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Lipoxygenase mutant with simultaneously-improved enzymatic activity and thermal stability, and preparation method thereof

A lipoxygenase and thermostability technology, applied in the field of enzyme engineering, can solve problems such as no lipoxygenase, achieve the effects of improving enzyme activity and thermostability, high expression, and solving poor thermostability

Inactive Publication Date: 2014-05-28
NANJING AGRICULTURAL UNIVERSITY
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  • 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 simultaneously-improved enzymatic activity and thermal stability, and preparation method thereof
  • Lipoxygenase mutant with simultaneously-improved enzymatic activity and thermal stability, and preparation method thereof
  • Lipoxygenase mutant with simultaneously-improved enzymatic activity and thermal stability, and preparation method thereof

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Embodiment 1

[0027] 1. Establishment of a high-throughput screening system for lipoxygenase

[0028] The crushing conditions of recombinant lipoxygenase genetically engineered bacteria were 1.5 mg / mL of lysozyme, 40 min of lysozyme treatment, 2.0 mmol / L of EDTA-2Na, 2% of Tween-60, and -70 °C. Thaw at 37°C, freeze and thaw 3 times. At the same time, the potassium iodide-starch method, which is simple, sensitive, and rapid, and the assay system has characteristic colors discernible to the naked eye, was used for high-throughput screening of LOX activity ( figure 1 ). The crude enzyme solution obtained by fermentation was passivated at 40 °C for 30 min, 45 °C for 20 min, 50 °C for 10 min, and 55 °C for 5 min, respectively, and the enzyme activity was detected in a 96-well plate by starch-potassium iodide method. Thermostability-enhancing mutant screening method integrated with instrument, 96-well plate, and ambient temperature.

[0029] 2. Construction of an error-prone PCR mutant librar...

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Abstract

The invention discloses a lipoxygenase mutant with simultaneously-improved enzymatic activity and thermal stability, and a preparation method thereof. The method utilizes a directed evolution technology to carry out directed evolution on anabaena lipoxygenase molecules, and after several rounds of error-prone PCR and DNA shuffling, obtains a lipoxygenase mutant with simultaneously-improved enzymatic activity and thermal stability, wherein the enzymatic activity of the lipoxygenase mutant is 21420 U / mL and is improved by 2.17 times, compared to that of wild lipoxygenase mutant, and the half-life period (t 1 / 2)of the lipoxygenase mutant at a temperature of 50 DEG C is improved by 1.9 times. The method can prominently improve the enzymatic activity and thermal stability of lipoxygenase, solves the problems of low enzymatic activity and bad thermal stability of lipoxygenase, and provides a fundament for industrial production of lipoxygenase. The improved lipoxygenase is more suitable for industrial production and application.

Description

technical field [0001] The invention relates to a lipoxygenase mutant with improved enzyme activity and thermal stability and a preparation method thereof, belonging to the technical field of enzyme engineering. Background technique [0002] Lipoxygenase (Lipoxygenase, EC 1.13.11.12, LOX) widely exists in animals and plants, and it is found in algae, baker's yeast, fungi and cyanogen bacteria. LOX can specifically catalyze the oxidation of polyunsaturated fatty acids containing 1,4-cis, cis-pentadiene double bond system to form hydroperoxides with conjugated double bonds. These hydroperoxides are active and important Chemical reaction intermediates have broad application prospects in food, medicine, chemical industry and other fields, especially in food processing, such as improving the quality of flour and its products, preparing flavor compounds, tea processing, etc. Although people have long recognized the potential application value of LOX in food processing, and LOX ex...

Claims

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Application Information

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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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