Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst as well as preparation method and application thereof

A technology of isopropanol dehydrogenase and isopropanol dehydrogenase crude enzyme, which is applied in the direction of organic compound/hydride/coordination complex catalyst, physical/chemical process catalyst, catalytic reaction, etc., which can solve cumbersome processing, Enzyme catalysts are difficult to recycle, reuse, and increase industrial production costs, etc., to achieve high catalytic efficiency, major industrial application value, and excellent stability

Pending Publication Date: 2020-09-22
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the carbonyl reductase used to catalyze the reaction and the isopropanol dehydrogenase used to regenerate the coenzyme in this report are both crude enzy...

Method used

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  • Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst as well as preparation method and application thereof
  • Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst as well as preparation method and application thereof
  • Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, preparation of carbonyl reductase / isopropanol dehydrogenase co-immobilized catalyst

[0039] Weigh 5g of polyvinyl alcohol, 3g of polyethylene glycol and 35mL of water into a reaction flask, heat until the solution is clear, cool down to below 50°C, add 10mL of carbonyl reductase crude enzyme solution (15% w / v) and 5mL of iso Propanol dehydrogenase crude enzyme solution (15% w / v), mix well. After mixing, drop the solution onto the polyethylene film with a syringe, and then place it in a blast oven at 35-40°C for 1 hour to obtain the carbonyl reductase / isopropanol dehydrogenase co-immobilized catalyst, which is stored in Store at 4°C for later use.

Embodiment 2

[0040] Example 2, Study on Thermal Stability of Carbonyl Reductase / Isopropanol Dehydrogenase Co-immobilized Catalyst

[0041] Get carbonyl reductase free enzyme (crude enzyme liquid), isopropanol dehydrogenase free enzyme (crude enzyme liquid) and the carbonyl reductase / isopropanol dehydrogenase co-immobilized catalyst that embodiment 1 makes, respectively in 20 ℃, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C for 30 minutes, and then measure the enzyme activity respectively, with the highest activity as 100%, measure in parallel three times, and make the corresponding temperature stability curve, as figure 1 and 2 shown.

[0042] Carbonyl reductase enzyme activity assay method: take a certain amount of carbonyl reductase free enzyme or immobilized enzyme, add to the phosphate buffer solution (100 mM, pH 7.5) containing substrate (10 mM), NADPH (0.24 mM) ), shake for 1 min, centrifuge, take the supernatant to measure the absorbance change at 340 nm, and calculate the enzyme activity p...

Embodiment 3

[0045] Example 3, Study on pH Stability of Carbonyl Reductase / Isopropanol Dehydrogenase Co-immobilized Catalyst

[0046] Get carbonyl reductase free enzyme, isopropanol dehydrogenase free enzyme and the carbonyl reductase / isopropanol dehydrogenase co-immobilized catalyst that embodiment 1 makes, add to pH 4,5,6,7,8 Incubate in a 30°C water bath for 5 minutes, and then measure the enzyme activity, with the highest activity as 100%, measure three times in parallel, and make the corresponding pH stability curve, as shown in image 3 and 4 shown.

[0047] Depend on image 3 and 4 It can be seen that, for isopropanol dehydrogenase, both the immobilized enzyme and the free enzyme lose their activity at pH=4, and in the case of pH=5-8, the pH stability of the immobilized enzyme is better than that of the free enzyme. improved. For carbonyl reductase, the pH stability of the immobilized enzyme was lower than that of the free enzyme under acidic conditions; however, the pH stabili...

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Abstract

The invention belongs to the technical field of biological pharmacy, and particularly discloses a carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst as well as a preparation method and application thereof. According to the invention, carbonyl reductase and isopropanol dehydrogenase are co-embedded in a solid phase carrier obtained from polyvinyl alcohol and polyethylene glycol; the obtained co-immobilized enzyme catalyst can realize efficient and high stereoselectivity reduction of 3-carbonyl-5-hexenoic acid ester to generate (R)-3-hydroxy-5-hexenoic acid ester through catalysis of carbonyl reductase and circulation of isopropanol dehydrogenase on coenzyme NADPH. The catalyst can be used for preparing (R)-3-hydroxy-5-hexenoic acid ester by taking 3-carbonyl-5-hexenoic acid ester as a substrate, and the co-immobilized catalyst prepared by the method disclosed by the invention is high in catalytic efficiency, good in stability, reusable, concise in process and low in cost, and has excellent actual industrial application value.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, in particular to a co-immobilized enzyme catalyst and its preparation method and synthesis ( R )-3-Hydroxy-5-hexenoate for application. Background technique [0002] ( R )-3-Hydroxy-5-hexenoate is an important chiral compound widely used in the synthesis of pharmaceutical and chemical intermediates including statins for hypolipidemic drugs. Its structural formula is shown in Figure (I) below, where R is C 1 -C 8 Alkyl or cycloalkyl, mono- or polysubstituted aryl or aralkyl. [0003] [0004] As the current main way to synthesize compound (I) and its structural analogues, chemical methods have their shortcomings, for example, extreme reaction conditions are often required, including -20 o C to -50 o C low temperature (US patent US6355822) and high hydrogen pressure (European patent EP1176135), etc. These harsh reaction conditions severely limit the industrial prospects of chem...

Claims

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Application Information

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IPC IPC(8): B01J31/06C07C67/31C07C69/732
CPCB01J31/003B01J31/06C07C67/31B01J2231/643C07C69/732
Inventor 陈芬儿黄则度胡辰
Owner FUDAN UNIV
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