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Enzyme protein for cutting sterol side chain and its application

A protein and fusion protein technology, applied in the direction of enzymes, introduction of foreign genetic material and peptides using vectors, etc., can solve the problems of expensive culture medium, unestablished production methods, and low productivity.

Active Publication Date: 2011-12-07
MITSUBISHI RAYON CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Animal-derived enzyme protein has low enzyme protein activity, and in addition, its production method has not been established due to factors such as expensive medium and slow growth rate for the culture of host cells
In addition, a method of transforming yeast with bovine-derived P450scc and converting cholesterol into pregnenolone in the presence of corticoferredoxin and corticoferredoxin reductase constituting the electron transport system has been disclosed, but Its productivity is very low, and productivity at an industrial level has not been obtained (Patent Document 1)

Method used

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  • Enzyme protein for cutting sterol side chain and its application
  • Enzyme protein for cutting sterol side chain and its application
  • Enzyme protein for cutting sterol side chain and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0150] Example 1: Obtaining DNA encoding P450scc (CYPSS204A) and ferredoxin derived from Sphingomonas subterraneanus NBRC16086

[0151] Sphingomonas subterraneanus NBRC 16086 (Department of Biogenetic Resources, Biotechnology Headquarters, Independent Administrative Agency Product Evaluation Technology Foundation) was inoculated in L medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% Glucose, pH 7.2), cultivate overnight at 30°C, and extract genomic DNA from the resulting cells. Genomic DNA was extracted using a DNA extraction kit ISOPLANT (manufactured by Wako Pure Chemical Industries, Ltd.). For this genomic DNA, polymerase chain reaction (PCR) was performed using LA Taq polymerase (manufactured by TAKARA Bio Co., Ltd.) using primer CYP-1F (SEQ ID NO: 5) and primer CYP-2R (SEQ ID NO: 6), A portion of the DNA encoding CYPSS (also sometimes referred to as the "CYPSS204A gene") is amplified. The temperature conditions at this time were: 94°C / 3 minutes, 30 cycles (94°C / 3...

Embodiment 2

[0153] Embodiment 2: Making the Escherichia coli expression strain BP215 of P450scc (CYPSS204A) derived from Sphingomonas subterranea NBRC 16086

[0154] From the Sphingomonas subterranean NBRC 16086 genomic DNA obtained in embodiment 1, utilize primer CYP-3F (SEQ ID NO: 7) and primer CYP-4R (SEQ ID NO: 8), use KODplus polymerase (Toyo (manufactured by Japan Textile Co., Ltd.) PCR was performed to amplify a DNA region including CYPSS204A and the ferredoxin gene present downstream of it. The temperature conditions at this time were: 95° C. / 5 minutes, 30 cycles of (98° C. / 30 seconds, 60° C. / 30 seconds, 68° C. / 2 minutes), and 68° C. / 10 minutes.

[0155] The amplified 1.8 kb DNA fragment was purified using the QIAquick PCR purification Kit (manufactured by QIAGEN), digested with Nde I and Spe I, and combined with T4 DNA ligase The Escherichia coli expression vector pT7NS-camAB (International Publication No. 2003 / 087381 pamphlet) was ligated and transformed into Escherichia coli D...

Embodiment 3

[0156] Example 3: Obtaining DNA encoding P450scc (CYP204A1) from Sphingomonas aromaticolyticus ATCC 700278 and making Escherichia coli expression strain BP172

[0157]Sphingomonas arolytica (ATCC 700278) was inoculated in L medium, cultured at 30° C. for 2 days, and genomic DNA was extracted from the obtained bacteria as in Example 1. Using primer CYP-5F (SEQ ID NO: 9) and primer CYP-6R (SEQ ID NO: 10), and LA Taq polymerase (manufactured by TAKARA Bio), amplified from the genomic DNA containing the CYP204A1 gene and present in Its downstream ferredoxin gene fragment. The temperature conditions at this time were: 94°C / 3 minutes, 30 cycles (98°C / 20 seconds, 63°C / 30 seconds, 68°C / 2 minutes), 72°C / 5 minutes. The 1.8 kb base sequence is shown in SEQ ID NO:4. Its base number 1-1422 is CYP204A1, and 1433-1765 is the ferredoxin gene.

[0158] The 1.8 kb DNA fragment amplified as a result of this reaction, including the CYP204A1 gene and the ferredoxin gene present downstream there...

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Abstract

P450scc enzyme protein is an important enzyme protein which can catalyze the first stage in an industrially useful steroid hormone biosynthesis. The invention aims to obtain P450scc enzyme protein having a high activity. Specifically disclosed is a sterol side chain-cleaving enzyme protein having the following physicochemical properties (1) and (2): (1) activity: the enzyme protein can act on a sterol represented by formula (I) defined in the description to cleave the bond between a carbon at position-20 and a carbon at position-22 in the sterol side chain moiety in the sterol, thereby producing a compound represented by formula (II) defined in the description; and (2) substrate specificity: when a microorganism capable of producing the enzyme protein is reacted in an aqueous solution containing 100 [mu]g / ml of 4-cholesten-3-one or cholesterol at 28 DEG C for 5 hours, the rate of conversion reaction from 4-cholesten-3-one into progesterone is 10% or more and the rate of conversion from cholesterol into pregnenolone is 10% or more.

Description

technical field [0001] The present invention relates to an enzyme protein that cleaves the bond at the 20th and 22nd positions of the sterol side chain to produce pregnenolone, etc., which are industrially valuable as medicine or pharmaceutical intermediates, DNA encoding the enzyme protein, and incorporating the DNA into A transformant obtained by using the vector, a method for producing pregnenolone and the like using the enzyme protein, and a method for producing cortisol and its derivatives, and the like. Background technique [0002] 11β, 17α, 21-trihydroxy-4-pregnene-3,20-dione (cortisol) and its precursors, pregnenolone, progesterone, and 7-dehydropregnenolone are all used as medicines Industrially valuable compounds and pharmaceutical intermediates. As a conventional production method of pregnenolone, a naturally-derived sterol compound such as cholesterol, diosgenin, and stigmasterol is used as a raw material and synthesized by a plurality of organic synthesis reac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K19/00C12N1/21C12N9/06C12P21/02C12P33/16
CPCC12N15/70C12P33/12C12N15/75C12N9/0077C12N9/0081
Inventor 山岸正博阪本刚上田诚伊藤将士藤井良和株本浩树有泽章藤井匡
Owner MITSUBISHI RAYON CO LTD
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