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Magnetic combined cross-linked enzyme aggregate biocatalyst and preparation method and application thereof

A biocatalyst and collective biotechnology, applied in the field of bioengineering, can solve problems such as difficult filtration and recovery, easy aggregation into blocks, and obstacles to the industrial application of CLEAs

Inactive Publication Date: 2020-04-07
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CLEAs are soft, poor in mechanical properties, easy to aggregate into agglomerates, and difficult to filter and recover. These defects hinder the industrial application of CLEAs.

Method used

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  • Magnetic combined cross-linked enzyme aggregate biocatalyst and preparation method and application thereof
  • Magnetic combined cross-linked enzyme aggregate biocatalyst and preparation method and application thereof
  • Magnetic combined cross-linked enzyme aggregate biocatalyst and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Enzyme activity assay

[0055] QNR enzyme activity assay:

[0056] Standard reaction mixture system: buffer solution B (PBS, pH7.2-7.4), 3 μmol 3-quininone, 0.3 μmol NADH, appropriate amount of enzyme QNR, total volume 1 mL. Changes in absorbance values ​​were measured at λ = 340 nm. Definition of enzyme activity unit: the amount of enzyme required to convert 1 μmol NADH within 1 min at 25°C.

[0057] GDH enzyme activity assay:

[0058]Standard reaction mixture system: buffer solution B (PBS, pH7.2-7.4), 10 μmol glucose, 1 μmol NAD + , an appropriate amount of enzyme GDH, in a total volume of 1 mL. Changes in absorbance values ​​were measured at λ = 340 nm. Enzyme activity unit definition: 1 μmol NAD converted within 1 min at 25°C + The amount of enzyme required.

Embodiment 2

[0060] Magnetic Fe 3 o 4 Preparation of nanoparticles:

[0061] FeCl with a mass ratio of 2:1 3 ·6H 2 O (10.8116g) and FeCl 2 4H 2 O (3.9762g) was dissolved in 100-200mL deionized water, under nitrogen protection, and mechanically stirred at a speed of 500-1500r / min for 0.5-1.0 hours. Add 10-30mL NaOH solution with a concentration of 8-12M dropwise, and mechanically stir at a speed of 1000-2000r / min for 1-1.5 hours. Raise the temperature to 70-100°C, mature for 0.5-2.0 hours; cool down to room temperature, magnetically separate and wash with deionized water for 3-5 times, dry to obtain magnetic Fe 3 o 4 nanoparticles;

[0062] Magnetic Fe 3 o 4 Amino-functionalization of nanoparticles:

[0063] Magnetic Fe 3 o 4 100-200 mg of nanoparticles, dispersed in 140-280 mL of ethanol / water solution with a volume ratio of 5:2, under nitrogen protection, stirred at 20-80 ° C, 400-1000 r / min for 1.0-3.0 hours. Add 2-4mL concentrated ammonia water, stir for 0.5-1.5 hours, add...

Embodiment 3

[0065] Joint cross-linking immobilization of QNR and GDH:

[0066] Get the amino-functionalized magnetic Fe prepared in Example 2 3 o 4 Nanoparticles 5mg, dispersed in 1.0mL phosphate buffered saline solution (PBS, 10mM, pH7.2-7.5) containing QNR (4.0mg / mL) and GDH (2.0mg / mL), at 4°C, speed 600r / min Stir for 0.5 hours. Add 9 volumes of ice-cold precipitant (saturated ammonium sulfate), and stir for 1.5 hours. Add 1.0 mL of glutaraldehyde with a concentration of 40 mM, and stir at 400 r / min for 8.0 h. Magnetic separation, washed 3 times with PBS, to obtain magnetic combi-CLEAs.

[0067] The magnetic combi-CLEAs prepared in embodiment 3 is carried out scanning electron microscope analysis

[0068] Drop the sample solution onto a clean cover glass, dry it in vacuum at 40°C, spray gold to cover the sample, and image it with a scanning electron microscope (SEM, S-3000N type). The analysis results are shown in the attached figure 1 . attached figure 1 It is combi-CLEAs micro...

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Abstract

The invention provides a magnetic combined crosslinked enzyme aggregate biocatalyst. The biocatalyst can produce diffraction peaks at diffraction angles 2 theta of 30.5+ / -0.2 degrees, 35.4+ / -0.2 degrees, 44.7+ / -0.2 degrees, 57.3+ / -0.2 degrees and 63.4+ / -0.2 degrees. A preparation method of the biocatalyst is simple, does not need special equipment, needs mild process conditions, is easy to operate, realizes a low cost and is suitable for industrialization. The biocatalyst is used for asymmetric synthesis of (R)-3-quinuclidinol, realizes synchronous carbonyl reduction and coenzyme NADH / NAD<+> in-situ regeneration, prevents diffusional limitation to a substrate , a coenzyme and a product, has good catalytic activity and high catalytic efficiency, greatly shortens reaction time and has obvious economic values.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a biocatalyst of a magnetic joint cross-linked enzyme aggregate, a preparation method and application thereof. Background technique [0002] (R)-3-quinine alcohol (molecular formula C7H13NO, molecular weight 127.18, CAS number: 25333-42-0) is a key chiral intermediate for the synthesis of drugs such as aclidinium bromide, solifenacin and ravatorate body. At present, the industry mainly uses chiral catalysts, such as: XylSkewphos / PICA-Ruthenium(II) complex or BINAP / IPHAN-Ru(II) complex, etc., to asymmetrically reduce 3-quinine to synthesize (R)-3- quinine alcohol, but this chemical synthesis method needs to screen chiral ligands; the transition metals used are expensive, highly toxic, and difficult to remove from the product; and the prepared product has low optical purity and needs further purification. Another method is racemic resolution, which has the disa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N9/04C12P17/18
CPCC12N9/0006C12N9/0008C12P17/182C12Y101/9901
Inventor 孙立力刘笃强李伟陈倩李晶
Owner CHONGQING MEDICAL UNIVERSITY
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