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Low-temperature lipase and its gene sequence

A technology of lipase and low temperature, applied in genetic engineering, plant gene improvement, hydrolytic enzymes, etc., can solve the problem of low activation energy of low temperature lipase

Inactive Publication Date: 2007-12-12
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(4) The activation energy of low temperature lipase is low

Method used

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  • Low-temperature lipase and its gene sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Extraction of Pseudomonas marine total DNA

[0030] 1. Using Pseudomonas sp isolated from the sea, get 20 g of its fresh wet thalli and suspend in 10 ml of 50 mM Tris buffer (pH 8.0).

[0031] 2. Add a small amount of lysozyme and 8ml 0.25mM EDTA (pH8.0), mix well and place at 37°C for 20min.

[0032] 3. Add 2ml of 10% SDS and place at 55°C for 5min.

[0033] 4. Extract once each with equal volumes of phenol and chloroform.

[0034] 5. Take the last supernatant, add 2 times the volume of ethanol, and recover the DNA.

[0035] 6. Wash with 70% ethanol and absolute ethanol respectively.

[0036] 7. Dissolve the precipitate in 0.5ml TE buffer (pH 8.0, 10mM Tris, 1mM EDTA), add 3μl of 10mg / ml RNase, and incubate at 37°C for 1h.

[0037] 8. Extract once with equal volumes of phenol and chloroform respectively, and add 2 times the volume of ethanol to the supernatant to recover DNA.

[0038] 9. Wash the precipitate with 70% ethanol and absolute ethanol respecti...

Embodiment 2

[0040] Cloning of embodiment 2 low temperature lipase gene

[0041] 1. Take 0.5 μl of the total DNA solution of Pseudomonas marine as a template, and use the following oligonucleotide sequences as primers (including restriction sites of BamHI and HindIII), amplify the desired DNA fragments by conventional PCR techniques.

[0042] The primer sequences are as follows:

[0043] Primer 1: 5'-CTAAGCTTGTATCATTTTCGCGTCGT-3'

[0044] Primer 2: 5'-AGGGATCCGATGACTGATACATCGGC-3'

[0045] 2. Recover the PCR product, perform double digestion with restriction endonucleases BamHI and HindIII, and connect with the DNA of the plasmid pET-28a that has undergone the same digestion under the action of T4 DNA ligase to obtain the recombinant plasmid pL-1.

[0046] 4. Transform competent Escherichia coli BL21 with the recombinant plasmid. Spread on LB solid medium containing 50 μg / ml Kan (kanamycin) and olive oil. Incubate at 37°C for 12 hours, and then incubate at 60°C for 1-5 hours. If there ...

Embodiment 3

[0049] Example 3 High-efficiency expression of low-temperature lipase gene in engineering bacteria BL21LIP

[0050] The engineered strain BL21LIP was cultured in LB liquid medium containing kanamycin at 37°C for 16 hours with shaking, and then transferred to a fermenter. When the OD value of the bacteria reached 0.6-1.0, the expression was induced by adding ITPG with a final concentration of 0.6mM.

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Abstract

The invention discloses a low- temperature lipase and its code gene and application. The gene of low- temperature lipase is 70% homologous to DNA squence according to SEQ No. 1 limit in sequence table, and is DNA squence of functinoal protein with same code. The protein of low- temperature lipase code is protein with SEQ No. 2 amnio acid squence in squence table or protein with same activity derived from squence 2. The invention aslo discloses a method for constructing low- temperature lipase engineering bacteria and method for preparing low- temperature lipase by using said engineering bacteria. The activity and stability of low- temperature lipase is pretty goos at low temeprature, and can be used in fields of washing agent industry, foodstuff industry, biological pharmacy and environmental biological technique. The application prospect is wide.

Description

technical field [0001] The invention relates to a low-temperature lipase isolated and purified from Pseudomonas sp and its coded gene sequence, the construction of a low-temperature lipase engineering bacterium, and a method for preparing the low-temperature lipase by using the engineering bacterium. Background technique [0002] Lipase (lipase, EC3.1.1.3), also known as glyceride hydrolase, refers to an enzyme that decomposes triglyceride ester bonds formed by higher fatty acids and glycerol, and is widely found in animal tissues, plant seeds and microorganisms. [0003] As an important industrial enzyme, lipase can be used in oil hydrolysis, improvement of food flavor and aroma, medical medicine, degreasing of leather and silk spinning, modification of low-grade oil, addition to detergents and cosmetics, etc. Especially in the oleochemical and organic synthesis industries, enzyme-catalyzed reactions have the advantages of mild conditions, low energy consumption, low raw ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N1/21C12N1/20C12R1/38C12R1/19
Inventor 林秀坤陈雷牛荣丽董平原王征宋春霞
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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