Preparation method of immobilized threonine aldolase, immobilized threonine aldolase and application

A technology of threonine aldolase enzyme and threonine aldolase enzyme liquid is applied in the field of immobilized threonine aldolase and its application, and the preparation field of immobilized threonine aldolase can solve the problem of Using resin and other problems to achieve huge economic and social benefits and high product yield

Inactive Publication Date: 2021-07-02
宁波泓森生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no suitable immobilized threonine aldolase product for the production of p-thymphenylphenylserine, and there is no immobilized threonine with both good batch reaction stability and high enzyme activity recovery. A report on acid aldolase
Moreover, after the immobilized enzyme is inactivated, both the resin and the enzyme will be discarded, and there is no reasonable strategy for reusing the resin in the existing reports of immobilized threonine aldolase

Method used

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  • Preparation method of immobilized threonine aldolase, immobilized threonine aldolase and application
  • Preparation method of immobilized threonine aldolase, immobilized threonine aldolase and application
  • Preparation method of immobilized threonine aldolase, immobilized threonine aldolase and application

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Embodiment 1

[0029] Embodiment 1: Preparation of free threonine aldolase enzyme solution

[0030] Use Escherichia coli BL21 engineering strains containing threonine aldolase gene (gene derived from Bacillus nealsonii, GenBank accession code WP_016204489.1) (for the construction method, please refer to the articles published by our laboratory W. Zheng, K. Chen, Z .Wang, X.Cheng, G.Xu, L.Yang, J.Wu, Organic Letters, 22(2020) 5763-5767.), Streak the plate in a clean bench and culture it in a 37°C incubator for 12h, Then pick the bacteria in the ultra-clean platform at 37 ° C, 200rpm shaker culture 2 ~ 3h, OD 600 At about 0.8 at 18°C, 200rpm induction culture for 16h to OD 600 At about 3, centrifuge at 4°C, 12000rpm for 10min, and discard the supernatant. Every 10 mL of bacterial liquid was centrifuged with 3 mL of 50 nM, pH 8.0 phosphate buffer to suspend, and then crushed at 400 W for 20 times to obtain free threonine aldolase enzyme liquid.

Embodiment 2

[0031] Embodiment 2: the pretreatment of resin

[0032] Wash a certain amount of amino resins NAA, HAA, HA, NH, ESQ-3 with 50mM, pH 8.0 phosphate buffer (when cleaning, use a glass rod to gently stir to prevent the resin from breaking) 2 or 3 times and no foam will appear . Mix the cleaned amino resin NAA and the glutaraldehyde crosslinking agent with a volume fraction of 0.4% at a ratio of mass (resin mass before cleaning): volume = 1:3, and activate it on a shaker at 25°C and 150rpm for 2-3h . The activated resin turns yellow from the original one, and the activated amino resin NAA is suction-filtered, and then washed with 50mM, pH 8.0 phosphate buffer for 2-3 times to remove the residual glutaraldehyde on the surface, then suction-filtered, and then used The freeze-dried and activated NAA was stored at 4°C for future use. Take a certain amount of D301, D301R, D354, and D314 and soak them in distilled water for 24 hours, then soak them in ethanol with a volume fraction of...

Embodiment 3

[0033] Example 3: Immobilization of threonine aldolase

[0034] Add the activated resin and free threonine aldolase enzyme solution to 10mL of 50mM, pH 8.0 phosphate buffer solution at a ratio of 0.6g carrier / 3mL free enzyme solution and place in a shaker at 30°C and 150rmp Immobilized for 4 to 5 hours. Then the obtained immobilized enzyme was washed 2 to 3 times to remove the residual enzyme on the surface, filtered by suction, freeze-dried and stored at 4°C for future use.

[0035]Sodium alginate immobilized enzyme: dissolve (pH 8.0 phosphate buffer solution) to prepare 7mL of sodium alginate with a concentration of 3%, place it in a bath at 40-50°C and heat to ensure that the sodium alginate is fully dissolved, add 3mL after cooling For the foam breaking solution, stir and dissolve evenly with a glass rod. Use a syringe to absorb the dissolved sodium alginate enzyme solution, and slowly add it dropwise to 3% 50mL calcium chloride solution to form sodium alginate embedding...

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Abstract

The invention discloses a preparation method of immobilized threonine aldolase, the immobilized threonine aldolase and application of the immobilized threonine aldolase. The preparation method comprises the following steps: (1) obtaining a threonine aldolase liquid; (2) activating an amino resin NAA; and (3) mixing and immobilizing the activated amino resin NAA and the threonine aldolase liquid to obtain the immobilized threonine aldolase. According to the immobilization method for preparing the threonine aldolase by using the amino resin NAA, the immobilized enzyme is high in enzyme activity recovery, operation stability and storage stability; when the immobilized enzyme is applied to synthesis of p-methylsulfonylphenylserine, the yield and de value of the immobilized enzyme are higher than those of the free enzyme after the immobilized enzyme and the free enzyme react in the same reaction system for the same time, wherein the yield and the de value of the immobilized enzyme are respectively 16.12 g / L and 98.2%, and the yield and the de value of the free enzymes are respectively 14.23 g / L and 95.8%, so that the selectivity is higher than that of the free enzyme, the yield of the obtained product is higher than that of the free enzyme, and huge economic benefits and social benefits are achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of immobilized threonine aldolase, immobilized threonine aldolase and application thereof. Background technique [0002] Threonine aldolase is a PLP-dependent enzyme that can catalyze the cleavage of threonine into glycine and acetaldehyde and its reverse reaction. This enzyme is widely found in various organisms and plays an important role in their metabolic pathways. effect. In production applications, because threonine aldolase has a high selectivity for cleaving substrates, the reverse reaction catalyzed by this enzyme is often used to synthesize β-hydroxy-α-amino acids from simple organic compounds in one step. Compared with organic synthesis with many steps, the biocatalytic process using the enzyme is simpler and more environmentally friendly. However, the free enzyme has poor stability, cannot be reused, and is difficult to separate from the product, wh...

Claims

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Application Information

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IPC IPC(8): C12N11/091C12N9/88C12P13/04
CPCC12N11/091C12N9/88C12P13/04C12Y401/02005
Inventor 徐刚陈开通郑文隆吴坚平杨立荣
Owner 宁波泓森生物科技有限公司
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