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Transaminase immobilization method

A transaminase and transaminase technology, which is applied in the direction of being fixed on/in an organic carrier, can solve problems such as unfavorable chemical bonds and immobilization of transaminase, and achieve the effects of not easily falling off, large amount of enzyme loading, and abundant holes.

Active Publication Date: 2015-07-15
四川百特芳华医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Polyvinyl alcohol sponge is obtained by acetalization of polyvinyl alcohol. It has excellent water absorption and mechanical properties, large specific surface area, and has the potential to be used as a transaminase immobilization carrier. However, polyvinyl alcohol sponge only contains hydroxyl active groups, which is not conducive to Immobilizing transaminases with chemical bonds

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The immobilization method of the transaminase of the present embodiment comprises the following steps:

[0024] a. Cut the polyvinyl alcohol sponge into 27mm 3 Take 10.0 grams of sponge particles and add them to a beaker, add 1000 ml of water, stir mechanically, add 0.010 g of ferrous sulfate, add 1.00 ml of 30% hydrogen peroxide, heat up to 60 ° C, and react for 4 hours. Pour the reaction solution into a cloth bag, squeeze the sponge particles in the cloth bag dry, soak the cloth bag in purified water for 5 minutes, then squeeze the sponge particles in the cloth bag to dry and dehydrate to obtain an oxidized polyvinyl alcohol sponge.

[0025] b. Put the oxidized polyvinyl alcohol sponge into a beaker, add 1000ml of water, stir mechanically, and add 10 grams of ATA-113 transaminase Add 1 gram of isopropylamine hydrochloride, add 0.50 gram of pyridoxal phosphate, adjust the pH to 7.5 with isopropylamine, heat to 40°C for 6 hours, pour the reaction solution into a cloth...

Embodiment 2

[0031] The immobilization method of the transaminase of the present embodiment comprises the following steps:

[0032] a. Cut the polyvinyl alcohol sponge into 27mm 3 . In a 2000 liter reactor, add 1000 liters of water, start stirring, add 10.00 kg of sponge particles, add 10 g of ferrous sulfate, add 1.00 liter of 30% hydrogen peroxide, heat up to 55 ° C, and react for 5 hours. The reaction solution was filtered with a three-legged centrifuge, washed with purified water, and centrifugally dehydrated to obtain an oxidized polyvinyl alcohol sponge.

[0033] b. In a 2000-liter reactor, add 1000 liters of water, start stirring, add oxidized polyvinyl alcohol sponge, and add 20 kg of ATA-117 transaminase Add 2.00 kg of isopropylamine hydrochloride, add 0.75 kg of pyridoxal phosphate, adjust the pH to 8.0 with isopropylamine, heat to 37 ° C for 8 hours, centrifuge the reaction solution, remove the solution, and obtain an aminated polyvinyl alcohol sponge .

[0034] c. Pour ami...

Embodiment 3

[0038] The immobilization method of the transaminase of the present embodiment comprises the following steps:

[0039] a. Cut the polyvinyl alcohol sponge into 27mm 3 . In a 2000 liter reactor, add 1000 liters of water, start stirring, add 10.00 kg of sponge particles, add 10 g of ferric chloride and 5 kg of ferrous chloride, add 4.00 liters of 10% hydrogen peroxide, heat up to 60 ° C, and react for 5 hours. The reaction solution was filtered with a three-legged centrifuge, washed with purified water, and centrifugally dehydrated to obtain an oxidized polyvinyl alcohol sponge.

[0040] b. In a 2000-liter reactor, add 1000 liters of water, start stirring, add oxidized polyvinyl alcohol sponge, and add 5 kg of ATA-117 transaminase Add 2.00 kg of isopropylamine hydrochloride, add 0.5 kg of pyridoxine phosphate and 0.5 kg of pyridoxamine phosphate, adjust the pH to 8.0 with isopropylamine, heat to 30 ° C for 8 hours, centrifuge the reaction solution, remove the solution, and ob...

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PUM

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Abstract

The invention provides a transaminase immobilization method. The method comprises the following steps: oxidizing a hydroxyl group on polyvinyl alcohol sponge, adding transaminase to convert a carbonyl group into an amino group, flocculating, and carrying out cross-linking immobilization by using polyaldehyde to obtain immobilized transaminase. The immobilization method has the advantages of high enzyme activity unit and enzyme purification.

Description

technical field [0001] The invention relates to a method for immobilizing transaminase. Background technique [0002] Transaminases convert carbonyl groups in compounds to amino groups. It can efficiently produce chiral amines, and because of its relatively cheap substrates, high product purity, and mild reaction conditions, it has great application value. [0003] The immobilized enzyme is easy to separate from the product and can be recycled and reused. It has better stability and longer operation cycle than free enzyme. There are many ways to immobilize enzymes or cells, such as physical adsorption, affinity binding, covalent cross-linking, flocculation embedding, etc. [0004] CN201280031237 uses resin as a carrier to immobilize transaminase, but using resin as a carrier has the disadvantages that the resin is expensive and the amount of enzyme loaded is low. Taking Examples 1 to 5 of this patent as an example, 1 gram of resin carrier can only immobilize 0.125 gram of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08
Inventor 黄清东张翔向世明
Owner 四川百特芳华医药科技有限公司
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