A coupling method for separation, purification and immobilization of penicillin acylase used in the synthesis of amoxicillin

A technology of penicillin acylase and crude enzyme of penicillin acylase, which is applied in the field of bioengineering, can solve the problems of insignificant impurity removal effect, cumbersome process steps, and low total enzyme yield, so as to achieve small amplification effect and shorten operation time , the effect of simplifying the operation steps

Active Publication Date: 2018-06-15
山西双雁生物科技有限公司
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AI Technical Summary

Problems solved by technology

[0005] The main problems in the above-mentioned process are as follows: (1) the process steps are too complicated and the operation time is long, resulting in low production efficiency; (2) more steps and some enzymes will be lost due to the long operation time, resulting in low overall yield of enzyme; ( 3) The main impurity removal methods are flocculation precipitation, ammonium sulfate precipitation, ultrafiltration and plate-and-frame filtration (or centrifugation). The separation means are relatively traditional, and the effect of impurity removal is not significant; (4) Solid-liquid separation and concentration are not considered from the perspective of process integration , impurity removal and other separation and purification and immobilization operations are interconnected, resulting in too many steps

Method used

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  • A coupling method for separation, purification and immobilization of penicillin acylase used in the synthesis of amoxicillin
  • A coupling method for separation, purification and immobilization of penicillin acylase used in the synthesis of amoxicillin
  • A coupling method for separation, purification and immobilization of penicillin acylase used in the synthesis of amoxicillin

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Effect test

preparation example

[0067] (1) Fermentation culture of penicillin acylase

[0068] Using recombinantly expressed E. coli strains, the method described in the existing literature (Lu Jianmei, the expression of penicillin acylase in E. coli for the synthesis of amoxicillin [D], master's degree thesis of Beijing University of Chemical Technology, 2013) was carried out Ferment and cultivate until the enzyme activity of penicillin acylase in the fermentation broth is 35.7IU / mL.

[0069] (2) Fermentation broth pretreatment:

[0070] (i) After the fermentation, centrifuge the fermentation broth at 4500g for 20min to collect the cells; then add 0.1mol / L pH 8.0 phosphate buffer to the cells according to the phosphate buffer / cell volume mass ratio of 2.5mL / g liquid, under 900 bar pressure, high-pressure homogenate and crush the cells for 80 minutes, homogenate twice, and take samples at intervals to measure the enzyme activity until the cell fragmentation is basically complete, and obtain the crude enzyme...

Embodiment 1

[0074] (1) Under the condition of 14°C, the phosphate (by K 2 HPO 4 .3H 2 O and KH 2 PO 4 Composition) be dissolved in the crude enzyme solution II of penicillin acylase that preparation example obtains in 90min and the pH value of adjusting solution in the process of adding is 8.0 (add KH first 2 PO 4 Adjust pH to 6.5, add K 2 HPO 4 .3H 2 O to adjust the pH to 8.3, and finally add KH 2 PO 4 Adjust the pH to 8.0), add salt while stirring to fully dissolve the salt, and the stirring speed is 250rpm; then centrifuge at 4500g for 20min, collect the supernatant, and obtain a mixed solution of inorganic salt;

[0075] (2) Mix the inorganic salt mixed solution obtained in step (1) with the mixed solution of tert-butanol and water recovered in step (3) according to the volume ratio of 1:0.6, vortex for 2 minutes until fully mixed, and then centrifuge at 3500g for 10 minutes, Form a three-phase distribution system consisting of an upper phase solution rich in tert-butanol, a...

Embodiment 2

[0081] (1) Under the condition of 12°C, the phosphate (by K 2 HPO 4 .3H 2 O and KH 2 PO 4 Composition) be dissolved in the penicillin acylase crude enzyme liquid I that preparation example obtains and the pH value of adjusting solution in the process of adding is 8.5 (add KH first 2 PO 4 Adjust pH to 7, add K 2 HPO 4 .3H 2 O to adjust the pH to 9 and finally add KH 2 PO 4 Adjust the pH to 8.5), stir while adding salt (stirring speed is 250rpm) to fully dissolve the salt, and obtain a mixed solution of inorganic salt;

[0082] (2) Mix the inorganic salt mixed solution obtained in step (1) with the mixed solution of tert-butanol and water recovered in step (3) according to the volume ratio of 1:0.4, vortex for 2 minutes until fully mixed, and then centrifuge at 2000g for 20 minutes, Form a three-phase distribution system consisting of an upper phase solution rich in tert-butanol, a middle phase solid, and a lower phase solution rich in salt and enzymes, and separate th...

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Abstract

The invention relates to the field of bioengineering, and particularly relates to a separation purification and immobilization coupling method for penicillin acylase used for amoxicillin synthesis. The method includes (1) preparing an inorganic slat mixed solution containing a crude penicillin acylase solution and an inorganic salt, with the concentration of the inorganic salt in the mixed solution being 0.05-0.3 g / mL; (2) mixing the mixed solution obtained in the step (1) with tertiary butanol, performing phase splitting to obtain a three-phase distribution system including a tertiary butanol-rich upper-phase solution, middle-phase solid and a low-phase solution rich in the salt and the penicillin acylase; and (3) adding a carrier into the lower-phase solution obtained in the step (2) to perform penicillin acylase immobilization. The method is simple and convenient to operate, and activity and the yield of the obtained penicillin acylase are high.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a separation, purification and immobilization coupling method of penicillin acylase used for synthesizing amoxicillin. Background technique [0002] Penicillin acylase is a key enzyme for the preparation of β-lactam antibiotics. In industrial production, this enzyme has been successfully applied to the synthesis of amoxicillin, mostly in the form of immobilized enzyme. In order to prepare the immobilized enzyme that meets the production requirements, the penicillin acylase is firstly separated and purified from the fermentation broth, and then the enzyme is immobilized. The expression vectors of penicillin acylase mainly include Escherichia coli, Bacillus subtilis, Bacillus megaterium and Pichia pastoris. [0003] Although the expression vectors are different, the separation and purification of penicillin acylase from the fermentation broth all face some common problems: (1) remova...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/84C12N11/00C12P37/02C12P37/04
CPCC12N9/84C12N11/00C12P37/02C12P37/04C12Y305/01011
Inventor 王旭东修志龙董悦生王启斌
Owner 山西双雁生物科技有限公司
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