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A method for extracting and purifying haloacid dehalogenase

A technology of haloacid dehalogenase and purification method, which is applied in the field of designing haloacid dehalogenase, can solve problems such as differences in enzyme properties and activities, and achieve the effect of eliminating hazards

Active Publication Date: 2016-04-13
中科榆林能源技术运营有限责任公司
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Problems solved by technology

The ocean is rich in halogen ions, and the halogen organic compounds produced by marine organisms living in a high-halogen concentration environment are also very high. However, factors such as bacterial species, climate, and sea environment will cause differences in the properties and activities of enzymes

Method used

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  • A method for extracting and purifying haloacid dehalogenase
  • A method for extracting and purifying haloacid dehalogenase
  • A method for extracting and purifying haloacid dehalogenase

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Embodiment 1

[0039] HypersilGOLDC18 column (5μm, 250mm×4.6mm, Thermo); mobile phase, acetonitrile-water (15:85, pH2.2), containing 0.1% (v / v) concentrated H in water 3 PO 4 (85%, w / v); flow rate, 1mL / min; column temperature, 30°C; detection wavelength, 210nm. The separation and purification of embodiment 1 haloacid dehalogenase

[0040] 16.9g of frozen Pseudomonas stutzeri thalline was mixed with 341mL buffer A (50mMK 2 HPO 4 -K 2 HPO 4 +0.65mMDTT, pH 7.5) resuspended, and after dispensing about 25mL in each tube, the cells were disrupted by ultrasonic (350w, ultrasonic 5s, interval 5s, 60 times as a cycle. A total of 2 cycles). After centrifugation (12000rpm×30min, 4°C), take the supernatant for use. Add solid ammonium sulfate to the supernatant to make 40% ammonium sulfate saturation, stir for 1 hour, and centrifuge (12000rpm×30min, 4°C) to obtain precipitate A. Add ammonium sulfate to the supernatant obtained after centrifugation until the saturation reaches 80%, stir for 1 hour,...

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Abstract

The invention discloses a separation and purification method of dehalogenase with stereoselectivity from a strain of marine bacteria and an application of the dehalogenase in biological resolution. According to the method, two types of 2-haloacid dehalogenase with stereoselectivity, namely L-2-haloacid dehalogenase and D-2-haloacid dehalogenase, are obtained from the strain of the marine bacteria, namely pseudomonas stutzeri by separation and purification. The apparent molecular weight of the enzyme of an enzyme subunit of the L-2-haloacid dehalogenase is about 26kDa, and the L-2-haloacid dehalogenase further has great reaction activity in the pH range of 8-11. D-hydroxy halogenated acid with configuration conversion is generated by selectively catalyzing the conversion of chiral L-2-haloacid through the enzyme. The D-2-haloacid dehalogenase can catalyze the conversion of D-2-haloacid and generate L-2-hydroxy acid, and further has good application prospects as a chiral resolution reagent.

Description

technical field [0001] The invention designs a haloacid dehalogenase, specifically a method for separating and purifying the haloacid dehalogenase in Pseudomonasstutzeri and its application. technical background [0002] Halogenated organic compounds are important raw materials and intermediates in fine chemicals and medicines. Dehalogenases are a class of enzymes that can release halogens from halogenated compounds. It not only plays an important role in the degradation of environmental organic halides, but also has good application prospects in chiral resolution and preparation of chiral compounds due to its stereoselective catalytic activity. [0003] At present, most of the bacteria that have been found to produce dehalogenase come from the terrestrial environment, and there are very few microorganisms from the marine environment. The ocean is rich in halogen ions, and marine organisms living in a high-halogen concentration environment produce very high halogen organic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12P7/42C12R1/38
CPCC12N9/14C12Y308/0101
Inventor 张锦友薛松曹旭鹏
Owner 中科榆林能源技术运营有限责任公司
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