D-psicose 3-epimerase production strain and immobilization method thereof

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 cannot be reused, etc., to achieve the effect of simplified separation and purification, good continuous transformation ability

Active Publication Date: 2018-06-01
JIANGNAN UNIV
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  • Abstract
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  • Claims
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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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  • D-psicose 3-epimerase production strain and immobilization method thereof
  • D-psicose 3-epimerase production strain and immobilization method thereof
  • D-psicose 3-epimerase production strain and immobilization method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Cloning of Clostridium cellulolyticum DPEase coding gene and construction of expression vector

[0031] Two pairs of primers P1 and P2, P3 and P4 with 15bp homology arms were designed according to the DPEase gene in the database and the Escherichia coli-Bacillus subtilis shuttle vector pHY300PLK sequence:

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

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

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

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

[0036] Using the plasmid with the DPEase gene and the pHY300PLK vector as templates, clone the target gene containing 15bp homology arms and the pHY300PLK vector, recover the PCR products separately, and use infusion for seamless connection. Connection system: target gene 2μL, pHY300PLK 0.5μL, infusion 1μL, ddH 2 O 1.5 μL. After mixing the above system, place it in a water bath at 50°C for incubation for 30 minutes, then immediately transfer it to a...

Embodiment 2

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

[0038] 1) Pick a single colony of Bacillus subtilis (CCTCC M2016536) on a fresh LB plate (LB solid medium g / L: peptone 10, yeast extract 5, NaCl 10, 0.2 agar powder) and inoculate it in 10 mL LB liquid medium, 37 Cultivate at 200rpm for 10.5h.

[0039] 2) Take 2.5 mL of the overnight culture and transfer it into 40 mL of LB medium added with 0.5 M sorbitol, culture at 37° C. with shaking at 200 rpm for 4.5 h.

[0040] 3) Cool the bacterial solution on ice for 10 minutes, then centrifuge at 5000 rpm at 4°C for 5 minutes to collect the bacterial cells.

[0041] 4) Wash the cells with 50 mL pre-cooled electroporation medium (sorbitol 0.5 M, mannitol 0.5 M, glucose 10%), centrifuge at 5000 rpm, 4° C. for 5 min to remove the supernatant, and rinse 4 times in this way.

[0042] 5) Resuspend the washed bacteria in 1 mL of electroporation medium, aliquot them into 1.5 mL EP tubes, and fill each tube with 2...

Embodiment 3

[0045] Embodiment 3: Shake flask fermentation produces enzyme

[0046] 1) Fermentation culture

[0047] The recombinant Bacillus subtilis BS-pHY300PLK-DPEase strain obtained in Example 2 was inoculated in LB medium, and after cultivating at 37°C for 8-10h, it was transferred to TB fermentation medium with 5% inoculum size, at 33°C, Incubate at 200 rpm for 48 hours to produce enzyme. After the fermentation, the wall was ultrasonically broken to obtain the crude enzyme liquid.

[0048] LB medium (g / L): peptone 10, yeast extract 5, NaCl 10

[0049] TB medium (g / L): peptone 10, yeast powder 24, glycerol 5, K 2 HPO 4 ·3H 2 O 16.43, KH 2 PO 4 2.31.

[0050] 2) Determination of enzyme activity

[0051] Add 200 μL of appropriately diluted enzyme solution to 800 μL of fructose-containing HEPES buffer (20 mM / L, pH 8.0, 0.1 mmol / L Co 2+ ), make the final concentration of fructose 80g / L. After shaking and mixing, place in a 55°C water bath to react for 10 minutes, and boil for ...

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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 invention relates to a D-psicose 3-epimerase production strain and an immobilization method thereof, belonging to the technical field of bioengineering. Background technique [0002] Rare sugars refer to a class of monosaccharides and their derivatives that exist in nature but are very low in content. There are 34 rare sugars that have been discovered so far. These rare sugars are new functional sweeteners, which have been Widely used in food, medicine and health care and other fields. D-psicose is an isomer at the C3 position of D-fructose, which is a kind of rare sugar. Its sweetness is 70% of that of sucrose, but its energy value is only 0.3%, so it has a variety of unique physiological and nutritional functions. [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 enoug...

Claims

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Application Information

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Patent Type & Authority Applications(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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