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Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof

A technique for Yersinia, purification methods, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc.

Inactive Publication Date: 2009-07-22
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In the prior art, there is no low-temperature bacterial strain Yersinia sp. (Yersinia sp.) KM1, and the low-temperature alkaline lipase resistant to organic solvent obtained by purifying the bacterial strain as a raw material, and the report of its separation and purification method

Method used

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  • Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof
  • Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof
  • Yersinia strain KM1, low temperature alkaline lipase prepared thereby and purification method thereof

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Embodiment 1

[0033] Isolation, screening and identification of Yersinia enterocolitica KM1 strain:

[0034] Yersinia enterocolitica (Yersinia enterocolitica) KM1 strain was isolated from the cold storage of Kunming East Railway Station slaughterhouse. Strain selection medium is (g / L): tryptone, 10; yeast extract, 5; NaCl, 10; olive oil emulsion, 2%; agar, 20; pH7.0-7.2.

[0035] Yersinia sp. KM1 strain was identified by bacterial system identification method. The morphological characteristics of the bacteria are as follows: the colonies are round, with neat edges, moist, milky white, sticky, easy to pick; the cells are short rod-shaped, Gram-negative bacteria. The pH range for enzyme production is 4.0-9.5, the optimum pH is 7.2; the temperature range for enzyme production is 4-37°C, the optimum temperature for enzyme production is 13°C; the time range for enzyme production is 18-66 hours, the optimum enzyme production The time is 54 hours. The physiological and biochemical characteristi...

Embodiment 2

[0039] Purification of cryogenic lipase

[0040] (1) Preparation of crude enzyme solution: the fermentation and culture conditions of Yersinia sp. KM1 strain were temperature 13° C., pH 7.2, and culture time 52 hours. The fermentation broth is centrifuged, and the supernatant is obtained as the crude enzyme solution.

[0041] (2) Ammonium sulfate precipitation: Add ammonium sulfate to the crude enzyme solution to 30-40% saturation, centrifuge to take the supernatant, continue to add ammonium sulfate to 70-80% saturation, centrifuge to discard the supernatant, precipitate at 25mM, pH7.0 -8.0 phosphate buffered saline overnight.

[0042] (3)Sephacry TM HRS-100 chromatography: Centrifuge the dialyzed enzyme solution, concentrate the supernatant, and load the Sephacry pre-equilibrated with 25mM, pH 7.0-8.0 phosphate buffer TMHRS-100 chromatographic column, the flow rate is 0.4ml / min, elution, the enzyme active part is collected, and the concentrated solution is collected by ce...

Embodiment 3

[0048] Characteristics of low temperature lipase:

[0049] ①Optimum enzyme activity temperature

[0050] Add the enzyme liquid to phosphate buffer (25mM phosphate buffer, pH7.2), use pNPB as substrate, and measure the enzyme activity of lipase produced by KM1 strain at different temperatures in the range of 0-70°C. The result is as figure 1 As shown, it shows that the optimum enzyme activity temperature of the low-temperature lipase is 37°C, and there is still nearly 20% activity at 0°C. When the temperature exceeds 40°C, the enzyme activity drops sharply, and the residual enzyme activity is less than 10% when the temperature reaches 70°C. %, this result shows that the lipase has the typical characteristics of low-temperature enzymes, that is, the reaction temperature of the enzyme is low.

[0051] ②Thermal stability

[0052] Add the enzyme solution to phosphate buffer (25mM phosphate buffer, pH 7.2), keep it warm in constant temperature water baths at different temperature...

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Abstract

The invention discloses a psychrotrophic bacteria strain Yersinia sp. KM1, a psychrotrophic alkaline lipase that is generated by the strain and resistant to organic solvents, and a separating and purifying method of the psychrotrophic alkaline lipase. The purifying method comprises the steps of ammonium sulfate precipitation of a fermented supernatant, centrifugal concentration with a 10KDa ultrafiltration pipe, Sephacry HRS-100 chromatography and Superdex G-75 chromatography, and finally obtains pure psychrotrophic alkaline lipase under electrophoresis. The purified psychrotrophic alkaline lipase has low reaction temperature, wider pH accommodation range and high resistance to organic solvents, and consequently has huge application potential in the fields of cleaning solvents, food processing, medicine production and biodiesel production, etc.

Description

Technical field: [0001] The invention belongs to the technical field of microbes, and in particular relates to a low-temperature bacterial strain, i.e. low-temperature bacterial strain Yersinia sp. Sexual lipase, and its separation and purification method. Background technique: [0002] Lipase (Lipase, EC 3.1.1.3) is a class of enzymes that catalyze the degradation of natural oils (triglycerides) into glycerol and free fatty acids at the oil-water interface. Low-temperature lipase is generally secreted by low-temperature microorganisms. The optimum reaction temperature of the enzyme is generally lower than 40°C, and it still has high enzyme activity at 0°C. Compared with high-temperature and medium-temperature lipase, low-temperature lipase has a wider pH adaptation range, which further broadens its industrial application range. [0003] Low temperature lipase has attracted extensive attention of scientists from various countries due to its good catalytic activity and low ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/20C12R1/01
Inventor 季秀玲陈贵元魏云林林连兵井申荣
Owner KUNMING UNIV OF SCI & TECH
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