RU2173708C2 - Dna fragment encoding polypeptide with activity of nitrile hydratase, recombinant plasmid dna pnhj20l encoding polypeptide, strain escherichia coli tg1/pnhj20l, method of preparing polypeptide with nitrile hydratase property - Google Patents

Abstract

FIELD: molecular biology, biotechnology, genetic engineering. SUBSTANCE: invention relates to amino acid sequences of α-and β-subunits of polypeptide with activity of nitrile hydratase and DNA fragments encoding this polypeptide. Also, invention relates to recombinant plasmid DNA pNHJ20L encoding polypeptide eliciting properties of nitrile hydratase, transformer containing recombinant plasmid DNA and method of nitrile hydratase preparing. DNA fragment encoding polypeptide with nitrile hydratase activity is inserted into plasmid vector, strain E. coli is transformed using plasmid pNHJ20L and the obtained strain TG1/pNHJ20L (FERM BP-2778) is able to produce polypeptide with properties of nitrile hydratase. EFFECT: increased yield of polypeptide of strain as compared with that in microorganisms used in common practice. 5 cl, 4 dwg

Description

 The invention relates to a DNA fragment obtained from Rhodococcus rhodochrous and encoding a polypeptide with nitrile hydratase activity that hydrolyzes nitrile to amide. The invention also relates to recombinant DNA containing the above DNA fragment, and a transformant containing recombinant DNA.

 The present invention also relates to a method for producing nitrile hydratase using a transformant.

Prior art
Nitrile hydratase or nitrilase is known as an enzyme that hydrolyzes nitriles to amides. Microorganisms forming nitrile hydratase include those belonging to the genus Bacillus, the genus Bacteridium, the genus Micrococcus, and the genus Brevibacterium (see JP-B-62-21517 / 1989, USP N 4001081), the genus Corynebacterium, and the genus Nocardia (see JP-B-56 -17918/1989, USP N 4248968), the genus Pseudomonas (see JP-B-59-37951 / 1984), USP N 4637982), the genus Rhodococcus, the genus Arthrobacter and the genus Microbacterium (see JP-A-61-162193 / 1986, EP-A-0188316) and Rhodococcus rhodochraus (see JP-A-2470/1990, EP-A-0307926).

 Nitrile hydratase is used to hydrolyze nitriles to amides. In the invention, microorganisms are transformed so that they contain multiple copies of recombinant DNA encoding a nitrile hydratase using biotechnology. The resulting recombinant produces nitrile hydratase at a significantly higher level compared to commonly used microorganisms.

 The creators of the present invention previously described a DNA fragment obtained from Rhodococcus N-774 (FERM BP-1936), which also encodes a polypeptide with nitrile hydratase activity JP-A-2-119778 / 1988).

 In contrast to the above link, the present invention uses a DNA fragment obtained from Rhodococcus rhodochrous J-1 to produce nitrile hydratase. We have isolated a gene encoding nitrile hydratase, the gene has been introduced into an acceptable plasmid vector by which the corresponding host has been transformed, i.e. successively obtained transformant producing nitrile hydratase with high activity also with respect to aromatic nitriles.

Summary of the invention
The present invention relates to:
(1) a DNA fragment ^(L) encoding a polypeptide with nitrile hydratase activity, the polypeptide comprising α ^(L) and β ^(L) subunits of amino acid sequences 1 and 2 (see the end of the description);
(2) recombinant DNA, including in the vector of DNA ^(L) according to paragraph (1);
(3) a transformant containing recombinant DNA according to paragraph (2);
(4) a method for producing nitrile hydratase, which consists in cultivating the transformant described in paragraph (3) and isolating nitrile hydratase from the culture.

Below the present invention is described in more detail. The invention is carried out by carrying out steps (1) to (8):
(1) Isolation and purification of nitrile hydratase and partial amino acid sequencing of nitrile hydratase.

 Two types of nitrile hydratase (designated H-type and L-type, respectively) are isolated and purified from Rhodococcus rhodochrous J-I (FERM BP-1478) and both enzymes are separated by HPLC into α and β subunits. A portion of the amino acid sequence of each subunit is determined (FIG. 1).

 (2) Obtaining a DNA probe for the nitrile hydratase gene.

 The DNA probe is obtained from JM105 / pV UK121 (FERM BP-1937) according to the procedure of JP-A-2-119778 / 1990, which is associated with a high degree of homology in the amino acid sequence between the nitrile hydratase of the β subunit of Rhodococcus N-774, described in this publication from the Official Gazette of Japanese Patents, and the subunits of Rhodococcus rhodochrous J-1. Plasmid pVUK121 containing the nitrile hydratase gene derived from Rhodococcus N-774 was obtained from the JM105 / pVU-K121 culture, pVU K-121 DNA was digested with SphI and SalI SphI-SalI — the fragment contains the Rhodococcus nitrile hydratase gene (Fig. N-7. 2) a DNA fragment is radiolabeled.

 (3) Detection of a DNA segment containing a nitrile hydratase gene from Rhodococcus rhodochrous J-I chromosomes. Chromosomal DNA is obtained from a culture of Rhodococcus rhodochrous J-1.

 Chromosomal DNA is hydrolytically digested with restriction enzymes and hybridized with a probe according to the Southern hybridization technique (Southern EM, J. Mol. Biol. 98, 503 (1975)).

 Two DNA fragments of different lengths are selected.

 (4) Construction of a recombinant plasmid.

 A recombinant plasmid is constructed by inserting a chromosomal DNA fragment of step (3) into a plasmid vector.

 (5) Transformation and selection of a transformant containing a recombinant plasmid.

 The transformant is obtained using the recombinant plasmid of step (4). The transformant containing the recombinant plasmid was selected using the probe of step (2) according to the hybrid colony technique (R Bruce Wallace et al., Nuc. Aci.Res. 9, 879 (1981)). In addition, the presence of the nitrile hydratase gene is confirmed by Southern hybridization. Selected plasmids are designated as pNHJ 20L.

 (6) Isolation and purification of plasmid DNA and construction of a restriction map.

 The plasmid DNA pNHJ20L obtained in step (5) is isolated and purified. A restriction map of this DNA is constructed (FIG. 3) to determine the region containing the nitrile hydratase gene.

 (7) DNA sequencing.

 The DNA fragment inserted into plasmid pNHJ20L was cleaved using the appropriate restriction enzyme and sequenced. The nucleotide sequence of the DNA fragment (FIGS. 4 and 5) indicates that it contains the sequence traced on the basis of the amino acid sequence of step (1).

 (8) Preparation of a nitrile hydratase transformant.

 The transformant of step (7) is cultivated.

 Rhodococcus rhodochrous J-1 was deposited at the Fermentation Research Institute of the Agency for Industrial Sciences and Technology, where it was assigned registration number FERM BP-1478. The TGI / NHJ20L transformant containing pNHJ20L of step (5) was deposited there and assigned the registration number FERM BP-2778.

 Any vectors can be used, including: a plasmid vector (e.g., AT153, pMP9, pHC624, pKC7, etc.), a phage vector (e.g., λgt II (toyobo), Charon 4A (Amersham), etc. ) Enzymes that can be used include: SphI, SalI, EcoRI, SeaI, BumHI and the like, manufactured by the industry (Takara Shuzo). A variety of recipients can be used for transformation, including, but not limited to: E.Coli, Jm105, and E.Coli TGI.

 The culture medium for the transform is usually used for such purposes environments.

 The conversion of nitriles to amides is carried out using nitrile hydratase, crude nitrile hydratase, a culture of a transform, isolated bacterial cells or treated cell material and the like obtained from a transform culture.

 Suitable nitriles for the invention include: aromatic nitriles with 4-10 carbon atoms in an aromatic moiety and aliphatic nitriles with 2-6 carbon atoms described in European Patent Publication No. 0307926. Typical examples of nitriles include: 4-, 3- and 2-cyanopyridine, benzonitrile, 2,6-difluorobenzonitrile, 2-thiophenecarbonitrile, 2-furonitrile, cyanopyrazine, acrylonitrile, methacrylonitrile, crotonitrile, acetonitrile and 3-hydroxypropionitrile.

 Technical Results

 The amino acid sequence of the α- and β-subunits of nitrile hydratase derived from Rhodococcus rhodochrous J-1 is described. A DNA fragment encoding a nitrile hydratase is inserted into an expression vector and a recombinant vector is used for transformation. The transformant contains many copies of the gene and is capable of producing nitrile hydratase at a much higher level compared to commonly used microorganisms.

 Explanations for the drawings.

 In FIG. 1 shows the N-terminal amino acid sequences of the α- and β-subunits of nitrile hydratase produced by Rhodococcus rhodochrous J-1.

 In FIG. 2 shows the DNA sequence of the Rhodococcus N-774 nitrile hydratase gene used as a DNA probe.

 In FIG. 3 shows a restriction map of the recombinant plasmid pNHJ20L.

 In FIG. Figure 4 shows the DNA sequence of a DNA fragment in pNHJ201 derived from Rhodococcus rhodochrous J-I; and the amino acid sequence corresponding to a given nucleotide sequence.

 The present invention is illustrated in detail by the following example, which is not intended to limit the invention.

The following abbreviations are used in the example:
TE - Tris-HCl (10 mm, pH 7, 8), EDTA (1 mm, pH 8),
TNE - Tris-HCl (50 mM, pH 8), EDTA (1 mM, pH 8), NaCl (50 mM),
STE - Tris-HCl (50 mM, pH 8), EDTA (5 mM, pH 8), sucrose (35 mM),
2xVT medium - 1.6% Tryptone, 1% yeast extract, 0.5% NaCl.

Example
(1) Isolation and purification of nitrile hydratase and partial amino acid sequencing of nitrile hydratase.

Rhodococcus rhodochrous J-1 is cultivated for 80 hours in medium (3 g / l yeast extract, 0.5 g / l KH ₂ PO ₄ 0.5 g / l K ₂ HPO ₄ , 0.5 g / l MgSO ₄ • 4H ₂ O, 0.01 g / l CoCl ₂ and 3 g / l crotonamide, pH 7.2). Bacterial cells are harvested. 50 g of bacterial cells are destroyed and fractionated with ammonium sulfate. The sample is dialyzed and the dialysate is centrifuged. The liquid portion is chromatographed on a DEAE-Cellophin column, a phenyl-Sepharose column, a Sephadex C-150 column, and an Octyl-Sepharose column. Two fractions with enzymatic activity are obtained and dialysed. The dialysate is transferred to a reversed-phase high performance liquid chromatography column (Senshu Pak VP-304-1251, Senshu Kadaku) and two subunits (α and β), respectively, are obtained. Using the protein sequencer model 470A applied biosystems determine the N-terminal amino acid sequence α $\genfrac{}{}{0ex}{}{\text{(H)}}{\text{1}}$ β $\genfrac{}{}{0ex}{}{\text{(H) -}}{\text{1}}$ , α $\genfrac{}{}{0ex}{}{\text{(L) -}}{\text{1}}$ and β $\genfrac{}{}{0ex}{}{\text{(L)}}{\text{1}}$ subunits. Amino acid sequences are shown in FIG. 1.

 (2) Obtaining a DNA probe for the nitrile hydratase gene.

B 100 ml of 2xUT medium containing 50 μg / ml ampicillin was cultured for 12 hours at 30 ^° C. E. Coli JM105 (FERM BP-1937) containing pYU121. Bacterial cells are harvested and TNE added. The cell suspension is then centrifuged. 8 ml of STE and 10 mg of lysozyme are added to the solid. The mixture was incubated for five minutes at 0 ^° C., after which 4 ml of 0.25 M EDTA was added. Then, 2 ml of 10% SDS and 5 ml of M NaCl are added to the mixture at room temperature. The resulting mixture was incubated for three hours at 0-4 ^o C and then ultracentrifuged. 1/2 volume of PEG 6000 is added to the liquid portion. The mixture is incubated for 12 hours at 0-4 ^° C. and centrifuged. TNE was added to the solid part, bringing the volume to 7.5 ml, after which CSCl was added to the suspension. The mixture is centrifuged to remove proteins. Then 300-500 mg / ml of ethidium bromide is added to the liquid part. The mixture is transferred to a centrifuge tube, the tube is sealed by heating and ultracentrifuged. Using a peristaltic pump, covalently closed circular DNA is extracted. To the extract is added isopropyl alcohol saturated with water in an amount slightly exceeding an equal amount, with the extraction of ethidium bromide. The sample is dialyzed according to TE and 3 ml of purified plasmid pVUK12L is obtained.

pVUK12I DNA is hydrolytically digested with SphI and SalI to give a 2.07 kbp DNA fragment containing a nitrile hydratase gene derived from Rhodococcus N-774. Fragment radiolabeled ³² P to obtain a probe. The nucleotide sequence of the probe is shown in FIG. 2.

 (3) Obtaining a DNA fragment containing the chromosomal nitrile hydratase gene.

B 100 ml of medium (10 g / l glucose, 0.5 g / l KH ₂ PO ₄ , 0.5 g / l K ₂ HPO ₄ , 0.5 g / l MgSO ₄ • 7H ₂ O, 1 g / l yeast extract, 7.5 g / l peptone, 0.01 g / l CoCl ₂ , 7.5 g / l urea, 1% glycine or 0.3 μg / ml ampicillin, 1 l water, pH 7.2) cultivate Rhodococcus rhodochrous J-1. Bacterial cells are harvested and the solid is washed with TNE. Then the solid part is suspended in 10 ml of TE. 4 ml of 0.25 M EDTA, 10-20 mg of lysozyme, 10-20 mg of achromoprotease and 10 ml of 10xSDS are added to the suspension. The suspension is incubated for three hours at 37 ^° C. and then 15 ml of phenol is added to it. The mixture was incubated for 15 minutes at room temperature and then centrifuged. The top layer was removed and 0.7 ml of 2.5 M sodium acetate and diethyl ether were added to the supernatant. The mixture is centrifuged and the top layer discarded. Two volumes of ethanol are added to the bottom layer and the DNA is extracted with a glass rod. DNA is rinsed for five minutes in each case with a mixture of TE-ethanol in a ratio of 2: 8, 1: 9 and 0: 9 (vol./vol.). Then, the DNA was resuspended in 2-4 ml TE (37 ^° C). 10 μl of a mixture of RNase A and T _{1 was} added to the suspension, and the mixture was incubated at 37 ^° C. An equal amount of phenol was added to the mixture, and then centrifuged. More than equal amounts of ether are added to the supernatant. The mixture is again centrifuged, the upper layer discarded, and the lower layer maintained. The lower layer is dialyzed for about a day in 2 L of TE containing a small amount of chloroform, and additionally dialyzed in fresh TE for 3-4 hours. 4 ml of crude chromosomal DNA are obtained.

To 15 μl of crude chromosomal DNA, 10 μl of TE, 3 μl of reaction buffer (10x) and 2 μl of SacI are added. The resulting mixture was incubated for 1 hour at 37 ^o C and subjected to agarose gel electrophoresis (60 V, three hours). Southern hybridization of chromosomal DNA is carried out using the probe of step (2). Fragments of about 6 kb were found. and 9.4 kb characterized by strong hybridization.

15 μl of chromosomal DNA is hydrolytically digested with SacI and subjected to agarose gel electrophoresis according to the above procedure. 6 kb DNA fragments were cut out in the gel. and 9 c.p. and each fragment is transferred into three volumes of 8 M NClO ₄ . After solubilization, each solution is spotted onto 6 mm diameter GF / C filter paper (Whatman). Ten drops (≈ 100 μl) of TE containing 6 M NaClO ₄ are added to the filter paper, and then ten drops (≈ 100 μl) of 95% ethanol. The paper is dried for 3 minutes in air and placed in an Eppendorf tube. 40 μl of TE is added to the tube and the whole is incubated for 30 minutes at 47 ^° C. The tube is then centrifuged. The result is about 40 μl of supernatant with 4.6 kV and 9.4 kV DNA fragments containing the chromosomal DNA nitrile hydratase gene.

 (4) Insertion of a chromosomal DNA fragment into a vector.

10 μl of TE, 3 μl of reaction buffer (10x) and 2 μl of SacI are added to 10 μl of pUC19 and the mixture is incubated for 1 hour at 30 ^° C. Then 2 μl of 0.25 M EDTA are added to the mixture. After that, 7 μl of Tris-HCl (1 M, pH 9) and 3 μl of bacterial alkaline phosphatase (BSF) are added to the mixture. The mixture was incubated for 1 hour at 65 ^° C. and then TE was added to a total volume of 100 μl. The mixture is extracted three times with an equal amount of phenol. An equal amount of ether is added to the extract. The lower layer was separated and 10 μl of 3 M sodium acetate and 250 μl of ethanol were added to it. The mixture was incubated for 30 minutes at -80 ^° C, centrifuged, dried and resuspended in TE.

5 μl of the resulting pUC19 DNA are mixed with 40 μl of the 9.4 kv DNA fragment of step (3). 6 μl of ligation buffer, 6 μl of ATP (6 mg / ml) and 3 μl of T4 DNA ligase are added to the mixture. The mixture was incubated for 12 hours at 4 ^° C. to obtain a recombinant plasmid containing a 9.4 kb DNA fragment at the pac19 SacI site.

 (5) Transformation and selection of transformants.

10 ml of 2xUT medium is inoculated with E. Coli TGI (Amersham) and incubated for 12 hours at 37 ^° C. After incubation, the resulting culture is added to a fresh 2xUT culture to a concentration of 1% and the resulting mixture is incubated for two hours at 37 ^° C. The culture is centrifuged and the solid part suspended in 5 ml of cold 50 mm solution of CaCl ₂ . The suspension is left on ice for forty minutes and then centrifuged. To the solid part, 0.25 ml of cold 50 mM CaCl ₂ solution and 60 μl of the recombinant DNA of step (4) are added. The mixture was incubated for 40 minutes at 0 ^° C, subjected to heat shock for two minutes at 42 ^° C, kept for five minutes on ice and added to 10 ml of 2xUT medium. The mixture was incubated for 90 minutes at 37 ^° C. with shaking and then centrifuged. The solid part is suspended in 1 ml of 2xUT medium and two aliquots of 10 μl of suspension are applied to a 2xUT agarose plate separately containing 50 μg / ml ampicillin. The plate is incubated at 37 ^o C. The grown colonies are transferred to a nitrocellulose filter and hydrolytically split. DNA is fixed on the filter and hybridizes with the probe of stage (2). The filter is subjected to radio autographs and then a recombinant colony is selected. In addition, the presence of the nitrile hydratase gene in the transform was confirmed by Southern hybridization.

 (6) Isolation and purification of a recombinant plasmid and construction of a restriction map of the inserted DNA fragments.

The transformant selected in step (5) was grown for 12 hours at 37 ^° C. in 100 ml of 2xVT medium containing 50 μg / ml ampicillin. Bacterial cells are harvested and TNE added. The cells are again harvested by centrifugation and 8 ml of STE and 10 mg of lysozyme are added to them. The mixture was incubated for five minutes at 0 ^° C. and then 4 ml of 0.25 M EDTA, 2 ml of 10% SDS (at room temperature) and 5 ml of 5 M NaCl were added. The mixture was incubated for three hours at 0-4 ^° C. and ultracentrifuged. 1/2 volume of 30% PEG 6000 was added to the supernatant, the resulting mixture was incubated for 12 hours at 0-4 ^° C and centrifuged again. TNE was added to the solid portion to bring the volume to 7.5 ml. CSCl was added to the suspension to remove proteins. Then, 300-500 mg / ml of ethidium bromide is added to the supernatant and the mixture is transferred to a centrifuge tube. The tube is sealed by heating and ultracentrifuged. Using a peristaltic pump, a covalently closed continuous circular DNA is removed to which a slightly larger than equal amount of isopropyl alcohol saturated with water is added to remove ethidium bromide. A DNA sample is dialyzed in TE to give 3 ml of purified recombinant DNA. The resulting recombinant plasmid containing a 9.4 kbp DNA fragment is designated pNHJ20L.

 The resulting plasmid DNAs are hydrolytically digested with EcoRI, BamHI, PsfI, SacI and SalI. Designed restriction maps are shown in FIG. 3.

 (7) DNA sequencing.

 The position of the nitrile hydratase gene in the DNA fragment of plasmid pNHJ20L is determined in accordance with designed restriction maps and Southern hybridization method. The additional segment in pNHJ20L was cleaved with EcoRI and SacI to give 9.4 kb from the DNA fragment. 1.73 kbp fragment

 The obtained DNA fragments are sequenced according to the method of Sanger (Sanger F., Science 214, 1205-1210 (1981)) using the phage vector M13. The nucleotide sequence of the 1.73 kb DNA fragment (pNHJ20L) is shown in FIG. 4.

 The amino acid sequence deduced from the nucleotide sequence was found to be completely identical to the amino acid sequence determined in step (1). Sequence analysis, in addition, revealed that the DNA fragment contains a sequence encoding the α and β subunits.

 (8) Preparation of nitrile hydratase using a transformant and conversion of nitriles to amides using nitrile hydratase.

10 ml of 2xUT medium containing 50 g / ml of ampicillin are inoculated with TG-I / pNHJ20L and incubated for 12 hours at 30 ^o C. 1 ml of the resulting culture is added to 100 ml of 2xUT medium (50 mg / ml of ampicillin, 0.1 g of CoCl ₂ • 6H ₂ O / L) and the mixture is incubated for 4 hours at 30 ^° C. IPTG is added to the mixture to a final concentration of 1 mM and incubated for 10 hours at 30 ^° C. After collecting the cells, they are suspended in 5 ml of 0.1 M phosphate buffer ( pH 7.5), the suspension is destroyed by ultrasound for 5 minutes and centrifuged for 30 minutes at 12000 g.

The resulting supernatant is used for a truss test. The enzyme test is carried out in a reaction mixture (12 ml) containing 50 mM potassium phosphate buffer (pH 7.5), 6 mM benzonitrile and the required amount of enzyme. The reaction is carried out for 30 minutes at 20 ^° C. and is stopped by the addition of 0.2 ml of 1 M HCl. The amount of benzamide formed in the reaction is determined by HPLC. For control use a mixture prepared according to the above method, but from E. coli TG-I. The level of nitrile hydratase activity in cell-free extracts of E. coli containing pNHJ20L is 6.99 • 10 ^-3 units / mg when cultured in 2xUT in the presence of CoCl ₂ and IPTG. Benzamide was detected in the reaction mixture TG-I / pNHJ20L, while benzamide was not found in the reaction mixture TG-I.

Claims (4)

1. A DNA fragment encoding a polypeptide with nitrile hydratase activity obtained by cloning a nitrile hydratase gene from Rhodococcus rhodochrous J-1 (FERM BP-1478), comprising the following nucleotide sequence (see graphic part). α ^(L) -subunit:
β ^(L) -subunit: (see graphic part). corresponding to the following amino acid sequences: α ^(L) -subunit: (see graphic part). β ^(L) -subunit: (see graphic part). 2. Recombinant plasmid DNA PNHJ20L encoding a polypeptide exhibiting nitrile hydratase properties, containing a -SacI-SacI fragment, including a 9.4 kbp nitrile hydratase gene from Rhodococcus rhodochrous J-1 (FERM BP-1478); -SacI-SacI fragment of the pUC19 DNA vector of 2.686 kbp; unique restriction sites: three EcoRI sites, two SacI sites, two PstI sites; genetic marker; Amp ^r - ampicillin resistance gene.  3. The strain Escherichia coli TGl / pNHJ20L (FERM BP-2778) is a producer of a polypeptide with nitrile hydratase properties.  4. A method of producing a polypeptide with the properties of nitrile hydratase, comprising culturing a strain, isolating and purifying the target product, characterized in that the bacterial strain Escherichia coli TGl / pNHJ20L (FERM BP-2778) is cultivated.

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