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Hydrogen production associated protein, coding genes thereof and application thereof

A protein and gene technology, which is applied in the field of hydrogen production-related proteins and their coding genes and applications, can solve the problem of no gene reporting, etc., and achieve the effects of improving glucose utilization ability, strong substrate utilization ability, and enhanced hydrogen production ability

Inactive Publication Date: 2010-01-27
WUXI RES INST OF APPLIED TECH TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are many reports about the hydrogen production of Enterobacter aerogenes, but most of them are in terms of technology and cultivation [E.Palazzi, B.Fabiano, P.Perego.Bioprocess Eng.22:205-213 (2000).]. Hydrogen-related genes have not been reported yet

Method used

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  • Hydrogen production associated protein, coding genes thereof and application thereof
  • Hydrogen production associated protein, coding genes thereof and application thereof
  • Hydrogen production associated protein, coding genes thereof and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0058] Cloning of embodiment 1, nuob gene

[0059] Sequence analysis of the Enterobacter NADH dehydrogenase gene sequence in NCBI, a pair of primers nuoB-fw and nuoB-rv were designed to target Enterobacter aerogenes (E.aerogenes IAM1183) (purchased from the Institute of Applied Microbiology, Tokyo University, Japan ( IAM) strain library) DNA as a template for PCR.

[0060] The results showed that a band with a molecular weight of 675bp was obtained. The PCR product was stored at 4°C and sent to Shanghai Invitrogen Corporation for sequencing. The sequencing results showed that the amplified nucleotide sequence was shown in sequence 2 in the sequence listing, and the encoded amino acid sequence was the protein in sequence 1 in the sequence listing. The protein shown in sequence 1 is named NuoB, and the nucleotide shown in sequence 2 is named nuob.

[0061] After online comparison and analysis, nuob has the highest homology with NADH dehydrogenase subunit B of Klebsiella pneumo...

Embodiment 2

[0062] Example 2, the acquisition of Enterobacter aerogenes E.aerogenes IAM1183-B mutant

[0063] Through sequence analysis, a pair of primers nuoB-tet-fw and nuoB-tet-rv were designed, and the underlined sequence in the primers was the homology arm of Enterobacter aerogenes (E. aerogenes IAM1183) ΔnuoB.

[0064] Transformation of pYM-red plasmid (Yu Mei, Zhou Jianguang, Chen Wei, Li Shanhu, Huang Cuifen, establishment of a transferable recombinant engineering system pYM-Red. Advances in Biochemistry and Biophysics. 2005, 32(4) .) electric shock into Enterobacter aerogenes (E.aerogenes IAM1183), carry out chloramphenicol (24mg / mL) resistance screening, extract pYM-red plasmid from positive bacterial strain and carry out identification, obtained the recombinant plasmid containing pYM-red Enterobacter aerogenes (E. aerogenes IAM1183).

[0065] With pACYC184 plasmid (Nanjing Shengbiao Biotechnology Co., Ltd., 70905-3) (Chang, A.C., and S.N.Cohen.1978.Construction and characteriz...

Embodiment 3

[0068] Embodiment 3, E.aerogenes IAM1183-B engineering bacteria growth and hydrogen production situation

[0069] In a 70mL serum bottle, add 20mL of glucose medium to culture the activated engineered bacteria E.aerogenes IAM1183-nuoB in shake flasks; at the same time, shake the activated wild bacteria E.aerogenes IAM1183 under the same conditions as a control. The specific operation steps are as follows:

[0070] (1) Seed culture: use a 15ml centrifuge tube, add 5ml of LB medium, inoculate from a solid plate, cultivate overnight at a temperature of 37°C and an air bath shaker at a speed of 170rpm.

[0071] (2) Anaerobic shake flask culture: adopt 70ml serum bottle anaerobic culture bottle, glucose medium filling capacity 20ml (use the air in the top of culture bottle and culture medium in advance with nitrogen replacement); The inoculation amount of seed bacteria 2.5% (v / v), the culture temperature is 37° C., and the rotation speed of the air bath shaker is 170 rpm. Incuba...

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Abstract

The invention discloses a hydrogen production associated protein, coding genes thereof and application thereof. The hydrogen production associated protein is a protein (a) or protein (b), wherein the protein (a) has an amino acid sequence represented by the No.1 sequence 1 in a sequence table; or (b) the protein has an amino acid sequence that has substituents of one or more amino acid residues, lacks one or more amino acid residues or has one or more additional amino acid residues and is associated with the hydrogen production and derived from the sequence 1. The invention also obtains a strain of engineering bacteria by inactivating the coding genes of the protein in the enterobacter aerogenes, and the engineering bacteria have the advantages of high growing speed and strong environmental adaptability, greatly improve the glucose utilizing ability and the hydrogen producing ability, can be applied to biological hydrogen production, and have great instruction significance.

Description

technical field [0001] The invention relates to a hydrogen production-related protein and its coding gene and application. Background technique [0002] The fossil energy system established since the world industrial revolution in the 18th century is now facing two major challenges: 1) The reserves of fossil energy are decreasing day by day, facing the danger of depletion; 2) The combustion of fossil fuels produces a large amount of pollutants, especially CO 2 The greenhouse effect caused by the emission of greenhouse gases has brought a huge threat to the environment on which human beings depend. Therefore, developing sustainable renewable clean energy and gradually replacing fossil energy is an important strategic issue related to national energy security, environmental security and social stability and peaceful development. [0003] Hydrogen energy, as one of the potential alternative clean energy sources in the future, is an efficient energy carrier with the characteris...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/11C12P3/00C12R1/01
Inventor 邢新会赵洪新马堃卢元张翀王立言杨程来奇恒
Owner WUXI RES INST OF APPLIED TECH TSINGHUA UNIV
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