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L-methionine gamma-lyase gene from deep sea metagenome and expression product thereof

A technology of methionine lyase and metagenomics, applied in lyase, genetic engineering, plant gene improvement, etc., can solve the problems of low enzyme yield and difficult scale production

Active Publication Date: 2012-06-13
广州市微生物研究所集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using naturally isolated strains or their mutant strains for enzyme preparation, the yield of enzymes is extremely low, and it is difficult to achieve profitable scale production; currently, the main tendency is to use genetically engineered strains for enzyme preparation

Method used

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  • L-methionine gamma-lyase gene from deep sea metagenome and expression product thereof
  • L-methionine gamma-lyase gene from deep sea metagenome and expression product thereof
  • L-methionine gamma-lyase gene from deep sea metagenome and expression product thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Acquisition of gene mgl

[0034] 1. Marine sediment metagenomic library construction

[0035] DNA was extracted from marine sediments from four sites in the South China Sea (water depth 1256-2893 m), and DNA from the four sources was combined. Separation by pulsed field electrophoresis and purification by 0.8% agarose gel electrophoresis, the resulting DNA fragment was ligated with the plasmid fosmid and transfected into the host strain E.coli EPI300, spread on LB plate containing 12.5 μg / mL chloramphenicol, 37°C After culturing overnight, 39,600 transformants were obtained, and the clone library was stored at 4°C and -80°C.

[0036] 2. Identification of library clones

[0037] Randomly pick 70 fosmid clones from the above-mentioned deep-sea metagene library and inoculate them in the LB culture solution containing antibiotics, and cultivate overnight at 37°C with shaking. After the culture was concentrated, a plasmid amplification inducer was added, shaken...

Embodiment 2

[0041] Example 2: Protein sequence analysis of gene mgl

[0042] Using the ExPASy-ProtParam tool of the Swiss Institute of Bioinformatics (SIB), some basic properties of the MGL protein sequence, such as molecular weight, isoelectric point and amino acid composition, were analyzed online ( http: / / www.expasy.ch / tools / protparam.html ). On-line analysis results (see Table 2) show that MGL protein is composed of 426 amino acids, its molecular weight is 46.024kDa, and its theoretical isoelectric point (pI) is 5.28. It is an acidic protein. The half-life in E. coli cells is more than 10 hours, and the half-life in E. coli cells is up to 30 hours. In Table 2, the amino acid sequence of MGL derived from this case is compared with the amino acid sequence of MGL from other sources with the highest similarity, and the results show the obvious differences between the two proteins. Amino acid composition analysis showed (see Table 3) that the protein had higher Ala and Leu contents, fol...

Embodiment 3

[0053] Example three: functional identification of gene mgl

[0054] 1. Cloning of gene mgl

[0055] Design primers for amplifying the full-length gene of mgl according to the known mgl sequence:

[0056] mgl F: 5'-TCAGCGT CCATGG AATCTGATGCG-3' (the underline is the Nco I restriction site)

[0057] mgl R: 5'-CATGAAGTTGTG GAATTC TGTCATG-3' (the underline is the EcoR I restriction site)

[0058] Inoculate the clone f in LB (containing 12.5 μg / mL chloramphenicol), prepare the recombinant plasmid fosmid DNA as a template according to item 2 of Example 1, carry out PCR amplification with the above primers, and use Nco I and EcoR I performs double digestion. At the same time, pET 32a plasmid was prepared, which was also digested with Nco I and EcoRI. The digested products of the two were ligated to construct a recombinant plasmid pET-mgl carrying mgl.

[0059] The recombinant plasmid pET-mgl was transformed into Escherichia coli BL21(DE3), and the recombinant carrying mgl w...

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Abstract

The invention relates to a novel gene from deep sea environment, in particular to a novel gene which is from deep sea metagenome and can produce L-methionine gamma-lyase activity. The novel gene is cloned, engineering Escherichia coli is constructed and an expression product is obtained. The L-methionine gamma-lyase gene from the deep sea metagenome has the size of 1,281bp and the sequence shown as SEQ ID NO.1, and codes 426 amino acids with the sequence shown as SEQ ID NO.2; and protein molecular weight is 46.024kDa and an isoelectric point is 5.28. The deep sea L-methionine gamma-lyase geneis cloned for the first time; the product of the gene is analyzed for the first time; a method for massively preparing zymoprotein in recombinant gene engineering is provided; and the zymoprotein enough for industrial application can be obtained.

Description

technical field [0001] The invention belongs to the field of microbial technology and genetic engineering, and relates to a new gene from deep-sea environment, especially a new gene that can produce L-methionine γ-lyase activity from deep-sea metagenome. Cloning, construction of engineering Escherichia coli and obtaining expression products. Background technique [0002] L-methionine γ-lyase (MGL or METase for short) is a 5’-pyridoxal phosphate (PLP)-dependent multifunctional enzyme. It mainly catalyzes the γ-elimination reaction of L-methionine to generate methyl mercaptan, ammonia and α-butyruvate, and the enzyme can also catalyze the β-elimination reaction of L-cysteine ​​and thio-L-cysteine And the γ, β-substitution reaction of L-methionine and L-cysteine ​​and their derivatives. The reaction equation for the γ-elimination of L-methionine catalyzed by MGL (see Ilya V. Manukhov et. al. Journal of Bacteriology. 2005, 187: 3889-3893). [0003] [0004] MGL is mainly f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N15/63C12N1/21C12N9/88C12Q1/527C12P13/04A61K38/51A61P35/00A23L1/226C12R1/19A23L27/20
Inventor 周世宁李慧贤黄雅丽陆勇军
Owner 广州市微生物研究所集团股份有限公司
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