JPH0731471A - Artificially fused enzyme - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme, having specific amino acid sequences, capable of enhancing the activity in the monatomic oxygenating reaction and useful for an oxidizing reactional process for a useful substance and the oxidizing removal, etc., of toxic substances in industrial waste water. CONSTITUTION:This enzyme has an amino acid sequence coding a human hepatic cytochrome P450-3A4 on the N-terminal side and an amino acid sequence of coding a yeast NADPH-cytochrome P450 reductase on the C-terminal side. Furthermore, a yeast strain having a plasmid, expressible in the yeast, containing an artificially fused enzymic gene having a base sequence coding the human hepatic cytochrome P450-3A4 on the N-terminal side and a base sequence coding the yeast NADPH-cytochrome P450 reductase on the C-terminal side and transduced thereinto is preferably cultured to produce this enzyme.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an artificial fusion enzyme and a method for producing the same, an artificial fusion enzyme gene thereof, a plasmid containing the artificial fusion enzyme gene and a yeast expression plasmid, and a yeast strain into which the artificial fusion enzyme gene is introduced. It is about.

[0002]

2. Description of the Related Art Cytochrome P-450 is a heme protein widely existing in the living world from microorganisms to mammals, and catalyzes a one-atom oxygenation reaction using a wide range of fat-soluble compounds as substrates. This broad substrate specificity of cytochrome P-450 is due to the molecular diversity of cytochrome P-450. That is, cytochrome P-450 has a large number of molecular species, each of which has a wide range of substrate specificity and overlaps with each other, and a wide range of fat-soluble compounds can be used as substrates. However, the pathway for supplying electrons to many cytochrome P-450s is common, and in liver microsomes, mainly NADPH-containing flavin adenine mononucleotide and flavin mononucleotide as coenzymes in the molecule.
Cytochrome P-450 reductase supplies electrons from NADPH to the substrate-bound cytochrome P-450. Generally, the amount of NADPH-cytochrome P-450 reductase in liver microsomes cannot be said to be sufficient, and electron transfer from NADPH-cytochrome P-450 reductase to cytochrome P-450 becomes the rate-determining monoatomic oxygenation reaction. , Low activity per cytochrome P-450 molecule.

[0003]

However, by utilizing cytochrome P-450, the oxidation reaction process of useful substances,
Further, in the case of practical application to oxidative removal of harmful substances in industrial wastewater, it was necessary to further increase the activity of the one-atom oxygen addition reaction of cytochrome P-450.

[0004]

[Means for Solving the Problems] In view of the above situation, the present inventors have conducted extensive studies in order to find a cytochrome P450 having more excellent one-atom oxygen addition activity, and as a result,
Yeast NADPH-cytochrome P4 and a gene having a nucleotide sequence encoding a species of cytochrome P450 derived from human liver
50 By connecting genes having a nucleotide sequence encoding a reductase into a single gene, the one-atom enzyme addition activity of certain cytochrome P450s and the supply of reducing power from NADPH possessed by NADPH-cytochrome P450 reductase Constructing an artificial fusion enzyme gene encoding an artificial fusion enzyme having the ability in the same molecule, obtaining a yeast expression vector relating to this gene, and further obtaining a yeast strain into which the yeast expression vector has been introduced, and adding the artificial fusion enzyme to the yeast strain. Succeeded in producing. It was confirmed that the produced artificial fusion enzyme has both functions of electron transfer and substrate oxidation in a single molecule and is an artificial new enzyme having excellent properties, and completed the present invention. . That is, the present invention has an artificial fusion enzyme having an amino acid sequence encoding human liver cytochrome P450 3A4 on the N-terminal side and an amino acid sequence encoding yeast NADPH-cytochrome P450 reductase on the C-terminal side, and a method for producing the same. The artificial fusion enzyme gene, a plasmid containing the artificial fusion enzyme gene and a yeast expression plasmid, and a yeast strain into which the artificial fusion enzyme gene is introduced are provided.

The gene having a nucleotide sequence encoding human liver cytochrome P450 3A4 used in the present invention is obtained, for example, by PCR from a commercially available human liver-derived cDNA library.
It can be isolated using a conventional method such as a method.

The gene having the nucleotide sequence encoding the yeast NADPH-cytochrome P450 reductase used in the present invention can be isolated by, for example, the method described in JP-A-62-19085.

[0007] The gene having the nucleotide sequence encoding the human liver cytochrome P450 3A4 encoding the above human liver cytochrome P450 3A4 and the gene having the nucleotide sequence encoding the yeast NADPH-cytochrome P450 reductase are connected by a usual gene manipulation method. As a single gene, human liver cytochrome
P450 3A4 has one-atom enzyme addition activity and yeast NADPH-
An artificial fusion enzyme gene encoding an artificial fusion enzyme having the ability to supply reducing power from NADPH possessed by cytochrome P450 reductase is constructed.

The promoter for expression in yeast is not particularly limited as long as it is a promoter used in ordinary yeast expression systems. For example, a promoter of yeast alcohol dehydrogenase gene (hereinafter referred to as ADH promoter and ), Glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as GAPDH promoter), phosphoglycerate kinase (hereinafter referred to as PGK).
Described as a promoter. ) And so on. The ADH promoter is, for example, a yeast expression vector containing the yeast ADH1 promoter and the terminator.
pAAH5 (Available from Washington Research Fundation, Ammerer et al., Method in Enzymology, 101part (p. 1
92-201)], and can be prepared by a usual gene manipulation method. The yeast ADH1 promoter is a product of the Washington Res
Included in earch Fundation's US Patent Application No. 299,733, for use in the United States for industrial and commercial purposes requires permission from the right holder.

[0009] The yeast expression plasmid containing the above-mentioned promoter for expression in yeast and the above artificial fusion enzyme gene can be constructed using a conventional gene recombination method. For example, a yeast expression vector pAAH5N having the ADH promoter and terminator described in Japanese Patent Laid-Open No. 2-211880 is used as the artificial fusion enzyme gene.
The method of constructing by inserting into the Hind III site of The yeast expression plasmid thus obtained, for example, a method using alkali metal (LiCl),
It is introduced into the yeast strain by a conventional method such as the protoplast method.

The artificial fusion enzyme of the present invention can be produced by culturing the yeast strain into which the expression plasmid in yeast thus obtained is introduced. The culture can be performed by a usual culture method.

The yeast strain used in the present invention is, for example, Saccharomyces cerevisiae.
myces cerevisiae) and the like. Preferably Saccharomyces cerevisiae AH22 strain (ATCC38626),
Saccharomyces cerevisiae SHY 3 strain, Saccharomyces cerevisiae NA87-11A strain and the like can be mentioned.

The artificial fusion enzyme of the present invention thus produced can be extracted and purified from the cultured cells by a conventional method. For example, the microbial cells are treated with a lysing enzyme such as zymolyase to form spheroplasts, which are then crushed by a mechanical method using ultrasonic treatment, French press, glass beads to prepare a microsome fraction. It can be purified by conventional methods such as ion exchange column chromatography such as DEAE-cellulose column chromatography and 2 ', 5'-ADP sepharose 4B column chromatography using the characteristics of yeast NADPH-cytochrome P450 reductase moiety. it can.

Examples will be described below in more detail, but the present invention is not limited to these examples.

Example 1 Acquisition of Human Liver Cytochrome P450 3A4 Gene and Construction of Yeast Expression Plasmid From a human liver-derived cDNA library (Clontech), FIG.
Two fragments of about 0.6 kb and about 0.9 kb were amplified using the primers shown in 1. The obtained fragment of about 0.6 kb was digested with SacI and subcloned into pUC118 vector. Then, it was digested with EcoRI, blunted, and XbaI linker was introduced into the 0.9 kb fragment digested with XbaI and SacI.
The two fragments were ligated. This plasmid was cleaved with SphI, blunted, and an XbaI fragment was excised from the one into which the XbaI linker had been introduced and inserted into the XbaI site of pUCANX. This is No
pAAH5N and pAHRR which were cut out at tI and similarly treated with NotI
Human liver cytochrome P450 3A4
Yeast expression plasmid p3A4 and yeast NADPH-cytochrome
A plasmid p3A4R co-expressed with P450 reductase was constructed
(See Figure 2).

Example 2 Construction of yeast expression plasmid containing artificial fusion enzyme gene A plasmid was constructed according to FIG. Plasmid p3
XbaI-X using A4 as a template and the primers shown in FIG.
The hoI fragment was obtained. In addition, plasmid pBFCR1 (Japanese Patent Application No. 4-20
The approximately 2.1 kb XhoI-HindIII fragment obtained from 9226) was inserted into the commercially available vector Blue Script (+) at the XhoI and HindIII sites and then co-digested with restriction enzymes XhoI and XbaI to obtain a fragment. The plasmid obtained by inserting both of these fragments into the XbaI site of vector pUCAN at the same time
Digestion with tI gave a fragment of approximately 5.6 kb. Approximately 10.5 kb of this fragment and the vector pAAH5N (JP-A-2-211880)
The desired yeast expression plasmid pF3A4 was obtained by ligating the tI fragments. The artificial fusion enzyme consists of 1156 amino acid residues, the structure of which is from the N terminus to human liver cytochrome P4.
The entire amino acid sequence encoding 503A4 (residue 503), the sequence derived from the linker (Ala-Arg-Ala), the yeast NADPH-cytochrome P450 reductase N-terminal from the 42nd to the C-terminal are continued.

Example 3 Introduction of yeast expression plasmid pF3A4 into yeast 1.0 ml of YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) was transformed into Saccharomyces cerevisiae.
AH22 strain was inoculated, shaken at 30 ℃ for 18 hours, and then centrifuged.
The cells were collected (5,000 × g, 10 minutes). The obtained bacterial cells
Suspend in 1.0 ml 0.2M LiCl solution, then centrifuge again
(5000 × g, 10 minutes), and add 20 μl of 1 to the resulting pellet.
M LiCl solution, 30 μl of 70% polyethylene glycol 400
0 (Wako Pure Chemical Industries, Ltd.) solution, about 1.0 μg of each of the various yeast expression plasmids obtained in Examples 1 and 2 was added in an amount of 10 μl. After mixing this well, incubate at 30 ℃ for 1 hour, and
140 μl of sterile water was added and stirred. This solution was added to an SD synthetic medium plate [2.0% glucose, 0.67% nitrogen source amino acid-free (Nitrogen base w / o amino acids, manufactured by Difco),
20 μg / ml histidine 2.0% agar], and incubated at 30 ° C. for 3 days to select a transformed yeast strain carrying the above yeast expression plasmid. In this way
Various yeast cells expressing human liver cytochrome P450 3A4 were prepared in yeast.

Example 4 Quantification of artificial fusion enzyme expressed in yeast 200 ml of a culture solution of yeast expressing the artificial fusion enzyme (SD synthesis medium, cell concentration 1.5 × 10 ⁷ cells / ml) was collected. , 10ml 100m
After suspending in M potassium phosphate buffer (pH 7.0), it was centrifuged (5000 × g, 10 minutes). The obtained pellet was newly suspended in 2.0 ml of 100 mM potassium phosphate buffer (pH 7.0), and 1.0 ml was dispensed into each of two cuvettes. After blowing carbon monoxide into the cuvette on the sample side, 5-10 mg of dithionite was added to both cuvettes, and the difference spectrum of 400-500 nm was measured while stirring and the artificial fusion enzyme concentration was calculated. The expression level of artificial fusion enzyme per cell is approximately
It was 1.5 × 10 ⁵ molecules.

Example 5 Preparation of Yeast Microsome Fraction A culture solution of each of the various yeast cells prepared in Example 3 (SD synthesis medium, cell concentration of about 1.0 × 10 ⁸ cells / ml) 3.
81 was collected and the cells were mixed with 400 ml of buffer solution A (10 mM Tris-HCl).
(pH7.5), 2M sorbitol, 0.1mM DTT, 0.2mM EDT
After suspending in A), 160 mg of ZYMOLYACE 100,000 (Zym
olyase 100T; Seikagaku Corporation)) was added and incubated at 30 ° C. for 60 minutes. The spheroplasts obtained by centrifugation (5000 xg, 10 minutes) were suspended in 100 ml of buffer solution A and then centrifuged (5000 xg, 10 minutes) again. The same centrifugation operation was repeated once again to wash the spheroplasts, and then the spheroplasts were washed with 200 ml of the buffer solution (10 mM).
Tris-HCl (pH 7.5), 0.65M sorbitol, 0.1 mM DT
T), and the suspension was sonicated (50w, 5 minutes). The supernatant obtained by centrifugation (10,000 xg, 20 minutes) was further subjected to ultracentrifugation (125,000 xg, 70 minutes) to recover the precipitate. 0.1M potassium phosphate buffer for the precipitation
A microsome fraction was obtained by adding 10 ml of (pH 7.4) and suspending.

Example 6 Measurement of Testosterone Hydroxylation Activity in Transformed Yeast Strains (Live Cells) Carrying Various Yeast Expression Plasmids Various yeast cells produced in Example 3 (artificial fusion enzyme expressing strains, Human liver cytochrome P450 3A4 and yeast NADPH-
Three types of strains, co-expressing cytochrome P450 reductase and control strain, each of the culture solution (SD synthesis medium,
Testosterone was added to 2 ml of the cell concentration of about 2.0 × 10 ⁷ cells / ml) so that the final concentration was 0.05 mM, and after incubation at 30 ° C. for 15 hours, 4 ml of dichloromethane was added to the reaction solution, and well. After stirring, the mixture was centrifuged (5000 xg, 10 minutes). The dichloromethane layer was recovered from the separated layer, and the recovered product was analyzed by HPLC. The analysis conditions are shown below. 1. Column: μBondapak C18 (φ4x300mm, Waters) 2. Elution condition: Acetonitrile 20% -70% aqueous solution Linear concentration gradient / 25 minutes 3. Flow rate: 1.5 ml / min 4. Detection: Absorbance at 254 nm As a result, testosterone hydroxide was detected in the artificial fusion enzyme-expressing strain and in the strain simultaneously expressing human liver cytochrome P450 3A4 and yeast NADPH-cytochrome P450 reductase. It was also found that the activity per artificial fusion enzyme molecule was about 30 times as high as the activity per human liver cytochrome P450 3A4 molecule in a strain that co-expressed human liver cytochrome P450 3A4 and yeast NADPH-cytochrome P450 reductase. on the other hand,
Various artificial fusion enzymes known in the art, such as rat liver cytochrome P450c and rat NADPH-cytochrome P450 reductase (DNA, Vol. 6, p31-39, 1987), bovine adrenal P4
In yeast expression of artificial fusion enzyme of 5017α and yeast NADPH-cytochrome P450 reductase (DNA Cell. Biol., Vol. 9, p27-36, 1990), the activity per artificial fusion enzyme molecule was The activity was about three times the activity per molecule of cytochrome P450 in the strain that co-expressed cytochrome P450 and NADPH-cytochrome reductase.

[0020]

INDUSTRIAL APPLICABILITY The yeast strain into which the artificial fusion enzyme gene of the present invention has been introduced remarkably enhances the activity of human liver cytochrome P4503A4 in the one-atom oxygenation reaction, and the oxidation reaction process of useful substances or harmful substances in industrial wastewater It has become possible to be applied practically to the oxidative removal of

[Brief description of drawings]

FIG. 1 is a diagram showing primers for cloning human liver cytochrome P450 3A4 gene.

FIG. 2 is a diagram showing a method for constructing a human liver cytochrome P450 3A4 yeast expression plasmid p3A4 and a yeast NADPH-cytochrome P450 reductase co-expression plasmid p3A4R.

FIG. 3 is a diagram showing a method for constructing a yeast expression plasmid pF3A4 containing an artificial fusion enzyme gene.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1: 865) (C12N 1/19 C12R 1: 865)

Claims (8)

[Claims] 1. An N-terminal side having an amino acid sequence encoding human liver cytochrome P450 3A4 and a yeast NA side at the C-terminal side.
An artificial fusion enzyme having an amino acid sequence encoding DPH-cytochrome P450 reductase. 2. A nucleotide sequence encoding human liver cytochrome P450 3A4 on the N-terminal side, and yeast NADPH- on the C-terminal side.
An artificial fusion enzyme gene having a nucleotide sequence encoding a cytochrome P450 reductase. 3. A plasmid containing the gene according to claim 2. 4. A yeast expression plasmid containing a promoter for expression in yeast and the gene according to claim 2. 5. pF3A4. 6. A yeast strain into which the gene according to claim 2 has been introduced. 7. Saccharomyces cerevisiae AH22 (pF3A
Four). 8. A yeast strain into which the yeast expression plasmid according to claim 4 has been introduced is cultured.
A method for producing the artificial fusion enzyme described.