A coupling method for separation, purification and immobilization of penicillin acylase for preparing 6-aminopenicillanic acid

A technology of penicillin acylase and aminopenicillanic acid, applied in the field of bioengineering, can solve the problems of insignificant impurity removal effect, cumbersome process steps, and low production efficiency, achieve small amplification effect, shorten operation time, and improve production efficiency effect

Active Publication Date: 2018-04-20
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 for preparing 6-aminopenicillanic acid
  • A coupling method for separation, purification and immobilization of penicillin acylase for preparing 6-aminopenicillanic acid
  • A coupling method for separation, purification and immobilization of penicillin acylase for preparing 6-aminopenicillanic acid

Examples

Experimental program
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Effect test

preparation example

[0067] (1) Fermentation culture of penicillin acylase

[0068] Using recombinantly expressed Pichia strains, adopt the method described in the existing literature (Luo Qian, high-density fermentation and immobilization of PGA produced by recombinant Pichia [D], master's degree thesis of Central South University of Forestry and Technology, 2013) Fermentation culture, test the enzyme activity of penicillin acylase in the obtained fermentation liquid is 83.2IU / mL.

[0069] (2) Fermentation broth pretreatment:

[0070] (i) After the fermentation is finished, add 0.01g / mL of ammonium sulfate according to the volume of the feed liquid, stir to dissolve, then add 0.008g / mL of cetyltrimethylammonium bromide according to the volume of the feed liquid, and stir for 25min; The temperature of the solution was raised to 45°C, and the pH was adjusted to 7.6 with 2mol / L NaOH solution at the same time; after the temperature and pH were adjusted in place, the temperature was maintained at 45°...

Embodiment 1

[0075] (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 Regulate pH to 8.0), obtain inorganic salt mixed solution;

[0076] (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 middle phase solid, and a lower phase solution rich in salt and penicillin acylase, and separate the three-phase solution;

[0077] (3) get the upper phase ...

Embodiment 2

[0082] (1) Under the condition of 12°C, the phosphate (by K 2 HPO 4 .3H 2 O and KH 2 PO 4 Composition) is 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 6 (add KH first 2 PO 4 Adjust pH to 5, add K 2 HPO 4 .3H 2 O to adjust the pH to 7 and finally add KH 2 PO 4 Adjust the pH to 6), stir while adding salt (stirring speed is 250rpm) to fully dissolve the salt, and obtain a mixed solution of inorganic salt;

[0083] (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 1:0.25, 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 penicillin acylase, and sep...

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Abstract

The invention relates to the field of bioengineering, in particular to a method for separation, purification and fixed coupling of penicillin acylase for preparing 6-amino penicillanic acid. The method comprises the following steps that 1, an inorganic salt mixed solution containing penicillin acylase crude enzyme and inorganic salt is prepared, wherein the inorganic salt concentration in the inorganic salt mixed solution is 0.05-0.35 g / Ml; 2, the inorganic salt mixed solution obtained in the step 1 is mixed with tertiary butanol, and the phase separation is performed to obtain a three-phase distribution system consisting of an upper-phase solution rich in tertiary butanol, a middle-phase solid and a lower-phase solution rich in salt and penicillin acylase; 3, a carrier is added into the lower-phase solution obtained in the step 2 to fixing the penicillin acylase. The method is simple and convenient to operate, and the obtained penicillin acylase is higher in activity and yield.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a coupling method for separation, purification and immobilization of penicillin acylase for preparing 6-aminopenicillanic acid. 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 preparation of 6-aminopenicillanic acid, 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 com...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/84C12N11/00C12P37/06
CPCC12N9/84C12N11/00C12P37/06C12Y305/01011
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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