Enzyme for catalyzing formaldehyde to synthesize hydroxyl acetaldehyde and application thereof

A formaldehyde and acetyl coenzyme technology, applied in the biological field, can solve problems that are difficult to apply

Active Publication Date: 2017-07-04
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the extreme sensitivity of this pathway to oxygen, it is difficult to apply it in other species

Method used

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  • Enzyme for catalyzing formaldehyde to synthesize hydroxyl acetaldehyde and application thereof
  • Enzyme for catalyzing formaldehyde to synthesize hydroxyl acetaldehyde and application thereof
  • Enzyme for catalyzing formaldehyde to synthesize hydroxyl acetaldehyde and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation of BFD mutant

[0035] The species source of the gene encoding the BFD enzyme is Pseudomonas putida, the amino acid of the BFD enzyme is shown in sequence 1, and the nucleotide sequence of the gene encoding the BFD enzyme is shown in sequence 2.

[0036] Single-point or multiple-point mutations were performed on the BFD enzyme to obtain the BFD mutants shown in Table 1.

[0037] The BFD mutant W86R-N87T is the mutation of tryptophan (Trp) at position 86 to arginine (Arg) in the BFD amino acid sequence (sequence 1), and the mutation of asparagine (Asn) at position 87 to threonine. Amino acid (Thr), other amino acid residues remain unchanged, the resulting sequence.

[0038] The coding gene of BFD mutant W86R-N87T is that the tgg at the 256-258th position of the BFD enzyme coding gene nucleotide sequence (sequence 2) is mutated into cgt, and the aac at the 259-261st position is mutated into acc, other bases Basis remains the same, the resul...

Embodiment 2

[0067] Example 2, Functional detection of BFD mutant protein

[0068] 1. BFD mutant protein catalyzes the condensation of formaldehyde to glycolaldehyde and 1,3-dihydroxyacetone

[0069] Detection method: The recombinant bacteria expressing BFD enzyme prepared in Example 1, the recombinant bacteria expressing BFD mutants and the recombinant bacteria expressing F / XPK were cultured in 200ml 2YT 37°C to OD 600 =0.6, 0.5mM IPTG induced at 16°C for 18h, centrifuged at 3500rpm for 15min, the medium was washed away with protein buffer, the cells were collected, and then resuspended in 20ml of protein buffer added with 0.5mM TPP. Take 500ul of resuspended bacteria and add 500ul of formaldehyde solution containing 0.5mM TPP with a final concentration of 5g / L prepared from protein buffer, react at 37°C and 750rpm for 2h, centrifuge to take the supernatant, and perform liquid phase detection. AMINEXHPX-87H, 300X7.8MM column, 5mM sulfuric acid was used as the mobile phase for liquid phas...

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Abstract

The invention discloses an enzyme for catalyzing formaldehyde to synthesize hydroxyl acetaldehyde and an application thereof. In the invention, through site-directed mutation of BFD, a mutant of the enzyme is found; and by means of the mutant of the enzyme, high-effect polymerization of the formaldehyde is achieved; meanwhile, through F / XPK, generation of acetyl phosphoric acid from the hydroxyl acetaldehyde or 1,3-dihydroxyacetone is achieved; with combination of phosphotransacetylase (Pta), a route from the formaldehyde to acetyl coenzyme A is achieved in three steps with the enzyme, thereby creating a new formaldehyde assimilation route, namely, synthesizing the acetyl coenzyme A from the formaldehyde in three steps. The route is short and is free of carbon loss and ATP input.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an enzyme that catalyzes the synthesis of glycolaldehyde from formaldehyde and its application. Background technique [0002] Methylotrophs in nature can use carbon-resources to synthesize metabolites necessary for growth. Methylotrophic bacteria mainly have three ways to assimilate formaldehyde, which are ribulose monophosphate pathway (RuMP), serine cycle pathway and Calvin-Benson-Bassham (CBB) cycle pathway after formaldehyde is completely oxidized. In the ribulose monophosphate pathway (RuMP), three molecules of formaldehyde are condensed to one molecule of pyruvate, which is then decarboxylated to form acetyl-CoA and CO 2 , The carbon utilization rate of this process is 67%. The serine cycle pathway requires an external supply of ATP to drive thermodynamically unfavorable reactions. Similarly, formaldehyde is completely oxidized to CO 2 CO was then fixed using the Calvin-Ben...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12P9/00C12P7/24C12P7/26C12P19/32
CPCC12N9/00C12P7/24C12P7/26C12P9/00C12P19/32C12N9/88C12Y401/01007C12P7/28
Inventor 江会锋刘玉万逯晓云杨晟缑俊冉杨一群卢丽娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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