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A d-psicose 3-epimerase production strain and its immobilization method

A technology of epimerase and psicose, which is applied to the production strain of D-psicose 3-epimerase and the field of immobilization thereof, can solve the problem of high cost, unreusable enzyme liquid cost, Free enzymes can not be reused, etc., to achieve the effect of simplified separation and purification, good continuous transformation ability

Active Publication Date: 2020-12-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] D-psicose is rarely found in nature. At present, D-psicose is mostly produced by enzymatic catalysis, but there are also shortcomings in enzymatic catalysis. For example, the poor stability of recombinant enzymes is not enough to meet the needs of industrial production, and the enzyme The liquid cannot be reused and the cost is high
[0005] Because DPEase is an intracellular enzyme in its natural state, breaking the wall to obtain a large amount of enzyme solution required for industrial production is not only time-consuming and labor-intensive, but also costly
In the process of industrial production of D-psicose, free enzymes cannot be reused, and free enzymes generally have poor thermal stability and are not suitable for industrial continuous production

Method used

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  • A d-psicose 3-epimerase production strain and its immobilization method
  • A d-psicose 3-epimerase production strain and its immobilization method
  • A d-psicose 3-epimerase production strain and its immobilization method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Cloning and Expression of Clostridium vector encoding gene DPEase

[0031] The database DPEase gene and E. coli - B. subtilis shuttle vector pHY300PLK sequences with two primer pairs designed 15bp homology arms P1 and P2, P3 and P4:

[0032] P1: 5'-TAAGGAGTGTCAAGAATGAAACATGGCATC-3 '

[0033] P2: 5'-TTTATTACCAAGCTTTTAGCTATGTTTATGA-3 '

[0034] P3: 5'-TCATAAACATAGCTAAAAGCTTGGTAATAAA-3 '

[0035] P4: 5'-GATGCCATGT TTCATTCTTGACACTCCTTA-3 '

[0036] Respectively, and the plasmid vector pHY300PLK with DPEase gene as a template, cloning the gene containing homology arms and 15bp vector pHY300PLK, the PCR products were gel recovered seamlessly with infusion. Connection architecture: the gene 2μL, pHY300PLK 0.5μL, infusion 1μL, ddH 2 O 1.5μL. After the above system was placed after mixing incubated at 50 ℃ water bath after 30min, was immediately transferred to the ice in an ice bath 5min, and the ligation product was transformed JM109 competent, ampicillin-resistant coati...

Embodiment 2

[0037] Example 2: Transformation of recombinant plasmid pHY300PLK-DPEase

[0038] 1) fresh LB plates (LB solid medium g / L: tryptone 10, yeast extract 5, NaCl 10,0.2 agar) pick Bacillus subtilis Bacillus subtilis (CCTCC M2016536) single colony was inoculated into 10 mL of LB broth, 37 ℃, 200rpm culture 10.5h.

[0039] 2) Take 2.5mL was transferred overnight cultures in LB medium access sorbitol was added 40mL of 0.5M, 37 ℃, 200rpm shaking culture 4.5h.

[0040] 3) The bacterial solution ice bath was 10min, then 5000rpm, 4 ℃, 5min cells were collected by centrifugation.

[0041] 4) pre-cooled transfer medium is electrically 50mL (sorbitol 0.5M, mannitol 0.5M, 10% glucose) to wash the cells, 5000rpm, 4 ℃, the supernatant was centrifuged 5min, rinsed four times so.

[0042] 5) The washed bacteria were resuspended in 1mL electroporation medium, aliquoted into 1.5mL EP tube, tubes per 200μL of competent cells.

[0043] 6) The competent cells were added 200μL 10μL recombinant plasmid, ...

Embodiment 3

[0045] Example 3: Shake flask fermentation enzyme

[0046] 1) Fermentation culture

[0047] The recombinant subtiline BS-PHY300 PLK-DPEASE strain obtained in Example 2 was inoculated in the LB medium, and after 8-10 h at 37 ° C, it was transferred to TB fermentation medium, 33 ° C, 200 rpm constant temperature culture 48h yield enzyme. After the fermentation is completed, the ultrasound wall acquires the crude enzyme solution.

[0048] Lb medium (g / L): protein 10, yeast paste 5, NaCl 10

[0049] TB medium (g / L): protein 10, yeast powder 24, glycerin 5, K 2 HPO 4 · 3h 2 O 16.43, kh 2 PO 4 2.31.

[0050] 2) Determination of enzyme

[0051] Add 200 μL of suitable diluted enzyme solution to 800 μl of HEPES buffer (20 mm / L, pH 8.0, 0.1 mmol / L CO) 2+ In the middle, the fructose final concentration is 80 g / L. After oscillating mixing, it was placed in a 55 ° C water bath to react 10 min, boiled 10 min lyase termination reaction. After the sample was centrifuged, the detector wa...

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Abstract

The invention discloses a D-psicose 3-epimerase production strain and an immobilization method thereof, and belongs to the technical field of bioengineering. According to the invention, recombinant bacillus subtilis pHY300PLK-DPEase for producing D-psicose 3-epimerase is constructed, and the recombinant bacterium (the recombinant bacillus subtilis) serves as the production strain; an enzyme activity recovery rate of immobilized cells reaches 64%, the optimum temperature of the immobilized cells is improved by 5 DEG C in comparison with that of whole cells and the optimum pH value of the immobilized cells is free from obvious change; and after continuous conversion with 300g / L of fructose as a substrate for seven times, a conversion rate can be still kept at 28% and residual enzyme activitycan be still kept at 81%. The immobilization method is simple and easy to implement and immobilized particles are excellent in performance; therefore, a quite high industrial application value is achieved.

Description

Technical field [0001] The present invention relates to a D- psicose 3-epimerase immobilization method of production strains and belongs to the technical field of biological engineering. Background technique [0002] Refers to a class of sugar rare in nature, but very small amounts as monosaccharides and derivatives thereof, sugar rare species has been found in 34 species of rare sugars which function as a novel sweetener, which has been widely used in diet, medicine and health care. D- psicose was isomer D- fructose on the C3 position, belonging to rare saccharides. It was 70% of the sweetness of sucrose, but its energy value only 0.3%, it has a number of unique physiological and nutritional functions. [0003] D- psicose rarely found in nature, most of the current produced by enzymatic catalysis D- psicose, Enzymatic but also shortcomings such as poor stability of the recombinant enzymes is insufficient to meet the needs of industrial production, and the enzyme It was unable to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/61C12N15/75C12N9/90C12P19/24C12P19/02C12R1/125
CPCC12N9/90C12N15/75C12P19/02C12P19/24C12Y501/03
Inventor 吴敬宿玲恰孙帆
Owner JIANGNAN UNIV
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