Method for efficiently preparing D-psicose 3-epimerase and use of D-psicose 3-epimerase

A technology for epimerase and psicose, which is applied in the efficient preparation of D-psicose 3-epimerase and the application field in the preparation of psicose, and can solve the problems of low reaction efficiency and the like

Inactive Publication Date: 2018-02-23
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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The current patent protection is only aimed at the expression and transformation of a si

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  • Method for efficiently preparing D-psicose 3-epimerase and use of D-psicose 3-epimerase
  • Method for efficiently preparing D-psicose 3-epimerase and use of D-psicose 3-epimerase
  • Method for efficiently preparing D-psicose 3-epimerase and use of D-psicose 3-epimerase

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Embodiment 1

[0073] Embodiment 1, construction Corynebacterium glutamicum recombinant strain expresses combined DPE operon with the form of episomal plasmid

[0074] 1. Construction of recombinant strain DPE1 of Corynebacterium glutamicum

[0075] First, construct the recombinant expression vector pEC-RPCDPE1 and design primers 1 and 2 according to the tuf promoter sequence (SEQ ID NO: 4) derived from Corynebacterium glutamicum, with the DPE gene sequence (SEQ ID NO: 1) derived from Paenibacillus senegalensis ) designed primers 3 and 4, designed primers 5 and 6 with the DPE gene sequence (SEQ ID NO: 2) derived from Ruminococcus sp., and designed primers 7 and 6 with the DPE gene sequence (SEQ ID NO: 3) derived from Clostridium cellulolyticum 8, wherein primers 2 and 3, primers 4 and 5, and primers 6 and 7 respectively contain a homologous region of about 40 bp for fusion between PCR fragments, primer 5 contains the H34 promoter sequence, and primer 7 contains the H36 promoter subsequence,...

Embodiment 2

[0109] The expression of the combined DPE operon of embodiment 2, plasmid form in Corynebacterium glutamicum

[0110] 1. Cultivation of recombinant strains of Corynebacterium glutamicum

[0111] Select 100mL BHI medium (brain heart extract powder 37g / L, kanamycin 25ng / mL), and cultivate the recombinant strains DPE1 and DPE2 of Corynebacterium glutamicum at 30°C and 200rmp for 12-24h, 4°C, Centrifuge at 8000rmp for 15min to collect the bacteria, and use ddH 2 O Concentrate the bacterial solution to 5 mL, and prepare a crude enzyme solution by ultrasonic crushing.

[0112] 2. Determination of enzyme activity

[0113] In order to measure the enzyme activity of crude enzyme, set up the following reaction system (500ul): D-fructose: 5%, crude enzyme: 0.1mg, MnCl 2 : 1mM; react for 10min, add NaOH to terminate the reaction, and perform liquid chromatography detection.

[0114] The crude enzyme activity of combined DPE and single DPE was analyzed respectively by using the above s...

Embodiment 3

[0115] Example 3, construction of recombinant strains of Corynebacterium glutamicum to express combined DPE operons in the form of chromosomal integration

[0116] 1. Construction of the recombinant strain DPE3 of Corynebacterium glutamicum

[0117] First, the integration site vector pK18-ldhA was constructed to integrate the DPE combined operon into the lactate dehydrogenase gene (ldhA) site of the chromosome, according to the upstream and downstream sequence information of the lactate dehydrogenase gene (ldhA) in Corynebacterium glutamicum , design primer 9, primer 10, primer 11 and primer 12, wherein primer 9 and primer 10 are used to amplify the upstream gene fragment of ldhA, primer 11 and primer 12 are used to amplify the downstream gene fragment of ldhA, primer 10 and primer 11 There is a homologous region of about 40 bp, which is used for the fusion of upstream and downstream fragments. Primer 9 and primer 12 contain EcoRI and HindIII restriction sites respectively. T...

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Abstract

The invention discloses a method for efficiently preparing D-psicose 3-epimerase and a use of the D-psicose 3-epimerase and provides a method for efficiently preparing an enzyme catalyst for catalyzing the same reaction through isozyme combined expression. The method utilizes corynebacterium glutamicum as a host cell to construct a recombinant strain expressed by plasmid dissociation and chromosomal integration, measures D-fructose catalytic efficiency, improves enzyme catalytic efficiency by 2-4 times than that of single expression of D-psicose 3-epimerase through combined expression and hasa conversion ratio of 29% when 70% of fructose is a substrate. The method improves D-psicose production efficiency and is suitable for industrial production of D-psicose. The invention also disclosesa novel use of the D-psicose 3-epimerase in psicose synthesis. The enzyme is derived from Paenibacillus senegalensis, has catalytic activity of about 25 U/mg and can be used to convert D-fructose intoD-psicose.

Description

technical field [0001] The invention relates to the field of industrial biotechnology, in particular to a method for efficiently preparing D-psicose 3-epimerase and its application in the preparation of allulose. Background technique [0002] D-psicose is an epimer of fructose, its sweetness is 70% of that of sucrose, but its energy value is only 0.007kcal / g, and its energy absorption efficiency is only 0.3% of that of sucrose. It is a low-calorie sweetener that can be used as a sucrose substitute in the food field; D-psicose has been proven to have a hypoglycemic effect, and can also inhibit the activity of liver fat synthase enzyme intestinal d-glucosidase, thereby reducing abdominal pain. The accumulation of fat also has high medical value in the treatment of neurodegenerative and atherosclerotic diseases. The U.S. Food and Drug Administration (FDA) officially approved D-psicose as a GRAS food in 2011, allowing it to be used in food, pharmaceutical preparations and dieta...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N15/61C12N1/21C12P19/24C12P19/02C12R1/15
CPCC12N9/90C12N15/77C12P19/02C12P19/24C12Y501/03
Inventor 孙媛霞杨建刚朱玥明门燕马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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