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D-psicose 3-epimerase mutant with improved thermal stability, and continuous production of d-psicose using same

A technology of epimerase and psicose, which is applied in the direction of racemase/epimerase, isomerase, and the use of vectors to introduce foreign genetic material, can solve problems such as side effects, and achieve extended half-life, Effects of reducing production time and cost and improving production efficiency

Active Publication Date: 2014-11-19
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, sugar alcohols (such as xylitol, etc.) that are widely used as sugar substitutes may have side effects such as diarrhea in case of excessive consumption

Method used

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  • D-psicose 3-epimerase mutant with improved thermal stability, and continuous production of d-psicose using same
  • D-psicose 3-epimerase mutant with improved thermal stability, and continuous production of d-psicose using same
  • D-psicose 3-epimerase mutant with improved thermal stability, and continuous production of d-psicose using same

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0056] Preparation of D-psicose 3-epimerase variants with improved thermostability by random mutagenesis

[0057] D-psicose 3-epimerase derived from Agrobacterium tumefaciens ATCC33970 (having an amino acid sequence identical to SEQ ID NO: 1 disclosed in Korean Patent Publication No. 10-2011-0035805A) was used A library of D-psicose 3-epimerase variants was constructed by error-prone polymerase chain reaction (error-prone PCR) as a template.

[0058] Specifically, a typical PCR mutagenesis kit that results in two to three mutations per 1000 base pairs is employed. As primers, oligonucleotides introduced into the restriction enzyme recognition site sequences of NcoI and PstI restriction enzymes were used to perform polymerase chain reaction to construct genes encoding D-psicose 3-epimerase variants library, the variants were then inserted into E. coli BL21.

[0059] LB medium containing 50 µg / ml ampicillin was used to culture Escherichia coli BL21 containing a plasmid contain...

example 2

[0068] Preparation of D-psicose 3-epimerase variants with improved thermostability using rational design

[0069] In a similar manner to Example 1, variants S213C and I33L were selected as variants with improved thermostability without significantly decreased activity. Based on the selected variants, amino acids at positions 33 and 213 of the amino acid sequence were subjected to site-directed mutagenesis.

[0070] Specifically, the polar and uncharged serine (Ser) at position 213 is replaced by polar and uncharged threonine (Thr), cysteine ​​(Cys) or methionine (Met), respectively; The nonpolar amino acid proline (Pro); glutamic acid (Glu), which has a negative charge; and lysine (Lys), which has a positive charge, are substituted.

[0071] The non-polar isoleucine at position 33 is replaced by polar and uncharged cysteine; non-polar valine (Val), leucine or proline; negatively charged valley amino acid; and a positively charged lysine substitution.

[0072] The relative a...

comparative experiment example 1

[0079] Comparison of the apparent melting temperatures of wild-type D-psicose 3-epimerase and D-psicose 3-epimerase variants according to Example 2

[0080] To determine the thermostability of wild-type D-psicose 3-epimerase and D-psicose 3-epimerase variants according to Example 2 (i.e. I33L, S213C and I33L-S213C) To measure the apparent melting temperature (T m ).

[0081] Therefore, it can be seen that the apparent melting temperatures arranged in ascending order are wild type figure 2 ). Specifically, the apparent melting temperature was increased by about 4.3°C in the case of the S213 variant and by about 7.6°C in the case of the I33L-S213C variant compared to the wild type.

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Abstract

The present invention relates to a D-psicose 3-epimerase mutant of which the thermal stability is improved by substituting an amino acid of a specific sequence number. In addition, the present invention relates to a recombinant vector comprising the gene of the D-psicose 3-epimerase mutant, and a recombinant strain transformed with the recombinant vector. Further, the present invention relates to an immobilized reactor prepared using the enzyme mutant or the recombinant vector, and a method for producing D-psicose using the immobilized reactor.

Description

technical field [0001] The present invention relates to a D-psicose 3-epimerase variant derived from Agrobacterium tumefaciens with improved thermostability and a method for producing D-psicose using the same. Background technique [0002] D-Psicose is a monosaccharide that is considered a rare sugar because it is rarely found in natural materials or exists in small amounts. D-Psicose has ultra-low energy density and sweetness similar to sugar, and thus is widely used as a functional sweetener. [0003] D-psicose is an epimer of fructose and has a sweetness and taste very similar to fructose. However, unlike fructose, D-psicose is hardly metabolized in the body and thus has almost zero calories. D-psicose is useful as an active ingredient of diet food because D-psicose has the ability to inhibit the activity of enzymes involved in lipid synthesis and reduce abdominal obesity. In addition, sugar alcohols (such as xylitol, etc.), which are widely used as sugar substitutes, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/92C12N15/61C12N15/77C12P19/02
CPCC12P19/02C12N9/90C12P19/24C12Y501/03C12N9/92C12N15/52C12N15/77
Inventor 金良姬金镇夏李英美洪暎镐金敏会金成俌朴承源吴承贤吴德根崔珍根
Owner CJ CHEILJEDANG CORP
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