A method for separating and recovering chloroperoxidase

A chloroperoxidase, separation and recovery technology, applied in the biological field, can solve the problems of complex composition of fermentation broth, low chromatographic purification products, large content of cell metabolites, etc., shorten the purification cycle, improve extraction interference, and volume requirements small effect

Inactive Publication Date: 2011-12-14
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] Usually, in order to obtain high-purity CPO, it is often necessary to comprehensively utilize the physical and chemical properties of the target product, and use a combination of multiple separation techniques. In the past CPO purification process, due to the extremely low concentration of the target protein, the composition of the fermentation broth is complex, and the content of cell metabolites is large. The impurity removal, extraction and purification procedures are complicated and there are many links, and the yield of the final chromatographic purification product is generally lower than 6%.

Method used

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  • A method for separating and recovering chloroperoxidase
  • A method for separating and recovering chloroperoxidase
  • A method for separating and recovering chloroperoxidase

Examples

Experimental program
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Effect test

Embodiment 1

[0023] (1) Preparation of CPO fermentation broth

[0024] Press the Caldariomyces fumago lawn cultivated on the PDA plate for 10 days by 2 cm 2 The inoculum of bacterial lawn / 250mL Erlenmeyer flask was inserted into the fermentation medium (including g / L: maltose 40, NaNO 3 2, KCl2, KH 2 PO 4 2. MgSO 4 ·7H 2 O 0.5, FeSO 4 ·7H 2 O 0.02, CaCl 2 0.09, plus 20% potato extract by volume, the initial pH is 7.0), the liquid volume in the shaker flask is 50ml / 250mL, the fermentation temperature is 25°C, the shaker speed is 240r / min, and the fermentation is 6d.

[0025] (2) Pretreatment of CPO fermentation broth

[0026] Centrifuge the fermented liquid at 4000g and 10°C for 10 minutes, or use vacuum filtration to remove mycelium and other solids, collect the supernatant containing melanin, and slowly add the supernatant to an amount equal to 6% of the supernatant in an ice bath. % PEG400 with constant stirring and mixing, let stand for 4 hours until the flocculation of PEG ...

Embodiment 2

[0032] The process is the same as in Example 1, except that step (2) uses PEG800.

[0033] The results show that the total enzyme activity of the sediment is 60% of the total enzyme activity of the stock solution, the total enzyme activity of the residual solution is 36% of the total enzyme activity of the stock solution, and the total enzyme activity loss is 4%. It shows that the recovery effect of CPO using PEG800 is still unsatisfactory, but it is significantly improved compared with using PEG400.

Embodiment 3

[0035] The process is the same as in Example 1, except that step (2) uses PEG4000.

[0036] The results show that the total enzyme activity of the sediment is 78% of the total enzyme activity of the stock solution, the total enzyme activity of the residual solution is 18% of the total enzyme activity of the stock solution, and the total enzyme activity loss is 4%. It shows that the use of PEG4000 has a better recovery effect on CPO, but the loss of total enzyme activity is greater.

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Abstract

The invention discloses a method for separating and recovering chloroperoxidase (Chloroperoxidase, EC 1.11.1.10, CPO), which utilizes co-precipitation and redissolution method, that is, hydrophilic polymer polyethylene glycol is precipitated and entrained at high salt concentration CPO molecules, so as to enrich the CPO protein concentration, and then use the two-phase extraction technology to efficiently separate and recover high-purity CPO, that is, to separate different proteins by using the different distribution coefficients of the separation target product and impurities in the hydrophilic polymer phase solution. The present invention proposes reasonable and feasible two-phase CPO extraction and purification conditions through research, summarizes the separation technology of micro-content bioactive substances, solves or improves the extraction interference, low recovery rate, high product price, and purification cycle existing in the current CPO production process. Long, can not scale production and other issues.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for separating and recovering chloroperoxidase. Background technique [0002] Chloroperoxidase (Chloroperoxidase, EC 1.11.1.10, CPO) molecule is a heme glycoprotein, its prosthetic group is ferric (IX) protoporphyrin, many of its spectroscopic and chemical properties are related to cytochrome P- 450 are highly similar, and peroxidase itself has a binding site similar to catalase. Therefore, CPO has a wide range of substrate adaptability and can catalyze a variety of substances for peroxidation reactions, such as: can catalyze halide ions , Aromatic compounds, aliphatic compounds and alcohol compounds, etc. undergo peroxidation reactions. At present, CPO has been applied to the synthesis of various chiral compounds and pharmaceutical production to reduce the loss of chiral isomers in the reaction process, remove the toxicity of chiral enantiomers of drugs, and reduce the pollu...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12R1/645
Inventor 陈军许甜甜张书翠诸葛俊贵
Owner SHANGHAI NORMAL UNIVERSITY
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