D-psicose 3-epimerase and method for preparing D-psicose using the same

Abstract

Provided are a novel D-psicose 3-epimerase and a method for preparing psicose using the same.

Claims

1. A method for preparing D-psicose, comprising: contacting a D-psicose 3-epimerase consisting of the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the D-psicose 3-epimerase, or a culture of the microorganism with D-fructose.

2. The method of claim 1, wherein the contacting is performed at pH of 5.0 to 9.0, at a temperature of 40° C. to 90° C., or for 0.5 to 48 hours.

3. The method of claim 1, further comprising: before, after or simultaneously with the contacting of the D-fructose, contacting the D-psicose 3-epimerase, the microorganism expressing the D-psicose 3-epimerase, or the culture of the microorganism with a metal.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram of a recombinant vector for expressing a psicose epimerase (KGDPE) consisting of amino acid sequence of SEQ ID NO: 1 of the present invention.

(2) FIG. 2 is HPLC analysis data on results of psicose preparation utilizing D-fructose as a substrate using the KGDPE of the present invention.

(3) FIGS. 3A and 3B are graphs showing a relative enzymatic activity of the psicose epimerase according to temperature, wherein FIG. 3A shows an activity of a D-psicose 3-epimerase (ATPE) derived from Agrobacterium tumefaciens in the prior art, and FIG. 3B shows an activity of KGDPE in the present invention.

(4) FIG. 4 is a graph showing relative enzymatic activity of the KGDPE of the present invention according to pH change.

(5) FIG. 5 is a graph showing relative enzymatic activity of the KGDPE of the present invention according to addition of various metals.

DETAILED DESCRIPTION OF EMBODIMENTS

(6) Hereinafter, the present invention will be described in more detail by the following Examples. However, the present invention is not limited to the Examples below, and it should be understood that various modifications and changes may be made by those skilled in the art within the scope and spirit of the present invention.

(7) Throughout the specification of the present invention, unless otherwise noted, “%” used to denote a concentration of a specific material refers to a solid/solid (weight/weight) %, a solid/liquid (weight/volume) %, and a liquid/liquid (volume/volume) %.

EXAMPLES

Example 1. Preparation of Transformed Strain that Prepares Psicose Epimerase Derived from Microorganism of Genus Kaistia

(8) A gene which was expected to have activity of a psicose epimerase that converts D-fructose to psicose from the microorganism of the genus Kaistia was selected, and a recombinant expression vector including the gene and a transformed microorganism were prepared.

(9) Specifically, a gene kgdpe of Kaistia granuli KCTC 12575, which was expected to be a psicose epimerase, was selected from the gene sequences of the microorganism of the genus Kaistia registered at Genbank, and a forward primer (SEQ ID NO: 3) and a reverse primer (SEQ ID NO: 4) were designed and synthesized based on the amino acid sequence (SEQ ID NO: 1) and the nucleotide sequence (SEQ ID NO: 2) of the gene. By using the synthesized primer, a gene was amplified by performing a PCR reaction (33 cycles: 1 cycle including 94° C. for 1 minute, 58° C. for 30 seconds, and 72° C. for 1 minute) using a genomic DNA of Kaistia granuli KCTC 12575 as a template. The amplified gene was purified using a PCR purification kit (Quiagen) and inserted into pET24a(+) (novagen, USA) using restriction enzymes NdeI and notI to construct a recombinant vector pET24a(+)-KGDPE (FIG. 1).

(10) The recombinant vector was transformed into Escherichia coli BL21 (DE3) by heat shock transformation (Sambrook and Russell: Molecular Cloning, 2001), and then stored frozen in 50% glycerol and used. The transformed strain was named E. coli BL21(DE3)/KGDPE, deposited on Oct. 20, 2016 in the Korean Culture Center of Microorganisms (KCCM) which is an international depository under the Budapest Treaty, and granted accession number KCCM11918P.

Example 2. Preparation and Purification of Psicose Epimerase

(11) To prepare the psicose epimerase from E. coli BL21(DE3)/KGDPE prepared in Example 1, E. coli BL21(DE3)/KGDPE was inoculated into 5 ml of LB-kanamycin medium, and was subjected to shake-culture at 37° C., 200 rpm until the absorbance measured at 600 nm reached 1.5. Then, the shake-cultured culture liquid was inoculated into 500 ml of LB-kanamycin medium, and when the absorbance at 600 nm was 0.7, 0.5 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and the cells were main-cultured at 16° C. and 150 rpm for 16 hours.

(12) The main-cultured culture liquid was centrifuged at 8000 rpm for 20 minutes to recover only the cells, and the cells were washed twice with 0.85% (w/v) NaCl and then lysed in a lysis buffer (50 mM Tris-HCl, pH 7.0 300 mM NaCl), and disrupted at 4° C. for 20 minutes using a sonic vibrator. The disrupted liquid was centrifuged at 4° C., 13,000 rpm for 20 minutes to recover the supernatant. Then, the supernatant was applied to a Ni-NTA column (Ni-NTA Superflow, Qiagen) previously equilibrated with the above lysis buffer, and a buffer solution (50 mM Tris-HCl, 300 mM NaCl, pH 7.0) containing 250 mM imidazole was sequentially flowed to obtain a purified psicose epimerase (hereinafter, referred to as KGDPE). The SDS-PAGE of the KGDPE confirmed that the size of the monomer was about 32 kDa.

Example 3. Confirmation of KGDPE Activity

(13) 3-1. Confirmation of Conversion Activity from D-fructose to Psicose

(14) To confirm whether the KGDPE prepares psicose using D-fructose as a substrate, KGDPE (50 mM Tris-HCl, pH 7.0) prepared in Example 2 was added to 50 mM Tris-HCl buffer (pH 8.0) containing 50 wt % D-fructose and 3 mM MnSO.sub.4, and reacted at 55° C. for 6 hours. Then, the reaction was stopped by heating at 100° C. for 5 minutes, and then the preparation of the psicose was confirmed by HPLC analysis. The HPLC analysis was performed using HPLC (Agilent, USA) Refractive Index Detector (Agilent 1260 RID) equipped with Aminex HPX-87C column (BIO-RAD), wherein a mobile phase solvent was water, a temperature was 80° C., and a flow rate was 0.6 ml/min.

(15) As a result, it was confirmed that the psicose could be prepared from the D-fructose using KGDPE (FIG. 2).

(16) 3-2. Confirmation of Conversion Activity from D-fructose to Psicose

(17) To confirm whether the preparation ability of the KGDPE is superior to that of the conventional psicose epimerase (ATPE, SEQ ID NO: 5, Korean Patent Laid-Open Publication No. 10-2011-0035805) used in the preparation of the psicose, a conversion rate from the D-fructose to the psicose was confirmed.

(18) Specifically, E. coli BL21 (DE3) transformed with the recombinant expression vector pET24a-ATPE was inoculated into LB medium containing kanamycin having a concentration of 10 μg/ml, and then the enzyme was expressed and purified in the same manner as in Example 2. The obtained enzyme was added to 50 mM Tris-HCl buffer (pH 8.0) containing 50 wt % D-fructose and 3 mM MnSO.sub.4 and reacted at 55° C. for 6 hours. Then, the reaction was stopped by heating at 100° C. for 5 minutes, and then the preparation of the psicose was confirmed by HPLC analysis. The HPLC analysis was performed under the same conditions as in Example 3-1. The conversion rate to psicose was calculated as the amount (mg/min) of the psicose prepared per minute by the enzyme, and the reaction rate of KGDPE was shown as a relative value, wherein the reaction rate value of ATPE was set to 100%.

(19) As a result, it was confirmed that the amount of the psicose prepared per minute when using the KGDPE was 117.6% as compared to when using the ATPE, and thus, the conversion rate from D-fructose to psicose was remarkably increased when the KGDPE was used (Table 1).

(20) TABLE-US-00001 TABLE 1 Enzyme KGDPE ATPE Relative conversion rate (%) 117.6 100

Example 4. Analysis of KGDPE Characteristics

(21) 4-1. Analysis of Enzyme Activity According to Temperature

(22) The KGDPE and the D-fructose substrate were reacted for 2 hours under various temperature conditions (40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C. and 75° C.), and enzyme activities according to the temperatures were compared. The above reactions were performed in the same manner as in Example 3-1 except for the temperature and the reaction time, and the enzyme activities were measured as the conversion rate from the D-fructose to the psicose. The conversion rate was calculated as the percentage of weight of the psicose prepared after the reaction relative to a weight of the substrate (D-fructose) before the reaction.

(23) As a result, the KGDPE exhibited a high conversion activity of 25% or more at all measurement temperature ranges, and it was confirmed that as the temperature increased, the activity increased and the maximum conversion rate was observed at the maximum temperature of 75° C. (Table 2).

(24) TABLE-US-00002 TABLE 2 Temperature (° C.) KGDPE (Conversion rate, %) 40 26.7 45 27.8 50 28.8 55 29.7 60 30.5 65 31.2 70 32.1 75 32.8

(25) 4-2. Analysis of Thermal Stability of Enzyme

(26) To compare thermal stability of the KGDPE with that of the conventional enzyme ATPE, the respective enzymes were heat-treated at various temperatures (55° C., 60° C. and 65° C.), and enzymatic treatment solutions were sampled for each heat treatment time (0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours) to determine residual activity of each enzyme. The reaction was performed for 30 minutes by changing the reaction time only in the same manner as in Example 3-1, and the residual activity of the enzyme was measured by the conversion rate from the D-fructose to the psicose.

(27) As a result, the reduction of the half life of the KGDPE according to the temperature increase was remarkably smaller than that of the ATPE, and thus, it was confirmed that the KGDPE had high thermal stability (FIGS. 3A and 3B).

(28) 4-3. Analysis of Enzyme Activity According to pH

(29) To determine the enzyme activity according to pH, the D-fructose substrate was reacted with KGDPE at various pHs. At this time, the reaction was performed in the same manner as in Example 3-1 except for the reaction time and pH.

(30) Specifically, the enzyme reaction was performed at 55° C. for 30 minutes by using 50 mM potassium phosphate at pH 5.0, pH 6.0, pH 6.5, pH 7.0, pH 7.5, and pH 8.0, and using a 50 mM Tris-HCl buffer at pH 8.0, pH 8.5, and pH 9.0. Then, the enzyme activity was measured as the conversion rate from the D-fructose to the psicose.

(31) As a result, it was confirmed that the KGDPE exhibited activity of 70% or more as compared to the maximum activity at pH 6 to pH 8.5, and exhibited the highest activity at pH 8.0 (Table 3, FIG. 4).

(32) TABLE-US-00003 TABLE 3 pH Relative conversion rate (%) 50 mM 5 63 potassium 6 91 phosphate 6.5 93 7 95 7.5 100 8 99 50 mM 8 76 Tris-HCl 8.5 71 9 73

(33) 4-4. Activity Analysis of Enzyme According to Addition of Metal

(34) To confirm the activity of the KGDPE according to a metal addition, under the same reaction conditions as in Example 3-1, MnSO.sub.4 was replaced with various metal salts (LiCl, Na.sub.2SO.sub.4, MgCl.sub.2, NaCl, FeSO.sub.4 and CaCl.sub.2) and added to a final concentration of 3 mM. Then, the enzyme activity was measured. The control group was not treated with the metal salts.

(35) As a result, it was confirmed that the addition of Li, Na, Mg, Fe and Ca as well as Mn increased the activity of the KGDPE as compared to the control group, and among them, it could be confirmed that Mn increased the enzyme activity the most (Table 4 and FIG. 5).

(36) TABLE-US-00004 TABLE 4 Metal Salt Relative enzyme activity (%) LiCl 91 Na.sub.2SO.sub.4 88 MgCl.sub.2 88 NaCl 88 FeSO.sub.4 95 MgSO.sub.4 90 MnSO.sub.4 100 CaCl.sub.2 96 no metal 79

(37) From the above description, it will be understood by those skilled in the art that the present invention can be made in other specific forms without modifying a technical idea or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be interpreted to cover all modifications or variations derived from the meaning and scope of the appended claims and their equivalents rather than the detailed description.