Medium-temperature bacterium for producing high-salinity tolerance ester hydrolase and application of medium-temperature bacterium

A hydrolytic enzyme and high-salinity technology, applied in the field of microorganisms, can solve the problems of restricting practical applications, difficulty in meeting practical application requirements, and inhibition, and achieve the effects of low cost, strong thermal stability, and high catalytic activity

Active Publication Date: 2021-05-11
SECOND INST OF OCEANOGRAPHY MNR +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, in some application scenarios with harsh hydrolysis conditions, such as high salinity, the enzymatic activity of most lipolytic enzymes in the prior art will be severely inhibited in these hydrolysis environments, thus limiting their practical application and difficult to meet the reality Application requirements

Method used

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  • Medium-temperature bacterium for producing high-salinity tolerance ester hydrolase and application of medium-temperature bacterium
  • Medium-temperature bacterium for producing high-salinity tolerance ester hydrolase and application of medium-temperature bacterium
  • Medium-temperature bacterium for producing high-salinity tolerance ester hydrolase and application of medium-temperature bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1: Activity detection of ester hydrolase Ala2 produced by mesophilic bacteria SIOC 00011

[0047] The activity of the purified ester hydrolase was determined by the p-nitrophenol hexanoate method. Specific operation: 1 ml reaction system includes 1 mM p-nitrophenol hexanoate, 100 mM NaH 2 PO 4 -Na 2 HPO 4 Buffer solution (pH=8.0) and 0.0016 μg of pure enzyme protein, using a UV-visible spectrophotometer (Beckman DU800, USA) to continuously measure the absorbance value A at 45 °C 405 2 min, use the inactivated enzyme solution as a control for zero adjustment. One unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 µmol p-nitrophenol from p-nitrophenol esters per minute. The measured esterase activity was 14586 U / mg.

Embodiment 2

[0048] Embodiment 2: Ester hydrolase Ala2 substrate specificity analysis

[0049] The substrate specificity analysis of hydrolase Ala2 uses the system (1 ml): 100 mM NaH 2 PO 4 -Na 2 HPO 4 Buffer (pH=8.0), 1 mM substrate, add 0.0016 μg pure enzyme protein, and measure the absorbance value A continuously at 45°C 405 2 min. The substrates used in the determination are: p-nitrophenol acetate (C2), p-nitrophenol butyrate (C4), p-nitrophenol hexanoate (C6), p-nitrophenol octanoate (C8) , p-nitrophenol caprate (C10), p-nitrophenol dodecanoate (C12), p-nitrophenol myristate (C14), p-nitrophenol palmitate (C16). It has been determined that Ala2 has higher catalytic activity for p-nitrophenol esters with shorter acyl carbon chains (C2, C4, C6 and C8), and the highest catalytic activity is when the substrate is p-nitrophenol hexanoate (C6). ( figure 1 ). The results showed that ester hydrolase Ala2 had better catalytic activity on esters with shorter acyl carbon chains, and it...

Embodiment 3

[0050] Embodiment 3: analysis of optimal reaction conditions of ester hydrolase Ala2

[0051] The optimal reaction pH of hydrolase Ala2 was determined in the range of 4.0 to 10.5. The specific operation is: add 1 mM p-nitrophenol hexanoate and 0.0016 μg pure enzyme protein to different pH buffers, and continuously measure the absorbance value A at 45°C 348 2 min. The buffer used for the determination is: 100 mM citric acid-sodium citrate buffer (pH 3.0~6.0), 100 mM potassium dihydrogen phosphate-sodium hydroxide buffer (pH 6.0~8.0), 100 mM Tris hydrochloric acid buffer ( pH 7.5~9.0) and 50 mM 2-cyclohexylaminoethanesulfonic acid-sodium hydroxide buffer (pH 9.0~10.5). The measurement results showed that the optimal reaction pH of Ala2 was 6.0, and it was active in the range of pH 5.0-10.5 ( figure 2 ).

[0052] The optimal reaction temperature of hydrolase Ala2 was determined in the range of 25-55 degrees Celsius. The specific operation is: in 1 ml reaction system, add 1...

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Abstract

The invention relates to the field of microorganisms, and discloses a medium-temperature bacterium for producing high-salinity tolerance ester hydrolase and application of the medium-temperature bacterium. According to the invention, a medium-temperature bacterium SIOC 00011 is separated from deep seawater of Western Pacific Ocean, and ester hydrolase produced by the strain reaches the maximum enzyme activity at 45 DEG C, keeps high activity when the pH value is 6.0-9.0, has strong tolerance to high salinity, and can tolerate metal ions such as Ba<2+>, Mg<2+> and the like. The Ala2 has high catalytic activity on short-chain fatty acid, and the most suitable substrate is p-nitrophenol hexanoate. The Ala2 coded by the gene has thermal stability and relatively high adaptability to high salinity, so that the Ala2 can be applied to industrial production under salt-containing conditions such as wastewater treatment, fine chemical engineering, pharmacy, environmental restoration and the like.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a mesophilic bacterium producing high-salinity tolerance ester hydrolase and its application. Background technique [0002] Ester hydrolases widely exist in microorganisms, animals and plants, and are a general term for a class of hydrolases that can catalyze the hydrolysis or synthesis of fatty acid ester bonds. Ester hydrolase participates in multiple metabolic processes of organisms, plays an important role in ester transport, cell structure construction and energy metabolism, and is one of the enzymes necessary to maintain the survival of living organisms. Family IV esterases are highly homologous to mammalian hormone-sensitive lipases (HSL), so they are also called HSL family esterases from bacteria. Studies have shown that most of the esterases derived from marine bacteria belong to this family. This family of esterases has a variety of different bacterial origins, exhibits ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/16C12R1/01
CPCC12N1/20C12N9/16
Inventor 吴月红施晓威许学伟孟凡旭周鹏程虹
Owner SECOND INST OF OCEANOGRAPHY MNR
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