Method for purifying polygalacturonase by galacturonic acid magnetic nanoparticles

A technology of magnetic nanoparticles and galacturonic acid, which is applied in the direction of glycosylase, biochemical equipment and methods, enzymes, etc., can solve the problems that have not been reported, and achieve cheap methods, low juice viscosity, and loss of enzyme activity small effect

Active Publication Date: 2021-04-30
SHANGHAI JIAO TONG UNIV +4
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] There is no relevant report on the method of affinity purification of exo-PG by magnetic nanoparticles

Method used

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  • Method for purifying polygalacturonase by galacturonic acid magnetic nanoparticles
  • Method for purifying polygalacturonase by galacturonic acid magnetic nanoparticles
  • Method for purifying polygalacturonase by galacturonic acid magnetic nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the optimization of Aspergillus flavus growth

[0036] Aspergillus flavus was obtained from the Applied Molecular Biotechnology Research Laboratory (AMBR), Punjab University, Lahore, Pakistan, on glucose-free pectin agar medium (1% NaNO 3 , 0.5% MgSO 4 .7H 2 O, 0.1% CaCl 2 , 1.0μM FeCl 3 , pH 7.2 and 2.0% agar) to keep the slope. Use about 2ml (1×10 10 / ml spores) of Aspergillus flavus liquid culture was inoculated with 50ml pectin medium in a 250ml flask. Pure apple pectin (1.0%) was added to different flasks containing agar-free medium. The inoculated culture was placed in an orbital shaker at 160 rpm at 30°C for 5 days. Every day, remove 1ml of medium and check exo-PG activity.

[0037]exo-PG activity was measured by the dinitrosalicylic acid (DNS) method. An assay mix containing 0.5 ml of 0.1% polygalacturonic acid (w / v) and 0.1 ml of culture supernatant (crude enzyme) prepared in 50 mM sodium acetate buffer (pH 5) was incubated at 30 °C for 1...

Embodiment 2

[0038] Example 2, the combination of magnetic nanoparticles (MNP) and galacturonic acid (Glt)

[0039] Suspend the magnetic nanoparticles (MNP) in double distilled water, and sonicate for 7-10 minutes, the particles that settle due to gravity are separated, and the remaining fine particles are suspended, and the particles are used to combine with galacturonic acid (0.4g), Dissolve in 120ml of distilled water and mix with 0.3g of MNP. The mixture was sonicated on ice for 4-5 min. Adjust with 1.M NaOH, pH 8, and incubate at 37°C for 15 minutes. 0.4 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) was added to the solution and adjusted with 1 M HCl, pH 6.4. The mixture was kept at 25°C for 6 hours with constant shaking. Finally 0.4 g of EDAC was added again and kept at 25°C for 20 hours with constant shaking. Bound nanoparticles were separated by applying an external magnetic field and washed five times with distilled water.

[0040] Before protein purification, mag...

Embodiment 3

[0045] Embodiment 3, the purification of exo-PG

[0046] The activity of exo-PG containing apple pectin (1%) was observed in glucose-free medium, which can be used for large-scale production of exo-PG. Inoculate 2ml of Aspergillus flavus dilution culture in about 250ml culture medium (10 10 / ml spores) and incubated in an orbital shaker at 30°C, 160rpm for 5 days. The medium was removed and filtered through sterile Whatman filter paper, and the clarified filtrate was used for purification of exo-PG.

[0047] exo-PG was purified directly from the culture filtrate. All purification steps were completed in an ice bath at 4 °C, about 1.0 g of Glt-MNP was mixed with 10 ml of culture filtrate, and incubated at 30 °C for 60 min with constant shaking. Glt-MNPs were removed from the solution by applying an external magnetic field and washed twice with PG buffer (10 mM Tris-Cl, pH 7.3). exo-PG bound to Glt-MNP was removed by mixing with 5 ml of elution buffer (0.3M NaCl in 10 mM Tri...

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Abstract

The invention relates to the technical field of food and enzyme engineering, in particular to a method for purifying polygalacturonase by galacturonic acid magnetic nanoparticles, which uses a GltMNP nano composite material to purify exo-PG from fermentation culture of apple pectin, and verifies successful combination of galacturonic acid (Glt) and magnetic nanoparticles (MNP) through FTIR research. Although many methods can purify exo-PG, the present invention can enhance the stability and homogeneity of the enzyme as compared to other methods that are inexpensive, efficient, reliable and purified with minimal enzymatic activity loss, the purified exo-PG emerging in a single band of 66 kDa. The purified exo-PG is used for fruit juice treatment of apples, peaches and guavas, the fiber texture of pulp can be further reduced, the viscosity of the juice is lower, and the purified exo-PG can be used for industrial-scale food processing.

Description

technical field [0001] The invention relates to the fields of food and enzyme engineering, in particular to a method for purifying polygalacturonase by galacturonic acid magnetic nanoparticles. Background technique [0002] Microbial enzymes involved in food processing have attracted researchers to study them due to their application in the agri-food industry. Cellulose, starch, lignin, xylan and pectin are important components of agricultural fruits and vegetables, among which, pectin is a very important agricultural material, and pectinase hydrolyzes pectin, accounting for 25% of the global food processing enzyme sales %, Clarity and purity of food products is a big issue in the food industry due to less degradation of the polymer matrix of plant materials. Pectinases are naturally produced by microorganisms and fungi in plants. Among them, fungi are considered to be the best sources for the commercial production of pectinases. [0003] Polygalacturonase (PG) is a depol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N13/00H01F41/00
CPCC12N9/2402C12N13/00H01F41/00C12Y302/01015
Inventor 魏冬青马迪哈·沙赫扎德·洛迪阿伊莎·沙欣穆罕默德·塔希尔·汗伊姆蒂亚兹·沙菲克扎胡尔萨姆拉王恒王艳菁
Owner SHANGHAI JIAO TONG UNIV
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