transgenic whole prepared according to one embodiment of the present invention, was deposited at the Korea Institute of Biotechnology and Biotechnology Center, Eoeun-dong, Yuseong-gu, Daejeon, Korea on January 2, 2007. On January 2, 2007, they were given accession number KCTC 11059BP.

Specifically, the transformant Escherichia coli BL21 (DE3) / pETNPul6xH (KCTC 11059BP) is derived from the Nostak strain ( Nostoc sp.), And reacted at 25 ℃ and pH 7 conditions to quantify the amount of glocose a malto-octahydro agarose K _{m /} Kcat value of malto cyclohepta OSU 2.5 times to 3.5 times than the K _{m /} Kcat value of, say buttocks Naos K _{m /} Kcat value of K _{m /} Kcat value of malto-octahydro OSU of measurement It is possible to produce amylofluranease having the amino acid sequence of SEQ ID NO: 4 which is 55 to 65 times.

Producing amylofluranease by culturing the transformant E. coli BL21 (DE3) / pETNPul6xH (KCTC 11059BP) using the transformant transformed with a recombinant protein for protein production It can be carried out by a conventional method for producing a protein, and in detail, the transformant is incubated in LB medium to which kanamycin is added at 600nm under conditions of 30 ℃ to 37 ℃ until the absorbance is 0.6, The temperature of the medium is lowered to 15 ° C. to 30 ° C., preferably 20 ° C. to 25 ° C., and IPTG is added at a final concentration of 0.1 to 10 mM to 4 to 30 ° C., preferably 20 ° C. to 25 ° C. After incubating for 10 hours, the cultured expressed intracellular amyloflurane can be purified by separation or purification, specifically, cell harvest, cell disruption or hemolysis (lysis), supernatant separation and chromatography.

For example, the culture of the transformant is incubated at 30 to 37 ℃ in LB medium added with kanamycin and cultured at 600nm until the absorbance is 0.6, the medium temperature is lowered to 15 to 25 ℃, IPTG It can be carried out by adding the final concentration of 0.1 to 10 mM incubated at 15 to 25 ℃ for 4 to 10 hours.

In addition, the step of producing the amylofluranease may further comprise the step of separating or purifying the amylofluranease from the culture medium in which the transformant is cultured, after culturing the transformant.

For example, crushing the transformant present in the culture solution, taking a supernatant after centrifuging the culture medium crushed the transformant, heat-treating the supernatant and the nickel supernatant to the heat-treated supernatant The method may further comprise performing chemical chromatography.

The chromatography may be specifically or an ion exchange chromatography performed in the order of nickel affinity chromatography (Ni-NTA affinity chromatography), DEAE-toyopearl chromatography, or gel permeation chromatography (GPC), preferably Nickel affinity chromatography.

In addition, the present invention provides a method for producing malto oligosaccharides containing maltooctaose by reacting amylofluranease derived from the Nostak strains to a substrate containing starch.

More specifically, the production method is derived from the Nostak strain, the substrate containing the starch, has alpha-1,4-glucoside binding and alpha-1,6-glucoside binding hydrolytic activity, pullulan Amylofluranease, capable of hydrolyzing alpha-1,6-glucoside bonds of and producing a malto-oligosaccharide mixture comprising maltooctaose as a main product based on starch, preferably SEQ ID NO: 4 Amylofluranease having an amino acid sequence of is reacted at 0 ° C. to 40 ° C., preferably 20 ° C. to 30 ° C., and pH 7.0 to pH 8.0, preferably pH 7.0 to pH 7.5, malto octane-containing maltose. It may be a method for preparing an oligosaccharide.

The amylofluranease derived from the Nostak genus strain has alpha-1,4-glucoside binding and alpha-1,6-glucoside binding hydrolytic activity, and in detail, pullulan is used as an optimal substrate. Decomposes the alpha-1,6-glucoside linkage of flulan to produce maltotriose, has hydrolyzability to the alpha-1,4-glucoside linkage of maltooligosaccharides, and has alpha- The 1,6 glucoside bond can be decomposed to produce maltooligosaccharides, and the long chain length malto oligosaccharides produced by the decomposition of the starch can be hydrolyzed, but the cyclodextrin cannot be hydrolyzed. It may be an enzyme. Preferably, the amylofluranease derived from the Notaxin strain may have an amino acid sequence of SEQ ID NO.

The maltooligosaccharide may include maltooctaose as a main component, preferably maltooctaose as a main component, and may further include maltoheptaose, maltononaose or a mixture thereof, and more preferably. Preferably, maltooctaose is included as a main component, and may further include one or more selected from the group consisting of glucose molecule number 7, and glucose molecule number 9 to 11 maltooligosaccharides, and most preferably maltooctaose.

In the present invention, the main component is at least 51% by weight based on the total malto oligosaccharide, if the malto oligosaccharide consists of two types of malto oligosaccharide, 40% by weight based on the total malto oligosaccharide In the case of the four kinds of maltooligosaccharides, it means a component which is 30% by weight or more based on the total maltooligosaccharides, preferably 30% by weight to 99.9% by weight, and more preferably 40% by weight to 90% by weight. it means.

For example, when maltooligosaccharides are composed of malto oligosaccharides having 7 to 11 glucose molecules, 30 to 80 wt% of the maltooctaose, which is the main component, based on the total content, more preferably 40 to 70 wt% Can be. In addition, when the malto oligosaccharide is composed of malto oligosaccharides having 7 to 9 molecules of glocose, 40% to 90% by weight of the maltooctaose, which is the main component, based on the total content, more preferably 45% to 80% by weight. Can be.

In addition, the malto oligosaccharide comprising maltooctaose as a main component, and further comprising maltoheptaose, maltononaose, or a mixture thereof, preferably, the content of maltoheptaose and maltooctaose in the malto oligosaccharide mixture is heavy 1: 3 to 1: 5 (maltoheptaose: maltooctaose) on the basis of the content of maltononas and maltooctaose 1: 2 to 1: 4 (maltononose: maltooctaose) by weight It may be a phosphorus mixture.

The content of maltoheptaose and maltooctaose is 1: 3 to 1: 5 (maltoheptaose: maltooctaose) by weight, preferably 1: 3.5 to 1: 4.75, more preferably 1: 3.75 to 1: 4.5, and the content of maltononaose and maltooctaose is 1: 2 to 1: 4 (maltononaose: maltooctaose) by weight, preferably 1: 2.25 to 1: 3.5, More preferably, it may be 1: 2.5 to 1: 3.25.

The starch refers to a polysaccharide produced by long linking of glucose (D-glucose) to cause a condensation reaction, and may be referred to as starch. The starch may preferably be at least one selected from the group consisting of amylose, amylopectin, soluble starch, rice starch, corn starch, potato starch and wheat starch. The substrate containing starch may preferably be an aqueous solution of starch, but may be appropriately selected depending on the type and use of the material to be produced, but is not limited thereto.

The enzyme reaction may be 10 ℃ to 40 ℃, preferably 20 ℃ to 30 ℃ and pH 7.0 to pH 8.0, preferably pH 7.0 to pH 7.5.

In addition, the addition amount and reaction time of the enzyme in the enzyme reaction is 1.5 to 14.6 mg, preferably 2.9 mg to 5.8 mg of the amyloflurane based on 5 mg of the substrate, preferably 1 hour to 48 hours. May be performed by reacting 2 hours to 24 hours, more preferably 3 hours to 15 hours.

The manufacturing method of preparing malto oligosaccharides containing maltooctaose may further include a step of pretreatment by adding a debranching enzyme to the substrate containing starch. The pretreatment step hydrolyzes the alpha-1,6 glucoside bonds of the starch and removes the alpha-1,6 glucoside bonds before reacting the amylofluranease to the substrate, thereby linearizing the starch. It may be a step of preparing to maltooligosaccharide.

The debranching enzyme refers to an enzyme that selectively hydrolyzes alpha-1,6 glucoside bonds, and preferably may be isoamylase.

The pretreatment step comprises 0.2 mg to 0.4 of the debranching enzyme based on 5 mg of the substrate at 40 ° C. to 70 ° C., preferably 60 ° C. to 65 ° C. and pH 4 to pH 5, preferably pH 4.3 to pH 4.5. mg, preferably 0.3 mg to 0.4 mg may be added to react for 10 hours to 80 hours, preferably 30 hours to 70 hours.

In addition, the manufacturing method for producing maltooligosaccharides containing maltooctaose may further comprise a maltooligosaccharide concentration step.

In detail, an organic solvent is added to a reaction solution prepared by adding amylofluranease to the starch and reacting, that is, malto-oligosaccharide including maltooctaose, to remove impurities, and to remove maltooctaose. It may further include a concentration step of increasing the content of maltooctaose in the malto oligosaccharide comprising.

The organic solvent may be an alcohol, preferably an alcohol having 1 to 4 carbon atoms, more preferably ethanol.

Concentration step of removing impurities by adding the non-solvent

Adding an organic solvent to maltooligosaccharide prepared by adding amylofluranease to the substrate containing starch and reacting the same, and performing centrifugation; And it may be a step of removing the precipitate by collecting only the supernatant obtained by the centrifugation. By the concentration step, maltooligosaccharide having a glucose number of 12 or more can be removed.

In addition, the step of adding an organic solvent to the supernatant and performing centrifugation and removing the supernatant obtained by the centrifugation to obtain a precipitate may be further performed. By removing the supernatant to remove the malto oligosaccharide having a glucose number of 6 or less, the glucose molecule number of 7 to 11 malto oligosaccharides, preferably the glucose molecule number of 7 to 9 malto oligosaccharides, more preferably the content of maltooctaose can be higher. have.

For example, in the concentration step, ethanol is added to the reaction solution prepared by adding amylofluranease to the starch and reacting with 5 times the volume of the reaction solution, followed by centrifugation to obtain a supernatant. By removing the precipitate precipitated by the centrifugation it is possible to remove maltooligosaccharide having a glucose molecule number of 12 or more. In addition, after centrifugation and additionally added 5 times the ethanol of the reaction solution prepared by adding the same volume, that is, amylofluranease to the starch and reacting to the supernatant, centrifugation was performed and the supernatant Remove it. The precipitate precipitated by the centrifugation contains malto oligosaccharides having 6 to 12 glucose molecules, and preferably malto oligosaccharides having 7 to 11 glucose molecules. Thus, the supernatant is removed to remove impurities such as malto oligosaccharides having 5 or less glucose molecules. Can be removed

On the other hand, the precipitate removed after the first centrifugation, that is, the precipitate consisting of malto oligosaccharide having a glucose number of 12 or more is solubilized with distilled water of 70 ℃ to 100 ℃, preferably 90 ℃ to 100 ℃, the amyl A malto oligosaccharide manufacturing step of preparing malto oligosaccharides including maltooctaose using rofluranease may be performed.

That is, the precipitate consisting of malto oligosaccharides having a glucose number of molecules of 12 or more can be reused as a substrate of the malto oligosaccharide manufacturing step including maltooctaose using the amyloflurane as shown in FIG. 1.

In addition, the malto oligosaccharide manufacturing method containing the maltooctaose may be further performed a purification process. The purification process may be a process of performing chromatography, preferably liquid chromatography or anion exchange chromatography. For example, the liquid chromatography may be performed using a gel permeation column. Purification using the chromatography may be carried out after the concentration.

The preparation method is through the purification process, a mixture of high purity malto oligosaccharide having 7 to 11 glucose molecules, preferably high purity maltoheptaose, maltooctase and maltononaose, more preferably high purity maltooctaose Can be prepared.

Malto oligosaccharide manufacturing method comprising maltooctaose from starch according to the present invention, malto oligosaccharides containing maltooctaose as a main product can be easily produced, malto oligosaccharides containing maltooctaose produced by the above method as a main product, Preferably maltooctaose comprising as a main component, and further comprises one or more selected from the group consisting of malto oligosaccharides of 7 and 9 to 11 glucose molecules, more preferably maltooctaose is a new reduced rice material It is widely available in food. In addition, the high-purity maltooctaose concentrate obtained through the separation and purification from this can be used for the measurement of amylase activity in the blood, it can be widely used as a raw material for diagnostic reagents.

As described above, the present invention easily accumulates maltooctaose from the starch in the reaction solution by using the hydrolysis rate difference according to the chain length of the maltooligosaccharide of the amylofluranease derived from the Notaxin strain, and maltooctaose. By preparing a malto-oligosaccharide mixed concentrate containing as a main product, it is not only easy and simple to prepare high-purity maltooctaos used in various fields, such as medicine, chemical reagents or food, but also the value used by conventional techniques. By replacing gamma-cyclodextrin, an expensive raw material, with starch, an economical raw material, maltooctaose concentrates can be produced at low cost, and the industrial value thereof is very high.

Hereinafter, examples of the present invention will be described. The following examples are only examples of the present invention in order to more clearly show the present invention, the protection scope of the present invention is not limited by the following examples.

Example 1 Amylofluranese Gene Cloning and Amylofluranese Production

1-1 Amylofluranease Cloning

Nostagux strain PCC73102 ( Nostoc sp. PCC73102, ATCC, USA) was transferred to ATCC medium 819 Blue-Green Nitrogen-fixing medium [0.04 g K ₂ HPO ₄ , 0.075 g MgSO ₄ 7H ₂ O, 0.036 g CaCl ₂ · 2H ₂ O, 6.0 mg Citric acid, 6.0 mg Ferric ammonium citrate, 1.0 mg EDTA, 0.02 g Na ₂ CO ₃ , 1 mL Trace metal Mix A5 (2.86 g H ₃ BO ₃ , 1.81 g MnCl ₂ 4H ₂ O, 0.222 g ZnSO ₄ · 7H ₂ O, 0.39 g Na ₂ MoO ₄ · 2H ₂ O, 0.079 g CuSO ₄ · 5H ₂ O, 49.4 mg Co (NO ₃ ) ₂ · 6H ₂ O], inoculated in 50 ml and incubated at 25 ° C. Cells were recovered by centrifugation and chromosomal DNA was isolated (Dubnau, D. and RD Abenson, 1971, J. Mol. Biol., 56, 209-221).

In other words, the recovered cells were treated with lysozyme to form protoplast cells, and then SDS (sodium dodecyl sulfate) was added to completely destroy the cells. Sodium chloride was added to the total reaction solution to precipitate the protein. After centrifugation, only the supernatant was taken, and a mixture of phenol: chloroform: isoamyl alcohol (ratio of the volume of each solution = 25: 24: 1) was added in the same volume as the supernatant to remove impurities such as protein in the solution. This process was repeated several times. A double volume of 99% ethanol was added to precipitate, which was wound up with a glass rod to separate and dissolved in 1 / 10xSSC buffer (3M sodium chloride, 0.3M sodium citrate).

As for the amylofluranease gene, PCR was performed by the method of Table 2 below using the primers of Table 1 to isolate the amylofluranease gene.

Forward primer Nos-NdeI (5'-AAA AAG TTA CAT ATG CAA ATT CAA ACA CCA-3 ', SEQ ID NO: 1) and reverse primer Nos-XhoI (5'-AAA GTC) to isolate the amylofluranease gene CTC GAG GTC CCC AGT CCC CAG GAT-3 ', SEQ ID NO: 2) were prepared, respectively, and the primers are shown in Table 1 below.

order SEQ ID NO: Forward Primer Nos-Ndd I 5'-AAA AAG TTA CAT ATG CAA ATT CAA ACA CCA-3 ' One Reverse primer Nos-Xho I 5'-AAA GTC CTC GAG GTC CCC AGT CCC CAG GAT-3 ' 2

The PCR product was subjected to polymerase chain reaction (PCR) using the forward primers and the reverse primers on the Nostak strain chromosomal DNA, and the PCR product was TNERM, and the electrophoresis confirmed that the size of the PCR product was about 1.5 kb. The conditions of the PCR are as described in Table 2 below.

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

The PCR product was treated with restriction enzymes Nde I and Xho I at 37 ° C. for 6 hours and ligated with a pET-29b expression vector (Novagen, Germany) treated with the same restriction enzyme to prepare pETNPul6xH. The cleavage map of the vector of the recombinant vector pETNPul6xH prepared through the ligation is shown in FIG. 3. The pETNPul6xH vector contains an amylofluranease gene and contains 8 additional amino acid residues (Leu-Glu-6His) at the 3 'end of the amylofluranease gene.

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

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

0.15 ml of the suspension and 500 ng of the ligated solution were mixed and allowed to stand for 1 hour in ice and then subjected to thermal shock at 42 ° C. for 2 minutes. 0.8 ml of LB liquid medium was mixed with the thermal shock mixture, followed by incubation at 37 ° C for 1 hour. The cultures incubated for 1 hour were plated on LB agar medium containing kanamycin (final concentration 20 ° C g / ml), and strains showing resistance were first selected.

The first selected strains were inoculated in 5 ml of LB liquid medium containing kanamycin, incubated at 37 ° C. for 12 hours, and centrifuged to obtain cells. The plasmid was separated from the cells recovered by the plasmid separation kit (Qiagen, USA) and cleaved under the same conditions using the same NdeI and XhoI restriction enzymes as described above, and contains an amylofluranease gene having a size of about 1.5 kb. It was confirmed.

Culture of 1-2 Transformants and Production of Amylofluranease

The pETNPul6xH vector prepared above was transformed into E. coli BL21 (DE3) in the same manner as above, and then kanamycin resistance was checked to select the strain transformed with the vector, that is, a transformant. Was inoculated into 2.5 L of LB liquid medium containing kanamycin and incubated at 37 ° C., when the absorbance became 0.6 at 600 nm, IPTG was added to a final concentration of 0.5 mM and incubated at 25 to 37 ° C. for 5 hours. After the IPTG addition, the expression amount of amylofluranease according to temperature is shown in FIG. 4.

As shown in FIG. 4, the expression level of amylofluranease according to the culture temperature was very low at the temperature of 30 ° C. or higher. there was.

1-3 tablets

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

5 shows electrophoresis images of samples of purification steps of amyloflurane derived from Nostak strains produced in E. coli BL21 (DE3) transformed with recombinant vector pETNPul6xH. 5 is a size marker, lane 1 is a supernatant obtained by ultrasonic grinding of E. coli BL21 (DE3) / pETNPul6xH transformant, and lane 2 is a Ni- sample of the supernatant obtained after the ultrasonic grinding. It is the amyloflurane purified by NTA affinity chromatography.

As shown in FIG. 5, amylofluranease (lane 2) was confirmed to be efficiently purified through a Ni-NTA affinity chromatography process, and the size of the amylofluranease on SDS-PAGE was approximately. The molecular weight of 55,000 Da was shown to be similar to the theoretical molecular weight inferred from the amino acid sequence of the amylofluranease.

MALDI-TOF MS analysis was performed to confirm the molecular weight of the amylofluranease purified by Ni-NTA affinity chromatography, and the MALDI-TOF MS analysis is shown in FIG. 6 (Maldi-T of Mass Spcetrometry). of Synthetic polymers, Harald Pasch and Wolfgang Schrepp, Springer-Verlag (2003).

The MALDI-TOF MS analysis result of amyloflurane shown in FIG. 6 was found to be 56584.39, and the analysis result corresponds to the binding of Histidine tag to amylofluranease, and thus, the MALDI-TOF MS analysis result. Showed almost the same value as 56,512 Da, the theoretical molecular weight inferred from the amino acid sequence.

Example 2: Characterization of Amylofluranease derived from Northtaxin strain

2-1 Enzyme Titer

To 125 µl of 1% pullulan dissolved in 50 mM sodium phosphate (pH7.0) buffer, 100 µl of 50 mM sodium phosphate (pH7.0) buffer and 30 µl of enzyme were prepared. The reaction solution without enzyme is preheated at 25 ° C. for 5 minutes, and the enzyme is reacted for 10 minutes. 750 μl DNS solution (10.6 g of 3.5-dinitrosalicylic acid, 19.8 g NaOH, 306 g Na, K-Tartrate, 7.6 ml phenol, 8.3 g Na-metabisulfite and 1416 ml of distilled water) was added to stop the reaction. The reaction without enzyme is used as a control. Boil the reaction solution for 5 minutes and cool with running cold water. Absorbance is measured at 570 nm. One unit is the amount of enzyme required to form 1 μmol of reducing sugar, which is produced for one minute under specific reaction conditions.

2-2 pH Characteristics

To determine the optimal reaction pH of the amylofluranease of the present invention, paranitrophenyl maltopentaose was added to each of the Robinson Robinson buffers (pH 2.0 to 11.0) in various pH ranges to add 1% (w / v) para. Nitro phenyl maltopentaose substrate solution was prepared, and 30 µl of amyloflurane of Example 1-3 was added to 300 µl of dissolved 1% (w / v) paranitrophenyl phenyl maltopenedos substrate solution at 25 ° C. After 4 minutes of reaction, 400 μl of ethanol and 100 μl of 2M Tris were added to stop the reaction. The absorbance of the reaction solution was stopped at 405 nm was measured. Relative activity was measured based on the highest absorbance values.

Figure 7 shows the enzyme activity according to the pH of amylofluranease, amylofluranease of the present invention has an optimum titer at pH 7.5 and maintained a titer of 50% or more at pH 7.0 to 8.0.

2-3. Temperature characteristic

In order to determine the optimum reaction temperature of amyloflurane of the present invention, paranitrophenyl maltopentaose was added to 50 mM sodium phosphate buffer (pH 7.0) to add 1% (w / v) paranitrophenyl phenyl maltopenta. An os substrate solution was prepared, and the substrate solution was heated to various temperature ranges of 5 to 60 ° C., and then absorbance was measured according to the method of Example 2-2 to determine relative enzyme activity.

Figure 8 shows the enzyme activity according to the temperature of amylofluranease, amylofluranease of the present invention has an optimum titer at 25 ℃ and maintained a titer of about 60% or more at 10 to 40 ℃.

2-4 Enzyme Substrate Specificity

To determine the enzyme substrate properties of amylofluranease, glucose (G1, glucose), maltose (G2, maltose), maltotriose (G3, maltotriose), maltotetraose (G4, maltotetraose), maltopenta as substrates Amylose using os (G5, maltopentaose), maltohexaose (G6, maltohexaose), maltoheptaose (G7, maltoheptaose), alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, soluble starch and flulan The degradation products by plulanase were analyzed.

Specifically, each substrate was dissolved in 50 mM sodium phosphate buffer (pH 7.0) at a concentration of 1% (w / v) to prepare 150 μl of substrate solution. 120 μl of the 50 mM sodium phosphate buffer solution was added to each of the prepared substrate solutions, and 30 μl (0.1 unit) of amylofluranease of Example 1-3 was added and reacted at 25 ° C. for 12 hours. The reaction of amylofluranease was heated in boiling water for 5 minutes to terminate the reaction. The reaction solution of each sample which terminated the reaction was loaded into 1 μl of Whatman K5F silica gel TLC plate, and isopropyl alcohol, ethyl acetate and distilled water 3: 1: 2 (isopropyl alcohol: ethyl acetate: distilled water, v / v / v) developed once in a mixed solution of v), dried and soaked in methanol containing 0.3% (w / v) N- (1-naphthyl) -ethylenediamine and 5% (w / v) sulfuric acid. After removal, color development was performed in an oven at 110 ° C. for 10 minutes. In order to confirm substrate specificity of the enzyme, degradation products by amylofluranease were analyzed using thin layer chromatography.

In Figure 9 it can be seen that the amylofluranease of the present invention shows the characteristics of a typical fluranease hydrolyzing the flulan to produce maltotriose. In addition, the amylofluranease has a length of 3 chains, that is, substrates having maltotriose (G3) having 3 glucose or less cannot hydrolyze, but alpha-1 of malto oligosaccharides having 4 or more glucoses It can be seen that the hydrolysis ability is shown for, 4-glycosidic bonds. In addition, the amylofluranease did not hydrolyze the alpha-1,4-glycosidic bonds of cyclodextrins, but showed activity against soluble starch, producing glucose, maltose and maltooctaose. there was.

2-5 Analysis of Starch Degradation Trend of Amylofluranease

In order to analyze the amylofluranease trend of starch degradation, the degradation products by Northtaxin strain flulanase were analyzed using amylopectin, amylose, soluble starch, wax starch, rice starch, corn starch and wheat starch as substrates. It was.

Specifically, each substrate and amylofluranease dissolved in 50 mM sodium phosphate buffer (pH 7.0) at a concentration of 1% (w / v) were reacted according to the method of Example 2-4, and the reaction was terminated. After completion of the reaction, the reaction solution of each sample was terminated by loading 1 μl of a Whatman K5F silica gel TLC plate, and isopropyl alcohol, ethyl acetate, and distilled water 6: 1: 3 (isopropyl alcohol: ethyl acetate: distilled water, v / v / v), then developed once in a mixed solution of mixed solution and dried to contain 0.3% (w / v) N- (1-naphthyl) -ethylenediamine and 5% (w / v) sulfuric acid After soaking in methanol and developing for 10 minutes in an oven at 110 ° C., in order to analyze the tendency of starch decomposition of the enzyme, the degradation products by amylofluranease were analyzed using thin layer chromatography. Shown in

In FIG. 10, M is maltooligosaccharide standard, lane 1 is amylopectin as a substrate, lane 2 is amylose as a substrate, lane 3 is soluble starch as a substrate, and lane 4 is wax starch as a substrate. Lane 5 uses rice starch as substrate, lane 6 uses corn starch as substrate, and lane 7 uses wheat starch as substrate.

As shown in FIG. 10, the amylofluranease of the present invention can be seen to show the property of hydrolyzing starch to produce glucose, maltose and maltooctaose as main products.

Example 3 Preparation of a Mixture Made mainly of Maltooctaose Using Amylopectin

The trend of amylopectin degradation of amyloflurane was analyzed using thin layer chromatography (TLC) and high performance anion exchange chromatography.

Analysis of Amylopectin Degradation Trend of Amylofluranease Using 3-1 Thin Film Chromatography

According to Example 2-4, amylopectin dissolved in 50 mM sodium phosphate buffer (pH 7.0) at a concentration of 1% (w / v) was reacted with amylofluranease, and the reaction was terminated. In the same manner as in Example 2-5, the result of analyzing the decomposition product by amylofluranease using thin layer chromatography is shown in FIG.

In FIG. 11, M is a maltooligosaccharide standard material, and lane 1 is a result of thin layer chromatography analysis of decomposition products obtained by decomposing amylopectin as a substrate using amylofluranease of the present invention.

As shown in FIG. 11, the amylofluranease of the present invention can be seen to exhibit the property of hydrolyzing amylopectin to produce glucose, maltose and maltooctaose as main products.

Analysis of Amylopectin Degradation of Amyloflurane Using 3-2 High Performance Anion Exchange Chromatography

The reaction solution obtained in Example 3-2 was analyzed using High-Performance Anion Exchange Chromatography (HPAEC) (Determination of saccharides in biological materials by high-performance anion-exchange chromatography with pulsed amperometric detection, Journal of Chromatography , 546, pp 297-309 (1991).

In detail, the sample, that is, the reaction solution, was diluted three times with ultrapure water, filtered through a 0.45 μm thin film filter paper, and 20 μl was injected, and the flow rate was 1 ml per minute. HPAEC analysis was carried out using a GP40 gradient pump (Dionex, USA), ED40 electrochemical detector (Dionex, USA) and CarboPac-Column (CarboPac PA1). As a solvent, 150 mM caustic soda solution was used. After 40 minutes of dissolving 600 mM sodium acetate in solvent A as the solvent B, the concentration gradient was linearly adjusted so that the solvent B became 0 to 40% for 40 minutes.

12 shows the results of analyzing the reaction product produced by reacting the amylopectin prepared in Example 3-1 by the high performance anion exchange chromatography with the amylofluranease of the present invention.

As shown in FIG. 12, the main constituents of the reactants produced by reacting amylopelurane of the present invention with amylopectin obtained in Example 3-1 indicate glucose, maltose and maltooctaose.

In addition, FIG. 12 confirms that the content of malto oligosaccharides having 7 to 9 glucose molecules, in particular maltooctaose (G8), is high in the total malto oligosaccharide in the reaction product. As a result of analysis of FIG. It was confirmed that the content ratio of maltooctaose was 3:13 (maltoheptaose: maltooctaose), and the content ratio of maltooctase and maltononaose was 13: 5 (maltooctaose: maltononaose).

From the above results, it was confirmed that by controlling the reaction time, reaction conditions and the amount of enzyme relative to the substrate of the amyloflurane of the present invention, it is possible to obtain a high purity maltooctaos simply by high yield by minimizing the production of by-products. .

Example 4 Preparation of a Mixture Containing Maltooctaose as a Main Product by Using Starches Pretreated with Isoamylase

4-1 Pretreatment of Starch

Amylopectin and corn starch were dissolved in 50 mM sodium acetate buffer (pH 4.3) to 0.5% (w / v), and then 0.34 mg of isoamylase was added based on 50 mg of the substrate for 60 hours at 60 ° C. I was. After the reaction, the reaction solution was heated in boiling water for 15 minutes to terminate the reaction.

4-2 Preparation of Mixed Liquid Containing Maltooctaose as Main Product

The substrate solution of starch, ie, linear maltooligosaccharide, pretreated according to Example 4-1, was adjusted to pH 7.0 using the same buffer 500 mM sodium phosphate buffer, and then preheated at 25 ° C. for 5 minutes. . Add 47.3mU of amylofluranease of Example 1-3 based on 50 mg of each substrate solution, and react for 7 hours at 25 ° C., and then heat the amylofluranease reaction solution in boiling water for 15 minutes. The reaction was terminated.

4-3 High Performance Anion Exchange Chromatography Analysis of Reactions

The reaction solution obtained in Example 4-2 was analyzed using High-Performance Anion Exchange Chromatography (HPAEC) (Determination of saccharides in biological materials by high-performance anion-exchange chromatography with pulsed amperometric detection, Journal of Chromatography , 546, pp 297-309 (1991).

In detail, the sample, that is, the reaction solution, was diluted three times with ultrapure water, filtered through a 0.45 μm thin film filter paper, and 20 μl was injected, and the flow rate was 1 ml per minute. HPAEC analysis was carried out using a GP40 gradient pump (Dionex, USA), ED40 electrochemical detector (Dionex, USA) and CarboPac-Column (CarboPac PA1). As a solvent, 150 mM caustic soda solution was used. After 40 minutes of dissolving 600 mM sodium acetate in solvent A as the solvent B, the concentration gradient was linearly adjusted so that the solvent B became 0 to 40% for 40 minutes.

The reaction product produced by reacting the amylofluranease of the present invention with a substrate pretreated with isoamylase prepared in Example 4-1 by the high performance anion exchange chromatography, that is, amylopectin and corn starch, was analyzed. The results are shown in FIG. 13-1 and 13-2 are results for amylopectin, and FIGS. 13-3 and 13-4 are results for corn starch.

As shown in FIG. 13, FIGS. 13-1 and 13-3 show starches obtained by treating the amylopectin and corn starch obtained in Example 4-1 with isoamylase, that is, linear maltooligosaccharides, have mainly a chain length. It is composed of maltooligosaccharides of 8 or more. 13-2 and FIG. 13-4 illustrate the main components of the reaction product produced by reacting amylofluranease of the present invention with starch pretreated with the isoamylase obtained in Example 3-1. Maltose, maltooctaose.

In addition, Figure 13 is a pre-treatment of starch that is a kind of debranching enzyme isoamase, that is, if the linear malto oligosaccharide prepared by removing the alpha-1,6 glycosidic bond of starch as a substrate, the number of glucose molecules 7 to 11 malto oligosaccharides, in particular maltooctaose not only shortens the reaction time of the reaction to produce, but also indicates that the reaction conditions can be easily adjusted to minimize the production of by-products. In addition, it was confirmed that the corn starch can easily produce malto oligosaccharides having 7 to 11 glucose molecules, in particular maltaoctase, using amylopectin as well as various forms of starch.

Example 5: Preparation of high purity maltooctaose concentrate through separation and purification of maltooctase

5-1 Fraction of Maltooctaose Using Ethanol

Cold ethanol was added to the maltooctaose concentrate prepared according to Example 4-2, 5 times the volume of the concentrate, and then centrifuged at 4 ° C. and 12,000 rpm for 20 minutes. Only the supernatant was obtained. The maltooligosaccharides above were removed. The same volume, that is, the ethanol is added to the supernatant again 5 times the concentration of the concentrate, and centrifugation is performed to remove the supernatant to recover malto oligosaccharides including maltooctaose and malto oligosaccharides having 6 to 11 glucose molecules. It was.

FIG. 14 shows the results of thin layer chromatography analyzing maltooctaose fractions by removing maltooligosaccharides having 12 or more glucose molecules and maltooligosaccharides having 5 or less glucose molecules using the ethanol.

As shown in FIG. 14, the fraction prepared by the fraction using ethanol is mainly composed of maltooctaose, including maltooctaose, maltooligosaccharide having 7 to 11 glucose molecules, and more specifically maltooligosaccharide having 7 to 9 glucose molecules. Could know.

High performance anion exchange chromatography analysis of 5-2 reaction solution

Maltooligosaccharide prepared in Example 4-1 was analyzed using high performance anion exchange chromatography in the same manner as in Example 3-3, and the analysis results are shown in FIG. 15.

As shown in FIG. 15, oligosaccharides having a chain length of 4 or less and maltooligosaccharides having a chain length of 12 or more could be removed relatively simply by fractionation using ethanol, and maltooctaose content of the whole maltooligosaccharides was 50. By weight%, maltooctaose could be separated and purified in high purity by the said method.

Table 3 shows the composition of the high purity maltooctaose concentrate prepared through Example 5-1, which was analyzed by high performance anion exchange chromatography.

Furtherance weight% Maltohexaose (G6) One Maltoheptaose (G7) 13 Maltooctaos (G8) 50 Maltononaos (G9) 17 Malto de Chaos (G10) 10 Maltoen de Chaos (G11) 7 Maltodo de Chaos (G12) 2 Sum 100

5-3 Analysis of Maltooctaose Content with Different Reaction Times

The method of Example 4-2 was carried out, but the amylopectin as a substrate, the reaction time of the substrate and the amyloflurane of Example 1-3 was carried out by reacting for 30 minutes to 24 hours.

The reaction product was analyzed using high performance anion exchange chromatography in the same manner as in Example 4-3, and the analysis results are shown in FIG. 16.

As shown in FIG. 16, when the substrate and amyloflurane were reacted for 1 hour, not only maltooctase but also maltooligosaccharides having 9 or more glucose molecules including maltononose were shown to be contained in a large amount. On the other hand, when the reaction time of the enzyme reaction was 5 hours, maltooctaose showed a higher content than other maltooligosaccharides, it was confirmed that the content of maltooligosaccharides having a glucose number of 10 or more is very low. In addition, when the reaction time of the enzyme reaction is 18 hours, the content of maltooligosaccharides having a glucose molecule number of 9 or more was very low, but the content of maltooligosaccharides having a glucose molecule number of 5 or less was increased, that is, the content of impurities was increased.

From the above results, it was confirmed that in preparing malto oligosaccharides having 7 to 11 glucose molecules, the reaction time can be adjusted to increase the production efficiency and lower the content of impurities to increase the purity.

Example 6: Confirmation of Substrate Specificity of Amylofluranease Using Kinetic Data

When amylofluranease of the present invention produces maltooligosaccharides from starch, in order to confirm that maltooctaose is produced as a main product, the type of substrate is divided into maltoheptaose, maltooctaose and maltononaose. The K _m value and K _{m /} Kcat value of the reaction were measured.

6-1 Preparation of Substrate

As described above, maltoheptaose, maltooctaose and maltononase were prepared to confirm the substrate specificity of amylofluranease.

Specifically, maltose was dissolved in 5 mM sodium acetate buffer solution (pH 7.5) by dissolving 5% soluble starch and 5% maltose to prepare a reaction solution.

After the prepared reaction solution was preheated to 70 ° C., Thermus aquaticus- derived alpha-glucanotransferase was added and reacted for 15 hours.

After the reaction was terminated by maintaining the reaction solution at 121 ° C. for 15 minutes, maltoheptaose (G7) and maltooctaose were extracted from the reaction solution using Preparative recycling HPLC (High performance liquid chromatography, JAI Co., Japan). (G8) and maltononaose (G9) were purified to prepare a reaction substrate.

6-2 Determination of Kinetics According to Substrate of Amylofluranease

100 μl of amylofluranease of Example 1-3 was added to each substrate prepared in Example 6-1, and the reaction was performed at 25 ° C. and pH 7 for 3 minutes.

As shown in FIG. 17, amylofluranease of the present invention hydrolyzes maltoheptaose (G7), maltooctaose (G8) and maltononaose (G9) in glucose units, thereby confirming substrate specificity. To determine the titer of K _m and K _{m /} Kcat values of maltoheptaose, maltooctaose and maltonanoose, the GOD-POD method (JD FOX and JF Robyt, Anal. Chem . 195 (1) 93-96 (1991)) and the results are shown in Table 4 below.

As shown in Table 4, when comparing the K _m value of each substrate, it was confirmed that the value of maltoheptaose is 7 times or 20 times larger than the values of maltooctase and maltononaose, compared to the value of Kcat In other words, it was confirmed that the value of maltononaose was about three times larger than the value of maltoheptaose. In addition, when comparing the K _{m /} Kcat, the value of maltooctaose is 2.5 to 3.5 times higher than the value of maltoheptaose, preferably about 3 times higher, and the value of maltononaose is higher than that of maltoheptaose. 55 times to 65 times, preferably 59 times to 63 times, and more preferably 60 times to 62 times higher.

From the above results, it was confirmed that maltononose is the optimum substrate among the three substrates, and from this fact, it was confirmed that amylofluranease of the present invention can selectively hydrolyze starch into maltooctaose units. .

Therefore, in the case of producing maltooctaose using starch from the above results, using the amylofluranease of the present invention, it is possible to produce a high concentration of maltooctaose in excellent yield, the amyloflurane of the present invention Naise was expected to be very effective industrially.

1 is a process for producing a high purity maltooligosaccharides maltooctaose is the main product from the substrate containing starch using a noxol strain derived from amyloflurane according to an embodiment of the present invention,

Figure 2 is a gene sequence of amylofluranease derived from Nostagusok strains according to an embodiment of the present invention,

3 is a cleavage map of the recombinant vector pETNPul6xH according to an embodiment of the present invention,

4 is a photograph showing the amount of expression of amyloflurane derived from Nostak strain according to the culture temperature after the addition of IPTG according to an embodiment of the present invention through activity measurement,

5 is a SDS-PAGE electrophoresis image according to the purification steps of the No. lococcal strain-derived amyloflurane produced in E. coli BL21 (DE3) transformed with the recombinant vector pETNPul6xH according to an embodiment of the present invention.

6 is a result of analysis of matrix associated laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) of purified Northtaxin strain-derived amylofluranease,

Figure 7 shows the enzyme activity according to the pH of the amylofluranease derived from the Notaxin strain according to an embodiment of the present invention,

Figure 8 shows the activity according to the temperature of the amylofluranease derived from Nostak strains according to an embodiment of the present invention,

FIG. 9 illustrates amylofluranease derived from a Notaxin strain according to an embodiment of the present invention, including glucose (G1, glucose), maltose (G2, maltose), maltotriose (G3, maltotriose), and maltotetraose (G4). , maltotetraose), maltopentaose (G5, maltopentaose), maltohexaose (G6, hexaose), maltoheptaose (G7), alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, soluble starch, flulan ( pullulan) and then analyzed by thin layer chromatography (Thin Layer Chromatography, TLC), the picture of Figure 9-means a control group without the flulanase, + means a test group to which the flulanase is added,

FIG. 10 is a photograph of TLC analysis of the result of reacting amylofluranease derived from Nostak strains according to an embodiment of the present invention to various starches.

FIG. 11 is a photograph of TLC analysis of the result of reacting amylofluranease derived from Notaxin strain according to an embodiment of the present invention to amylopectin,

12 is a result of analyzing a resultant obtained by reacting amyflulurane derived from Notaxin strain according to an embodiment of the present invention to amylopectin using high-performance anion exchange chromatography (HPAEC),

FIG. 13 is a result of analysis of a reaction solution in which amylofluranease derived from Northtaxin strain is acted on starch pretreated using isoamylase according to an embodiment of the present invention. ,

FIG. 14 shows the fractionation of maltaoctase using ethanol in a reaction solution in which amylopectrane-derived amylofluranease is reacted with amylopectin pretreated using isoamylase according to an embodiment of the present invention. A fraction of the fractions was analyzed by thin layer chromatography,

FIG. 15 shows the fractionation of maltaoctase using ethanol in a reaction solution in which amylopectrane-derived amylofluranease was reacted with amylopectin pretreated using isoamylase according to an embodiment of the present invention. One fraction was analyzed by high performance anion exchange chromatography,

FIG. 16 is a reaction obtained by timely reacting amylofluranease derived from Northtaxin strain to amylopectin pretreated using isoamylase according to an embodiment of the present invention. The fractions of maltooctaose fractions using ethanol in the solution were analyzed by performance anion exchange chromatography.

17 is a reaction obtained by timely acting amylofluranease derived from the Notaxin strain on maltoheptaose, maltooctata, and maltononaose according to an embodiment of the present invention according to an embodiment of the present invention The solution is analyzed by high performance liquid chromatography.

<110> SEOUL NATIONAL UNIVERSITY INDUSTRY FOUNDATION <120> NOSTOC SP-DERIVED AMYLOPULLULANASE AND PREPARATION METHOD OF          MALTOOLIGOSACCHARIDE WITH THE SAME <130> dpp20071349kr <150> KR10-2007-0010473 <151> 2007-02-01 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Nod NddI-For <400> 1 aaaaagttac atatgcaaat tcaaacacca 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Nox-XhoI-Rev <400> 2 aaagtcctcg aggtccccag tccccaggat 30 <210> 3 <211> 1467 <212> DNA <213> Artificial Sequence <220> <223> amylopullulanase derived Nos sp. <400> 3 atgcaaattc aaacaccaga ttgggtaaag cacgctgttt tctaccaaat tttcccagat 60 cgctttgcca gaagcaaaca gccgcgcaaa cggttattgc aagaagcccg ttgggaagat 120 tgggactcta tgccaaccct ccagggttat aaaggtgggg atttatgggg catcatggag 180 gatttagact acatccagaa tttggggatt aacgcgattt atttcacacc catctttcaa 240 tccgccagta atcaccgcta tcatacccac gattattatc aagttgaccc gatgttaggg 300 ggcaacgagg cttttaaaga attgcttgat gcagcccatc aacggaatat caaagtcgtt 360 ctggatgggg tgtttaacca ttccagccgt ggctttttct ttttccacga cgttttagaa 420 aatggccccc attcaccttg ggtaaattgg ttcaaaatag aaggctggcc cctttcacct 480 tataacggtg aattccctgc aaactacgtg ggttgggcgg gaaatcgcgc cttgccagaa 540 tttaaccacg ataatccaga agtacgagaa tatattatgg aaattgccga atattggctt 600 aaattcggca tcgacggttg gcgattagat gtcccatttg aaataaaaac tccgggtttt 660 tggcaggaat tccgcgatcg cactaaagcc atcaatcccg aagcatatat tgtgggcgaa 720 gtgtggggag attcccgcca gtggttggat ggaacgcaat ttgacggcgt aatgaattat 780 ttatttgctg ggccgacaat tgcttttgca gctggcgatc gcgtagtctt agaacaagtc 840 caaagccgcg actatcaacc ctacccaccc ttatttgctg ccgagtacgc caccaaaatc 900 caagaagtac tgcaacttta cccttgggaa attcagctaa ctcaactcaa tttgttagca 960 agtcacgata cagcccgatt gatgaccatt gcaggcggtg atatagcgag tgtagaatta 1020 tcgactttac tgttactcac ctttcccggt gcccccagca tttactatgg tgatgaagtc 1080 ggtttacctg gtggcataga tcccgactca aggcgtggct ttcctttaga ggctaactgg 1140 aatcaagaaa ttttcaatac tcatcgtcaa ttaattacca tccgccaaac atacccagcc 1200 ttgcgtacag gtgattacca agtcctctat gctcaagggc aactttatct ctttgcacga 1260 actttaggca cagaagaatt gataattgct ataaacgctg gcacaagttc ggcaaccgca 1320 aatgtagatg tggctagttt gcatactcaa cccaacaagc tgttatatgg tactgctgaa 1380 gctgagtgga atggtgaaga aggaactcag caattgtcat taactcttcc cgcacgcagt 1440 ggctgcatcc tggggactgg ggactag 1467 <210> 4 <211> 488 <212> PRT <213> Artificial Sequence <220> Amylopullulanase-derived Nos sp. <400> 4 Met Gln Ile Gln Thr Pro Asp Trp Val Lys His Ala Val Phe Tyr Gln   1 5 10 15 Ile Phe Pro Asp Arg Phe Ala Arg Ser Lys Gln Pro Arg Lys Arg Leu              20 25 30 Leu Gln Glu Ala Arg Trp Glu Asp Trp Asp Ser Met Pro Thr Leu Gln          35 40 45 Gly Tyr Lys Gly Gly Asp Leu Trp Gly Ile Met Glu Asp Leu Asp Tyr      50 55 60 Ile Gln Asn Leu Gly Ile Asn Ala Ile Tyr Phe Thr Pro Ile Phe Gln  65 70 75 80 Ser Ala Ser Asn His Arg Tyr His Thr His Asp Tyr Tyr Gln Val Asp                  85 90 95 Pro Met Leu Gly Gly Asn Glu Ala Phe Lys Glu Leu Leu Asp Ala Ala             100 105 110 His Gln Arg Asn Ile Lys Val Val Leu Asp Gly Val Phe Asn His Ser         115 120 125 Ser Arg Gly Phe Phe Phe Phe His Asp Val Leu Glu Asn Gly Pro His     130 135 140 Ser Pro Trp Val Asn Trp Phe Lys Ile Glu Gly Trp Pro Leu Ser Pro 145 150 155 160 Tyr Asn Gly Glu Phe Pro Ala Asn Tyr Val Gly Trp Ala Gly Asn Arg                 165 170 175 Ala Leu Pro Glu Phe Asn His Asp Asn Pro Glu Val Arg Glu Tyr Ile             180 185 190 Met Glu Ile Ala Glu Tyr Trp Leu Lys Phe Gly Ile Asp Gly Trp Arg         195 200 205 Leu Asp Val Pro Phe Glu Ile Lys Thr Pro Gly Phe Trp Gln Glu Phe     210 215 220 Arg Asp Arg Thr Lys Ala Ile Asn Pro Glu Ala Tyr Ile Val Gly Glu 225 230 235 240 Val Trp Gly Asp Ser Arg Gln Trp Leu Asp Gly Thr Gln Phe Asp Gly                 245 250 255 Val Met Asn Tyr Leu Phe Ala Gly Pro Thr Ile Ala Phe Ala Ala Gly             260 265 270 Asp Arg Val Val Leu Glu Gln Val Gln Ser Arg Asp Tyr Gln Pro Tyr         275 280 285 Pro Pro Leu Phe Ala Ala Glu Tyr Ala Thr Lys Ile Gln Glu Val Leu     290 295 300 Gln Leu Tyr Pro Trp Glu Ile Gln Leu Thr Gln Leu Asn Leu Leu Ala 305 310 315 320 Ser His Asp Thr Ala Arg Leu Met Thr Ile Ala Gly Gly Asp Ile Ala                 325 330 335 Ser Val Glu Leu Ser Thr Leu Leu Leu Leu Thr Phe Pro Gly Ala Pro             340 345 350 Ser Ile Tyr Tyr Gly Asp Glu Val Gly Leu Pro Gly Gly Ile Asp Pro         355 360 365 Asp Ser Arg Arg Gly Phe Pro Leu Glu Ala Asn Trp Asn Gln Glu Ile     370 375 380 Phe Asn Thr His Arg Gln Leu Ile Thr Ile Arg Gln Thr Tyr Pro Ala 385 390 395 400 Leu Arg Thr Gly Asp Tyr Gln Val Leu Tyr Ala Gln Gly Gln Leu Tyr                 405 410 415 Leu Phe Ala Arg Thr Leu Gly Thr Glu Glu Leu Ile Ile Ala Ile Asn             420 425 430 Ala Gly Thr Ser Ser Ala Thr Ala Asn Val Asp Val Ala Ser Leu His         435 440 445 Thr Gln Pro Asn Lys Leu Leu Tyr Gly Thr Ala Glu Ala Glu Trp Asn     450 455 460 Gly Glu Glu Gly Thr Gln Gln Leu Ser Leu Thr Leu Pro Ala Arg Ser 465 470 475 480 Gly Cys Ile Leu Gly Thr Gly Asp                 485  

Claims (11)

Derived from Nostoc sp., Amylofluranease having the amino acid sequence of SEQ ID NO: 4 and having Leu-Glu-6His added to the 3 'end of the sequence. Derived from Nostoc sp., Km _/ Kcat value of maltooctaose measured by quantifying the amount of glocose reacted at 25 ° C. and pH 7 was 2.5 to 3.5 times that of Km _/ Kcat of maltoheptaose, and maltonona has the amino acid sequence of the trehalose in the K _{m /} Kcat value of malto-octahydro OSU of K _{m /} 55 times the Kcat value to 65 times as SEQ ID NO: 4, fluorene with the 3 'end of the sequence is Leu-Glu-6His added amyl Lannaise. No Stark sp (Nostoc sp.) Being derived from, by reacting under the conditions of 25 ℃ and pH 7, yi K _{m /} Kcat value of malto-octahydro agarose measured by quantifying the amount of global coarse of malto cyclohepta agarose K _{m /} compared with the Kcat value of 2.5 times to 3.5 times, the end of the buttocks Naos of K _{m /} Kcat value has a 55 times to 65 times the SEQ ID NO: 4 amino acid sequence of the K _{m /} Kcat value of malto-octahydro agarose, 3 of the sequence, Escherichia coli (KCTC 11059BP) producing amylofluranease with Leu-Glu-6His added at the end. Amylofluranease derived from Nostoc sp. On a substrate containing starch, having an amino acid sequence of SEQ ID NO: 4, and having Leu-Glu-6His added to the 3 'end of the sequence 10 A method for producing maltooligosaccharide comprising maltooctaose by reacting at the conditions of ℃ to 40 ℃ and pH 7.0 to pH 8.0. The malto oligosaccharide of claim 4, wherein the malto oligosaccharide comprises maltooctaose in an amount of 30 wt% to 99.9 wt% based on the total malto oligosaccharide, and further comprises maltoheptaose, maltononaose, or a mixture thereof. Oligosaccharide Preparation Method. According to claim 4, The malto oligosaccharide comprises maltooctaose 30% by weight to 80% by weight based on the total content, and further comprises one or more selected from the group consisting of malto oligosaccharides of 7 to 11 glucose molecules Phosphorus maltooligosaccharide manufacturing method. The method according to any one of claims 4 to 6, wherein the substrate is at least one aqueous solution selected from the group consisting of amylopectin, amylose, soluble starch, rice starch, corn starch, potato starch and wheat starch. The method according to any one of claims 4 to 6, wherein prior to the step of reacting the amylofluranease, a debranching enzyme is reacted with a substrate containing the starch to bind alpha-1,6 glucoside of starch. Method for producing maltooligosaccharide further comprising the step of hydrolyzing. The method according to any one of claims 4 to 6, wherein the malto oligosaccharide production method comprises reacting the amylofluranease with the organic solvent to the malto oligosaccharide prepared by reacting the amylofluranease. Malto oligosaccharide manufacturing method further comprising the step of removing impurities by addition. The malto oligosaccharide of claim 4, wherein the malto oligosaccharide comprises maltooctaose in an amount of 40 wt% to 90 wt% based on the total malto oligosaccharide, and further comprises maltoheptaose, maltononaose, or a mixture thereof. Oligosaccharide Preparation Method. The method according to claim 4, wherein the malto oligosaccharide comprises maltooctaose 40% to 90% by weight based on the total content, and further comprises one or more selected from the group consisting of malto oligosaccharide having 7 to 9 glucose molecules Phosphorus maltooligosaccharide manufacturing method.

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