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Gamma-butyrobetaine hydroxylase originated from neurospora crassa

一种碱羟化酶、丁基的技术，应用在γ-丁基甜菜碱羟化酶领域，能够解决副作用等问题

Active Publication Date: 2007-08-08
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this method can cause side effects in the body, because it contains D-carnitine (Curr.Ther.Res.28,195-198,1980)

Method used

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  • Gamma-butyrobetaine hydroxylase originated from neurospora crassa
  • Gamma-butyrobetaine hydroxylase originated from neurospora crassa
  • Gamma-butyrobetaine hydroxylase originated from neurospora crassa

Examples

Experimental program
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example 1

[0038] Example 1: Construction of Neurospora crassa cDNA library.

[0039] To obtain the cDNA of N. crassa, mRNA was first isolated from N. crassa, and cDNA was synthesized from the isolated mRNA by PCR (polymerase chain reaction) using polythymidine (polyT) primers. The cDNA was inserted into the EcoR1 / XhoI site of the AD5 cloning vector, and a cDNA library was constructed in the plasmid as follows. Escherichia coli BNN322 strain was cultured overnight in LB medium supplemented with kanamycin and 0.2% maltose, collected by centrifugation, and dissolved in 1ml of 10mM MgSO 4 in suspension. The bacterial suspension was mixed with 3.5×10 of the constructed cDNA library 7 The lambda phages were incubated together for 30 minutes at 30°C without stirring. Add 2 ml of LB medium to the culture, and incubate the infected strain at 30 °C for 60 min while stirring. The final culture was plated on LB plates containing ampicillin (75 μl- / ml). Plasmids were isolated from the grown col...

example 2

[0040] Example 2: Preparation of primers for obtaining γ-butylbetaine hydroxylase gene.

[0041] The amino acid sequence of γ-butylbetaine hydroxylase (γ-BBH) produced by N. crassa was compared with that of γ-BBH produced by human, rat and Pseudomonas ( FIG. 3 ). SEQ ID NO: 1 represents the amino acid sequence of γ-BBH produced by Neurospora crassa, SEQ ID NO: 2 represents the amino acid sequence of γ-BBH from human, SEQ ID NO: 3 represents the amino acid sequence of γ-BBH from murine, and SEQ ID NO: 4 represents the γ-BBH from Pseudomonas - the amino acid sequence of BBH. The sequence homology results are as follows (starting with a pairwise alignment):

[0042] Sequence (1:2) permutation, score: 11%;

[0043] Sequence (1:3) alignment, score: 11%;

[0044] Sequence (1:4) alignment, score: 10%;

[0045] Sequence (2:3) alignment, score: 88%;

[0046] Sequence (2:4) alignment, score: 29%;

[0047] Sequence (3:4) alignment, score: 29%.

[0048] The γ-BBH produced by Neuros...

example 3

[0054] Example 3: Obtaining the gene encoding γ-BBH.

[0055] From the Neurospora crassa cDNA library prepared in Example 1, the γ-BBH gene was amplified by the PCR method using a pair of primers prepared in Example 2. The PCR product was electrophoresed on an agarose gel, and a band was observed at about 1.4 kb. The nucleotide sequence of the amplified gene was determined using an automatic DNA sequencer. At the same time, the BLAST program of NCBI Company was used to perform homology search on the determined nucleotide sequences. As a result, a gene 100% identical to the amplified gene was found in the genome sequence of Neurospora crassa, and the function of the translation product of the found gene was only a hypothetical protein. The PCR product was then digested with EcoRI and Sal I, ligated with pUC19 digested with the same restriction enzymes, and introduced into E. coli DH5. Transformants were identified using blue / white screening. When the plasmid was isolated fr...

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Abstract

Disclosed is a polynucleotide encoding gamma-butyrobetaine hydroxylase ( gamma-BBH) originating from Neurospora crassa. Also disclosed are a recombinant vector comprising the polynucleotide, a transformant transformed with the recombinant vector, gamma-BBH encoded by the polynucleotide, and a method of preparing L-carnitine, which comprises hydroxylating gamma-butyrobetaine using gamma-BBH encoded by the polynucleotide.

Description

technical field [0001] The present invention relates to a gamma-butylbetaine hydroxylase (gamma-BBH) produced by Neurospora crassa. In particular, the present invention relates to a polynucleotide encoding γ-butylbetaine hydroxylase produced by Neurospora crassa, a recombinant vector comprising the polynucleotide, and a transformant transformed with the recombinant vector, obtained by The γ-butyl betaine hydroxylase encoded by the polynucleotide, and a γ-butyl betaine hydroxylase encoded by the polynucleotide to make γ-trimethylammonium butyric acid (γ-butyrobetaine ) Hydroxylation method for preparing L-carnitine. Background technique [0002] L-carnitine (3-hydroxy-4-trimethylammonium-butyric acid), also known as vitamin Bt, is a natural vitamin analogue that is very important in human metabolism. L-carnitine was first isolated from bovine muscle tissue by two Russian scientists Gulewitsch and Krimberg in 1905, and its chemical structure was determined in 1932. L-Carnit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/55C12N9/02C12P13/00
CPCC12N9/0071C12P13/007B23Q39/00B23Q5/043B23Q2039/002
Inventor 钟孙欧李宏克康万库久杰永寇运孙帕孙斯帕永宏
Owner CJ CHEILJEDANG CORP
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