The dna fragment that encodes a polypeptide with the activity of nitrilimines, recombinant plasmid dna Рnhj20l encoding a polypeptide, an escherichia coli strain tg1/Рnhj20l, a method of obtaining a polypeptide with properties of nitrilimines

 

(57) Abstract:

The invention relates to biotechnology and relates to amino acid sequences - and-abedini polypeptide with the activity of nitrilimines and DNA fragments encoding the polypeptide. Also the invention concerns recombinant plasmid DNA pNHJ20L that encodes a polypeptide exhibiting the properties nitrilimines, transformants containing recombinant plasmid DNA, and the method of obtaining nitrilimines. The DNA fragment that encodes a polypeptide with the activity of nitrilimines, is inserted in a plasmid vector. Transform the E. coli strain using recombinant plasmids pNHJ20L. The resulting strain E. coli TG1/pNHJ20L (FERM BP-2778) capable of producing the polypeptide with properties of nitrilimines at a much higher level than that normally applied to the microorganisms. 4 S. p. f-crystals, 4 Il.

The invention relates to a DNA fragment derived from Rhodococcus rhodochrous and codereuse polypeptide with nitrilgidrataznoi activity, which hydrolyzes the nitrile into amide. The invention also relates to a recombinant DNA containing the above DNA fragment, and transformant containing recombinant DNA.

The present invention also relates to trihydrates or nitrilase known as an enzyme, hydralicious NITRILES to amides. The microorganisms that form nitrilimines include organisms belonging to the genus Bacillus genus Bacteridium the genus Micrococcus and 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, genus Microbacterium (see JP-A-61-162193/1986, EP-A-0188316) and Rhodococcus rhodochraus (see JP-A-2470/1990, EP-A-0307926).

Nitrilimines used for the hydrolysis of NITRILES to amides. In the invention, the microorganisms convert so that they contain multiple copies of a recombinant DNA that encodes nitrilimines, the use of biotechnology. The resulting recombinant produces nitrilimines on a significantly higher level compared with the commonly used microorganisms.

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

In contrast to the above references in this invention is a DNA fragment derived from Rhodococcus rhodochrous J-1, for producing nitrilimines. We have isolated the gene encoding nitrilimines, gene embedded within an acceptable plasmid vector, which retasu with high activity and also in relation to aromatic NITRILES.

Summary of the invention

The present invention relates to:

(1) the DNA fragment(L), codereuse polypeptide with nitrilgidrataznoi activity, the polypeptide includes(L)and(L)-subunit amino acid sequences 1 and 2 (see end of description);

(2) a recombinant DNA comprising a vector DNA(L)under paragraph (1);

(3) transformant containing recombinant DNA according to item (2);

(4) the method of obtaining nitrilimines, which lies in the cultivation of transformant described in paragraph (3), and the allocation of the culture nitrilimines.

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 nitrilimines and partial amino acid sequencing of nitrilimines.

From Rhodococcus rhodochrous J-I (FERM BP-1478) isolate and purify two types of nitrilimines (denoted, respectively, as H-type and L-type) and both enzymes separated by HPLC on - and-subunits. Determine the portion of the amino acid sequence of each subunit (Fig. 1).

(2) Obtaining a DNA probe for the gene nitrilimines.

DNA probes derived from JM105/pV UK1 between nitrilimines-subunit Rhodococcus N-774, described in the specified publication of the Official Gazette of patents Japan, and subunits of Rhodococcus rhodochrous J-1. Plasmid pVUK121 containing gene nitrilimines derived from Rhodococcus N-774, from a culture JM105/pVU-C, DNA pVU K-121 hydrolytically cleaved with SphI and SalI SphI-SalI fragment contains the gene nitrilimines Rhodococcus N-774 (Fig. 2) a DNA fragment of radiometal.

(3) Detection of a segment of DNA containing the gene of nitrilimines of the chromosomes Rhodococcus rhodochrous J-I. Chromosomal DNA from a culture of Rhodococcus rhodochrous J-1.

Chromosomal DNA hydrolytically cleaved using restriction enzymes and hybridized with probe hybridization technique of Southern (Southern E. M., J. Mol. Biol. 98, 503 (1975)).

Selected two DNA fragments of different lengths.

(4) Construction of recombinant plasmids.

Recombinant plasmid construct insertion of chromosomal DNA fragment of step (3) in a plasmid vector.

(5) Transformation and selection of transformants containing the recombinant plasmid.

The transformant obtained using the recombinant plasmid of step (4). Containing the recombinant plasmid of transformant selected using the probe of step (2) by the method of hybrid colonies (R Bruce Walla is RNA. Selected resulting plasmid is designated as pNHJ 20L.

(6) isolation and purification of plasmid DNA and construction of restriction maps.

Isolate and purify obtained in step (5) of plasmid DNA pNHJ20L. Construct a restriction map of this DNA (Fig. 3) to determine areas that contain the gene nitrilimines.

(7) DNA Sequencing.

Built-in plasmid pNHJ20L DNA fragment otscheplaut using the appropriate restriction enzyme and is sequenced. The nucleotide sequence of the DNA fragment (Fig. 4 and 5) shows that it contains a sequence are tracked on the basis of the amino acid sequence of step (1).

(8) the Obtaining by means of transformant nitrilimines.

Culturing the transformant of step (7).

Rhodococcus rhodochrous J-1 deposited at the research Institute of fermentation Agency of industrial science and technology, where he is assigned a registration number FERM BP-1478. The transformant TGI/NHJ20L containing pNHJ20L stage (5), deposited there, and he assigned a registration number FERM BP-2778.

Can be used any vectors, including plasmid vector (e.g., AT153, RMR, pHC624, pKC7, and so on), phage vector (on the, umHI, etc. manufactured by the industry (Takara Shuso). For transformation can be used a variety of recipients, including, but without limitation only by them: E. Coli Jm105 and E. Coli TGI.

Cultural environment for transformant are commonly used for such purposes environment.

The transformation of NITRILES into amides is carried out using nitrilimines, crude nitrilimines, culture transformant allocated bacterial cell or a treated material of the cell, etc., obtained from the culture of transformant.

Acceptable to the invention, the NITRILES include: aromatic NITRILES with 4-10 carbon atoms in the aromatic fragment and aliphatic NITRILES with 2-6 carbon atoms, as described in published European patent N 0307926. Typical examples of the NITRILES include: 4-, 3 - and 2-cyanopyridine, benzonitrile, 2,6-diferential, 2-thiophenecarbonitrile, 2-furonitrile, cyanovirin, Acrylonitrile, Methacrylonitrile, crotonates, acetonitrile and 3-hydroxypropionitrile.

Technical results.

Described amino acid sequence of - and-subunits of nitrilimines derived from Rhodococcus rhodochrous J-1. The DNA fragment encoding nitrilimines is inserted into vecto is and is able to produce nitrilimines at a much higher level compared to the commonly used microorganisms.

Explanation of drawings.

In Fig. 1 shows the N-terminal amino acid sequence of - and-subunits of nitrilimines produced Rhodococcus rhodochrous J-1.

In Fig. 2 shows the DNA sequence of the gene nitrilimines Rhodococcus N-774, used as a DNA probe.

In Fig. 3 shows the restriction map of recombinant plasmid pNHJ20L.

In Fig. 4 shows the DNA sequence of the 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 in the following example, which is not intended to limit the invention.

In this example we used the following abbreviations:

TE - Tris-HCl (10 mm, pH 7, 8), add (1 mm, pH 8),

TNE - Tris-HCl (50 mm, pH 8), add (1 mm, pH 8), NaCl (50 mm)

STE - Tris-HCl (50 mm, pH 8), add (5 mm, pH 8), sucrose (35 mm)

Wednesday 2xVT - 1.6% Tryptone, 1% yeast extract, 0.5% of NaCl.

Example

(1) isolation and purification of nitrilimines and partial amino acid sequencing of nitrilimines.

Rhodococcus rhodochrous J-1 cultivated 80 hours in medium (3 g/l yeast extract, 0.5 g/l Kabirat bacterial cells. 50 g of bacterial cells and destroy fractionary with ammonium sulfate. Sample cialiswhat and dialysate centrifuged. The liquid part chromatographic on a column of DEAE-Zerafina, a column of phenyl-Separate, a column of Sephadex C-150 and the column with Octyl-Separate. Receive and cialiswhat two fractions with enzymatic activity. Dialysate transferred to column high-performance liquid chromatography with reversed phase (Senshu Pak VP-304-1251, Senshu the Www.enl.ee) and receive respectively two subunits ( and ). Using protein sequencing machine model 470A applied Biosystems determine N-terminal amino acid sequence(H)1,(H)-1,(L)1and(L)1-subunits. Amino acid sequence shown in Fig. 1.

(2) Obtaining a DNA probe for the gene of nitrilimines.

B 100 ml of medium hut containing 50 μg/ml ampicilin, cultured for 12 hours at 30oC E. Coli JM105 (FERM BP-1937), containing pYU121. Bacterial cells are harvested and added to them TNE. The cell suspension is then centrifuged. 8 ml of STE and 10 mg of lysozyme is added to the solid part. The mixture is incubated for five minutes at 0oC, then add 4 ml of 0.25 M EDTA. Then to the mixture at room temperature is so To the liquid add 1/2 volume of PEG 6000. The mixture is incubated for 12 hours at 0-4oC and centrifuged. The solid part of the add TNE with bringing the volume up to 7.5 ml, after which the suspension was added CSCl. The mixture is centrifuged to remove proteins. Then to the liquid part of the add 300-500 mg/ml ethidium-bromide. The mixture is transferred to a centrifuge tube, the tube is sealed by heating and ultracentrifuging. Using a peristaltic pump with covalently closed circular DNA. To the extract add saturated water isopropyl alcohol in a quantity slightly greater than an equal amount, with the extraction of the ethidium-bromide. Sample cialiswhat on THOSE and get 3 ml of purified plasmids pVUK12L.

pVUK12I DNA hydrolytically cleaved with SphI and SalI and get a DNA fragment in 2,07 etc., of O., containing the gene for nitrilimines derived from Rhodococcus N-774. Fragment of radiometal32P with the receiving probe. The nucleotide sequence of the probe shown in Fig. 2.

(3) Obtaining a DNA fragment containing a chromosomal gene nitrilimines.

B 100 ml of medium (10 g/l glucose, 0.5 g/l KH2PO4, 0.5 g/l K2HPO4, 0.5 g/l MgSO47H2O, 1 g/l yeast extract, 7.5 g/l peptone, 0.01 g/the toes cells are collected and the solid part was washed with TNE. Then the hard part is suspended in 10 ml of TE. To the suspension is added 4 ml of 0.25 M EDTA, 10 to 20 mg of lysozyme, 10-20 mg chromoprotein and 10 ml 10xSDS. The suspension is incubated for three hours at 37oC and then to it was added 15 ml of phenol. The mixture is incubated for 15 minutes at room temperature and then centrifuged. The top layer is removed and the supernatant liquid add 0.7 ml of 2.5 M sodium acetate and diethyl ether. The mixture is centrifuged and the upper layer is discarded. To the bottom layer, add two volumes of ethanol and the DNA is extracted with a glass rod. DNA rinsed for five minutes in each case the mixture of the ethanol in the relationship 2:8, 1:9 and 0:9 (about./vol.). Then the DNA is again suspended in 2-4 ml of THE 37oC). To the suspension is added 10 μl of a mixture of RNase A and T1and the mixture is incubated at 37oC. To the mixture is added an equal amount of phenol, and then centrifuged. To the supernatant add more than an equal amount of ether. The mixture is again centrifuged, the upper layer is discarded and the lower layer remain. The bottom layer cialiswhat about a day in 2 l of THOSE containing a small amount of chloroform, and optionally cialiswhat in THOSE fresh for 3-4 hours. Receive 4 ml of crude chromosomal DNA.

To 15 μl of crude chromosomal DNA, add 10 mcafferty on the agarose gel (60 B, three hours). Hybridization of chromosomal DNA by Southern carried out using a probe of step (2). It was found that the fragments in approximately 6 T. p. O. and 9.4 T. p. O. characterized by a strong hybridization.

15 μl of chromosomal DNA hydrolytically cleaved with SacI and subjected to electrophoresis on agarose gel according to the aforementioned method. Gel cut DNA fragments in 6 K. p. O. and 9 K. p. O., and each fragment is transferred into a three volume 8 M NClO4. After solubilization each solution is applied in the form of spots on filter paper GF/C (Whatman) with a diameter of 6 mm To filter paper, add ten drops ( 100 µl) containing 6 M NaClO4and then ten drops ( 100 µl) of 95% ethanol. The paper is dried for 3 minutes in air and placed in an Eppendorf tube. In a test tube, add 40 ál of THOSE and all is incubated for 30 minutes at 47oC. Then the tube is centrifuged. The result is about 40 µl of the supernatant with DNA fragments 4.6 kV and 9.4 kV, containing the gene nitrile-hydratase chromosomal DNA.

(4) the Introduction into the vector fragment of chromosomal DNA.

To 10 μl of pUC19 add 10 ál of THOSE, 3 μl of reaction buffer (10x) and 2 μl SacI and the mixture is incubated for 1 hour at 30oC. Then to terminate the reaction, to the mixture was added 2 μl of the camping incubated for 1 hour at 65oC and then add THOSE to the full volume of 100 μl. The mixture was thrice extracted with an equal amount of phenol. To the extract add an equal quantity of ether. The bottom layer was separated and to it add 10 ál of 3 M sodium acetate and 250 μl of ethanol. The mixture is incubated for 30 minutes at - 80oC, centrifuged, dried and re-suspended in TE.

Mix 5 ál of the resulting DNA pUC19 and 40 μl of the DNA fragment of 9.4 kV phase (3). To the mixture add 6 μl of ligation buffer, 6 μl of ATP (6 mg/ml) and 3 μl of T4 DNA ligase. The mixture is incubated for 12 hours at 4oC preparation of recombinant plasmid containing the DNA fragment size of 9.4 kV in the SacI site of pUC19.

(5) Transformation and selection of transformants.

10 ml of medium hot inoculant E. Coli TGI (Amersham) and incubated for 12 hours at 37oC. After incubating the resulting culture was added to fresh culture hut to a concentration of 1% and the mixture is incubated for two hours at 37oC. the Culture is centrifuged and the solid part is suspended in 5 ml of cold 50 mm solution of CaCl2. The suspension is left on for forty minutes on ice and then centrifuged. The solid part of the add 0.25 ml of cold 50 mm solution of CaCl2and 60 μl of recombinant DNA of step (4). The mixture is incubated for 40 mine is added to 10 ml of medium hot. The mixture is incubated for 90 minutes at 37oC with shaking and then centrifuged. The hard part is suspended in 1 ml of medium hot and two aliquots of 10 μl of the suspension is applied on hot agarose plate, separately containing 50 μg/ml ampicilin. The plate is incubated at 37oC. the Grown colonies are transferred to a nitrocellulose filter and hydrolytically cleaved. The DNA fixed to the filter's hybrid with the probe of step (2). The filter is subjected to autoradiography and then select recombinant colony. In addition, the presence of gene nitrilimines in transformant confirmed by hybridization of Southern.

(6) isolation and purification of recombinant plasmids and construction of restriction map of the introduced DNA fragments.

Selected in step (5) of the transformant is grown for 12 hours at 37oC in 100 ml of medium 2xVT containing 50 µg/ml ampicillin. Bacterial cells are harvested and added to them TNE. Cells are again collected by centrifugation and add 8 ml of STE and 10 mg of lysozyme. The mixture is incubated for five minutes at 0oC and then add 4 ml of 0.25 M EDTA, 2 ml of 10% SDS (at room temperature) and 5 ml of 5 M NaCl. The mixture is incubated for three hours at 0-4oC and ultracentrifuging. To the supernatant idcategory part add TNE with bringing the volume up to 7.5 ml. To remove proteins to the suspension was added CSCl. Then the supernatant liquid add 300-500 mg/ml ethidium-bromide and the mixture is transferred to a centrifuge tube. The tube is sealed by heating and ultracentrifuging. Using peristalticheskogo pump remove covalently closed continuous ring of DNA to which to remove the ethidium-bromide add a few more than an equal amount of isopropyl alcohol saturated with water. The DNA sample cialiswhat in THOSE with 3 ml of the purified recombinant DNA. The resulting recombinant plasmid containing the DNA fragment size of 9.4 T. p. O., designated as pNHJ20L.

The obtained plasmid DNA hydrolytically cleaved with EcoRI, BamHI, PsfI, SacI and SalI. Designed restriction map shown in Fig. 3.

(7) DNA Sequencing.

The position of the gene nitrilimines in the DNA fragment of the plasmid pNHJ20L determined in accordance with the designed restriction mapping and hybridization of Southern. The extension segment in pNHJ20L otscheplaut with EcoRI and SacI to obtain a DNA fragment 9.4 T. p. O. fragment 1.73 T. p. O.

The resulting DNA fragments is sequenced by the method of Sanger (F. Sanger, Science, 214, 1205-a respectively in Fig. 4.

Amino acid sequence deduced from the nucleotide sequence was found to have been completely identical amino acid sequence determined in step (1). The sequence analysis also revealed that the DNA fragment contains a sequence encoding a - and a-subunit.

(8) the Obtaining by means of transformant nitrilimines and the conversion of NITRILES to amides using nitrilimines.

10 ml of medium hut containing 50 g/ml ampicillin, inoculant TG-I/pNHJ20L and incubated for 12 hours at 30oC. 1 ml of the obtained culture is added to 100 ml of medium hot (50 mg/ml ampicillin, 0.1 g CoCl26H2O/l) and the mixture is incubated for 4 hours at 30oC. To this mixture IPTG to a final concentration of 1 mm and incubated for 10 hours at 30oC. After collecting the cells are suspended in 5 ml of 0.1 M phosphate buffer (pH 7.5), suspension destroy action within 5 min of ultrasound and centrifuged for 30 min at 12000 g.

The obtained supernatant is used for permisiunea tests. Fermentive the test is carried out in the reaction mixture (12 ml) containing 50 mm califofnia buffer (pH 7.5), 6 mm benzonitrile and the required amount of enzyme. eghosa in the reaction of benzamide determined using HPLC. For control, a mixture prepared by the above method, but from E. coli TG-I. Level nitrilgidrataznoi activity in cell-free extracts of E. coli containing pNHJ20L is 6,99 10-3units/mg under cultivation in the environment hut in the presence of CoCl2and IPTG. In the reaction mixture TG-I/pNHJ20L found benzamid, while in the reaction mixture TG-I benzamid not found.

1. The DNA fragment that encodes a polypeptide with the activity of nitrilimines obtained by cloning the gene nitrilimines from Rhodococcus rhodochrous J-1 (FERM BP-1478), comprising the following nucleotide sequence (see graphic part).(L)-subunit:

(L)-subunit: (see graphic part).

correspond to the following amino acid sequences: (L)-subunit: (see graphic part).

(L)-subunit: (see graphic part).

2. Recombinant plasmid DNA PNHJ20L encoding a polypeptide exhibiting the properties nitrilimines containing-SacI-SacI fragment containing the gene of nitrilimines from Rhodococcus rhodochrous J-1 (FERM BP-1478) size of 9.4 T. p. N.; -SacI-SacI fragment DNA vector pUC19 size 2,686 T. p. N.; unique restriction sites: three sites EcoRI, two SacI site, two sites > PST; 78) producing the polypeptide with properties of nitrilimines.

4. A method of obtaining a polypeptide with properties of nitrilimines providing for the cultivation of the strain, isolation and purification of the target product, wherein the cultured bacterial strain Escherichia coli TGl/pNHJ20L (FERM BP-2778).

 

Same patents:

The invention relates to structures of nanometer size used to construct the microscopic and macroscopic structures

The invention relates to the complementarity determining regions (CDR, hypervariable regions) and variable regions (V regions) of murine monoclonal antibodies to human interleukin-8 (IL-8), human/mouse chimeric antibody to human IL-8, and reconstructed human antibodies, and region, defining a complementary variable region, a human light chain (L-chain) and variable regions of the heavy chain (H-chain) of the person replaced the CDR of mouse monoclonal antibodies to human IL-8

The invention relates to molecular biology and biotechnology

The invention relates to biotechnology

The invention relates to biotechnology

The invention relates to a synthetic single-stranded RNA molecules with highly specific for their endonuclease activity

The invention relates to genetic engineering, in particular to a technology for obtaining high-yielding strains Eserichia coli - produced recombinant human proteins used in modern medicine as thrombolytic agents

The invention relates to the preparation of the interest of the enzymes in the seeds of transgenic plants and application of the thus prepared seed and production processes in the absence of the required extraction or isolation of the enzyme

The invention relates to biotechnology and concerns bacteriocin from Micrococcus varians, a nucleotide fragment encoding the bacteriocins, strains of Micrococcus varians CNCM 1-1586 and CNCM 1-1587 producing the specified bacteriocins, as well as method of obtaining bacteriocin and its use to obtain food and cosmetics as an agent, active against pathogens
Up!