Strain in <i>Microbacterium </i>and method for producing psicose using same

Abstract

The present invention relates to a newly isolated bacterium belonging to the genus Microbacterium, a composition for producing psicose comprising the strain, and a method for producing psicose using the same.

Claims

1. A method for producing a psicose from a fructose-containing substrate using a bacterium belonging to the genus Microbacterium having a psicose conversion activity, comprising reacting the fructose-containing substrate with at least one selected from the group consisting of a microbial cell of the bacterium, a culture of the bacterium, and a lysate of the bacterium in the presence of at least one metal ion selected from the group consisting of manganese ion and cobalt ion, wherein the bacterium belonging to the genus Microbacterium is Microbacterium phyllosphaerae, wherein the Microbacterium phyllosphaerae is a strain deposited as an accession number KCCM12034P.

2. The method for producing a psicose of claim 1, wherein the step of reacting is performed by contacting the fructose-containing substrate with a support immobilized with at least one selected from the group consisting of a microbial cell of the bacterium, a culture of the bacterium, and a lysate of the bacterium.

3. The method for producing a psicose of claim 1, wherein the fructose-containing substrate contains fructose at a concentration of 40 to 75% (w/w).

4. The method for producing a psicose of claim 1, wherein the method is characterized by not using a buffer solution.

5. The method for producing a psicose of claim 1, wherein the method is performed under the temperature condition of 40 to 80 C.

6. The method for producing a psicose of claim 1, wherein the method is performed for 1 to 48 hours.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a graph showing a chromatogram confirming the psicose produced from the high concentration of fructose by High-Performance Liquid Chromatography (HPLC) in one example of the present invention.

(2) FIG. 2 is a graph showing relative activities of producing psicose of Microbacterium oxydans strain isolated in one example of the present invention according to the temperature.

(3) FIG. 3 is a graph showing relative activities of producing psicose of Microbacterium phyllosphaerae strain isolated in one example of the present invention according to the temperature.

(4) FIG. 4 is a graph showing relative activities of producing psicose of Microbacterium oxydans strain isolated in one example of the present invention according to reaction time.

(5) FIG. 5 is a graph showing relative activities of producing psicose of Microbacterium phyllosphaerae strain isolated in one example of the present invention according to reaction time.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(6) The present invention will be described in more detail by the following examples. However, the following examples are desirable examples of the present invention, and the present invention is not limited thereto.

Example 1. Isolation of Microorganism Converting Fructose into Psicose

(7) To isolate a strain converting a fructose into a psicose, 1% (w/v) psicose-added Mineral salt broth (KH.sub.2PO.sub.4 2.4 g/L, K.sub.2HPO.sub.4 5.6 g/L, (NH.sub.4).sub.2SO.sub.4 2.6 g/L, MgSO.sub.47H.sub.2O 0.1 g/L, yeast extract 1 g/L) was used.

(8) Each 1 g sample of soils obtained from orchard or fertile land, cheese, milk, edible flower, broccoli and ginseng was selected, and 1 g of each food was collected and added to MSP broth, and then cultured at 30 C. with agitation for 24 hours, thereby implementing enrichment. Then, 100 L (microliter) of culture solution was collected and smeared on an agar medium, and then cultured at 30 C. until the colony was confirmed. Colonies having different shapes and sizes among colonies formed in the agar medium were selected and inoculated into MSP broth, and then shaking cultured at 30 C. for 24 hours and centrifuged to recover only microbial cells. The recovered microbial cells were put into 100 L of 50 mM PIPES (piperazine-N, N-bis(2-ethanesulfonic acid)) buffer solution (pH 7.0) and suspended, and lysated by using a ultrasonic processor (ColepParmer) to collect a lysate solution. After centrifuging the lysate solution by 12,000 rpm at 4 C. for 10 mM, the supernatant was recovered and used as an enzyme solution (crude enzyme), and the enzyme solution was reacted at 30 C. for 12 hours with 10 mM fructose and psicose as substrates.

(9) Whether the psicose was converted into the fructose in the reaction solution was confirmed by Thin Layer Chromatography (TLC) analysis. The thin layer chromatography analysis was performed by using a developing solvent of a stationary phase of silica gel (Silica gel 60F254 (Merck, Germany)) of width 20 cm, height 10 cm and a mobile phase mixing acetonitrile and water at the volume ratio of 85:15 and developing by 3 times for 10 min.

(10) The strains in which a psicose was converted to a fructose confirmed by the TLC analysis were sorted and inoculated into 0.1% (w/v) psicose-added MS broth and shaking cultured at 30 C. for 24 hours, and after centrifugation, only microbial cells were recovered. The recovered microbial cells were washed with 0.85% (w/v) NaCl, and then suspended by putting 400 g/L fructose and 1 mM manganese ion-added 50 mM PIPES buffer solution (pH 7.0), and reacted at 70 C. for 1 hour.

(11) Then, after recovering the supernatant by centrifuging the reaction results, High-Performance Liquid Chromatography (HPLC) analysis was carried out. The liquid chromatography analysis was performed by using RID (Refractive Index Detector, Agilent 1260 RID) of HPLC (Agilent, USA) equipped with Aminex HPX-87C column (BIO-RAD). Water was used as a mobile phase and the temperature was 80 C., and the flowing rate was 0.6 mL/min. The obtained result was shown in FIG. 1, and two strains which produced psicose the most among several thousand kinds of strains was finally selected.

Example 2. Identification of Strain Having Psicose Conversion Activity

(12) The sequence of 16S ribosome RNA was confirmed to identify the strain isolated in Example 1. It was confirmed that the sequences (5.fwdarw.3) of 16S ribosome RNA of isolated strains were shown in SEQ ID NO: 1 or SEQ ID NO: 2, respectively.

(13) The strain with 16S rRNA shown in SEQ ID NO:1 was 78.76% of nucleotide sequence identity to Microbacterium oxidans DSM20578, and it was named Microbacterium oxidans SYG-A1. The strain was deposited to Korean Culture Center of Microorganisms (Address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depository authority, on May 26, 2017 under the provisions of the Budapest Treaty and the accession number KCCM12033P was given.

(14) The strain with 16S rRNA shown in SEQ ID NO:2 was 100% of nucleotide sequence identity to Microbacterium phyllosphaerae P369/016(NP_025405.1), and it was named Microbacterium phyllosphaerae SYG-A2. The strain was deposited to Korean Culture Center of Microorganisms (Address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depository authority, on May 26, 2017 under the provisions of the Budapest Treaty and the accession number KCCM12034P was given.

Example 3. Establishment of Optimum Temperature Condition Using Microbial Cell Reaction of the Strains

(15) For the isolated strains, microbial cells and a substrate were reacted under various conditions of temperature and metal ion, and the psicose conversion activities were compared.

(16) To confirmed the optimum temperature for producing a psicose, two strains isolated in Example 1 in the concentration of microbial cells of 5 mg(dcw)/mL was reacted in 400 g/L fructose and 1 mM manganese metal ion-added 50 mM PIPES buffer solution (pH 7.0) for 1 hour as changing the temperature in the range of 55 to 80 C., and after the reaction was finished, the production of psicose was measured through HPLC analysis with the same method as Example 1, and the obtained results were shown in FIG. 2 (M.oxydans SYG-A1). FIG. 3 (M.phyllosphaerae SYG-A2) and the following Table.

(17) TABLE-US-00001 TABLE 1 Reaction M. oxydans SYG-A1 M. phyllosphaerae SYGA2 temperature ( C.) Relative activity (%) Relative activity (%) 50 59 65 60 95 95 70 100 100 80 45 55

(18) As shown in FIG. 1, FIG. 2 and Table 1, it was confirmed that the relative activity of M.oxydans SYG-A1 and M.phyllosphaerae SYGA2 was increased as increasing the reaction temperature by 70 C. and decreased at 80 C. In addition, the isolated strains exhibited the maximum activity at the temperature of 70 C.

Example 4. Test for Psicose Productivity

(19) The maximum psicose productivity according to the reaction time was tested. The activities over the reaction time were measured under the conditions of 20 mg/mL of the concentration of microbial cells of strains isolated in Example 1, 400 g/L of the fructose concentration, the temperature of 70 C. and pH 7.0. The reaction was performed for 12 hours and the psicose productivity was confirmed by HPLC analysis in the 2 hour interval. The result was shown in FIG. 4, FIG. 5 and following Table.

(20) TABLE-US-00002 TABLE 2 Reaction M. oxydans SYG-A1 M. phyllosphaerae SYG-A2 Time (hr) Relative activity (%) Relative activity (%) 1 5.9 6 3 6.5 8 5 7.5 10.5 7 8.8 15 18 10.3 18.8

(21) As shown in Table 2 and FIG. 4, M.oxydans SYG-A1 showed the psicose conversion rate increased as the reaction time passed, and in particular, showed the maximum psicose conversion rate as approximately 10.3%, after the reaction at 70 C. for 18 hours, in which the produced psicose was approximately 28 g/L. In addition, as shown in Table 2 and FIG. 5, M.phyllosphaerae SYG-A2 showed the psicose conversion rate increased as the reaction time passed, and in particular, showed the maximum psicose conversion rate as approximately 18.8%, after the reaction at 70 C. for 18 hours, in which the produced psicose was approximately 52 g/L.