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Allulose epimerase variant, method for producing same, and method for producing allulose using same

A technology of epimerase and psicose, applied in the field of D-psicose 3-epimerase, can solve the problems of poor thermal stability, low conversion rate, unsuitable for industrialization, etc. The effect of preventing pollution, high conversion activity and reducing production cost

Active Publication Date: 2021-08-24
大象(株)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the conversion rate of fructose to psicose of the wild-type D-psicose 3-epimerase derived from microorganisms is not high, and the thermostability is poor under the optimal activation temperature conditions, especially not suitable for industrialization

Method used

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  • Allulose epimerase variant, method for producing same, and method for producing allulose using same
  • Allulose epimerase variant, method for producing same, and method for producing allulose using same
  • Allulose epimerase variant, method for producing same, and method for producing allulose using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Searching for amino acid substitution positions that improve the conversion rate and thermostability of D-psicose 3-epimerase

[0036] To improve the fructose-to-psicose conversion rate and thermostability of D-psicose 3-epimerase derived from Flavonifractor plautii, amino acid substitution positions were selected based on protein structure prediction. D-psicose 3-epimerase (or D-psicose 3 -epimerase). The D-psicose 3-epimerase derived from Flavonifractor plautii consists of the amino acid sequence shown in SEQ ID NO: 1, and has the maximum activity of converting fructose into psicose under neutral pH conditions , and it is possible to mass-produce psicose from fructose with high yield in a short period of time, but there is a problem in that when the enzyme reaction is performed under high temperature conditions to prevent contamination and the like, the activity rapidly decreases. In order to improve the conversion rate and thermal stability of fructose t...

Embodiment 2

[0037] Example 2: Preparation of recombinant vectors and recombinant strains overexpressing D-psicose 3-epimerase derived from Flavonifractor plautii

[0038] Based on the wild-type polynucleotide of the psicose epimerase derived from Flavonifractor plautii, nucleotides encoding five enzyme variants (I21C, A82R, L133D, G216S and A276R) were generated using overlap extension polymerase chain reaction. A polynucleotide fragment of an amino acid sequence.

[0039] First, a PCR reaction was performed using the primers shown in Table 1 below to prepare a gene encoding a psicose epimerase variant derived from Flavonifractorplautii. Specifically, 1 pM of the oligonucleotides in Table 1 was used as primers, and 100 ng of the wild-type polynucleotide (SEQ ID NO: 7) derived from the psicose epimerase variant of Flavonifractor plautii was used as a template. These two were mixed into a reaction solution to which 100 μM of deoxynucleoside triphosphate (dATP, dCTP, dGTP, dTTP) was added. ...

Embodiment 3

[0048] Example 3: Expression and purification of D-psicose 3-epimerase variants derived from Flavonifractor plautii

[0049] 1 ml of recombinant E. coli prepared in Example 2 was inoculated into a 1 L shake flask containing 150 ml of a protein expression medium having the composition of the following Table 3 (based on 1 L medium), and was incubated at 32° C. in a shaking incubator. Incubate for 24 hours while maintaining shaking conditions at 32° C. and 140 rpm. 1% lactose was included in the medium to induce overexpression of psicose epimerase variants.

[0050] table 3

[0051] Medium composition quantity Medium composition quantity glycerin 9.0% by weight MgSO 4

[0052] Then, overexpressed psicose epimerase variants were isolated by the following method. First, the culture solution of recombinant E. coli was centrifuged at 4100×g and 4° C. for about 15 minutes to remove the supernatant, and cells of the recombinant E. coli strain were recovered...

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Abstract

The present invention provides: a novel allulose epimerase variant in which glycine (Gly), which is an amino acid residue present at position 216 of the amino acid sequence of a wild-type D-allulose 3-epimerase derived from Flavonifractor plautii, is substituted with serine (Ser); and various uses thereof. Since the novel allulose epimerase variant according to the present invention has a higher conversion activity of fructose to allulose than wild-type D-allulose 3-epimerase derived from Flavonifractor plautii, and has excellent thermal stability especially under high temperature conditions of 60 DEG C or higher, contamination during an industrial-scale enzyme conversion reaction for the mass production of allulose can be prevented, the production time can be shortened, and the production cost can be reduced.

Description

technical field [0001] The present invention relates to a kind of psicose epimerase variant etc., and especially relates to a kind of allulose epimerase variant and various inventions derived therefrom, and derived from Flavonifractor plautii D- Compared with psicose 3-epimerase, it has improved conversion rate of fructose to psicose and thermostability. Background technique [0002] D-allulose (D-allulose) is the epimer of the third carbon of fructose, also known as D-psicose. Compared with sugar, D-psicose has a sweetness of 70% (Oshima 2006), but only 0.3% of calories, so it is a functional monosaccharide that can be used as a low-calorie sweetener in therapeutic foods (Matsuo et al. 2002). In addition, D-psicose has the function of absorbing glucose and suppressing blood sugar, so it can be applied to food for diabetics, health food Wait. Since it can inhibit the accumulation of abdominal fat by inhibiting the activity of enzymes involved in lipid synthesis in the l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P19/24C12P19/02C12R1/19
CPCC12N9/90C12N15/70C12P19/24C12P19/02C12Y501/03C12R2001/01
Inventor 尹炯燮崔银硕李芝河金硕洙
Owner 大象(株)