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Psicose epimerase and composition for conversion to psicose using same

A technology of epimerase and psicose, applied in racemase/epimerase, isomerase, introduction of foreign genetic material using a carrier, etc., can solve the problem of slow reaction speed, unsuitable for industrialization, Stability reduction and other issues

Active Publication Date: 2015-07-08
SAMSANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Not suitable for industrialization due to reaction under alkaline conditions inducing non-specific reactions and sugar browning
In addition, conventional enzymes have problems in that the production cost of psicose used in industrial production is increased due to the decrease in stability at high temperature or the slow reaction rate.

Method used

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  • Psicose epimerase and composition for conversion to psicose using same
  • Psicose epimerase and composition for conversion to psicose using same
  • Psicose epimerase and composition for conversion to psicose using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1. Production of recombinant strains producing D-psicose 3-epimerase

[0079] In order to optimize the expression in Escherichia coli, Bioneer Co., Ltd. (Bioneer.Co.Korea) was commissioned to synthesize a polynucleotide encoding the amino acid sequence No. 1 from the original genus Treponema ZAS-1. (serial number 2).

[0080] Use restriction enzyme NdeI and XhoI (NEB) to insert the polynucleotide of sequence number 2 synthesized into the identical restriction enzyme site of expression vector pET21a (Novagen), make recombinant vector pET21a / psicose 3-epimerase ( pET-TDPE) (refer to figure 1 ). Afterwards, according to the heat shock (heat shock) method, (Sambrook and Russell: Molecular cloning method) is transformed into Escherichia coli BL21 (DE3) (invitrogen) with the recombinant vector produced, and produces the aminoacid sequence that contains sequence No. 1 and encodes Recombinant strains of polynucleotides. The manufactured recombinant strain was named ...

Embodiment 2

[0083] Example 2. D-psicose 3-epimerase purification and property confirmation

[0084] In order to produce psicose using D-psicose 3-epimerase, a refined enzyme should first be used to precisely determine the characteristics of the enzyme.

[0085] (1) Purification of D-psicose 3-epimerase

[0086] After dispersing the bacteria recovered from the above Example 1 in the lysis buffer (50mM Tris_HCl 300mM NaCl pH8.0, 10mM imidazole), use a sonic vibrator (Ultrasonic processor. ColepParmer) at 4°C for 20 minutes to disrupt The crushed solution was centrifuged at 13000rpm for 20 minutes, and after only the supernatant was collected, it was put into a Ni-NTA column (Ni-NTA Superflow.Qiagen) equilibrated with a lysis buffer in advance, and then flowed through successively at 50mM A buffer containing 20mM imidazole in Tris_HCl300mM NaCl (pH8.0) and a buffer containing 200mM imidazole. By flowing through 50mM Tris_HC1300mM NaCl (pH8.0) and 200mM imidazole as the final step, the targe...

Embodiment 3

[0106] Example 3. Production of psicose using D-psicose 3-epimerase

[0107] (1) Production of allulose from high-concentration fructose through biotransformation

[0108] In order to utilize the D-psicose 3-epimerase obtained in Example 2 to produce high-concentration psicose, under the optimal conditions obtained above at 60°C and 50mM PIPES pH 7, make 400g / L The fructose reacts with 5-50 units / ml of enzyme. As a result, a final psicose of 128 g / L was produced at 50 units / ml of enzyme, and a conversion rate of 30.2% of psicose was obtained even at a high concentration of fructose. (refer to Figure 6 )

[0109] (2) Production of psicose by bacterial reaction including enzymes

[0110] In order to produce psicose using the bacterial cells collected in Example 1 above, the reaction was carried out at 60° C. and 50 mM PIPES pH 7, the optimum conditions for the enzyme, as in (1) above. Make 5.4 or 2.7mg-thalline dry weight / ml reaction volume Escherichia coli react with 400 ...

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Abstract

A D-psicose 3-epimerization enzyme of the present invention is an enzyme with excellent activity for producing psicose by epimerizing a third carbon location of fructose. By applying the enzyme based on the wide pH range useful for industries and stability at high temperatures, the psicose with high economic feasibility can be mass-produced. The enzyme can be applied for producing functional saccharide, and for health food materials, medicines, and cosmetics using the same.

Description

technical field [0001] The present invention relates to a psicose 3-epimerase derived from the primitive genus Treponema primitia, a composition for converting into psicose using the same, and a method for producing psicose. Background technique [0002] D-psicose (D-psicose) is the third carbon epimer (epimer) of fructose (fructose), which has 70% sweetness when compared with granulated sugar (Oshima 2006), but With only 0.3% energy, it is a functional monosaccharide that can be used as a low-calorie sweetener for diet foods (Matsuo et al. 2002). In addition, since it has the function of suppressing blood sugar by inhibiting the absorption of glucose, it can be applied to food and beverages for diabetic patients, food and beverages for slimming, etc.; because it has the function of inhibiting the enzyme activity related to lipid synthesis in the liver , so it can inhibit the accumulation of abdominal fat, so it can be used in various functional foods such as health foods (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12N9/90
CPCC12N9/90C12N15/70C12P19/02C12P19/24C12Y501/03
Inventor 崔祯尹金玟廷金慧贞朴钟辰李康杓许振率
Owner SAMSANG CORP
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