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Sarcosine oxidase affinity medium and method for synthesizing and purifying sarcosine oxidase

A sarcosine oxidase pro- and sarcosine oxidase technology, which is applied in the fields of bioengineering and diagnostic enzyme production, can solve the problems of low protein and activity recovery rate, long purification time, etc., and achieves increased purification efficiency and great economy. Benefit, effect of large industrial application potential

Active Publication Date: 2013-04-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Too many purification steps lead to longer purification time and lower recovery of protein and activity

Method used

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  • Sarcosine oxidase affinity medium and method for synthesizing and purifying sarcosine oxidase
  • Sarcosine oxidase affinity medium and method for synthesizing and purifying sarcosine oxidase
  • Sarcosine oxidase affinity medium and method for synthesizing and purifying sarcosine oxidase

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Embodiment 1

[0037] Embodiment 1: Synthesis and application of Sepharose-4-aminopyrrole-2-carboxylic acid

[0038]Sepharose CL 4 B (100 g) was washed with 10 times the volume of deionized water and dried into a wet cake; suspended in 50 mL activation buffer (0.8 M NaOH, 20% dimethyl sulfoxide, 10% epichlorohydrin ) Shake at 40°C for 2.5 h, then pour it into a glass frosted funnel, wash with 10 times the volume of distilled water each time under suction filtration until the pH of the washing solution reaches neutral, and then drain to form a wet cake. The activated Sepharose CL 4 B medium was suspended in 100 mL of deionized water, ethylenediamine was added at a molar ratio of 1:2, and the gel was kept at 60 °C for 12 h under stirring (200 rpm). Rinse with deionized water. NH 2 -Sepharose CL 4 B was resuspended in 50mL buffer system (0.8 M NaOH, 20% dimethyl sulfoxide, 10% epichlorohydrin) at 40°C for 2.5 h on a shaker, then poured into a glass frosted funnel, Wash with 10 times the volu...

Embodiment 2

[0050] Embodiment 2: Synthesis and application of polychitosan-4-aminopyrrole-2-carboxylic acid

[0051] Polychitosan (100 g) was washed with 10 times the volume of deionized water and dried into a wet cake; suspended in 50 mL activation buffer (0.8 M NaOH, 20% dimethyl sulfoxide, 10% epichlorohydrin ) Shake at 40°C for 2.5 h, then pour into a glass frosted funnel, wash with 10 times the volume of distilled water each time under suction filtration, until the pH of the washing solution is neutral, and then drain to form a wet cake. The activated polychitosan medium was suspended in 500 mL of 0.1M NaOH solution, 20 mL of ethylenediamine solution was added, and the gel was kept at 30 °C for 12 h under stirring (200 rpm). Rinse and dry with deionized water to obtain amino-polychitosan. Amino-polychitosan was suspended in 50 ml buffer system (0.8 M NaOH, 20% dimethyl sulfoxide, 10% epichlorohydrin) and shaken at 40 °C for 2.5 h, then poured into a glass frosted funnel, After wash...

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Abstract

The invention discloses a sarcosine oxidase affinity medium and a method for synthesizing and purifying sarcosine oxidase, which belong to the technical field of biologic engineering. The sarcosine oxidase affinity medium disclosed by the invention is prepared by connecting a sarcosine oxidase affinity ligand with a chromatography medium; preferably, the sarcosine oxidase affinity ligand is a substrate analog 4-amino pyrrole-2-carboxylic acid, and the chromatography medium is Sepharose or poly-chitosan; the invention further provides a synthesis method of the sarcosine oxidase affinity medium, and a method for purifying the sarcosine oxidase in a large scale by using the sarcosine oxidase affinity medium. By utilizing the sarcosine oxidase affinity medium disclosed by the invention, the sarcosine oxidase can be rapidly separated and purified in a large scale, and a sarcosine oxidase pure product can be obtained under the condition of one step affinity chromatography; and the whole purification step is short in time, the protein and activity recovering rate is high, and thus being applicable to large-scale popularization and application.

Description

technical field [0001] A sarcosine oxidase affinity medium and a method for synthesizing and large-scale purification of sarcosine oxidase belong to the technical field of bioengineering, in particular to the technical field of diagnostic enzyme production. Background technique [0002] Sarcosine oxidase (Sarcosine oxidase, E.C.1.5.3.1), referred to as SOX, is the third enzyme in the creatinine degradation metabolic pathway, which can specifically catalyze the substrate sarcosine to generate glycine. Microbes are the most important source of creatine oxidase, which is widely used in clinical diagnosis. Determination of creatinine content in serum by sarcosine oxidase coupled with creatinase and creatinase is an important indicator for judging kidney function. It is a reference index and has a good application prospect in the field of medicine. [0003] Bacillus (Bacillus), Streptomyces sp. (Streptomyces sp.), Corynebacterium (Corynebacterium), Arthrobacter (Arthroba...

Claims

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Application Information

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IPC IPC(8): B01J20/26B01D15/38C12N9/06C08B37/08
Inventor 杨海麟辛瑜仝艳军王武张玲
Owner JIANGNAN UNIV
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