Construction method of recombinant Bacillus subtilis expressing d-psicose 3-epimerase based on d-alanine-deficient selection marker

A technology of Bacillus subtilis and epimerase, applied in the field of enzyme genetic engineering, can solve the problems of difficult separation and purification, complicated process, many by-products, etc.

Active Publication Date: 2018-03-30
山东星光首创生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The chemical synthesis method has problems such as complicated process, many by-products, difficult separation and purification, and food safety.

Method used

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  • Construction method of recombinant Bacillus subtilis expressing d-psicose 3-epimerase based on d-alanine-deficient selection marker
  • Construction method of recombinant Bacillus subtilis expressing d-psicose 3-epimerase based on d-alanine-deficient selection marker
  • Construction method of recombinant Bacillus subtilis expressing d-psicose 3-epimerase based on d-alanine-deficient selection marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: D-alanine-deficient Bacillus subtilis 1A751 ( dal - ) Build

[0024]

[0025] Using plasmid p7S6 as a template, the primer pair P3 / P4 will lox 71- spc - lox 66 Antibiotic resistance gene fragments were amplified and recovered. D-alanine racemase gene on chromosome 1A751 of Bacillus subtilis dal The 800-900bp length fragments on both sides are selected as the homology regions, and the homology regions on both ends are amplified and recovered with primer pairs P1 / P2 and P5 / P6 respectively.

[0026] Use primer pair P1 / P6 to combine the homology arm fragments at both ends with antibiotics lox 71- spc - lox 66 fused together by PCR technology.

[0027] PCR reaction system: Add the following reagents in order to 0.2mL PCR tube: 1.5μL each of the upstream and downstream primers; 5μLhusion HF buffer (5×); 2μL 10mM dNTP mix (2.5mM each); 2μL upstream homology arm; 0.5μL lox 71- spc - lox 66; 2μL downstream homology arm; 0.5μL Phusion high-fidelity DNA polymerase; add d...

Embodiment 2

[0032] Example 2: Construction of food-grade safe plasmid pUB-P43-DPE-dal

[0033]

[0034] To enhance expression, a strong promoter P43 is fused upstream of the DPE enzyme gene to form a P43-DPE expression unit. The P43-DPE fragment was amplified using primers P7 / P9. Using pUB110 as the starting vector, using the primer pair P8 / P10, the vector backbone pUB was amplified. Then the P43-DPE and pUB fragments were subjected to PCR to form multimers.

[0035] PCR reaction system: Add the following reagents in order to 0.2mL PCR tube: 5μL Phusion HF buffer (5×); 2μL 10mM dNTP mix (2.5mM each); 2μL P43-DPE; 2μL pUB; 0.5μL Phusion high-fidelity DNA polymerization Enzyme; add distilled water to a final volume of 50μL.

[0036] PCR amplification conditions: pre-denaturation at 98°C for 30s; denaturation at 98°C for 30s, annealing at 55°C for 15s, extension at 72°C for 1 min (30 cycles); extension at 72°C for 10 min.

[0037] The PCR product was directly transformed into host 1A751 and scree...

Embodiment 3

[0039] Example 3: Fermentation of recombinant bacteria

[0040] 1. DPE enzyme activity determination method: In a 1mL reaction system, add 800μL of 100g / L D-fructose dissolved in phosphate buffer (50mM, pH7.0), 200μL of fermentation broth, incubate at 55°C for 10min, then Boil for 10 minutes to stop the enzyme reaction.

[0041] 2. Detect the amount of D-psicose produced by HPLC and calculate the enzyme activity. Enzyme activity unit (U): The amount of enzyme required to catalyze the production of 1μmol D-psicose per minute.

[0042] 3. Use an inoculating loop to pick fresh colonies from the plate and inoculate them into the seed culture medium at 37°C, 200 rpm, and incubate for 12 to 14 hours. Inoculate the fermentation medium with an inoculum of 3%, 37°C, 200 rpm, and sample regularly to determine OD 600 , Enzyme activity data ( image 3 ).

[0043] Seed medium: tryptone (10g / L), yeast extract (5g / L), NaCl (10g / L), prepared with purified water.

[0044] Fermentation medium: glucose...

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Abstract

The invention discloses a method for constructing a recombinant Bacillus subtilis expressing D-psicose 3-epimerase based on a D-alanine-deficient selection marker, belonging to the technical field of enzyme gene engineering. Taking Bacillus subtilis 1A751 as the starting strain, knocking out the D-alanine racemase gene (dal) on its chromosome to obtain D-alanine-deficient 1A751 (dal-); the DPE of Clostridium ATCC 35704 The enzyme gene was used as a parent, and its upstream fusion P43 promoter was constructed into P43‑DPE, which was constructed into plasmid pUB110 (NCBI‑Gene ID: 9507338) to obtain pUB‑P43‑DPE, and the antibiotic resistance on pUB‑P43‑DPE The sex genes Kan and Blm were replaced by dal to construct pUB-P43-DPE-dal; then transformed into 1A751(dal-) to obtain recombinant Bacillus subtilis 1A751-pUB-P43-DPE-dal, which was named as Bacillus subtilis ( Bacillus subtilis) SK38.001, deposit number CCTCC NO:M 2015257. The total enzyme activity of its fermented liquid reaches 16U / mL, which has important industrial application value.

Description

Technical field [0001] The present invention is a method for constructing a recombinant Bacillus subtilis expressing D-psicose 3-epimerase based on a D-alanine defective selection marker, and relates to a food containing DPE enzyme in Bacillus subtilis Grade expression belongs to the technical field of enzyme genetic engineering. Background technique [0002] D-psicose 3-epimerase (DPE enzyme) belongs to the D-tagatose 3-epimerase (DTE) family of enzymes, which can catalyze the C3 epimerization of a variety of ketoses It is a good biocatalyst for the production of rare sugars. It can synthesize a variety of carbohydrates alone or in conjunction with other enzymes, and is widely used in chemistry, food and pharmaceuticals. At present, DPE enzyme can catalyze the conversion between D-fructose and D-psicose, and use D-fructose to produce D-psicose. [0003] With the outbreak of a series of chronic diseases caused by excessive high-energy food intake worldwide, the dietary structure ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12N15/61C12P19/02C12R1/125
Inventor 江波沐万孟何伟伟张涛
Owner 山东星光首创生物科技有限公司
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