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Novel high-temperature-resisting lipase gene and coding product thereof

A lipase gene, high temperature-resistant technology, applied in genetic engineering, plant genetic improvement, microorganism-based methods, etc., to achieve the effect of large industrial production and application potential, high catalytic activity and thermal stability

Inactive Publication Date: 2012-10-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 99% of the microorganisms existing in nature cannot be cultivated, so the method of screening new biocatalysts from microorganisms isolated by traditional culture techniques is very limited

Method used

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  • Novel high-temperature-resisting lipase gene and coding product thereof
  • Novel high-temperature-resisting lipase gene and coding product thereof
  • Novel high-temperature-resisting lipase gene and coding product thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Acquisition of a novel lipase gene lip34

[0038]The random clone sequencing analysis and screening method was used to randomly select 4 positive clones for sequencing. The sequencing was completed by Shanghai Yingjun Biotechnology Co., Ltd., and then the obtained sequences were analyzed using software. Only plasmid No. 34 has a complete lipase gene of 825 bp, named lip34.

[0039] 2. Design primers according to the sequence of lipase gene lip34 as follows:

[0040] lip34-F: 5'-CGCGGATCCATGAGACAATTGAC-3'

[0041] lip34-R: 5'-CCGCTCGAGTTAATTGTTAGTGTCCC-3'

[0042] 3. PCR amplification

[0043] The reaction system is as follows:

[0044] 4. The PCR program is as follows:

[0045]

[0046]

[0047] 5. Gel recovery of lip34 gene amplification product

[0048] Follow the steps in the E.Z.N.A gel extraction kit manual for gel recovery.

[0049] 6. Double enzyme digestion and product recovery of the lip34 gene PCR product. The enzyme digestion product was det...

Embodiment 2

[0095] 1. Purification of recombinant lipase Lip34

[0096] Purified by nickel column affinity chromatography. Take 600mL of the induced bacterial liquid, centrifuge at 4°C, 5000×g for 10min, collect the bacterial cells; add the lysis buffer at a ratio of 20:1 (V:V); ultrasonicate 40 times at a power of 300W (each time / 3s, The interval time is 3s); then centrifuged at 4°C and 10000×g for 30min, and the supernatant was taken; the supernatant was added to a micro-gravity chromatography column, and the effluent liquid was collected; the column was washed twice with washing buffer, 4mL each time, and collected Effluent liquid; wash column 4 times with elution buffer, 0.5 mL each time, collect all effluent liquids respectively; take 10 μ L of components collected each time for SDS-PAGE analysis ( Figure 6 ).

[0097] 2. Determination of lipase activity

[0098]The determination of lipase activity utilizes the turbidimetric method provided by the kit: 50mmol / L Tris-HCl is used a...

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Abstract

The invention discloses a novel high-temperature-resisting lipase gene which is named as lip34 and which has a nucleotide sequence shown as SEQ ID NO.1. Also, the invention discloses a preparation method of the novel high-temperature-resisting lipase gene, a recombinant plasmid pET32a-lip34 containing the novel high-temperature-resisting lipase gene, and an expression vector thereof. The invention further discloses a high-temperature-resisting recombinant lipase and a preparation method thereof. The novel high-temperature-resisting lipase gene lip34 is excessively expressed in an escherichia coli prokaryotic expression system, and the production period of lipase is substantially shortened. The recombinant lipase obtained with the method has relatively high catalytic activity and thermal stability, and has good industrialized production and application prospects.

Description

technical field [0001] The invention relates to a novel lipase gene and its coded product, in particular to a novel lipase gene and its coded product from mangrove soil microorganisms. Background technique [0002] Lipase is an enzyme that widely exists in animals, plants and microorganisms, and plays an important role in lipid metabolism. At the oil-water interface, lipase catalyzes the hydrolysis of the ester bonds of triacylglycerols, releasing glycerides or glycerol and fatty acids with fewer ester bonds. In addition, there are various enzymatic activities, such as catalyzing the hydrolysis, synthesis and resolution of racemic mixtures of various esters. Lipase has mild reaction conditions, excellent stereoselectivity, and does not cause environmental pollution. Therefore, it is widely used in many industrial fields such as food, leather, medicine, feed, and detergent. [0003] 99% of the microorganisms existing in nature cannot be cultured, so the method of screening ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/10C12N15/70C12N1/21C12N9/20C12R1/19
Inventor 陆勇军龙思梅庞柳何磊
Owner SUN YAT SEN UNIV
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