KR20020059122A - Thermostable maltogenic amylase producing branched oligosaccharides, gene coding the same, and microorganism producing the same amylase - Google Patents

Abstract

PURPOSE: Provided are thermostable maltogenic amylase having thermo stability and producing branched oligosaccharides from starch, its coding gene, and a microorganism, Bacillus thermoalkalophilus ET2, producing the amylase. CONSTITUTION: The producing method of amylase by using Bacillus thermoalkalophilus ET2(KCTC 0084BP) comprises the steps of: manufacturing a plasmid vector including DNA sequence consisting of DNA sequence of SEQ ID NO:1 or DNA sequence of its allele and coding amylase; transforming a host with the vector; and culturing the transformed host.

Description

Heat-resistant maltogenic amylase that produces branched oligosaccharides, genes encoding it and microorganisms that produce it {Thermostable maltogenic amylase producing branched oligosaccharides, gene coding the same, and microorganism producing the same amylase}

The present invention relates to heat resistant maltogenic amylases and genes encoding them and microorganisms producing them.

Examples of maltogenic amylases that have specific enzymatic properties that hydrolyze starch to produce mainly maltose, break down pullulan to produce mainly pannose, and strongly degrade cyclodextrins are described in Korean Patent No. 186716. , Amylase produced from Bacillus stearothermophilus KFCC-10896, Bacillus licheniformis ATCC 27811 in US Pat. No. 5,583,039, Thermus sp in Korean Patent Publication No. 2000-47164 Amylase isolated from IM6501 KCTC 0527BP), or an enzyme having substantially the same properties as those thereof, and an enzyme produced by genetic recombination technology having substantially the same properties as these, and liquefied starch of the above-mentioned maltogenic amylase. Treatment with can yield branched oligosaccharides.

Amylase isolated from Thermos, a high-temperature strain of Korean Patent Application Laid-Open No. 2000-47164, decomposes acarbose and heat-resistant amylase that can transfer acarbosecin-glucose produced by decomposing acarbose to various sugar receptors. It is reported that the optimum operating temperature is 60 ℃, which is the most heat resistant among the amylase having the above characteristics.

Acarbose is a pseudotetrasaccharide composed of unsaturated cyclitol molecules, amino sugars, and two molecules of glucose, which have been reported to be no longer degraded by carbohydrate degrading enzymes. Acabiocin-glucose obtained by heating at 100 ° C. with dilute sulfuric acid for about 1 hour is pseudotrisaccharide (PTS), a pseudotrisaccharide in which a molecule of glucose at the reducing end of acarbose is hydrolyzed and removed. Known as

It is an object of the present invention to provide maltogenic amylases with very high optimum operating temperatures.

It is another object of the present invention to provide a gene encoding a maltogenic amylase.

Another object of the present invention is to provide a microorganism producing maltogenic amylase having a very high optimum operating temperature.

Another object of the present invention is to provide a recombinant microorganism comprising a gene encoding a maltogenic amylase.

1 is a temperature characteristic curve of amylase of the present invention when using β-cyclodextrin, starch, pullulan as a substrate (− ○ − soluble starch, − ▼ −: β-cyclodextrin).

Figure 2 is a pH characteristic curve of amylase of the present invention when using a β-cyclodextrin as a substrate (-●-: 50mM sodium acetate buffer,-○-50mM phosphate buffer,-▲-: 50mM Tris buffer solution Δ-: 50 mM glycine sodium hydroxide buffer solution.

3 is the result of thin layer chromatography showing the mode of action of amylase of the present invention on various substrates. In Fig. 4, reference material (M1): G1 to G7: maltodextrin, reference material (M2): PTS: pseudotrichsaccharide, Acb: acarbose, IAcb: isocarbose, substrate: A: β- Cyclodextrin, B: starch, C: flurane, D: acarbose.

Figure 4 is a result of high-speed ion chromatography showing the acarbiosine-glucose transition properties of the amylase of the present invention (α-MG: alpha-methyl glucopyranoside, G1: glucose, α-1,6, α-1 , 4, α-1,3: disaccharides linked by alpha-1,6, alpha-1,4, alpha-1,3 glucosidic bonds, PTS: pseudotrisaccharide, Acb: acarbose).

5 is a result of high-speed ion chromatography showing the form of amylase of the present invention to produce branched oligosaccharides (G1: glucose, IM: isomaltose, G2: maltose, IP: isopanose, Pan: pannos, BDP4: Branched tetrasaccharide).

The present invention relates to a novel heat resistant maltogenic amylase.

Maltogenic amylase according to the present invention hydrolyzes starch to produce mainly maltose and a small amount of glucose, decomposes pullulan to produce pannose, and strongly decomposes cyclodextrin to produce mainly maltose and a small amount of glucose. do. Amylase of the present invention specifically hydrolyzes acarbose, known as an inhibitor of amylase, to glucose and acarbiocin-glucose and transfers acarbiocin-glucose to various sugar receptors to produce various forms of transition products and also liquefy. Treating this enzyme with starch can produce branched oligosaccharides.

The amylase of the present invention has an optimum pH of about 8.0 and an optimum operating temperature of about 70 ° C when using β-cyclodextrin as a substrate. The molecular weight is about 69,045 Daltons as measured by Maldi Tof / Mass (Matrix aided laser desorption ionization time of flight / Mass spectroscopy).

The present invention also relates to a gene encoding a novel heat resistant maltogenic amylase.

The gene according to the present invention comprises a DNA sequence substantially identical to that described in SEQ ID NO: 1, a DNA sequence which is an allele of SEQ ID NO: 1, a sequence encoding an amylase functionally identical to an amylase encoded by SEQ ID NO: 1, and these sequences. As part of, it comprises a DNA sequence encoding acarbose degradable amylase. Such genes of the present invention can be isolated from, for example, Bacillus thermoalophilus ET2, or isolated from other microorganisms using SEQ ID NO: 1 or part thereof, or synthesized in whole or in part.

The invention also relates to microorganisms producing novel heat resistant maltogenic amylases. The present inventors searched for high-temperature strains having starch decomposition properties in swamps of Michigan, USA, and selected and identified excellent high-temperature strains, named Bacillus thermoalkalophilus ET2, Daejeon, Korea on October 31, 1998. It was deposited with the Korean Collection for Type Cultures, an affiliated biotechnology research institute, located at 52 Eun-dong, Yuseong-gu, Korea, and was assigned a deposit number (KCTC 0884BP).

The present invention also relates to a transformant comprising an amylase gene according to the present invention.

The transformant according to the present invention inserts the amylase gene of the present invention into a suitable vector according to a known method to produce a recombinant vector, introduces the produced vector into a host compatible with the recombinant vector, and acarbose. It can be prepared by selecting a transformant that produces a degradable amylase. The type of vector used at this time is not limited, and various known vectors may be used. The type of host used is also not limited, and may be introduced into a variety of known hosts that are compatible with recombinant vectors.

As a specific example, E. coli MC1061 is used as a host, and the plasmid p6xHis119 (Kim et al., 1999, Appl. Environ. Microbiol., 65 (4): 1644-1651) uses the maltose-generating heat resistant amylase gene of the present invention. The recombinant DNA (p6xHBTMA) obtained by insertion can be introduced into a host to produce a transformant ( E. coli BTMA).

In addition, the present invention relates to a method for mass production of amylase gene according to the present invention derived from Bacillus thermoalophilus ET2, the method of the present invention is derived from Bacillus thermoalkalophilus ET2 and degrades acarbose Preparing a plasmid vector comprising a DNA sequence encoding an amylase and comprising a portion of a DNA sequence that is substantially identical to SEQ ID NO: 1 or an allele of SEQ ID NO: 1, transforming a host cell with the vector, and transforming Culturing the prepared host cells.

In the case of producing maltose-resistant heat resistant amylase using the transformant according to the present invention, the cell culture period is 1-2 days and the culture temperature is 55 ° C. when the parent strain is used. Lowering the culture temperature to 37 ℃ can reduce the cost of microbial culture, it can also significantly increase the productivity of the enzyme. In addition, the acarbose degradable amylase according to the present invention is very high as the optimum temperature of the reaction 70 ℃, and excellent thermal stability, it is possible to complete the reaction in a short time by the enzymatic reaction at a high temperature, the fermentation process of other microorganisms Contamination can be prevented.

As used in the method of the present invention, a method for separating a chromosome from Bacillus thermoalophilus ET2, a method for cutting an isolated chromosome into an appropriate size, a method for linking to a desired vector, a method for introducing the vector into a cell, and the like Methods well known in the art, for example Molecular Cloning 1, 2, 3; A laboratory manual (J. Sambrook, E. F. Fritsch, T. Maniatis) can be carried out with reference to the method described.

The composition of the medium and the solution used described in the specification of the present invention are described below.

LBS medium (pH 7.0): 10 g / l bacto-tryptone, 5 g / l yeast extract, 5 g / l NaCl, 5 g / l soluble starch

LBA medium (pH 7.0): 100 μg / ml of ampicillin added to LB medium

Gene Donor Strains: Bacillus Thermoalkalophilus ET2

Host cell: E. coli MC1061 (supE44 ΔlacU169 (Φ 80 lacZΔM15) hsdR17 recAl endA1 gyrA96 thi-1 relA1)

Vector: p6xHis119

Composition of the solution used for transformation

Solution 1: 1% glucose, 50 mM Na ₂ EDTA, 25 mM Tris buffer, pH 8.0, 5 mg / ml lysozyme

Solution 2: 0.2 N sodium hydroxide, 1% SDS

Solution 3: 60ml 5M potassium acetate, 11.5ml glacial acetic acid, 28.5ml distilled water

Transformation solution 1: 50 mM calcium chloride solution

Transformation solution 2: 100 mM calcium chloride solution

TE solution: 10 mM Tris buffer (pH 8.0), 1.0 mM Na ₂ EDTA (pH 8.0)

The present invention also relates to an amylase encoded by a DNA sequence that is substantially identical to SEQ ID NO: 1 or an allele of SEQ ID NO: 1 or an enzyme that is functionally identical to an amylase encoded by SEQ ID NO: 1.

The present invention can be carried out as described in Korean Patent Publication No. 2000-25033, the contents of which are included in the contents of the present application specification.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by these Examples.

Example 1 Microbial Screening and Identification

The collected soil samples were suspended in sterile water, followed by centrifugation to take only the supernatant, which was plated on LBS solid medium containing 1.5% agar and incubated at 55 ° C. for 12 hours. Each solid medium was treated with iodine solution (I ₂ 0.203%, KI 5.2%) to select a strain having starch resolution for one week and identified using Bacillus thermoalkalophilus ET2 using its microbiological and physiological characteristics. Named it. The optimum growth temperature of this strain was 55 ° C.

Example 2. Transformant Preparation

Bacillus thermoalophilus ET2 (hereinafter referred to as parent strain) was inoculated in 50 ml of LBS medium, cultured at 55 ° C, and centrifuged to recover the cells. Dubnau, D. and R. D. Abenson, 1971, J. Mol. Chromosomal DNA was isolated based on the method described in 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. This was centrifuged and 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. Two volumes of 99% ethanol were added to precipitate, which was wound up with a glass rod to separate and dissolved in 1/10 × SSC buffer (3M sodium chloride, 0.3M sodium citrate).

E. coli with plasmid p6xHis119 and E. coli with plasmid pUC18 were respectively inoculated in 5 ml of LBA medium and incubated at 37 ° C. to log phase, and the cells were recovered by centrifugation of 1.5 ml of the culture medium. 100 μl of Solution 1 was added to the recovered cells, shaken vigorously, and left at room temperature for 5 minutes. 200 μl of Solution 2 was added thereto, mixed, and left to stand in ice for 5 minutes. 150 μl of Solution 3 was added thereto, mixed well, and then left in ice for 5 minutes, followed by centrifugation at 4 ° C. for 15 minutes to transfer only the supernatant to a clean tube. Add the same volume of phenol mixture (phenol: chloroform: isoamyl alcohol = 25: 24: 1 (volume ratio)) to the supernatant to remove protein, and then add 2 volumes of ice-cold ethanol and centrifuge at 4 ° C for 15 minutes. The plasmids were separated and precipitated and dissolved in 5 μl of TE solution (Molecular Cloning 1, 2, 3; A laboratory manual (J. Sambrook, et al.).

Two oligonucleotide primers MA-II (5'-GACGGYTGGCGBYTNGATGT-3 '), MA-IV (5'-TCATGACTGCCSAGCARRTT-, which specifically binds maltogenic amylases to the chromosomal genes of the parent strain obtained in Example 1 3 ') was used to perform polymerase chain reaction. From this, a gene fragment of about 300 bp was amplified, and the nucleotide sequence of the fragment was analyzed to confirm that it was a part of the maltogenic amylase gene. 5'-GTTCAGCACGGATTTCGTCTT-3 ').

Meanwhile, the chromosomal DNA of the parent strain obtained in Example 1 was cut with BamHI, a restriction enzyme, and ligated to plasmid pUC18, which was cut completely with the same enzyme, to prepare a chromosomal DNA library. Polymerase chain reaction with BS1822C and universal forward sequencing primer (5'-GTAAAACGACGGCCAGT-3 ') on the BamHI chromosomal DNA library yielded a DNA fragment of about 2.3 kb, which was returned to pUC18. Cloning to determine the sequence.

In addition, a chromosomal DNA library digested with SalI of the parent strain chromosomal DNA was prepared, and a DNA fragment of about 1.9 kb was obtained using BS1822N and universal forward sequencing primers to determine the base sequence. To isolate the maltogenic amylase gene, we devised BTMA5 (5'-GTGGCAAAGCATATGTTGAAAGAAGCC-3 '), BTMA3 (5'-GCCGAAGCGGAAGCTTACATCGAACG-3') primers, which contain coding regions of the genes from the sequence information of the two DNA fragments. The polymerase chain reaction was performed on the parent strain chromosomal DNA to obtain a product of about 2.2 kb. The amplified gene segments were treated with NdeI and HindIII and ligated with p6xHis119 vector digested completely with the same enzyme. For transformation, 5 ml of LB medium was inoculated with E. coli MC1061, a host cell, and incubated at 37 ° C. for 12 hours, and then 1.0 ml of the culture was inoculated in 50 ml of fresh LB medium and the absorbance was 0.5 at 600 nm. The cells were recovered by centrifuging 1.5 ml of the culture solution at 7000 xg for 5 minutes at 4 ° C., and then, 0.75 ml of the transformed solution 1 was added thereto, and the cells were suspended for 30 minutes in ice. Cells were recovered again by centrifugation at 6000 xg for 2 minutes at 4 ° C. 0.15ml of the transformed solution 2 was added to the recovered cells, and the cells were suspended and left in ice for 30 minutes.

0.15 ml of the suspension and about 500 ng of the ligated solution were mixed and left to stand in ice for 1 hour and then subjected to thermal shock at 42 ° C. for 2 minutes. 1 ml of LB medium was incubated at 37 ° C. for 1 hour, and then plated on 1.5% agar-containing LBA solid medium to select strains showing resistance to empicillin.

The first selected transformants were inoculated in LBA solid medium containing 1% soluble starch and incubated at 37 ° C. for about 12 hours, and 0.6% agar medium containing 3 mg of D-cycloserine was added thereto. 5 ml each of the transformants was poured into a culture medium and cultured again at 37 ° C. for 12 hours. Iodine solution was poured into the solid medium, and a strain showing a transparent ring around the bacteria was finally selected from the amylase producing strain (Esherichia coli MC1061 / p6xHBTMA).

Example 3 Amylase Gene Sequencing

Plasmid p6xHBTMA was isolated from the strain selected in Example 2 (Esherichia coli MC1061 / p6xHBTMA), and DNA fragments of about 3 kb containing the amylase gene were treated with various restriction enzymes and subcloned into plasmid pUC18 at appropriate sizes. Each plasmid DNA sequence thus prepared was determined using an ABI377 PRISM autosequencer (Perkin-Elmer Co.) (SEQ ID NO: 1). Amylase gene can be seen that consists of the DNA sequence of the base 1 ~ 1791 of SEQ ID NO: 1.

Example 4 Purification of Acarbose Degradable Amylase

Maltose-producing amylase producing strain (Esherichia coli MC1061 / p6xHBTMA) was inoculated in LBA medium and incubated at 37 ° C. for 10 hours and centrifuged to obtain cells. The recovered cells were suspended in 50 mM Tris buffer solution at pH 7.5, disrupted by ultrasonication, and centrifuged to obtain supernatant. Since six histidine residues were bound to the amino terminus of amylase expressed from p6xHBTMA, the supernatant was purified by passing through a Ni-NTA column (Stratagene Co., USA) and enzyme titers were measured.

Enzyme titer was added to 0.05 ml of enzyme solution, 0.2 ml of 50 mM glycine sodium hydroxide buffer solution at pH 8.0 and 0.25 ml of 1% (w / v) β-cyclodextrin solution. The reaction was stopped and then boiled for 5 minutes to develop color, and then determined by measuring absorbance at 575 nm. When the absorbance difference is 1.0, it is determined by 1 unit.

The molecular weight was about 69.045 Daltons as a result of analysis using Malditof / Mass, PerSeptive Biosystem Framingham, USA.

Example 5 Enzymatic Properties of Maltose-Generated Heat-Resistant Amylase

(1) temperature characteristics

To determine the optimum temperature for the purely purified enzyme, 0.25 ml of 50 mM glycine sodium hydroxide buffer solution at pH 6.0 containing 1% β-cyclodextrin and 30% liquefied corn starch as substrates and substrate After mixing 0.2 ml of the same buffer solution, which was not added, and preheating the mixture at each reaction temperature, 0.05 ml of the enzyme solution obtained in Example 4 was added to measure the enzyme titer. The results are shown in Fig. 1 (-○-soluble starch,-▼-: β-cyclodextrin).

(2) pH characteristics

In order to determine the effect of pH on the enzyme activity, the enzyme solution was preheated at 60 ° C. for 5 minutes by mixing 0.2 ml of the buffer solution with different pH and 0.25 ml of distilled water containing 1% of β-cyclodextrin, and Example 4 The enzyme solution obtained above was reacted for 30 minutes by adding 0.05 ml of the enzyme solution diluted with each pH buffer solution, and the enzyme activity was measured by the method described in Example 4. The results are shown in Fig. 2 (-●-: 50 mM sodium acetate buffer,-○-50 mM phosphate buffer,-▲-: 50 mM Tris buffer solution-Δ-: 50 mM glycine sodium hydroxide buffer solution). The buffer solution for each pH section is as follows.

pH 4.0-pH 6.0: sodium acetate

pH 6.0-pH 8.0: sodium phosphate

pH 8.0-pH 10.0: glycine sodium hydroxide

Example 6 Substrate Degradation Characteristics

50 mM glycine sodium hydroxide buffer solution at pH 8.0 and 0.5% (w / v) of pullulan, β-cyclodextrin, starch, and acarbose were used as substrates at 5 CU and 70 ° C. After reacting for 12 hours, each product was collected and analyzed by thin layer chromatography. This is shown in FIG. 3 (Standards (M1): G1 to G7: Maltodextrin, Standards (M2): PTS: pseudotrichsaccharide, Acb: Acarbose, IAcb: Isoacabose, Substrate: A: β-cyclodextrin, B: starch, C: flurane, D: acarbose). As can be seen in Figure 4, the amylase according to the present invention acts on pullulan to produce mainly panose, trace amounts of glucose and maltose, and break down β-cyclodextrin to produce a large amount of maltose and a small amount of glucose. It also breaks down acarbose to produce glucose and acabiocin-glucose. The starch, pullulan, and β-cyclodextrin degradability of the maltose-producing heat resistant amylase of the present invention were found to be 22: 100: 1297.

Example 7 Current Transfer Characteristics

To determine the enzyme's ability to transfer sugar, 5% (W / V) acarbose and 10% (W / V) alpha-methyl glucopyranoside were used as donors and acceptors, respectively. After reacting for 18 hours at 70 ° C. in 0.5 ml of sodium hydroxide buffer solution (pH 8.0), each product was analyzed by high-speed ion chromatography and shown in FIG. 4 (α-MG: alpha-methyl glucopyranoside, G1: glucose, α-1,6, α-1,4, α-1,3: disaccharides linked by alpha-1,6, alpha-1,4, alpha-1,3 glucosidic bond, respectively, PTS: Pseudotrisaccharide, Acb: acarbose). In addition, the reaction of the enzyme solution in 0.5 ml of glycine sodium hydroxide buffer solution containing 30% (W / V) starch was carried out under the same conditions by adding 40 U per gram of substrate. The result was analyzed by high-speed ion chromatography (G1). : Glucose, IM: isomaltose, G2: maltose, IP: isopanose, Pan: pannose, BDP4: branched tetrasaccharide).

It can be seen from FIGS. 4 and 5 that the amylase has a glycolysis capacity and produces a large amount of branched oligosaccharides.

Maltogenic amylase according to the present invention is excellent in heat resistance and can be used to synthesize a large amount of branched oligosaccharides from starch, and also by using the gene according to the present invention, a gene encoding an enzyme having a similar function to the enzyme of the present invention. Can be separated from other microorganisms.

Claims (4)

A DNA sequence encoding an enzyme substantially identical to SEQ ID NO: 1 or an allele of SEQ ID NO: 1 or an enzyme functionally identical to an amylase encoded by SEQ ID NO: 1. Amylase derived from Bacillus thermoallocophilus ET2 and having the following properties and having an acarbose resolution: i) A molecular weight of about 69,045 Daltons as measured by Maldi Tof / Mass ii) optimum temperature of reaction: about 70 ° C, iii) optimum pH: about 8.0 A plasmid vector that encodes an amylase that is derived from Bacillus thermoalophilus ET2 and degrades acarbose and comprises a DNA sequence that is substantially the same as SEQ ID NO: 1 or an allele of SEQ ID NO: 1 or a portion thereof. Preparing a; Transforming a host with said vector; And A method for producing amylase capable of acarbose resolution, comprising culturing the transformed host cell. Bacillus thermoalkalophilus ET2 KCTC 0884BP