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Immobilized Serratia lipase, preparation method and application for catalyzing and splitting diltiazem chiral precursor

A technology of Serratia marcescens and lipase, applied in biochemical equipment and methods, immobilized on/in organic carriers, microorganism-based methods, etc., can solve the problem of expensive carriers and high cost of immobilized enzymes , reaction and separation operations are inconvenient, etc., to achieve the effects of mild reaction conditions, easy preparation, and good industrial application development prospects

Inactive Publication Date: 2009-09-16
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of epoxy resin covalent coupling method for enzyme immobilization, the carrier price is relatively expensive, and the cost of immobilized enzyme is high; and the use of diatomaceous earth adsorption method for immobilization, although the immobilization method is very simple, the enzyme upload capacity It is also larger, but the stability of the immobilized enzyme is still not high enough, and the reaction and separation operations are not very convenient. These problems limit the industrial application of these two immobilization methods.

Method used

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  • Immobilized Serratia lipase, preparation method and application for catalyzing and splitting diltiazem chiral precursor
  • Immobilized Serratia lipase, preparation method and application for catalyzing and splitting diltiazem chiral precursor
  • Immobilized Serratia lipase, preparation method and application for catalyzing and splitting diltiazem chiral precursor

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Chitosan-immobilized Serratia lipase

[0031] Get 0.5g chitosan respectively, prepare chitosan gel and chitosan bead, concrete preparation method is as follows:

[0032] Chitosan gel: Dissolve 0.5g chitosan in 50ml 1% acetic acid solution, slowly add 1% NaOH solution under magnetic stirring until the solution is alkaline, and collect the separated chitosan flocs by suction filtration. Wash with water until neutral, set aside.

[0033] Chitosan pellets: Dissolve 0.5g of chitosan in 20ml of 2.5% acetic acid solution, drip it dropwise into 20% NaOH solution with a syringe, collect the pellets obtained by suction filtration, wash with water until neutral, and set aside.

[0034] Put the obtained chitosan carrier into 50ml of 1% glutaraldehyde aqueous solution, shake at 30°C for 12h, suction filter and wash. Put into 50ml Serratia lipase fermentation liquid (5000U / L), stir and immobilize at 4°C for 2h, suction filter and wash. The results of enzyme immobilizatio...

Embodiment 2

[0037] Example 2 Chitosan immobilization of Serratia lipase

[0038] In 10L of hot water, add 100g of chitosan and 100ml of acetic acid, stir to dissolve the chitosan, and slowly add 1mol / L NaOH dropwise until the solution becomes weakly alkaline. Suction filtration, the chitosan gel that washes out, the gel that obtains is added in 10L Serratia fermented liquid (about 5000U / L of lipase activity), add 50ml glutaraldehyde (50%), room temperature stirring reaction After 2 hours, filter and wash with suction to remove unreacted glutaraldehyde and protein, and obtain an immobilized enzyme with a wet weight of about 1.5kg and an activity of 21U / g.

Embodiment 3

[0039] Example 3 Chitosan-immobilized Serratia lipase catalytic resolution of (±)-MPGM in isopropyl ether-water two-phase system

[0040] Take 10g chitosan floc immobilized Serratia lipase, the total activity is 230U, add it to a 250ml three-necked flask, add 50ml isopropyl ether containing 1.04g (±)-MPGM and 50ml water, 30°C, 200rpm The reaction was stirred, and ammonia water was added dropwise to control the pH of the reaction solution to be constant at 8.5. Sampling was taken intermittently, and the remaining substrate concentration and ee value were analyzed. The reaction was terminated when the ee value of the remaining substrate in each batch of reaction reached more than 99.9%, the immobilized Serratia lipase was collected by suction filtration, washed with water, and the next batch of reaction was restarted. The immobilized Serratia lipase was repeatedly used for 30 batches, and still retained a higher activity ( figure 1 ).

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Abstract

The invention discloses an immobilized Serratia lipase, a preparation method and application for catalyzing and splitting diltiazem chiral precursor. The immobilized lipase can catalyze and split the diltiazem chiral precursor-racemic p-methoxyphenyl glycidic acid methyl ester ((plus or minus)-MPGM). The invention uses chitosan as immobilized carrier of Serratia lipase, uses glutaradehyde as crosslinker and binds the Serratia lipase on the immobilized carrier in a covalent manner. The immobilized lipase is applied to reaction of enzymatic hydrolysis and splitting of the (plus or minus)-MPGM, and can be repeatedly used for a plurality of times, thus showing good operation stability and practical application feasibility.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and relates to the immobilization of Serratia marcescens lipase, the immobilization of Serratia marcescens extracellular lipase, and the application of the immobilized lipase in the production of medical precursors. technical background [0002] Diltiazem (Diltiazem) is a benzothiapine calcium ion channel blocker discovered and synthesized in the 1970s. It acts on the vascular smooth muscle and atrioventricular node of coronary vessels and peripheral vessels, and has a blocking effect on calcium channels. It can inhibit the influx of calcium ions into cells, reduce the concentration of free calcium ions in vascular smooth muscle cells and relax vascular smooth muscle; it can effectively expand small arteries, increase coronary blood flow, reduce coronary venous oxygen difference, and reduce heart rate and Blood pressure effect. It can be used in the treatment of supraventricular arrhythmia, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10C12P41/00C12P7/62C12R1/425
CPCY02P20/588
Inventor 潘江赵丽丽许建和陆雅臣
Owner EAST CHINA UNIV OF SCI & TECH
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