Recombinant bacterium with granular methane monooxygenase activity and application thereof

A methane monooxygenase, granular technology, applied in the field of recombinant bacteria, can solve problems such as interruption of cell metabolic pathways, affecting cell metabolic activity, etc., and achieve the effect of easy cultivation

Inactive Publication Date: 2007-08-01
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If a special chemical reagent is added to selectively inhibit the activity of methanol dehydrogenase, although the accumulation of methanol is temporarily promoted, the metabolic pathway of the cell is interrupted, which seriously affects the metabolic activity of the cell and prevents the methane oxidation reaction.

Method used

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  • Recombinant bacterium with granular methane monooxygenase activity and application thereof
  • Recombinant bacterium with granular methane monooxygenase activity and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1, the acquisition of recombinant bacteria with granular methane monooxygenase activity

[0027] Cultivate Rhodococcus erythropolis LSSE8-1 (the bacterium was purchased from the General Microbiology Center of China Microbiology Culture Collection Management Committee) with a medium containing the following components: 2.44g KH per liter of solution 2 PO4, 14.03g Na 2 HPO4·12H 2 O, 0.3589g MgCl 2 ·6H 2 O, 0.001g CaCl 2 2H 2 O, 0.001g FeCl 3 ·6H 2 O, 0.004g MnCl 2 4H 2 O, 1.45ml glycerol and 2.00g NH 4 Cl, 0.1mmol / L dimethyl sulfoxide, pH7.0. Among them, 0.1mmol / L dimethyl sulfoxide was used as the only sulfur source. After culturing for 2-4 days, take 3 ml of the bacterial solution, collect the bacterial cells by centrifugation, and extract the genomic DNA. Using the genomic DNA as a template, use primer 1: 5'-AC AAGCTT GCACGGCTCCGGGCAGTTC 3' (the underlined nucleotide sequence is the recognition sequence of HindIII) and primer 2 5'-AC GAATTC AT...

Embodiment 2

[0041] Example 2. Acquisition and induction of recombinant bacterial cells with granular methane monooxygenase activity

[0042] The above-mentioned recombinant strain LSSE8-1 / pBS305-dsz-pmoCAB cultured on the nutrient slant (medium 1) was picked and inserted into 25 ml of basal medium. The composition of the basic medium: deionized water 1000 ml, KH 2 PO 4 2.44 g; Na 2 HPO 4 12H 2 O 14.03 g; NH 4Cl 2.00 g; MgCl 2 ·6H 2 O 0.36 g; CaCl 2 2H 2 O 0.001 g FeCl 3 ·6H 2 O 0.001 g MnCl 2 4H 2 O 0.004 g; Glucose 10 g, Na 2 SO 4 1mmol / L, pH7.0. After culturing at 30°C and 150 rpm for 24-48 hours, then inoculated into 500 ml of basal medium, and after 48-72 hours, centrifuged to obtain the bacteria. After washing the thalli with physiological saline, use sulfur-free medium (1000 milliliters of deionized water, KH 2 PO 4 2.44 g; Na 2 HPO 4 12H 2 O 14.03 g; NH 4 Cl 2.00 g; MgCl 2 ·6H 2 O 0.36 g; CaCl 2 2H 2 O 0.001 g FeCl 3 ·6H 2 O 0.001 g MnCl 2 4H 2 O 0.004...

Embodiment 3

[0043] Embodiment 3, recombinant bacterium converts ethane into ethanol

[0044] With the LSSE8-1 / pBS305-dsz-pmoCAB thalline of embodiment 2 (thalline is in logarithmic phase, OD 600 >20 to collect the bacteria) added to 5mM MgCl 2 In 10 milliliters of -20mM phosphate buffer solution, add gas at a ratio of ethane: air=1:1, react at 150 rpm at 30°C for 6 hours, centrifuge and get the supernatant for gas chromatography analysis, the results are shown in Figure 3 Show. In Figure 3, the dotted line is the standard 1% ethanol aqueous solution, and the solid line is the supernatant of the LSSE8-1 / pBS305-dsz-pmoCAB reaction solution. This result shows that there is ethanol in the LSSE8-1 / pBS305-dsz-pmoCAB reaction solution .

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Abstract

The present invention discloses one kind of recombinant bacterium with granular methane monooxygenase activity and its application. Fusion gene comprising the promoter sequences of coding gene and desulfurizing gene of granular methane monooxygenase is introduced into Rhodococcus, and after screening, the strain expressing granular methane monooxygenase or the recombinant bacterium with granular methane monooxygenase activity is obtained. The promoter sequence of the desulfurizing gene is located in the upstream of the granular methane monooxygenase coding gene. The recombinant bacterium avoids the effect of methanol dehydrogenase radically, makes methane oxidation stop in methanol reaction step, and is superior to wild methane oxidizing bacteria and easy to culture. The recombinant bacterium can convert alkane in natural gas into liquid alcohol, convert methane into methanol and oxidize propylene into 1, 2-epoxy propane.

Description

technical field [0001] The invention relates to a recombinant bacterium with granular methane monooxygenase activity and application thereof. Background technique [0002] Methanol is an important chemical raw material, its annual consumption ranks fourth after ethylene, propylene and benzene. Products such as propylene (MTP), olefins (MTO), dimethylformamide, methyl formate, methylamine, dimethyl carbonate and ethylene glycol can be prepared from methanol. With the gradual depletion of petroleum resources, methanol has begun to replace traditional fossil fuels in many ways, and is used to produce gasoline fuel, dimethyl ether fuel and gasoline additives. Therefore, the market demand for methanol is increasing. At present, the production of methanol is mainly obtained through the oxidation of methane. Since the methane molecule is quite stable, the C-H bond energy is 104 kcalmol -1 , Therefore, the oxidation of methane to methanol requires very harsh conditions. After ye...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/62C12N15/74C12N9/00C12P7/02
Inventor 缑仲轩罗明芳邢新会
Owner TSINGHUA UNIV
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