High throughput screening method for high-yield lipase bacteria

A technology of lipase bacteria and screening method, applied in the biological field, can solve the problems of calculation result error, labor consumption, material resources, multiple disposable plates, etc., and achieve the effects of accurate results, convenient operation and material saving.

Inactive Publication Date: 2017-05-31
南京河海环境研究院有限公司
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  • Abstract
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Problems solved by technology

Some problems in the traditional method-plate method, take the plate method, it takes 6 days (fungi), too many disposable plates and raw materials are needed (at least 5mL medium for a single plate), and a total of 750mL medium (with 50 Inoculum calculation), and manual measurement is required for calculation, which consumes a lot of manpower, material resources, and wastes a lot of time, and there are certain errors in the calculation results

Method used

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  • High throughput screening method for high-yield lipase bacteria

Examples

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Embodiment 1

[0025] Example 1: 96-well plate method

[0026] A high-throughput screening method was used to screen the mutagenized strains. Add 200ul of fermentation broth and a certain volume of bromophenol blue to a 96-well plate, mix it evenly, and measure its maximum absorption wavelength with an ultraviolet spectrophotometer. The maximum absorption wavelength of this solution is 570nm. According to the final result of physical radiation, single colony spores under the specific lethality induced by ultraviolet mutagenesis were selected and inserted one by one into the 96-well plate equipped with fermentation broth and bromophenol blue (3 parallel samples, wherein both sides were used as blanks) , cultivated in a constant temperature shaker at 30°C and 160r / min, and measured values ​​with a microplate reader at different time periods. When using a microplate reader for detection, the wavelength needs to be adjusted to 570nm and preheated for 30 minutes in advance. The bacteria produce...

Embodiment 2

[0027] Embodiment 2: plate method

[0028] Pour the configured culture medium into a disposable flat plate. The average flat plate contains 5mL solution. According to the final result of physical radiation, select the single colony spores under the specific lethality induced by ultraviolet mutagenesis and insert them into the flat plate (3 cultured in a constant temperature shaker at 30°C and 160r / min, and observed regularly until the plate on which it was located was all yellow (bromocresol purple color), and the full diameters of colonies and discolorations were measured with a ruler respectively. Among them, it takes 6 days to screen by the plate method, and the final result obtained by the detection is consistent with that of the 96-well plate method.

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Abstract

The invention provides a rapid screening method for high-yield lipase bacteria, belonging to the field of biotechnology. The invention further provides a high throughput screening method by which a discoloration reaction is carried out on a bromcresol purple acid-base indicator by utilizing the pH change of a fatty acid and establishes bacterial strains for generating lipase in a 96-well plate. The method is characterized in that the operation is simple, fast, accurate, high-throughput, etc. The invention relates to a method for rapidly screening the lipase bacteria. The technological difficulties are that 1, the prior art needs a manual measurement method, and adopts a diameter ratio calculation to obtain target bacteria that we need, and 2, in the process of cultivating induced bacteria, observation needs to be conducted from time to time so as to avoid exceeding the time limit (in general, 6 days is needed). The solution is that 1, a 96-well plate screening method is adopted so long as a microplate reader is used for measurement at the maximum absorption wavelength position thereof, and the data are more accurate, and 2, when the high throughput screening method is adopted, only 35 hours are needed from the beginning to the end, the process is simple and convenient, and observing at all times is not needed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly, conveniently and high-throughput screening of bacteria capable of producing high-yield lipase through the color change reaction of bromocresol purple acid-base indicator by using the pH change of fatty acids. The method has the characteristics of simple operation, rapidity, accuracy and high throughput. Background technique [0002] Lipase has a very important position in the living body, and the research on lipase has been reported for nearly 200 years. Because of its mild reaction conditions, high catalytic rate, good stereoselectivity, wide substrate specificity, easy extraction and potential unlimited supply, it has been widely used in a wide range of industrial fields such as food, energy, medicine, agriculture, fine Chemical and bioremediation industries. [0003] Microbial mutation breeding refers to the breeding method of using various phys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N13/00C12R1/80
CPCC12N15/01C12N13/00
Inventor 胡家富蔡金燕
Owner 南京河海环境研究院有限公司
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