KR100467956B1 - Novel Bacillus polymixa MGL21 strain, DNA producing cycloinulooligosaccharide fructanotransferase, method for producing cycloinulooligosaccharide fructanotransferase from it and for cyclofructan thereof - Google Patents

Abstract

The present invention relates to a novel microorganism that produces cycloinolooligosaccharide fructanotransferase and a method for producing cyclofructan using the same.

The present invention produces cyclofructan (CF) from inulin protein with Cycloinulooligosaccharide fructano transferase (CFTase) enzyme activity derived from Bacillus polymyxa MGL21 It is used in the enzymatic process to provide high value added sugar from plant waste resources.

Description

Novel Bacillus polymyxa MG21 strain producing cycloinolo oligosaccharide fructanotransferase, From this method of producing cycloinolo oligosaccharide fructanotransferase and cyclo fructan using the same Novel Bacillus polymixa MGL21 strain, DNA producing cycloinulooligosaccharide fructanotransferase, method for producing cycloinulooligosaccharide fructanotransferase from it and for cyclofructan

The present invention relates to novel microorganisms from soil that produce cycloinolooligosaccharide fructanotransferase enzyme.

Cyclofructan (CF) is a material in which 6 to 8 fructosyl groups are cyclically bonded by beta-2,1-bonds, and the hydrophobic part is hydrophilic on the outside of the molecule and hydrophobic on the inside. Because it can contain and dissolve poorly soluble substances, it can be used as a physical property improving agent such as stabilizing unstable substances and masking bitter taste, and can be widely used in various fields such as food, medicine, agriculture, chemical industry, etc. Known (Takai et al. J. Org. Chem ., 59, 2967, 1994).

Particularly, when comparing cyclodextrin and cyclofructane, which are generally used as physical property improving agents, cyclodextrin is a physical property improving agent that is decomposed by bacteria such as intestinal bacteria and stabilizes the clathrate when ingested because the structural unit is glucose. While its purpose is halved, cyclofrutan is fructose, which is stable to intestinal bacteria and is expected to be more effective than cyclodextrin as a physical improvement agent.

Meanwhile, inulin is a carbohydrate containing about 80% of the bulbs of compositae plants such as Jerusalem artichoke, chicory, and dahlia. -It is a compound connected in a straight chain by binding and glucose is α-1,2 at the end thereof. It is decomposed into fructose by exo-inulinase, and endo-inulinase. It is known to be decomposed into inulin oligosaccharide and to be decomposed into cyclo fructan by cycloinolo- oligosaccharide fructanotransferase (CFTase).

Oligosaccharides produced by these inulin hydrolase have similar sensitivity and physical properties as sugar, and can be used as an alternative sweetener that can solve side effects such as tooth decay, obesity, diabetes and arteriosclerosis caused by excessive intake of sugar. In addition, it has received a lot of attention recently because it has unique physiological functions such as low calorie, improvement of intestinal bacteria, prevention of tooth decay, and blood sugar strengthening.

Therefore, cyclofructane itself has fructo-oligosaccharide as a fructo-oligosaccharide, has a caries prevention effect, enterobacterial improvement effect, and can be widely used as a functional oligosaccharide because it is useful as a physical property improving agent and does not cause the above problems. .

However, cyclofrutan is produced in Bacillus Circulans (Kushibe et al Biosci. Biotrchnol. Biochem ., 58, 1136, 1994) and Bacillus maserans (Kim et al. J. Microbiol. Biotechnol. 6, 397, 1996). Although a method of obtaining a inulin decomposition product using CFTase has been reported, cyclofrutan is low in productivity and is not currently used industrially.

Accordingly, the present inventors have identified a new microorganism having high productivity of CFTase, and provided a culture method for increasing the efficiency of enzyme production, thereby completing the present invention to produce a large amount of cyclo fructan.

Accordingly, an object of the present invention is to provide a novel microorganism that produces CFTase having high inulin resolution and high specificity, and a culture method for increasing CFTase production efficiency from the microorganism, and a method for producing cyclo fructan.

Figure 1 shows the reaction of Bacillus polymix MGL21 strain culture with inulin, the production of cyclofructane (CF) by time is shown by the thin layer chromatography results.

Figure 2 shows the nucleotide sequences from the 5 'end and the 3' end of 16rRNA of Bacillus polymix MGL21.

Figure 3 shows the graphical representation of the enzyme production curve according to the cell growth of Bacillus polymix MGL21.

Figure 4 shows the molecular weight of the purified CFTase by the method of SDS-PAGE.

5 (A) is a graphical representation of the activity of CFTase according to the pH change, (B) is a graphical representation of the activity of CFTase according to the temperature change.

6 is a result of the reaction of the inulin and CFTase and the resulting sugar components analyzed by high-performance liquid chromatography.

Figure 7 shows the reaction product reacted with CFTase and inulin by thin layer chromatography, (B) shows the reaction product by thin layer chromatography with CFTase and GF4, (C) the reaction product reacted with CFTase and CF6 Is represented by thin layer chromatography.

In order to achieve the above object, the present invention provides a Bacillus polymyxa MGL21 KACC 91047 to produce a cycloinolo oligosaccharide fructanotransferase.

In addition, in order to achieve another object of the present invention, the present invention is a cycloinolooligosaccharide fructanot comprising culturing the microorganisms in the medium, and obtaining a cycloinolooligosaccharide fructanotransferase from the culture medium Provided is a process for preparing lanceferase.

Furthermore, the present invention provides a method for producing cyclo fructan by reacting cycloinolooligosaccharide fructanotransferase prepared by the above method with inulin.

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

1. Isolation and Identification of New Microorganisms

The present invention was to study a new strain of Bacillus genus that can be used in the production process of the enzyme by obtaining a high inulin degrading enzyme, high specificity of inulin degrading enzyme, as a result, it was isolated from the soil new microorganisms suitable for the purpose of research, It was named Bacillus strain MGL21. As a result of the identification, it was confirmed that the species is very close to Bacillus polymixa .

2. Cultivation of Bacillus Polymix MGL21

In order to increase the production of CFTase in Bacillus sp. MGL21 of the present invention, 2% of yeast extract and 2% of yeast extract and 0.5% of phosphoric acid in case of organic nitrogen as a carbon source and 2% as inulin in a medium Diammonium {(NH ₄ ) ₂ HPO ₄ } was added and the inorganic salts were added together with 0.05% of MgSO ₄ · 7H ₂ O, 0.05% of MnCl ₂ · 4H ₂ O and 0.001% of FeSO ₄ · 7H ₂ O. It is preferable to incubate at a temperature of 37 ℃ at pH 7.0.

3. Bacillus polymix yarn CFTase Gene Cloning from MGL21

Chromosome DNA of B. polymyxa MGL21 isolated from EcoR I and pUC18 (Takara) previously isolated from Eco R I were ligated, transformed into E - coli DH5α, and then screened for DNA sequencing by microorganisms containing CFTase. Cloning the gene by the shotgun method, the open reading frame (ORF), which encodes the gene of CTFase, consists of 3,999 nucleotides and encodes 1,333 amino acids with an expected molecular weight of about 149,375 Daltons.

In addition, CFTase is composed of five subunits by sequencing, and it is characterized by repeating three portions at the N- and C-terminals.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to specifically describe the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

(Example 1)

Isolation and Identification of Strains

(1) Experimental method

Bacillus strains of the present invention was isolated from the soil collected in Busan, South Korea as follows. First, soil samples were separated from a flat plate (1% inulin, 0.5% bactopeptone, 0.8% (NH ₄ ) 2 HPO ₄ , 0.4% NH ₄ H ₂ PO ₄ , 0.05% KCl, 0.05% MgSO ₄ 7H ₂ O, 0.001% FeSO ₄ · 7H ₂ O and 1.5% agar) were incubated at 37 ° C. for 1 day to isolate strains.

The colonies formed here were incubated for one day at 37 ° C. in a plate for separation again (TTC staining, 2, 3, 5-Triphenyl tetrazolidium chloride) (O. Gabriel et al, Anal. Biochem., 27, 545, 1969) Strains forming red rings around colonies were selected first. The selected strains were shaken for 1 day at 37 ° C using a liquid medium for separation, and then the supernatant was developed by thin-layer chromatography (TLC) to determine whether or not cyclo fructan was produced. (See FIG. 1). TLC was applied to the TLC plate 10 μl of the culture supernatant and developed 2-3 times at 50 ° C. using normal butanol: isopropanol: water (3: 12: 4 (v / v%)) as a developing solvent. After development, the detection of reducing sugars and oligosaccharides was developed by the urea spray method. Urea spray was prepared by the addition of urea in EtOH and 3g 5㎖ in _{1M H 3 PO 4 (water-} saturated n-BuOH 93.22㎖, H 3 PO 4 6.78㎖.

Secondarily selected strains were shaken and cultured at 37 ° C. for 1 day using a separating liquid medium, followed by centrifugation (13,000 rpm, 10 min), and the culture supernatant was used as an enzyme solution and 50 mM phosphate buffer solution containing 50% sugar ( pH 6.0) to perform the enzyme reaction at 37 ℃. After completion of the reaction, the reaction product was identified by high-performance liquid chromatography (HPLC) to finally isolate the strain producing oligosaccharides. To identify these isolated strains, we analyzed the biochemical properties of Bergey's Manual (Sneath et al. Bergey's Manual of systematic Bacteriology, U.S.N. 1986) and the nucleotide sequence of 16S rRNA.

The 16S rRNA gene was amplified with AmpliTag DNA polymerase (Takara Shuzo, Kyoto, Japan) with GenAMP PCR system 2400 (perkin Elmer, FosterCity, CA, USA).

PCR amplification primers used were 16S-7 (5'-AAGAGTTTGATCATGGC-3 ') and 16S-1510 (5'-AGGAGGTGATCCAACCGCAG-3'). E. coli ( see accession no. It corresponds to the common region of genes. Chromosomal DNA used as a template was prepared by the Sarcos method and purified by ultracentrifugation using an equilibrium density gradient of cesium chloride.

(2) Experiment result

The selected new strains were Gram-positive monobacterium, which had morphological characteristics of motility and endogenous spores, and formed circular colonies with white pigments in the solid medium for separation. In addition, it is confirmed that the aerobic and catalase activity has an optimal culture temperature of 37 ° C. or 20 ° C. to 45 ° C., and the optimal growth range is 6.0-8.5, but the optimum growth pH is neutral bacteria. It was. In addition, in hydrolytic activity, starch and gelatin are decomposed, and in the presence of 2% NaCl, growth is impossible, indole formation is not possible, and it shows a positive reaction in the Bogs-proskauer test. Confirmed.

In addition, 16S rRNA gene clone of 1.5kb DNA fragment was cloned from Bacillus strain MGL21, and sequence analysis of 3 'end and 5' end showed high homology of 98% or more with Bacillus polymyxa .

Therefore, this strain was named Bacillus polymix MGL21, and the strain was deposited on May 26, 2003 to the Agricultural Biotechnology Research Institute under the Agricultural Promotion Agency (KACC 91047).

The results of physiological and biochemical properties are shown in Table 1, and the nucleotide sequence of 16S rRNA of Bacillus polymix MGL21 is shown in FIG. 2.

TABLE 1

(Example 2)

Characteristics of Bacillus Polymix MGL21

1. Growth conditions

(1) Experimental method

Bacillus polymix MGL21 of the present invention for separating liquid medium (2% inulin, 2% Yeast extract, 0.5% (NH ₄ ) ₂ HPO ₄ , 0.2% NH ₄ H ₂ PO ₄ , 0.05% MnCl ₂ · 4H ₂ O, pH 7.0) cells were inoculated in 500 ml and cultured with stirring at 120 rpm at 37 ° C., and cell growth, enzyme production, and pH change are shown in FIG. 3. After inoculation, the culture medium was collected at regular time intervals, and the pH was measured by P. MET. The culture medium was determined by measuring the absorbance (OD ₆₆₀ ) at ₆₆₀ nm.

In addition, the enzyme activity was centrifuged at 10,000 rpm for 10 minutes, the supernatant was used as the enzyme solution, and the enzyme reaction was performed at 45 ° C. for 10 minutes with 50 mM phosphate buffer solution (pH 7.0) containing 2% inulin. After the reaction was completed, the enzyme reaction was stopped by heating in boiling water at 100 ° C. for 5 minutes, and the reaction solution was analyzed by high performance liquid chromatography (HPLC) to measure the amount of cyclo fructan produced.

High-speed liquid chromatography uses Watts Alliance 2690 (Waters Co. USA), a detector with a RI detector, and a column with Tosso's amide-80 (TSK-GEL AMIDE-80, 4.6 ×). 250 mm, TOSOH, Japan) was used, the column temperature was measured at 65 ℃, the solvent degassed acetonitrile: water (70: 30, v / v), flow rate 1 ml per minute.

Under the above conditions, the amount of enzyme that reacts with inulin buffer for 1 minute to produce 1 micromole of cycloinulohexaose (CF6) was defined as 1 unit.

(2) Experimental results

As a result of culturing Bacillus polymix MGL21 of the present invention at 37 ℃ to examine the cell growth, enzyme production and pH change, the pH in the medium gradually decreased from the initial 7.0 to pH 6.0 at 10 hours and then gradually increased again to 48 PH 7.0 was shown at time. The growth of the bacteria was the highest after the inoculation, which increased rapidly to 6.3 (OD ₆₆₀ ) at 12 hours, and then rapidly decreased to about 2.7 (OD ₆₆₀ ) at 20 hours of culture. Enzyme production according to the growth of the bacteria continued to increase after inoculation, which was the most produced at 24U / ml for 28 hours, and then gradually decreased.

3. Optimization of enzyme production medium

(1) carbon source

Different carbon sources in liquid media for separation (2% Yeast extract, 0.5% (NH ₄ ) ₂ HPO ₄ , 0.2% NH ₄ H ₂ PO ₄ , 0.05% MnCl ₂ 4H ₂ O, pH 7.0; basic separation medium) Inoculate the bacteria into 20 ml medium containing 1% of Destrose, Fructose, Maltose, Sugar, Lactose, Galactose, Xylose, Inulin, Soluble Starch and Cellulose, and incubate at 37 ℃ for 18 hours. After centrifugation (13,000 rpm, 10 min), the supernatant was used as the enzyme solution, and the enzyme activity was measured. The method for measuring enzyme activity was performed in the same manner as described above.

As a result, as shown in Table 2, the growth of the cells was well cultured in lactose, galactose, inulin, and soluble starch, and the absorbance at 660 nm was about 3 to 4, and double inulin was 11 units / in enzyme production. It showed high activity over ml.

Therefore, it was confirmed that the addition of inulin was effective in all aspects of cell growth and enzyme production.

TABLE 2

In addition, cell growth and enzyme production were analyzed by varying the concentration of inulin to 0.5, 1, 2, 4, 6, 8, and 10%. As shown in Table 3, cell growth increased as the concentration of inulin increased. At concentrations up to 2%, enzyme production increased with the highest activity of 20 units / ml at 2%, but at higher concentrations, enzyme production decreased. The results are shown in Table 3 below.

TABLE 3

(2) Influence of nitrogen source

Yeast extract and polypeptone were removed from the basic separation medium containing 2% of inulin as a carbon source, and 0.5% (NH ₄ ) ₂ SO ₄ , 0.5% (NH ₄ ) ₂ HPO ₄ , 0.5% (NH ₄₎ ) H ₂ PO ₄ , 0.5% NH ₄ Cl, 0.5% KNO ₃ , 0.5% NaNO ₃ , 0.5% NH ₄ NO ₃ , 0.5% peptone as organic nitrogen source, 0.5% corn steep liqual (CSL), 0.5% meat extract, Bacteria were inoculated into 20 ml medium containing 0.5% tryptone, 0.5% yeast extract, 1% yeast extract, 2% yeast extract, and 2,5% yeast extract, and incubated at 37 ° C for 18 hours, followed by centrifugation. The supernatant was measured for activity after the enzyme reaction under the same conditions as the aforementioned carbon source.

As a result, the inorganic nitrogen source (NH ₄ ) ₂ HPO ₄ was the most enzyme production (7.7 unit / ㎖) (see Table 4), and the organic nitrogen source as high as 8.8 units / ㎖ when 2% yeast extract was added Enzyme activity was shown (see Table 5).

(3) Influence of inorganic salts

Inorganic salts in the enzyme production medium with carbon and nitrogen sources determined 0.05% KCl, 0.05% MgSO ₄ · 7H ₂ O, 0.001% FeSO ₄ · 7H ₂ O, 0.05% MnCl ₂ · 4H ₂ O, 0.05% MnSO ₄ · 7H ₂ O was added to each other, and cultured at 37 ° C. for 18 hours. After centrifugation, the supernatant was measured for the activity after the enzyme reaction under the same conditions as the aforementioned carbon source.

As a result, the enzyme production is _{MgSO 4 · 7H 2 O, FeSO} 4 · 7H 2 O than when 0.05% MnCl ₂ · was the enzyme production the most common the addition of 4H ₂ O, MnCl ₂ · only the addition 4H ₂ O, When MnCl ₂ · 4H ₂ O was added together, the best growth and enzyme activity were 28.9 (OD ₆₆₀ ) and 28 units / ml, respectively. The results are shown in Table 6 below.

(4) influence of pH

Enzyme production was confirmed after 18 hours of incubation at 37 ° C. by adjusting the pH from 6 to 9 in 0.5N NaOH and 0.5N HCl. As a result, the bacterial growth was high at pH 6, but the enzyme activity was very low. The bacterial growth was low at pH 7, but the enzyme production was the highest at 36 units / ml.

The experimental results are shown in Table 7 below.

(5) influence of temperature

In order to examine the effect of the culture temperature on the enzyme production, the enzyme activity of the culture supernatant after cultivation by varying the culture temperature from 28 ℃ to 50 ℃ in the optimum enzyme production medium, as shown in Table 8 below, 45 ℃ In the above, cell growth was completely inhibited, and high cell growth and enzyme production were shown at 37 ° C, and 37 ° C was confirmed as the optimum temperature for enzyme production.

(4) Molecular Weight of Purified CFTase

The supernatant used for enzyme activity determination was purified on a metal chelating column (Amersham Biosciences), washing with at least 10 times the resuspension buffer corresponding to the column (20 mM Tris-HCl, pH 7.5 and 0.5 M NaOH) and Finally, the fused protein is eluted using a high-speed protein analyzer (FPLC system) at a concentration gradient of imidazole buffer concentration of 0-300 mM. At this time, the flow rate was 1 ml / min.

As a result of analyzing the purified protein by SDS-PAGE method, it was confirmed that the molecular weight of CFTase is about 150kDa (see FIG. 4).

As described and demonstrated in detail above, the present invention provides a novel fructose transferase cycloinuro oligosaccharide fractanotransferase isolated from a novel microorganism, namely Bacillus polymyxa MGL21.

Claims (3)

Bacillus polymyxa MGL21 KACC 91047 to produce cycloinolooligosaccharide fructanotransferase. A method for producing a cycloinolooligosaccharide fructanotransferase, comprising culturing the microorganism of claim 1 in a medium and obtaining a cycloinolooligosaccharide fructanotransferase from the culture. A method of producing cyclofructan by reacting the cycloinolooligosaccharide fructanotransferase prepared in claim 2 with inulin.