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Thermophil lipase gene, engineering bacterium, thermophil lipase and application thereof

A technology of thermophilic fat and enzyme gene, applied in genetic engineering, application, plant genetic improvement, etc., can solve problems such as inability to adapt

Inactive Publication Date: 2008-07-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] So far, the vast majority of lipases are derived from normal temperature microorganisms or general thermophilic microorganisms. Although many lipases have good catalytic activity, they cannot adapt to the high temperature reaction system in industry, but are derived from hyperthermophilic microorganisms. The lipase has the characteristics of good thermal stability and catalytic activity at high temperature. Therefore, the development and research of thermophilic lipase has very good industrial application prospects.

Method used

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  • Thermophil lipase gene, engineering bacterium, thermophil lipase and application thereof
  • Thermophil lipase gene, engineering bacterium, thermophil lipase and application thereof
  • Thermophil lipase gene, engineering bacterium, thermophil lipase and application thereof

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Effect test

Embodiment 1

[0034] Embodiment 1: Construction of thermophilic lipase engineering bacteria and expression of its enzyme

[0035] (1) Cultivation of hyperthermophilic bacteria Fervidobacterium changbaicum CBS-1 and extraction of chromosomal DNA.

[0036] The composition of the culture medium of every liter of Fervidobacterium changbaicum CBS-1 is as follows: (NH 4 ) 2 SO 4 , 1.3g; KH 2 PO 4 , 0.28g; MgSO 4 ·7H 2 O, 0.25g; CaCl 2 , 0.07g; FeSO 4 ·7H 2 O, 0.028g; MnCl 2 , 1.8 mg; Na 2 B 4 o 7 , 4.5mg; ZnSO 4 ·7H 2 O, 0.22 mg; CuSO 4 2H 2 O, 0.05 mg; NaMoO 4 2H 2 O, 0.03 mg; CoCl 2 , 0.01 mg; yeast extract, 1 g; peptone, 2 g; 0.1% (v / v) resazurin.

[0037] Subpackage the medium after high-temperature and high-pressure sterilization, and inject 0.5ml of sterilized 10% Na into every 100ml of medium 2 S (nutrient salt) solution.

[0038] Add 1ml of the above culture medium to the stem cells of Fervidobacterium changbaicum CBS-1 (strain preservation number DSM17883, JCM13353)...

Embodiment 2

[0063] Example 2: Characteristics of Recombinant Thermophilic Lipase

[0064] (1) Optimum reaction temperature

[0065] Reaction temperature is an important factor affecting the catalytic activity of enzymes. Generally speaking, the reaction activity of thermophilic lipase at high temperature is much higher than that of low temperature, but the stability near the optimum temperature is greatly reduced, and the temperature range detected for the catalytic activity of thermophilic lipase described in this patent is 20~90℃.

[0066] Using p-nitrophenol octanoate as the reaction substrate, the lipase activity was measured in the temperature range of 20-90°C, and the relative activity of the enzyme activity was plotted against the temperature. It can be seen from Figure 5 that its activity increases with the increase of temperature in the temperature range of 20-78°C, and decreases above 78°C, and the optimum temperature is 78°C.

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Abstract

The invention belongs to the technical field of biological engineering and in particular relates to a thermophile lipase gene which is derived from super-thermophilic bacterias Fervidobacterium changbaicum CBS-1, a thermophile fat engineering bacteria which is expressed by the gene recombinant vector in a normal temperature host, a thermophile lipase which is constructed through utilizing the engineering bacteria and the wide application of the thermophile lipase in the fields of biological chemical industry, biological diesel, oil processing, tool enzyme and the like. The thermophile lipase gene which is obtained by the invention has the nucleotide sequence which is shown in SEQ ID NO.1, the thermophile lipase has amino acid sequence which is shown in SEQ ID NO.2, the thermophile fat engineering bacteria is preserved in the Ordinary Microbial Centre of China Microbial Strains Preservation Commission on October 15, 2007, the preservation code is CGMCC No.2195, and the classified name is Escherichia coli (Escherichia coli).

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a thermophilic lipase gene derived from hyperthermophilic bacteria Fervidobacterium changbaicum CBS-1, a thermophilic lipase engineering bacterium expressed in a normal temperature host by the gene recombination vector, and using the engineering The thermophilic lipase constructed by bacteria and the application of the thermophilic lipase in industry. Background technique [0002] The in-depth research and wide application of biocatalysis technology is the "third wave" and an important pillar of biotechnology development after biopharmaceuticals and biotechnology agriculture. The core of this technology is the development of biocatalysts. As a biocatalyst, enzymes have been widely used in chemical industry, food, medicine, light industry and other fields. However, the contradiction between harsh conditions required for industrial production and enzyme stability has been...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/70C12N9/20C12N1/21C12P7/64C12R1/19
Inventor 冯雁蔡锦刚谢渊宋搏
Owner JILIN UNIV