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Immobilization L-asparaginase reactor

A technology of asparaginase and reactor, which is applied in the direction of enzyme production/bioreactor, biochemical instrument, method of supporting/immobilizing microorganisms, etc. It can solve the problems of poor storage stability, small specific surface area of ​​carrier, and enzyme inactivation, etc. Achieve good biocompatibility, improve the utilization rate and efficiency of enzymes, and simplify the preparation process

Inactive Publication Date: 2012-12-12
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the shortcomings of traditional immobilized enzyme carriers such as small specific surface area, easy to cause enzyme inactivation, and poor storage stability, the present invention provides a novel nano-gold immobilized L-asparaginase reactor and its preparation method

Method used

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  • Immobilization L-asparaginase reactor

Examples

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preparation example Construction

[0029] The preparation method of nano-gold used in the examples is as follows: After boiling chloroauric acid (1.0mM, 10.0mL), quickly add 1.0mL of newly prepared trisodium citrate (38.8mM) solution, and heat while stirring rapidly , the solution turned dark green, and then turned into wine red, and then continued to stir for 10 minutes, and the obtained nano-gold solution was diluted 3 times with triple distilled water for later use.

[0030] The prepared nano-gold has an average particle size of 20nm and a maximum absorption wavelength of 522nm.

[0031] Mix the nano-gold solution and L-asparaginase solution at a ratio of 1:1 (v / v), stir at room temperature for 1 hour, and then place it in a refrigerator at 4°C for 24 hours to obtain L-asparaginase-nano Gold Aggregate Solution.

[0032] Clean capillary tubes (5-50cm) of different lengths: wash with 1.0M sodium hydroxide solution for 2 hours, wash with water for 10 minutes, wash with 1.0M hydrochloric acid solution and water...

Embodiment 1

[0036] Embodiment 1, preparation nano-gold immobilized L-asparaginase reactor

[0037] 1) Mix the nano-gold solution and L-asparaginase solution (concentration 225.0 U / mL) at a ratio of 1:1 (v / v), stir at room temperature for 1 hour, and then place it in a refrigerator at 4°C for 24 hours to obtain L - Asparaginase-gold nano-aggregate solution.

[0038]2) Rinse the 30cm capillary with 1.0M sodium hydroxide solution for 2 hours, water for 10 minutes, 1.0M hydrochloric acid solution and water for 10 minutes, organic solvent acetone for 5 minutes and blow dry with nitrogen. Mix (3-mercaptopropyl)-trimethoxysilane and methanol (volume ratio 1:4), and then the solution flows through the capillary column under negative pressure. After standing at room temperature for 12 hours, it is washed repeatedly with methanol and water to obtain the inner wall modified mercapto group. Capillary column of silane coupling agent, spare.

[0039] 3) The gold nanometer solution-L-asparaginase solu...

Embodiment 2

[0043] Embodiment 2, preparation nano-gold immobilized L-asparaginase reactor

[0044] 1) Mix the nano-gold solution and L-asparaginase solution (4.5U / mL) at a ratio of 1:1 (v / v), stir at room temperature for 1 hour, and then place it in a refrigerator at 4°C for 24 hours to obtain L- Asparaginase-gold nano-aggregate solution.

[0045] 2) Wash the 5cm capillary with 1.0M sodium hydroxide solution for 2 hours, rinse with water for 10 minutes, wash with 1.0M hydrochloric acid solution and water for 10 minutes in sequence, rinse with organic solvent acetone for 5 minutes, and then blow dry with nitrogen. Mix (3-mercaptopropyl)-trimethoxysilane and methanol (volume ratio 1:4), and then the solution flows through the capillary column under negative pressure and fully contacts with its inner wall. After standing at room temperature for 12 hours, rinse with methanol and water repeatedly , that is, a capillary column with an inner wall modified mercaptosilane coupling agent is obtain...

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Abstract

The invention discloses a novel nanometer immobilization L-asparaginase reactor and a preparation method thereof. The enzyme reactor is prepared through a method including the following steps that nanometer gold prepared through a sodium citrate method is used as an enzyme immobilization material, characteristics of large specific surface area and good biocompatibility of the nanometer gold are used, enzyme is absorbed to the surface of the nanometer gold, and aggregation of enzyme-nanometer gold is prepared; and sulfydryl silane coupling agent is decorated on an inner wall of a capillary pipe, effects of sulfydryl and the nanometer gold are used, the aggregation of enzyme-nanometer gold is immobilized on an inner wall of the capillary pipe, and immobilization of L-asparaginase is achieved. The method combines the characteristics of large specific surface area of the nanometer gold, and enzymolysis efficiency of L-asparaginase on L-glutamine is effectively improved. The immobilization enzyme reactor is simple in preparation process, improves use rate and efficiency of enzyme, is favorable for developing a novel dosing method of the L-asparaginase, and provides a research basis for diagnosis and treatment of diseases.

Description

technical field [0001] The invention relates to an immobilized L-asparaginase reactor. Background technique [0002] L-asparaginase is a drug extracted from Escherichia coli, used in the treatment of acute lymphoblastic leukemia (Krejci O, Starkova J, Otova B, Madzo J, Kalinova M, Hrusak O, Trka J (2004) Leukemia 18:434-441). Since 1967, L-asparaginase has been used as an important component drug in the treatment of childhood acute lymphoblastic leukemia. This enzyme can hydrolyze L-asparagine in serum, leading to apoptosis and impairing protein synthesis. However, this enzyme has serious side effects as a drug, including nephrotoxicity, allergic reaction, pancreatitis and central nervous system toxicity, etc., and it will reduce the synthesis of coagulation factors. In order to avoid these side effects, L-asparaginase can be chemically modified, embedded or immobilized on a carrier. Among them, immobilizing enzymes on certain materials and establishing an extracorporeal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/40C12N11/14
CPCC12M25/16C12M21/18
Inventor 齐莉乔娟李雅萍木肖玉
Owner INST OF CHEM CHINESE ACAD OF SCI
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