KR101978565B1 - Pullulan degrading enzyme from Ruminococcus bromii and use thereof - Google Patents

Abstract

The present invention relates to a pullulan degrading enzyme derived from Ruminococcus bromii and a use thereof. More specifically, the present invention relates to a pullulan degrading enzyme derived from Ruminococcus bromii derived from Pannus The present invention also relates to a method for producing pannocyte using the composition, and to an α-amylase derived from ruminococousbrome having an activity of degrading pullulan to pananose.
The α-amylase derived from the lumino cortex of the present invention produces only panhorn without any other by-products from pullulan, so that the α-amylase can be usefully used for producing panhorns.

Description

[0002] Pullulan degrading enzyme from Ruminococcus bromii and use thereof [

The present invention relates to a method for treating Ruminococcus bromyl- derived pullulan degrading enzyme and its use, and more particularly to a composition for producing an α-amylase protein derived from Luminococcus bromi and a panose comprising the same, The present invention relates to a manufacturing method of a plate nose.

Panose is a trisaccharide carbohydrate composed of three glucose residues and can be used in a variety of applications. For example, inhibiting the metabolism of the harmful bacteria such as salmonella or E. coli and, intestinal yuikgyun the bifidobacteria can be used as prebiotics (prebiotics) promoting bacteria (Bifidobacterium) and Lactobacillus (Lactobacillus) growth of the microorganism of the genus. It has also been reported that carbohydrates can be used to prevent cavities because they are not metabolized by oral bacteria such as Streptococcus mutans (Microbiol Immunol. 1988; 32 (1): 25-31). In addition to these pharmacological functions, it can be used as a sweetener as a foodstuff function, and has a function of preventing color change of food, and is expected to be usefully used in the food and beverage industry.

On the other hand, the method of plate North production using enzymes, Aureobasidium (Auerobasidium) or Aspergillus (Aspergillus) glucosyl transmitter buffer race in origin substrate to maltose (maltose) using (glucosyltransferase, EC 2.4) A method in which sugar is transferred and biosynthesized; A method in which maltose and sucrose are used as a substrate to obtain pananose and fructose as products using dextransucrase (EC 2.4.1.5); Or a process for producing pananose from pullulan using Neopullulanase (EC 3.2.1.135); And a method for producing Pananus using microorganisms belonging to the genus Aureobasidium (Japanese Patent Laid-Open No. 1996-023989) have been developed. However, the above-mentioned known methods have a disadvantage in that by-products such as isomaltose, fructose, glucose, maltose and the like are produced together with pannose.

Under these circumstances, the present inventors have made extensive efforts to develop a method for producing pananose without by-products. As a result, it has been found that Ruminococcus amylase originating from the bromide- derived α-amylase was found to decompose pullulan to produce only pananose without any other by-products, thereby completing the present invention.

One object of the present invention is to provide a method of treating Ruminococcus amylase protein derived from a bromine- derived α-amylase protein.

It is another object of the present invention to provide a method for producing pananose comprising the step of reacting an α-amylase protein derived from L. gracilis with a pullulan.

It is another object of the present invention to provide an α-amylase derived from Luminococcus bromi, which has an activity of degrading pullulan to pannose.

It is still another object of the present invention to provide a polynucleotide encoding α-amylase derived from Luminococcus bromi, an expression vector comprising the polynucleotide, and a transformant comprising the expression vector.

In order to achieve the above object, one aspect of the present invention provides a composition for preparing panose, which comprises an α-amylase protein derived from Ruminococcus bromii .

In the present invention, it was confirmed that the α-amylase protein derived from Luminococcus bromy only produces pannus in high purity without any other by-products through the pullulanase activity of degrading pullulan.

The term " Ruminococcus " bromide "means an anaerobic and gram-positive bacterium belonging to the genus Luminocausus . However, it is not known that α-amylase derived from Luminocauscus broxima can produce pananose by degrading pullulan, The present invention is characterized in that the α-amylase derived from the above-mentioned LUMINOKOCUS BROOMY is capable of decomposing pullulan to produce only pananose without producing any by-products.

The term " panose " of the present invention refers to the formula C ₁₈ H ₃₂ O ₁₆ , a trisaccharide carbohydrate composed of three glucose residues. Three glucose molecules are linked via alpha-1, 4 and alpha-1, 6 glucosidic bonds. Pananos can be used for a variety of purposes, such as prebiotics, food additives, or tooth decay.

The term " -amylase " of the present invention is a kind of amylase, generally an endo-type enzyme that hydrolyzes amylose and amylopectin from the inside, and mainly produces texturin. Amylase because the reducing residue of the resulting saccharide is of the? -Pyranose type. In the present invention,? -Amylase is derived from Luminococcus brodii and may have? -Glucosidase activity, but is not limited thereto.

The α-amylase derived from the above-mentioned Luminococcus bromi can be named in combination with 'Amy 5'.

For the purpose of the present invention, the? -Amylase may be one having an activity of pullulanase activity, specifically degrading pullulan to pananose.

The term " pullulanase " of the present invention is a protein having a pullulanase activity, Or an enzyme that specifically hydrolyzes an? -1,6 glycoside bond to produce maltotriose. As a specific example, pullulanase (pullulan-6-glucanohydrolase) and type II pullulanase are known.

The term " pullulan " of the present invention refers to alpha-1,4-; Or a maltotriose unit, also known as? -1,6-glucan. Three maltotriose glucose units are linked by α-1,4 glycosidic bonds, and successive maltotriose units are linked by α-1,6 glycosidic bonds. Pullulan is a black yeast ( Aureobasidium pullulans (DE BARY) ARN.), which is generally used as a food additive for improving the physical properties, texture or sweetness of foods.

In the present invention, the α-amylase derived from Ruminococcus bromi may have the amino acid sequence of SEQ ID NO: 1.

The sequences used in the present invention are interpreted to include sequences showing substantial identity with the sequences listed in the sequence listing, considering mutations having biologically equivalent activity. The term " substantial identity " means aligning the sequence of the present invention to any other sequence as closely as possible and, when analyzing the aligned sequence using algorithms commonly used in the art, Means a sequence showing 60% homology, more specifically 70% homology, even more specifically 80% homology, most specifically 90% homology.

The term " homology " refers to the percentage of identity between two polynucleotides or polypeptide mimetics. The homology between sequences from one moiety to another can be determined by known techniques. For example, homology can be determined by aligning the sequence information and directly aligning the sequence information between two polynucleotide molecules or two polypeptide molecules using a computer program that is readily available. The computer program may be BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlign (DNASTAR Inc) or the like, but any program capable of determining homology can be used without limitation.

Therefore, the sequence having high homology with the sequence of SEQ ID NO: 1 according to the present invention, for example, the homology thereof is 70% or more, specifically 80% or more, more specifically 90% or more, still more specifically 95% Sequence having a high homology with the nucleotide sequence of the present invention should be construed as being included in the scope of the present invention.

The composition for preparing pananose according to the present invention is useful as a microorganism for producing an α-amylase protein derived from Luminococcus bromi; A culture solution of the microorganism; A disruption of said microorganism or said culture; Or a combination thereof.

The microorganism producing the protein refers to a microorganism that is modified so as to intrinsically produce the? -Amylase of the present invention or to enhance the activity of degrading pullulan into pananus compared to the intrinsic activity.

The " culture solution " is a nutrient substance used for the growth of each of the above-mentioned biomass, and includes, but is not limited to, a microorganism producing the protein, a culture medium containing the microorganism, And a culture filtrate containing the microorganisms.

As the culture solution, a culture solution known in the art can be used, and if it is possible to cultivate Luminococcus brothy, the concentration of C, H, O, N, P, K, S, Ca, Mg, Fe, Mn , Inorganic nutrients such as Cu, Zn, B, and Mo; Organic nutrients such as carbohydrates, plant growth regulators, and vitamins; Amino acids such as myo-inositol, glycine and L-glutamine; A carbon source such as glucose, fructose, mannose, ribose, xylose, sucrose, melibiose, cellobiose, lactose, amylose, carbohydrate, raffinose, sorbitol, mannitol and glycerol may or may not be added.

The " crushed product " means a product obtained by disrupting the microorganism, its culture, etc. by any known method.

Since the microorganism contains an α-amylase protein derived from LUMINOKOCUS BROOMIE having a phlorulanase activity, the culture solution of the microorganism, or the lysate of the microorganism or the culture solution contains the α-amylase protein, .

In a specific embodiment of the present invention, when the α-amylase derived from LUMINOKOCUS BROOMUS of the present invention is reacted with pullulan, the by-products such as glucose, maltose and isomalt are not present in the reaction product thereof, (Fig. 6).

This suggests that the composition of the present invention containing the α-amylase derived from the above-mentioned Luminocauscus broth can be usefully used for the production of pananose.

Another embodiment provides a method for producing pananose comprising the step of reacting the α-amylase protein from Luminococcus bombii with pullulan.

At this time, the definitions of the above-mentioned " Luminococus bacteria, " " alpha amylase, " " pullulan and "

In the present invention, the above reaction can be carried out under the condition that the α-amylase protein derived from L. cv.

Specifically, the above reaction is not limited as long as the α-amylase protein derived from the Luminocarbacterium has a plulo-lanease activity, but it can be carried out under pH conditions at a pH of 4.5 to 7.5, more specifically at a pH of 5 to 7 , Wherein pH 5 is sodium acetate; And pH 6-7 can be adjusted using, but not limited to, sodium phosphate buffer.

The temperature conditions may be 30 to 50 ° C, more specifically 35 to 45 ° C, but are not limited thereto.

Also, the time may be 1 to 5 hours, more specifically 3 hours, but is not limited thereto.

In a specific embodiment of the present invention, the activity of the α-amylase derived from Luminococcus bromi of the present invention is maximized under the condition that the pH is adjusted to 5 to 7 by sodium acetate or sodium phosphate buffer, It was confirmed that high activity lasted for a long time (Figs. 2 to 4). Also, when the α-amylase and pullulan were reacted in a sodium phosphate buffer at pH 7 for 3 hours at 35 ° C., no byproducts such as glucose, maltose and isomaltose were present in the reaction products thereof, (Fig. 6).

This suggests that the production method of the present invention, including the step of reacting pullulan with the α-amylase derived from Luminococcus bromi, can be usefully used for the production of pananose.

Another object is to provide an α-amylase derived from Luminococcus bromi, which has an activity of degrading pullulan to pananose.

Herein, the definitions of "pullulan", "pananose", "luminoccus broth" and "α-amylase" are as described above.

Specifically, the α-amylase derived from Ruminococcus bromi may be composed of the amino acid sequence of SEQ ID NO: 1 and may also have a pullulan degrading activity, ie, a pullulanase activity.

The? -Amylase of the present invention decomposes pullulan to produce only pananose without any other by-products, and thus can be usefully used for compositions and methods for preparing pananose.

Another aspect of the present invention provides a polynucleotide encoding an α-amylase protein derived from Luminococcus bombii, an expression vector comprising the polynucleotide, and a transformant comprising the expression vector.

Herein, the definitions of "pullulan", "pananose", "luminoccus broth" and "α-amylase" are as described above.

The polynucleotide of the present invention may be one which codes for an α-amylase protein derived from Luminococcus bromi. Specifically, the polynucleotide encoding the protein may be selected from a range that does not change the amino acid sequence of the protein expressed from the coding region, considering the codon preference in the organism to which the protein is to be expressed due to codon degeneracy The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 may be any of the nucleotide sequences of SEQ ID NO: 1, 2, 3, and 4, but may be specifically the nucleotide sequence of SEQ ID NO:

An expression vector comprising a polynucleotide of the invention refers to a polynucleotide construct comprising an essential control element operably linked to the expression of the polynucleotide insert, which is an expression vector capable of expressing the desired protein in a suitable host cell. By transforming or transfecting the prepared recombinant vector into a host cell, desired proteins can be obtained.

The expression vector containing the polynucleotide provided by the present invention is not particularly limited, but plasmids derived from Escherichia coli (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis -derived plasmids (pUB110 and pTP5) - derived plasmids (YEp13, YEp24 and YCp50) and Ti-plasmids which can be used for Agrobacterium-mediated transformation. Specific examples of phage DNA include lambda-phages (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11 and lambda ZAP). In addition, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus, double-stranded plant viruses (e.g., CaMV), single-stranded viruses Or viral vectors derived from germinavirus may also be used.

As a vector of the present invention, a fusion plasmid (for example, pJG4-5) to which a nucleic acid expression-activating protein (for example, B42) is ligated can be used. In order to facilitate the purification of the recovered target protein in the present invention, the plasmid vector may further include other sequences as necessary. Such fusion plasmids may include GST, GFP, His-tag, Myc-tag, etc. as tags, but the fusion plasmid of the present invention is not limited by these examples.

Further, in the production of the fusion protein, it may include a chromatography process, and the fusion protein may be purified by affinity chromatography in particular. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of the enzyme, can be used. When hexahistidine is used, Ni-NTA His-conjugated resin column (Novagen, USA) Can be easily recovered.

In order to insert the polynucleotide of the present invention into a vector, a method of inserting the purified DNA into a restriction site or a cloning site of a suitable vector DNA by cleaving the DNA with an appropriate restriction enzyme can be used.

The polynucleotide encoding the α-amylase protein derived from Luminococcus bromi of the present invention may be operably linked to a vector. The vector of the present invention may further comprise a promoter and a nucleic acid of the present invention in addition to a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker ), A ribosome binding sequence (SD sequence), and the like. Examples of selectable markers include, but are not limited to, chloramphenicol resistant nucleic acids, ampicillin resistant nucleic acids, dihydrofolate reductase, neomycin resistant nucleic acids, etc., but limiting the additional components operatively linked by the above examples no.

In the present invention, " transformation " refers to a phenomenon in which DNA is introduced as a host and DNA can be cloned as a factor of chromosome or by integration of chromosome integration, thereby introducing an external DNA into a cell to cause an artificial genetic change do.

By such transformation, an expression vector comprising a polynucleotide encoding the α-amylase protein derived from Luminococcus bromi of the present invention or a part of the expression vector can be introduced into a host cell, Means a part of an expression vector comprising a polynucleotide moiety encoding an α-amylase protein derived from Luminococcus bromi so as to confer the activity of the α-amylase protein derived from the Luminococcus bombii in the host cell. For example, but not by way of limitation, T-DNA of a Ti plasmid transferred into a host cell in an Agrobacterium-mediated transformation method.

Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art. Generally, the transformation methods include the CaCl ₂ precipitation method, the Hanahan method which uses a reducing material called DMSO (dimethyl sulfoxide) for the CaCl ₂ method, the electroporation method, the calcium phosphate precipitation method, the protoplasm fusion method, Agrobacterium-mediated transformation, transformation with PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods. The method for transforming the vector comprising the polynucleotide encoding the α-amylase protein derived from the luminoccus bombii of the present invention is not limited to the above examples, and a transformation or transfection method commonly used in the art Can be used without limitation.

The kind of the host cell that can be used in the production of the transformant in the present invention is not particularly limited as long as it is capable of expressing the polynucleotide of the present invention. Specific examples of the host which can be used in the present invention include bacteria belonging to the genus Escherichia such as E. coli ; Bacteria of the genus Bacillus such as Bacillus subtilis ; Bacteria of the genus Pseudomonas such as Pseudomonas putida ; Saccharomyces S. cerevisiae ), skiing investigation, Schizosaccharomyces yeasts such as pombe ; Animal cells, plant cells, and insect cells. Specific examples of the Escherichia coli strain that can be used in the present invention include CL41 (DE3), BL21 or HB101, and specific examples of the Bacillus subtilis strain include WB700 or LKS87.

The transformant into which the expression vector comprising the polynucleotide of the present invention has been introduced may be in the form of a transformed cell or an organism.

The transformant into which the expression vector containing the polynucleotide encoding the α-amylase protein derived from the Luminococaine bacterium of the present invention transformed by the above-mentioned method was introduced had a pullulanase activity for degrading pullulan to pananose I have.

In a specific embodiment of the present invention, Escherichia coli was transformed with the? -Amylase gene derived from the Luminococcus sp. Strain encoded by the nucleotide sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 1 Amylase recombinant protein consisting of the sequence was purified and purified (Example 1). As a result of reacting the protein with pullulan, it was confirmed that there was no by-product such as glucose, maltose, isomaltose and the like in the reaction product thereof, and pannosman was present (FIG. 6).

This is because a polynucleotide encoding an α-amylase derived from the above-mentioned Luminococcus bacterium strain, an expression vector containing the polynucleotide, and a transformant containing the expression vector can be usefully used for the production of Pananus .

The α-amylase derived from the lumino cortex of the present invention produces only panhorn without any other by-products from pullulan, so that the α-amylase can be usefully used for producing panhorns.

Figure 1 is a graphical representation of Ruminococcus is an SDS-PAGE image showing the result of purification of α-amylase (hereinafter referred to as "Amy 5") derived from bromide by Ni-NTA affinity chromatography. Inclusion body refers to the inclusion body, Crude refers to the crude protein of the cells, Crude Passing through refers to the crude protein of the cells filtered through chromatography, and Elute refers to the protein eluted from the chromatography resin.
FIG. 2 is a graph showing the activity of Amy 5 according to pH. FIG.
FIG. 3 is a graph showing the activity of Amy 5 according to temperature. FIG.
4 is a graph showing the thermal stability of Amy 5.
FIG. 5 is a chromatogram of TLC showing the result of analyzing the enzyme reaction and the product according to the substrate of Amy 5. G1-7 standard is based on malto-oligosaccharide polymerized with 1 to 7 glucose units (glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose G5), maltohexaose (G6), and maltoheptaose (G7)); ? -CD,? -CD and? -CD are? -cyclodextrin,? -cyclodextrin and? -cyclodextrin, respectively; And G3- [beta] -CD refer to G3- [beta] -cyclodextrin.
6 is a chromatogram of HPAEC analyzing the reaction product of Amyl 5 with pullulan.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example  One. Pullulan  Digestion enzyme production

Example  1-1. α- Amylase (? -amylase) gene Cloning cloning method

In order to prepare a pullulanase, α-amylase gene derived from Ruminococcus bromii was used.

Specifically, as shown in the following Table 1, forward and reverse primers were prepared for the chromosomal DNA of Luminococcus broccius ATCC 27255 strain. PCR was performed on the above chromosomal DNA using the above primers and KOD polymerase under the conditions shown in Table 2 below to obtain a PCR product of 1,572 bp. At this time, the nucleotide sequence of the obtained PCR product, that is, the base sequence of? -Amylase derived from Luminococcus brothy was named as SEQ ID NO: 2.

primer direction Base sequence F_NheI_rb_amy5 Forward 5'-gct agc TCA AAA TCA GAT TCA TCC GAC GGA AA-3 '
(SEQ ID NO: 3) R_XhoI_rb_amy5 Reverse 5'-ctc gag TTC AGC AGA CTT AAT GAT TAC CGT TGA-3 '
(SEQ ID NO: 4)

denaturalization
(denaturation) Initial denaturation
(initial denaturation) 95 ° C / 5 min Amplification
lt; / RTI > Denaturation 95 ° C / 30 seconds 30 times Annealing 60 ° C / 30 seconds Extension 72 ° C / 2 min Final Expansion
(final extension) Final extension 72 ° C / 5 min

The obtained PCR product (gene fragment) was A-tailed using Taq polymerase and then ligated with T-vector at 16 ° C for 3 hours. Each linkage mixture was transformed into E. coli DH10B strain and blue / white screening was used to obtain a construct in which the gene fragment was inserted.

Each construct was treated with restriction enzymes of Nhe I / Xho I and then single fractions were obtained by gel extraction. After separating the fragments of various lengths through agarose electrophoresis, connected with the same restriction enzymes pET21a expression vector (containing 6 × His tag) was produced by treatment with a recombinant expression vector pET- rb _ amy5. In this case, the pET- rb _ _ amy5 vector rb includes amy5 gene, it was confirmed that contains eight additional amino acid residues (Leu-Glu-His × 6 ) the 3 'end of the gene.

Example  1-2. α- Amylase  A method for producing a transformant containing a gene

The pET-Rb_amy5 vector prepared in Example 1-1 was transformed into E. coli BL21 (DE3) codon plus strain to prepare a transformant that finally expressed an? -Amylase gene. The transformant was inoculated into a liquid LB medium containing 100 μg / ml of ampicillin and cultured until an OD value of 600 nm reached 0.5. Then, 0.5 mM IPTG Was added. In order to purify the recombinant protein bound to the produced 6xHis-tag, the culture was centrifuged at 4,000 rpm at 4,000 rpm for 30 minutes to collect the transformant cells.

Example  1-3. α- Amylase  How to purify proteins

The cells recovered in Example 1-2 were suspended in a dissolution buffer (50 mM NaH ₂ PO ₄ , pH 7.0, 300 mM NaCl, 10 mM imidazole, pH 7.0) and sonicated. The crushed liquor was centrifuged at 4 DEG C for 12 minutes at 12,000 rpm for 20 minutes to obtain crude protein. The crude protein was injected into Ni-NTA affinity chromatography to purify the amylase recombinant protein. Thereafter, as shown in Fig. 1, it was confirmed that the recombinant protein was expressed and purified through SDS-PAGE. At this time, the amino acid sequence of the obtained recombinant protein, that is, the amino acid sequence of? -Amylase derived from Luminococcus brothy was named as SEQ ID NO: 1.

Example  2. Rumino Caucus Bromy  Derived α- Amylase  Character analysis

Example  2-1. Analysis of Enzyme Activity by pH

(Hereinafter referred to as "Amy 5") derived from Ruminococcus bromi expressed and purified through the above Example 1.

First, 100 μl of 50 mM sodium phosphate (pH 7) solution containing 10 μl of Amy 5 and 1 mM of γ-cyclodextrin (CD) was reacted at 45 ° C. for 10 minutes, (dinitrosalicylic acid) solution was added, heated for 5 minutes, and then cooled. Then, the reaction solution formed by the reducing end of the glucose and the degraded polymer produced through the reaction was measured at 550 nm absorbance using a spectrophotometer (DU730, Beckman Coulter, Pasadena, Calif., USA). The measured absorbance was set to one unit in terms of the amount of enzyme that produced 1 탆ole of reducing sugar per minute compared to the maltose standard curve.

To confirm the optimum pH of the Amy 5, 2 units of Amy 5 were dissolved in 50 mM sodium acetate buffer (pH 4 to 5), sodium citrate buffer (pH 5 to 6), pH A solution of 0.5% solubilized starch dissolved in a sodium phosphate buffer of 6-8, a tris-HCl buffer of pH 7-9, or a glycine-NaOH buffer of pH 9-10 soluble starch) at 45 ℃ for 5 min, the relative enzyme activity of Amy5 was compared.

As a result, as shown in Fig. 2, it was confirmed that the relative activity of Amy 5 was maximized as about 100% when sodium acetate of pH 5 was used as a buffer. In addition, it was confirmed that the sodium phosphate buffer having a pH of 6 or 7 exhibited a high activity of 80% or more than 60%, respectively.

From the above results, it was found that the use of sodium acetate at pH 5 or a sodium phosphate buffer at pH 6 or 7 for the enzyme reaction using α-amylase derived from Luminococcus bromi could maximize the activity of the enzyme Respectively.

Example  2-2. Enzyme activity analysis by temperature

In order to confirm the optimum temperature of Amy 5, 2 units of Amy 5 were reacted with 0.5% soluble starch dissolved in 50 mM of pH 7 sodium phosphate buffer at 30 to 55 ° C for 10 minutes to determine the relative enzyme activity of Amy 5 Were compared.

As a result, as shown in FIG. 3, it was confirmed that the relative activity of Amy 5 at 45 ° C was maximized as about 100%. It was also confirmed that the activity was 90% or more even at 35 to 40 ° C.

From the above results, it was confirmed that the enzyme reaction using the α-amylase derived from Luminococcus bromi was able to maximize the activity of the enzyme by using the temperature condition of 35 to 45 ° C.

Example  2-3. Analysis of thermal stability of enzyme

To confirm the thermal stability of the Amy 5, 2 units of Amy 5 were reacted with 0.5% soluble starch dissolved in 50 mM sodium phosphate buffer at pH 7 at 35, 40 or 45 ° C for 10 minutes, Enzyme activities were compared.

As a result, as shown in FIG. 4, it was confirmed that the relative activity of Amy 5 at 35 ° C. was stable even after 120 minutes of exposure, the activity decreased after 20 minutes at 40 ° C., Respectively.

From the above results, it was confirmed that using the temperature condition of 35 ° C for the enzyme reaction using α-amylase derived from Luminococcus bromi can maintain the activity of the enzyme for a long time.

Example  3. Rumino Caucus Bromy  Derived α- Amylase  Used Panose  Produce

Example  3-1. Enzyme reactions and analysis of products

0.5% amylose, soluble starch, pullulan, or amylopectin as a substrate of Amy 5; Or 10 mM of? -CD,? -CD,? -CD, G2-β-CD and G3-β-CD G3-β-cyclodextrin) and G5-β-CD (G5-β-cyclodextrin). 4 units of Amy 5 were added to a pH 7 sodium phosphate buffer containing each of the above substrates and reacted at 35 ° C for 3 hours. After the reaction, the reaction solution was heated in boiling water for 5 minutes to terminate the reaction.

Then, TLC (Thin-Layer Chromatography) analysis was performed on each of the reaction products in a 3: 1: 1 solvent mixture of isopropyl alcohol: ethyl acetate: water. TLC was developed using a Whatman K5F plate and dried. Then, color development was performed using a dip solution (methanol 950 ml, sulfuric acid 50 ml, 1-naphtol 3 g) and dried at 110 ° C to determine the enzyme reaction product Respectively.

As a result, as shown in FIG. 5, it was confirmed that α-amylase derived from LUMINOKOCUS BROSIS degrades each substrate, and in particular, when pullulan is used as a substrate, To produce panose.

Example  3-2. Amy 5 and Pullulan  Analysis of reaction products

It was confirmed through Example 3-1 that α-amylase derived from Luminococcus bromi generates pannose from pullulan. To confirm more precisely, the reaction product was analyzed by HPAEC (High Performance Anion Exchange Chromatography) Respectively.

Specifically, using a solvent A (50 mM NaOH) and a solvent B (50 mM NaOH, 100 mM NaOAc), a linear gradient reaching 40% of solvent B up to 50 minutes was applied to a CarboPAC PA-1 column , And qualitatively analyzed using an integrated pulsed amperometric detector. Further, the detection results of the glucose, maltose, isopanose or pananose standards and the detection results of the above reaction products were compared.

As a result, as shown in FIG. 6, it was confirmed that there was no by-product such as glucose, maltose, isomaltose or the like in the reaction product using pullulan as a substrate, and pananus was present only in the reaction product.

From the above results, it was confirmed that when the α-amylase derived from LUMINOKOCUS BROOMI used pullulan as a substrate, pullulan was decomposed to produce only PANNOS in high purity without any other by-products.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Pullulan degrading enzyme from Ruminococcus bromii and use          the <130> KPA170083-KR <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 524 <212> PRT <213> Ruminococcus bromii <400> 1 Ser Lys Ser Asp Ser Ser Asp Gly Asn Thr Ala Lys Val Ala Asp Pro   1 5 10 15 Ile Glu Gly Val Lys Ala Thr Ala Ser Thr Asp Lys Tyr Arg Asn Tyr              20 25 30 Tyr Glu Ile Phe Val Asn Ser Phe Cys Asp Ser Asn Gly Asp Glu Thr          35 40 45 Gly Asp Leu Gln Gly Ile Ile Ser Gln Leu Asp Tyr Leu Asn Asp Gly      50 55 60 Asp Pro Asn Ser Gly Asp Asp Leu Gly Val Asp Ala Ile Trp Leu Thr  65 70 75 80 Pro Ile Met Pro Ser Lys Ser Tyr His Lys Tyr Asp Val Glu Asp Tyr                  85 90 95 Tyr Asn Ile Asp Pro Asp Phe Gly Thr Leu Asp Asp Phe Asp Lys Leu             100 105 110 Ile Glu Glu Cys His Lys Arg Gly Ile Asn Val Ile Leu Asp Leu Val         115 120 125 Leu Asn His Ala Ser Ser Lys Asn Pro Leu Phe Thr Lys Ala Val Glu     130 135 140 Glu Val Ala Asp Asn Lys Leu Asp Gly Asn Ala Glu Tyr Phe Glu Ile 145 150 155 160 His Lys Ala Ser Tyr Phe Asp Ser Asn Thr Gln Thr Ile Ser Leu Gly                 165 170 175 Asn Gly Tyr Ala Cys Glu Ala Asn Phe Ser Gln Glu Met Pro Glu Trp             180 185 190 Asn Leu Asn Ser Lys Lys Thr Arg Glu Glu Phe Thr Lys Ile Ala Lys         195 200 205 Phe Trp Leu Asp Arg Gly Val Asp Gly Phe Arg Leu Asp Ala Cys Lys     210 215 220 Tyr Phe Thr Asn Lys Glu Thr Asp Gly Thr Glu Phe Leu Lys Trp Phe 225 230 235 240 Tyr Asp Thr Cys Lys Gly Ile Lys Glu Asp Val Tyr Met Val Gly Glu                 245 250 255 Asn Trp Thr Asp Asp Ser Asp Ile Gln Glu Leu Tyr Lys Ser Gly Ile             260 265 270 Asp Ser Gln Phe Ala Phe Lys Phe Ser Thr Ser Thr Gly Thr Ile Ile         275 280 285 Ser Asn Ile Ser Ser Gln Gly Gly Met Ala Thr Ala Lys Lys Ile Met     290 295 300 Asn Tyr Asp Asn Lys Met Ala Glu Ser Asn Pro Asn Ala Ile Asn Ala 305 310 315 320 Met Phe Leu Ser Asn His Asp Gln Val Arg Ser Gly Asn Ala Leu Glu                 325 330 335 Ser Gln Gly Leu Ser Ser Gln Lys Leu Ala Ala Ala Val Tyr Met Leu             340 345 350 Ala Pro Gly Asn Pro Phe Ile Tyr Tyr Gly Glu Glu Ile Gly Ile Lys         355 360 365 Ala Pro Asn Thr Thr Asn Asp Ala Ala Tyr Arg Thr Gln Met Val Phe     370 375 380 Asp Ser Glu Asn Leu Pro Asp Ile Tyr Val Asn Gly Ile Gly Asp Glu 385 390 395 400 Pro Asp Val Pro Thr Gly Gly Gly Val Lys Gln Gln Leu Ala Asp Lys                 405 410 415 Asp Ser Leu Leu Asn Tyr Tyr Arg Arg Ile Ile Thr Ile Lys Asn Gln             420 425 430 Asn Pro Glu Ile Ala Arg Gly Arg Ile Val Gly Thr Gln Gly Phe Asp         435 440 445 Asp Lys Thr Val Gly Ala Tyr Tyr Val Glu Tyr Glu Asp Ser Lys Leu     450 455 460 Leu Ile Ile His Asn Phe Ser Lys Asn Asp Ala Lys Glu Leu Thr Ile 465 470 475 480 Thr Asp Asp Met Ile Lys Asn Ala Thr Leu Arg Ala Asp Leu Ile Pro                 485 490 495 Glu Ser Ser Lys His Thr Glu Leu Lys Asp Gly Lys Ile Ser Val             500 505 510 Pro Pro Gln Ser Thr Val Ile Ile Lys Ser Ala Glu         515 520 <210> 2 <211> 1575 <212> DNA <213> Ruminococcus bromii <400> 2 tcaaaatcag attcatccga cggaaatact gcaaaagttg ccgacccgat tgaaggcgtt 60 aaggcaactg catcaaccga taaatacaga aactactacg aaattttcgt aaactctttc 120 tgcgattcaa acggtgacga aacaggcgac cttcagggaa tcatctcaca gcttgactac 180 cttaatgacg gcgatccgaa ttcaggtgac gacctcggtg ttgacgcaat ctggcttact 240 ccgattatgc cgtcaaagtc atatcataag tatgatgttg aagattatta caatattgat 300 cccgacttcg gtacgctcga cgattttgac aagcttattg aagaatgtca caagcgtggc 360 ataaatgtaa ttcttgacct tgttttgaat cacgcatctt caaaaaatcc gcttttcaca 420 aaggctgttg aagaggttgc cgataataag cttgacggca atgccgaata ttttgaaatt 480 cacaaggcct catattttga cagcaataca cagactatca gtcttggaaa cggttatgcc 540 tgtgaggcga acttttctca ggagatgccc gaatggaatc tcaattctaa aaagacgaga 600 gaagaattta caaaaattgc gaaattctgg cttgaccgag gagttgacgg cttcagactt 660 gacgcttgca aatactttac aaataaggaa accgacggaa cagaatttct caagtggttc 720 tacgacacct gcaagggtat taaggaagat gtctatatgg ttggcgagaa ctggaccgat 780 gattccgata ttcaggagct ttacaaatcc ggcatcgaca gccagtttgc atttaagttt 840 tcaacctcaa caggcacaat tatcagcaac atcatttcgc agggcggtat ggctactgcc 900 aaaaaaatta tgaactacga taacaaaatg gcagagtcaa atcccaatgc tatcaatgca 960 atgttccttt caaaccacga tcaggttcgt agcggaaacg cacttgaatc tcagggactt 1020 tcttctcaga aacttgcagc cgcagtttat atgcttgcgc cgggtaatcc gttcatctat 1080 tacggagagg aaatcggcat taaggctccg aataccacaa acgatgcggc atacagaaca 1140 cagatggttt ttgacagtga gaaccttccc gatatttatg taaacggtat cggagatgag 1200 ccagatgtgc ctaccggcgg cggtgtaaaa cagcagcttg ccgacaagga ttctctcctc 1260 aactactatc gtagaattat cacaatcaaa aatcagaacc ccgaaattgc aaggggcaga 1320 attgtaggca cacagggctt tgatgataag acagtcggcg cttactatgt tgagtatgag 1380 gattcaaagc tcctcattat tcacaacttc agcaagaatg acgccaagga gcttacaatt 1440 acagacgata tgattaagaa tgcaactctc cgtgcagacc ttatccccga gagcagttca 1500 aagcataccg agcttaaaga cggaaaaatc tctgtaccgc cacagtcaac ggtaatcatt 1560 aagtctgctg aatga 1575 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > F_NheI_rb_amy5 Forward primer <400> 3 gctagctcaa aatcagattc atccgacgga aa 32 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F_NheI_rb_amy5 Reverse primer <400> 4 ctcgagttca gcagacttaa tgattaccgt tga 33

Claims (12)

1. A composition for the production of panose comprising an alpha -amylase protein consisting of the amino acid sequence of SEQ ID NO: 1, wherein the protein has pullulanase activity. delete delete The composition of claim 1, wherein the composition for preparing panhose comprises a microorganism producing the protein; A culture solution of the microorganism; A disruption of said microorganism or said culture; Or a combination thereof. A method for producing a panose comprising reacting an α-amylase protein consisting of the amino acid sequence of SEQ ID NO: 1 with a pullulan, wherein the protein is pullulanase, &Lt; / RTI &gt; 6. The process according to claim 5, wherein the reaction is carried out at a pH of 4.5 to 7.5. 6. The process according to claim 5, wherein the reaction is carried out at 30 to 50 &lt; 0 &gt; C. 6. The process according to claim 5, wherein the reaction is carried out for 1 to 5 hours. An α-amylase consisting of the amino acid sequence of SEQ ID NO: 1, having an activity of degrading pullulan into panose. A polynucleotide encoding the alpha -amylase of claim 9. An expression vector comprising the polynucleotide of claim 10. A transformant, excluding human, comprising the expression vector of claim 11.

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