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Lipoxygenase mutant with improved enzyme activity and thermal stability and preparation method

A technology of lipoxygenase and thermostability, which is applied in the field of enzyme engineering, can solve the problems of no lipoxygenase, etc., achieve the effects of improving enzyme activity and thermostability, high-efficiency expression, and solving low yield

Inactive Publication Date: 2016-04-20
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

No relevant literature and patent reports on the directed evolution of lipoxygenase molecules were found after searching

Method used

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  • Lipoxygenase mutant with improved enzyme activity and thermal stability and preparation method
  • Lipoxygenase mutant with improved enzyme activity and thermal stability and preparation method
  • Lipoxygenase mutant with improved enzyme activity and thermal stability and preparation method

Examples

Experimental program
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Embodiment 1

[0026] The crushing conditions of the reorganized fat oxygenase gene engineering fungus are 1.5 mg / ml, the processing time of solverase enzymes is 40min, EDTA-2NA adds 2.0mmol / L, Twen-60 add 2%,-70 ℃ frozen frozen frozen37 ℃ melt and freeze 3 times.At the same time, potassium iodide-starch method with a simple, sensitive, fast, and determined system with the characteristics of the naked eye-starch method is used for LOX activity high-throughput screening ( figure 1 To.After the fermented crude enzyme solution is 40 ° C 30min, 45 ° C 20min, 50 ° C 10min, 55 ° C 5min passivation treatment, the starch-potassium iodide method is used to detect enzyme activity in the 96-hole plate to determine the enzyme-based meter, 96 microThe thermal stability of the pore board and the environmental temperature is improved.

[0027] 2. Erotic errors and the construction of the PCR mutant library and the screening of the mutant

[0028] With wild-type fishy algae fat oxygen enzymes PET-32A-Nana-LOX a...

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Abstract

The invention discloses a lipoxygenase mutant with improved enzyme activity and thermal stability and a preparation method thereof. The directional transformation of Anabaena lipoxygenase molecules is carried out by using directed evolution technology, and after multiple rounds of error-prone PCR and DNA ? Shuffling, obtained a lipoxygenase mutant with improved enzyme activity and thermostability at the same time, and the enzyme activity was 21420? U / mL is 2.17 times higher than the wild type, and its half-life t1 / 2 at 50°C is 1.9 times higher. Using this strategy can significantly improve the enzyme activity and thermal stability of lipoxygenase, solve the problems of low lipoxygenase activity and poor thermal stability, and provide a basis for its industrial production. The modified lipoxygenase is more suitable for industrial production and application.

Description

Technical field [0001] The invention involves enzyme activity and thermal stability at the same time, which is also increasing with fat oxygen enzymes and preparation methods. It belongs to the field of enzyme engineering technology. Background technique [0002] Fatoxygenase, EC1.13.11.12, LOX) is widely existed in animal and plant, and it is found in algae, bread yeast, fungi, and cyanococcus.LOX can catalyze the polyunsaturated fatty acid oxidation of a polyunsaturated fatty acid in the dual-bonding of the dual-bonding of the dual-bonding dual-bond. These hydrogen oxides are lively.Chemical reaction intermediates have a wide range of application prospects in the fields of food, medicine, chemical industry, especially in food processing, such as improved quality of flour and its products, preparation of flavor compounds, tea processing, etc.Although people have already recognized the potential application value of LOX in food processing and LOX exists in nature, it is directly ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70
CPCC12N9/0069C12N15/1027C12N15/1031C12N15/70C12Y113/11012
Inventor 吕凤霞陆兆新郭芳芳张充别小妹赵海珍
Owner NANJING AGRICULTURAL UNIVERSITY
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