Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis

A technology of Serratia marcescens and lipase, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of less research on the separation of chiral drugs, and achieve the effect of high tolerance

Active Publication Date: 2013-08-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, lipase has achieved rapid development mainly in food processing, daily chemical products and other industries, while there are few researches on the separation of chiral drugs and biodiesel.

Method used

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  • Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis
  • Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis
  • Method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1: Construction of recombinant B.subtilis168 bacterial strain

[0043] 1) Cloning of the complete lipase (lipA) gene sequence: The lipA gene primers were designed according to the Serratia marcescenslipA gene sequence published on the GENBANK website and the restriction site on the pMA-5 plasmid. The sequences of the primers are as follows (wherein the underlined part in italics is the restriction site, and the primers were synthesized by Saibaisheng Company).

[0044] lipA F 5'-ATC GAATTC ATGGGCATCTTTTAGCTATA-3'

[0045] lipA R: 5'-TGACT GCGCCGGC TTAGGCCAACACCACCTG-3'

[0046] Serratia marcescens chromosomal DNA was extracted and prepared according to the instruction manual of Shanghai Sangong Company Small Chromosomal DNA Extraction Kit (bacteria), and the extracted chromosomal DNA was placed in a -20°C refrigerator for use. Using the prepared Serratia marcescens chromosomal DNA as a template and lipAF and lipAR as primers, the entire sequence of lipA...

Embodiment 2

[0053] Embodiment 2: Optimization of fermentation conditions of recombinant bacterial strain B.subtilis168 / pMA-5-lipA

[0054] Optimization of medium conditions for recombinant strain B.subtilis168 / pMA-5-lipA:

[0055] a) According to the seed growth curve, it is determined that the seeds that have grown for 13-15 hours are the best seed age.

[0056] b) Choose to add soluble starch, maltose, cyclodextrin, glucose, sucrose, and lactose as carbon sources to analyze the impact on the cell mass and lipase production. The concentration is 2%, and the initial fermentation medium is LB medium. Since there is no strict carbon source in the LB medium, an alternative carbon source can be directly added as the only carbon source, and other components remain unchanged. The inoculum amount was 2%, cultured at 37°C and 160rpm for 36 hours, the cell mass was measured and the lipase activity was determined. It was found that when sucrose was the carbon source, the lipase yield was the high...

Embodiment 3

[0063] Embodiment 3: the enzyme activity assay of recombinant bacterial strain

[0064] The strain was cultured in LB medium for 36h, centrifuged at 8000rpm for 10min, and the supernatant was taken. Solution A: 16.5mmol / L isopropanol solution of p-nitrophenol palmitate (p-NPP). Solution B: 50 mmol / L Tris-HCl buffer solution (pH 8.0) containing 0.4% Triton X-100 and 0.1% gum arabic. During the measurement, the corresponding liquid A and liquid B were mixed at a ratio of 1:9 (volume ratio), and 100 μL of crude enzyme solution was added to 900 μL of the above mixed solution, and reacted at 40° C. for 10 min. Immediately add 1 mL of absolute ethanol to terminate the reaction, and measure the light absorbance at 410 nm. Enzyme activity (U) unit definition: The amount of enzyme needed to decompose p-NPP to produce 1 μmol p-nitrophenol (yellow) per minute is defined as 1 enzyme activity unit.

[0065] The primary bacteria were transferred to the initial fermentation medium at an i...

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Abstract

The invention relates to a method for cloning and expressing Serratia marcescens lipase by utilizing recombinant Bacillus subtilis, belonging to the fields of gene engineering and enzyme engineering. The method comprises the following steps of: firstly acquiring gene amplification of Serratia marcescens lipase (lipA), and then realizing overexpression of genes in type strain Bacillus subtilis 168 for the first time by utilizing pMA-5 plasmids. According to the method, Bacillus subtilis engineering strains which produce the Serratia marcescens lipase are constructed for the first time, after carrying out research on enzyme activities and fermenting properties of the strains and optimization on culture medium components and culture conditions, the activity of the lipase is 98.6 U / mL which is equal to 12 times of that, namely 8.57 U / mL, of a starting strain, and compared with the starting strain, the activity of the lipase is remarkably improved; and the method plays an active role in guiding the industrial production of the lipase by utilizing a microbiological fermentation method and can be applied to the field of food industry.

Description

technical field [0001] A method for cloning and expressing Serratia marcescens lipase by using recombinant Bacillus subtili, especially a safe and efficient method for expressing lipase which can be used in food industry, belongs to the field of genetic engineering and enzyme engineering. technical background [0002] Lipase (Lipase, full name Triacylglycerol acylhydrolase) EC3.1.1.3, is a special class of ester bond hydrolase. It can catalyze the hydrolysis of triacylglycerols into fatty acids, diglycerides, monoglycerides and glycerol, and its natural substrates are generally long-chain fatty acid acyl esters that are insoluble in aqueous solution. Lipase exists widely in nature. It not only plays an important physiological role in metabolism, but also has great industrial value, including the synthesis of some special organic substances, the hydrolysis of fats and oils, the improvement of spices in the development of the food industry, and chemical analysis etc. Lipase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N1/21C12N15/75C12R1/125
Inventor 饶志明司冠儒徐美娟
Owner JIANGNAN UNIV
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