Refractory S-adenosylmethionine synthetase gene and its polypeptide coded by it and preparing process

A technology for adenosylmethionine and high temperature resistance, which is applied in the field of high temperature resistant S-adenosylmethionine synthetase gene sequence and encoded polypeptide, and can solve the energy consumption of S-adenosylmethionine And other issues

Inactive Publication Date: 2002-11-13
HUADA GENE RES & DEV CENT HANGZHOU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Every time a molecule of S-adenosylmethionine is generated, the three phosphate groups of ATP must be hydrolyzed, which is also a common feature of ATP-dependent group transfer reactions, so S-adenosylmethionine The generation of is very energy-intensive

Method used

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  • Refractory S-adenosylmethionine synthetase gene and its polypeptide coded by it and preparing process
  • Refractory S-adenosylmethionine synthetase gene and its polypeptide coded by it and preparing process
  • Refractory S-adenosylmethionine synthetase gene and its polypeptide coded by it and preparing process

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Construction of a sequencing library

[0033] The sequencing library was constructed using the genome-wide shotgun method. Firstly, Tengchong thermophilic anaerobic bacteria were cultured according to (Yanfen Xue, 2000) modified MB medium (Balch et al., 1979), bacteria were collected according to Marmur (1961) method, and total DNA was extracted. In order to ensure the randomness of sequencing library construction and avoid the problem of hot spots of breakage to the greatest extent, a variety of methods and principles of library construction under different conditions were adopted. Firstly, physical shearing method (including ultrasonic method and shearing with Hydroshear Machine) was used, and then AluI was selected for random partial enzyme digestion according to the genome characteristics of the bacteria. Different intensities were used to process samples during physical shearing, and samples were processed by setting enzyme gradients during enzyme diges...

Embodiment 2

[0034] Example 2: Genome Sequencing

[0035] When sequencing the genome of thermophilic anaerobic bacteria in Tengchong, two automatic sequencers were mainly used: ABI377 and MegaBACE 1000. Both sequencers use the principle of electrophoresis for sequencing (see figure 2 ), 96 samples can be completed each time. ABI377 is a product of PE Company, which is a kind of ABI series. It belongs to the slab gel electrophoresis sequencer. MegaBACE 1000 is a product of Pharmacia, which belongs to capillary gel electrophoresis sequencer.

Embodiment 3

[0036] Example 3: Basecalling and sequencing quality monitoring

[0037] The so-called basecalling refers to the process of obtaining the correct base sequence from the raw data file obtained on the sequencer. Since the sequencer obtains the intensity change traces (traces) of light of different wavelengths corresponding to the four bases A, T, G, and C, it is necessary to use a computer to adopt a certain algorithm to correctly identify the bases corresponding to the different traces. . We used Phred software (Ewing B, Hillier L, 1998) because its results are more reliable and its output is more convenient for further analysis by other programs in the same software package.

[0038] The algorithm principle of Phred's basecalling is to judge the base type based on the shape, distance, and signal-to-noise ratio of each peak in the trajectory, and at the same time give the credibility information for this base, that is, the sequencing quality of the base. In large-scale sequen...

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Abstract

A process for preparing refractory S-adenosylmethionine synthetase gene and the polypeptide for coding it and the said gene used for preparing its transgenic microbe or animal and plant and recovering the enzyme coded by it, and in addition, the amino acid sequence of the said polypeptide with the activity of the said refractory S-adenosylmethionine synthetase and its functional analog, and the process based on the whole genom sequencing and analyzing of thermophilic anaerobe for preparing, separating and purifying the said polypeptide are disclosed. It also relates to coding of the separatedDNA with activity or its function identical variant and use of the recombinant DNA technique with the said DNA to produce the above-mentioned polypeptide or its function identical variant.

Description

technical field [0001] The invention relates to mutation or genetic engineering, in particular to a gene sequence of a high-temperature-resistant S-adenosylmethionine synthetase, a coded polypeptide and a preparation method. Background technique [0002] S-adenosylmethionine synthetase, S-adenosylmethionine synthetase (EC 2.5.1.6), which can be abbreviated as MAT, catalyzes L-methionine and adenosyl triphosphate (ATP) to generate S-adenosylmethionine Amino acid (AdoMet or SAM), simultaneously generating pyrophosphate and phosphoric acid. AdoMet is a ubiquitous methyl donor. The official name of S-adenosylmethionine synthetase is methionine adenosyltransferase. Every time a molecule of S-adenosylmethionine is generated, the three phosphate groups of ATP must be hydrolyzed, which is also a common feature of ATP-dependent group transfer reactions, so S-adenosylmethionine The generation of is very energy-intensive. [0003] 120 out of 130 methyltran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63
Inventor 李松岗王俊林霞汪建杨焕明
Owner HUADA GENE RES & DEV CENT HANGZHOU
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