Recombinant laccase of ligninolytic fungus trametes sp and method of its obtaining
SUBSTANCE: plasmid contains a DNA fragment encoding laccase C1 of ligninolytic fungus Trametes hirsuta or a DNA fragment hybridised with SEQ ID N0:1 in tough conditions, under control of promoter functioning in this cell. The resulting cell is a producer of laccase C1. The invention also refers to method of laccase obtaining using to the indicated cell.
EFFECT: obtaining laccase with high catalytic activity.
6 cl, 3 dwg, 1 tbl, 5 ex
The technical field
The present invention relates to microbiological, genetic engineering and biotechnology, in particular to a method for producing recombinant laccase ligninolytic fungus Trametes sp. Recombinant laccase can be used in such industrial processes such as bleaching paper pulp delignification of cellulose paste and improve the properties of paper and textile materials, cleaning the environment from pollution and dirt, staining materials, production of aldehydes and ketones from suitable substrates and others.
Description of the prior art,
Laccase (EC 188.8.131.52 para-diphenol: oxygen oxidoreductase) is polyphenoloxidases belonging to the family of blue oxidase. Laccase catalyzes the oxidation of the reducing substrate for reducing molecular oxygen to water. The products of reactions - free radicals reducing substrate and water. The usual substrates of laccase are: o-, m-, p-phenols with razlichnyi substituents, polyphenols, aromatic amines. Been recently opened the reaction of lackas with the participation of mediators - low molecular weight compounds that increase the efficiency of laccase in the biodegradation of lignin, resistant compounds of various structures, including xenobiotics and environmental pollutants.
Known methods for producing La is KAZ from Coprinus cinereus (U.S. patent 6242232; 6008029), Myceliophthora thermophila (U.S. patent 5981243; 5925554; 5795760), Scytalidium thermophilum (U.S. patent 5925554; 5843745; 5750388), Rhizoctonia solani (U.S. patent 5480801), Pycnoporus cinnabarinus (French patent FR 2865216) Phlebia (PCT application WO 9201046) using strains-producers of different species and genera.
Describes a method for the recombinant laccase fungus Polyporus (Trametes) villosa using Aspergillus and Fusarium (U.S. patent 5770418).
T. versicolor secretes several isoforms of laccase, which may seek to do this basidiomycete different functions (Reinhammar Century Copper Proteins and Copper Enzymes, V.3 / Lonti R., ed. CRC Press, Boca Raton, FL. 1-35, (1984)). Whether the producer of several isoforms of the enzyme makes cleanup individual isoenzyme.
To date, the facts described in the expression of laccase from Trametes trogii in Pichia pastoris (Colao M.S. et al., Microbial Cell Factories, v.5, No.1, p.31, (2006)); laccase from Trametes versicolor in Pichia pastoris (L.J. Jonsson et al., Current Genetics, 32(6), 425-430 (1997)); laccase from Trametes sanguinea in yeast (H. Hoshida et al., Journal of biosciences and bioenGln,eering, 92(4), 372-80 (2001)) and laccase from Trametes versicolor in Saccharomyces cerevisiae (Cassland, P. et al., Applied Microbiology and Biotechnology, 52(3), 393-400 (1999)).
The main physico-chemical characterization of the major laccase isoforms, Sekretareva fungus Trametes hirsuta 072, have been recently described, this gene isoform was cloned, and its nucleotide sequence was determined (Rebrikov, DSC. and other Applied biochemistry and Microbiology, 42, 6, 645-653 (2006)).
Od is ako currently no messages, describing individual recombinant laccase C1 ligninolytic fungus Trametes hirsuta, which has high catalytic activity, using strains of filamentous fungi, such as Penicillium canescens, allowing to produce high heterologous expression ligninolytic enzymes in active form.
Brief description of the invention
The purpose of the present invention are creating a strain of filamentous fungus - producer individual isoforms of the enzyme laccase, in particular laccase C1 ligninolytic fungus Trametes hirsuta, capable of efficient secretion of the target protein into the culture fluid, and the provision of a method of obtaining individual recombinant laccase C1. The task was solved by constructing recombinant plasmids pBGlac containing the gene encoding the laccase C1 ligninolytic fungus Trametes hirsuta, and strain Penicillium canescens PCA-10(niaD-)/BGlac for synthesis and production in Sekretareva soluble form laccase C1 ligninolytic fungus Trametes hirsuta.
The aim of the present invention is the provision of expressional cassette containing a DNA fragment encoding the laccase C1 ligninolytic fungus Trametes hirsuta (SEQ ID NO:1) or its variant.
Another objective of the present invention is the provision of recombinant plasma is IDA, containing the DNA fragment encoding the laccase C1 ligninolytic fungus Trametes hirsuta (SEQ ID NO:1) or its variant under the control of a promoter functional in the eukaryotic cell.
Another objective of the present invention is the provision of recombinant plasmids described above, where the plasmids used plasmid pBGlac.
Another objective of the present invention is the provision of eukaryotic cells, transformed the previously described plasmid, producer laccase from C1 ligninolytic fungus Trametes hirsuta.
Also the aim of the present invention is the provision described above in eukaryotic cells, the eukaryotic cells use cell filamentous fungus Penicillium canescens.
Another objective of the present invention is the provision of a strain of Penicillium canescens PCA-10(niaD-)/BGlac producer laccase C1 ligninolytic fungus Trametes hirsuta.
Another objective of the present invention is the provision of a method of obtaining a recombinant laccase C1 ligninolytic fungus Trametes hirsuta, including the stage of growth described above eukaryotic cells in a nutrient medium and isolation of recombinant laccase C1 from the culture fluid.
Also the aim of the present invention is the provision of the above-described method, characterized in that it is the number of eukaryotic cells use cell filamentous fungus.
Also the aim of the present invention is the provision of the above-described method, characterized in that as the cells of filamentous fungus used a strain of Penicillium canescens PCA-10(niaD-)/BGlac.
Another objective of the present invention is the provision of recombinant laccase C1 ligninolytic fungus Trametes hirsuta, obtained as described above.
Brief description of drawings
Figure 1 shows a diagram of the vector plasmid pBG containing regulatory elements of the gene of β-galactosidase Penicillium canescens.
Figure 2 shows the structural part of the gene laccase C1 Trametes sp.,
including its own start codon ATG, the signal peptide, the inner part with nitrone and a stop codon at the 3'-end.
Figure 3 shows a diagram of recombinant plasmids pBGlac used to create producer strain laccase C1 Trametes sp.
Detailed description of the present invention
For the implementation of the present invention, the main technical challenge was the creation of a strain of filamentous fungus - producer of recombinant laccase C1 ligninolytic fungus Trametes hirsuta, capable of efficient production and secretion of active target protein into the culture fluid. The task was solved by constructing recombinant plasmids pBGlac containing the gene encoding the synthesis of laccase C1 lignicola the ical fungus Trametes hirsuta, and strain Penicillium canescens PCA-10(niaD-)/BGlac for synthesis and production in Sekretareva soluble form laccase C1 ligninolytic fungus Trametes hirsuta.
The term "expression cassette" means a DNA fragment containing all of the necessary genetic elements for expression embedded in her gene such as a promoter, terminator. Specific examples of the genetic elements necessary for the expression of recombinant laccase comprising the expression cassette according to the present invention, include, but are not limited to, the promoter and terminator of β-galactosidase Penicillium canescens.
The DNA fragment, encoding the laccase C1 ligninolytic fungus Trametes hirsuta according to the present invention is, for example, a gene of the major laccase described in article Rebrikov D.N. and others (Applied biochemistry and Microbiology, 42, 6, 645-653 (2006)). Structure of the gene encoding the laccase C1 ligninolytic fungus Trametes hirsuta, are presented in figure 2. The laccase gene sequences S1 ligninolytic fungus Trametes hirsuta presented in the sequence Listing under the number SEQ ID NO:1. Amino acid sequence of laccase C1 are presented in the sequence Listing under the number SEQ ID NO:2.
The term "variant protein" in the sense in which it is used in the present invention means a protein which has the ISM is in in amino acid sequence, namely, deletions, insertions, additions or substitutions of amino acids, provided that it retains the desired activity of the protein, for example at least 10% of the activity of native laccase. A number of changes in the variant protein is dependent on the position or the type of amino acid residue in the three-dimensional structure of the protein. The amount of change can be from 1 to 30, preferably from 1 to 15 and most preferably from 1 to 5 changes in protein sequence 2 (SEQ ID NO:2). These changes can occur in regions of the protein which are not critical to its function. This is possible due to the fact that amino acids have a high homology with each other, and therefore the tertiary structure or activity of the protein is not violated by such a change. Therefore, as a variant protein can be a protein which has homology of not less than 70%, preferably not less than 80%, more preferably at least 90% and most preferably not less than 95% relative to the complete amino acid sequence represented by SEQ ID NO:2, provided that the activity of the protein is maintained. Homology between amino acid sequences can be established using well-known methods, for example using sequence alignment in computer the agreement the program BLAST 2.0, which calculates three parameters: the account, the identity and similarity.
Substitution, deletion, insertion, addition or substitution of one or several amino acid residues will represent a conservative mutation or conservative mutations, provided that the activity of the protein is maintained. An example of a conservative mutation(s) is a conservative substitution(s). Examples of conservative substitutions include the substitution of Ala for Ser or Thr, substitution of Arg for Gln, His or Lys, substitution of Asn for Glu, Gln, Lys, His or Asp, substitution of Asp for Asn, Glu or Gln, replacement of Cys to Ser or Ala, substitution of Gln for Asn, Glu, Lys, His, Asp or Arg, substitution of Glu for Asn, Gln, Lys or Asp, substitution of Gly to Pro, replacing His to Asn, Lys, Gln, Arg or Tyr, the substitution of Ile for Leu, Met, Val or Phe, substitution of Leu for Ile, Met, Val or Phe, substitution of Lys for Asn, Glu, Gln, His or Arg, substitution of Met for Ile, Leu, Val or Phe, substitution of Phe to Trp, Tyr, Met, Ile or Leu, substitution of Ser to Thr or Ala, substitution of Thr for Ser or Ala, substitution of Trp to Phe or Tyr, the substitution of Tyr for His, Phe or Trp, and replacement of Val to Met, Ile or Leu.
DNA fragments that encode essentially the same functional protein can be obtained, for example, by modifying the nucleotide sequence of the DNA fragment (SEQ ID NO:1)encoding a laccase, for example, by the method of site-directed mutagenesis, so that one or more amino acid residues at a specific site will be deleterows, substituted, inserted or added. The DNA fragments, modified as described the above, can be obtained using traditional processing methods with the aim of obtaining mutations.
DNA fragments that encode essentially the same functional protein laccase, can be obtained by expression of the DNA fragments with mutations described above in an appropriate cell, and determine the activity of the expressed product. DNA fragments that encode essentially the same functional protein laccase can also be obtained by separating the DNA fragments that hybridize with probes having a nucleotide sequence that contains, for example, the nucleotide sequence shown in the sequence Listing under the number SEQ ID NO:1, in hard conditions, and encodes a protein having the activity of laccase. "Stringent conditions"in the meaning ascribed to that expression in the framework of the present invention, means such of the conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, demonstration of stringent conditions may be conditions under which the DNA fragments with high homology, for example, DNA fragments having homology of not less than 50%, preferably not less than 60%, more preferably not less than 70%, even more preferably not less than 80%, even more preferably n is less than 90% and most preferably not less than 95% can gibridizatsiya with each other, and the DNA fragments having homology lower than the above, are not able to gibridizatsiya with each other. In addition, demonstration of stringent conditions can be such the conditions under which the DNA fragments hybridize with concentration equivalent to the conditions of single washing with hybridization by Southern, which are 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS, at 60°C. Duration of washing depends on the type of membrane used for blotting and, as a rule, recommended by the kit manufacturer. For example, the recommended duration of washing for membrane Hybond™ N+nylon (Amersham, USA) in harsh conditions is approximately 15 minutes. Preferably, the washing is repeated 2-3 times. As the probes can also be used incomplete nucleotide sequence shown in the sequence Listing under the numbers SEQ ID NO:1. The probes can be prepared by PCR using primers synthesized based on the nucleotide sequence provided in the sequence Listing under the number SEQ ID NO:1, and on the basis of fragments of DNA containing the nucleotide sequence shown in the sequence Listing under the number SEQ ID NO:1 as a matrix. In those cases, when the DNA fragment having a length of about 300 BP is used as the probe, the terms of which tlyvci in hybridization include, for example, 50°C., 2×SSC and 0.1% SDS.
Substitution, deletion, insertion or addition of nucleotides as described above, also includes mutations that occur in nature (mutant or variant), for example due to species variability.
Indicators of functional activity, in which it is considered that the resulting protein has properties of laccase is determined by its ability to oxidize various phenolic substrates of nature, such as o-, m-, p-phenols with different substituents, polyphenols, aromatic amines. Also laccase is able to increase the efficiency of biodegradation of lignin and resistant aromatic xenobiotics in the presence of mediators. For example, the laccase activity can be detected spectrophotometrically at 410 nm using a chromogenic substrate of 10 mm solution of pyrocatechin in 0.1 M Na-acetate buffer, pH of 4.9, "unit of activity" accept the increase in optical density in a 1 ml reaction mixture for 1 min, recalculated per 1 mg of protein insertion (Rebrikov D.N. and other Applied biochemistry and Microbiology, 42, 6, 645-653 (2006)).
The activity of laccase can also be determined by measuring the initial velocity of the enzymatic reaction using a Clark oxygen electrode in a cell with a volume of 0.7 ml at 25°C and constant stirring. As organic with the strata use a 10 mm solution of pyrocatechin in 0,015 M phosphate buffer, the pH of 4.9. The oxygen concentration in the solution is equal to 260 μm (according to the value of the coefficient Henry 773 ATM/mol × kg water). The activity of laccase expressed in mcmash of oxygen consumed per 1 mg of protein per 1 minute
It is believed that the variant protein has properties of laccase, provided that the activity specified option is not less than 10% activity of native laccase.
The recombinant plasmid according to the present invention contains a DNA fragment encoding the laccase C1 ligninolytic fungus Trametes hirsuta (SEQ ID NO:1) or its variant, under the control of a promoter functional in the eukaryotic cell.
As recombinant plasmids according to the present invention can be used any bacterially plasmid, such as, for example, pBGlac, but the list of such plasmids is not limited to it.
Recombinant plasmid pBGlac was constructed by cloning the gene encoding the laccase C1, plasmid pBG (), containing the sequence of the promoter, signal peptide, and terminator gene β-galactosidase Penicillium canescens, instead of the structural part of the gene of β-galactosidase.
Recombinant plasmid pBGlac contains the gene encoding the laccase C1, comprising expression cassettes under the control of a regulated promoter of the gene of β-galactosidase Penicillium canescens, providing a high level of transcription of CE is avago gene, and terminator gene β-galactosidase Penicillium canescens. The structure of the expression cassette comprises a nucleotide sequence containing the promoter of the gene of β-galactosidase, the sequence of the signal peptide of the gene of β-galactosidase Penicillium canescens; a DNA sequence encoding a protein having the activity of laccase, or option; and the scope termination of transcription of the gene of β-galactosidase Penicillium canescens. With the introduction of this plasmid into the cell achieved a high level of transcription of the gene encoding the laccase, highly efficient promoter of the gene of β-galactosidase.
Using a plasmid can be transformed to any eukaryotic cell that is susceptible to a similar transformation specified by the plasmid. The choice of cells is not critical, because the methodology and methods of transformation are well known to the person skilled in the art. Although depending on the type of cells and culture conditions the received transformant the level of expression of laccase may vary, the fact that expression of the target protein will be subject to the successful transformation of the cells of the recipient.
"Transforming cells with plasmid" means the introduction of plasmids into the cell using methods well known to the person skilled in the art. Transformation of this plasmid bring the increased gene expression, encoding the protein according to the present invention, and to increased activity of the protein in the bacterial cell. Methods of transformation include any of the standard methods known to a person skilled in the technical field, for example the method described in Example 3.
Transformation of a cell can also be implemented by a linear DNA fragment containing the gene encoding the laccase C1 ligninolytic fungus Trametes hirsuta (SEQ ID NO:1) or its variant, under the control of a promoter that functions in such a eukaryotic cell.
According to the present invention "eukaryotic cell producing laccase" means a eukaryotic cell that is capable of production and allocation of laccase according to the present invention in a nutrient medium, when the eukaryotic cell according to the present invention are grown in a specified medium. Used herein, the term "eukaryotic cell producing laccase" also means a cell which is capable of production of laccase and causes the accumulation of laccase in a nutrient medium in large quantities, compared to the natural or parent strain, and preferably means that the specified cell is capable of accumulating in the environment of the laccase in the amount of not less than 0.1 standard units activity in 1 ml of reaction mixture (the definition of "conventional unit activity is using a chromogenic substrate pyrocatechin given above).
It is preferable to use cells of filamentous fungi as recipients for transformation with recombinant plasmid containing the DNA fragment encoding the laccase.
An example of a strain of the recipient to obtain the producer of recombinant laccase according to the present invention is, but not limited to, a strain of filamentous fungi Penicillium canescens PCA-10(niaD-) (Aleksenko A.Y. et al., Curr. Genet., 28, 474-478 (1995)).
A method of obtaining a recombinant laccase C1 ligninolytic fungus Trametes hirsuta includes the stage of growth of eukaryotic cells transformed with the plasmid according to the present invention in a nutrient medium suitable for growing Kazannik eukaryotic cells and isolation of recombinant laccase C1 from the culture fluid.
Using the above method allows the synthesis and production in Sekretareva soluble form laccase C1 ligninolytic fungus Trametes hirsuta activity not less than 0.1 standard units activity in 1 ml of the reaction mixture.
Recombinant laccase C1 ligninolytic fungus Trametes hirsuta 072 obtained by the method according to the present invention has the following characteristics: molecular weight of 70±2 kDa, isoelectric point of 4.0, the carbohydrate content is 12%, the composition of the carbohydrate chain - mannose and N-acetylglucosamine, the number is in the copper ion - 4, a pH optimum of 4.5 to 5.0, a temperature optimum of 55±3°C.
The present invention will be described in more detail below with reference to the following not limiting the present invention to the examples.
Example 1. Cloning of laccase C1 ligninolytic fungus Trametes hirsuta.
Drugs total DNA Trametes sp., as described previously (Rebrikov D.N. and other Applied biochemistry and Microbiology, 42, 6, 645-653 (2006)). The DNA fragment from the genome of laccase amplified using DNA Thrametes sp. 072 as template and primers Ecolac (SEQ ID NO:3) and Xholac (SEQ ID NO:4)complementary to the sequence 5'- and 3'-ends of the laccase gene and containing the recognition sites of restrictase Eco52I and XhoI, respectively. Amplification was performed by PCR in the amplifier Gradient Palm-Cycler in special conditions. The reaction mixture contained 5 µl sample of chromosomal DNA, 1-fold reaction buffer Pwo DNA Polymerase (Roche), 200 μm dNTP (deoxynucleotides), 0.3 μm primers, the total volume of the mixture of 50 ál. Temperature range: denaturation 95°C for 5 min; 95°C for 20 sec; 56°C for 20 sec; 72°C - 2 min, for 30 cycles; extension 72°C - 5 min reaction Products were separated by gel electrophoresis in agarose. The DNA fragment of viselli from agarose and purified using the set Wizard SV Gel and PGR Clean-Up System.
Example 2. Construction of recombinant plasmids pBGlac.
Clone f is armenta vector plasmid pBG produced by sites ApaI and XhoI. Correct insertion was confirmed by sequencing the obtained plasmid constructions. The nucleotide sequence was determined using an automatic sequencing machine Beckman - Coulter CEQ-2000 in accordance with the Protocol recommended by the manufacturer. Videlenie and purification of plasmid DNA was performed using the set Wizard Plus Minipreps DNA Purification System. Procedure cloning was performed according to standard techniques (Sambrook J., Fritsch E., Maniatis T. Molecular cloning: a laboratory manual. New York, Cold Spring Harbor Lab Press, 1989).
The resulting plasmid was named pBGlac.
Example 3. Obtaining strains producing laccase C1 ligninolytic fungus Trametes hirsuta.
For transformation of filamentous fungi used a strain of P. canescens x-ray 10(niaD-) (Aleksenko A.Y., et al., Curr. Genet., 28, 474-478 (1995)).
P. canescens PC A-10 (niaD-) were grown for 16 hours at 30°C in complete culture medium: peptone is an enzymatic - 3 g/l, yeast extract 2 g/l, glucose 10 g/l, NH4Cl - 10 mM, 50x solution of mineral salts - 20 ml/l Solution of mineral salts (50x) had the composition: KCl - 26 g/l, MgSO4×7N2O - 26 g/l, KH2PO4-76 g/l, trace elements - 50 ml/l 50x solution of mineral salts included CuSO4×5H2About 400 mg/l FeSO4×5H2About 800 mg/l, MgSO4×2H2About 800 mg/l, Na2MoO4×2H2About 800 mg/l ZnSO4×7N2About 800 mg/ l, B4Na2O740 mg/L. Transferred mycelium in a solution of 1.2 M MgSO4and 10 mm NaH2PO4pH 5.8 was added lytic enzyme of T. harzianum (5 mg/ml). Protoplasmatologia was carried out for 2 hours at 30°C under the conditions of mixing. The suspension was transferred into a centrifuge tube and layered 1-2 cm solution: 0.6 M sorbitol, 10 mM CaCl, 10 mM Tris-HCl. After centrifugation at 3000 rpm and 4°C for 10 min was selected the interphase containing the protoplasts. The protoplasts were washed 2 times in a stabilizing solution SCT (1.2 M sorbitol, 10 mm Tris, pH 7.5, 10 mm CaCl2and resuspendable it to a concentration of 108protoplasts/ml Transformation was carried out as follows: 200 μl of the suspension of protoplasts was added 10 μg of transforming DNA pBGlac, incubated in ice bath for 20 min, and then held osmotic shock for 5 min in PCT (50% polyethylene glycol, 10 mm CaCl2, 10 mm Tris-HCl) and protoplasts were planted in the upper layer on agar selective minimal medium containing 1.2 M sorbitol. For selection of recombinant clones was applied system cotransformation niaD gene controlling the synthesis of nitrate reductase (Aleksenko A.Y., et al., Curr. Genet., 28, 474-478 (1995)). As a selective marker was the ability to utilize nitrate. As recipients were used strains defective in nitroreductase, unable to grow on media with up is the making of nitrates. For transformation niaD-sign was used nitrate reductase gene of A. niger, cloned in the plasmid pSTA10 (23) (Unkles et al., Gene, 78, 157-166 (1989)). The protoplasts transformed with a mixture of DNA plasmids pSTA10 and DNA plasmids pBGlac with laccase gene Thrametes sp. at a ratio of 1:10. As the marker gene and the gene laccase not United in a common plasmid, and were added separately, for the formation of transformant laccase gene was required cotransformation where protoplast should get both plasmids. Cotransformation is more rare than transformacija single plasmid. About 40% of cotransformation, able to grow on medium containing nitrates, also contained a plasmid pBGlac with laccase gene that was detected by the presence of laccase activity in the culture fluid.
Thus were selected transformants: 21 clone of P. canescens, named R. canescens PCA-10(niaD-)/BGlac.
For selection of clones producing a laccase, were fermentation culturing and testing culture fluids for the presence of the corresponding enzyme activity.
Example 4. Determination of productivity of strains producing laccase.
Fermentation cultivation of selected clones was performed according to the following protocols. The strain of P. canescens PCA-10(niaD-)/BGlac to obtain the inoculum was grown on Harisov the authorized medium at 30°C for 5-7 days. A water suspension of conidia was inoculable 100 ml of medium (final concentration of 4×10-5conidia/ml). Cultivation was performed in a fermentation medium on an orbital shaker at 240-250 rpm katalozhnyh flasks 750 ml for 140 hours. At the end of the fermentation culture liquid was separated from the mycelium and the solid residues of the medium by centrifugation (10000 g, 10 min). As fermentation medium for P. canescens used medium having the following composition: sugar beet pulp - 30 g/l, peptone - 50 g/l, KH2PO4- 25 g/L. Aarizona medium had the following composition: 50x solution of mineral salts - 20 ml/l, glucose 10 g/l NaNO31 M, 10 ml/l, agar, bacto - 20 g/l Solution of mineral salts (50x) had the composition: KCl - 26 g/l, MgSO4×7N2O - 26 g/l, KH2PO4-76 g/l, trace elements - 50 ml/l 50x solution of mineral salts included CuSO4×5H2About 400 mg/l FeSO4×5H2About 800 mg/l, MgSO4×2H2About 800 mg/l, Na2MoO4×2H2About 800 mg/l ZnSO4×7N2About 800 mg/ l, B4Na2O740 mg/L.
The most productive clone of P. canescens PCA-10(niaD-)/BGlac has produced a laccase with a total activity of 3,4 conventional units activity in ml. Effective expression of laccase was observed during the cultivation of the producer strain in a medium containing as the carbohydrate component of beet is beet pulp.
Example 5. Determination of biological activity of recombinant laccase.
The laccase activity in the culture fluid strains were detected by spectrophotometry (wavelength 410 nm), using as a chromogenic substrate of 10 mm solution of pyrocatechin (PCH) in 0.1 M Na-acetate buffer pH of 4.9. Pyrocatechin was pre-purified by sublimation in vacuum. The culture fluid of strain R. canescens PCA-10(niaD-) was used as negative control. The results of measurement of laccase activity in the culture liquid obtained by culturing the strain-producer R. canescens PCA-10(niaD-)/BGlac at different temperatures, are shown in Table 1.
It is known that gene expression in heterologous systems is often not the correct splicing of introns. However, in this case, the presence of active laccase in the culture fluids selected recombinant strains indicates the correct processing of mRNA transcribed from the gene laccase in strains of P. canescens.
|The activity of laccase at different temperatures cultivation of the producer strain laccase .canescens PCA-10(niaD-)/BGlac.|
|The cultivation temperature, °C||The activity of laccase, conventional units of activity /ml|
1. Cell filamentous fungus belonging to the genus Penicillium, transformed with the plasmid containing the DNA fragment encoding the laccase C1 ligninolytic fungus Trametes hirsuta (SEQ ID NO:1), or a DNA fragment, which hybridizes with SEQ ID NO:1 in harsh environments, under the control of a promoter functional in said cell, producing laccase from C1 ligninolytic fungus Trametes hirsuta.
2. The cell according to claim 1, characterized in that, as specified cells using the cells of the filamentous fungus Penicillium canescens.
3. A strain of Penicillium canescens PCA-10(niaD-)/BGlac producing laccase C1 ligninolytic fungus Trametes hirsuta.
4. A method of obtaining a recombinant laccase C1 ligninolytic fungus Trametes hirsuta, including the stage of growth of the cells of the filamentous fungus belonging to the genus Penicillium, according to claim 1 in a nutrient medium and isolation of recombinant laccase from C1 culture is fluid.
5. The method according to claim 4, characterized in that as these cells using the cells of the filamentous fungus Penicillium canescens.
6. The method according to claim 5, characterized in that as the cells of filamentous fungus using cells of a strain of Penicillium canescens PCA-10 (niaD-)/BGlac.
SUBSTANCE: method of producing cyclopropyl-condensed inhibitors of dipeptidyl peptidase IV involves using BOC-protected amine with structural formula (3) , obtained through reductive amination of acid with formula (1) by treating the said acid with ammonium formate, nicotinamide adenine dinucleotide, dithiothreitol and partially purified concentrate of phenyl alanine dehydrogenase and formate dehydrogenase (PDH/FDH) enzymes and without separation - by treating the obtained amine of formula (2) with ditertbutyl dicarbonate, obtaining BOC-protected amine.
EFFECT: cutting on costs.
13 cl, 7 ex
FIELD: chemistry, biochemistry.
SUBSTANCE: method includes crashing and homogenisation of sprouted horse radish roots, extraction of homogenisate of horse radish roots with 0.15±0.01 M solution of sodium chloride. Ballast proteins are separated and peroxidase is precipitated with ammonium sulphate salts of 45-48% saturation and 85-90% saturation respectively. Gel filtration of peroxidase solution is carried out on Sephadex G-100 with eluting with 0.5-0.2 M sodium chloride solution. Chromatographic purification is carried out on carboxymethylcellulose. Liophyl drying of target peroxidase is carried out. Dialysis against sodium-acetate buffer with pH 4.4-5.0 and dialysis against potassium-phosphate buffer with pH 8.0±0.1 is carried out. Concentration with additional purification on DEAE-cellulose with elution with the same buffer with further dialysis against deionised water is carried out. Method allows to obtain peroxidase with high specific activity and high output. Peroxidase output constitutes 2.52-3.50 g/kg of horse radish roots, specific activity is 640-700 A.U./mg protein.
EFFECT: obtaining peroxidase with high specific activity and high output.
6 cl, 2 dwg, 1 tbl, 2 ex
SUBSTANCE: method of obtaining laccase enzyme medicine involves sustaining cultivation parametres depending on dissolved oxygen concentration in medium. Temperature of 25-27°C and pH within 3.0-7.0 are maintained from the start of cultivation until dissolved oxygen concentration falls to 50%. After further decrease of dissolved oxygen concentration to 5% temperature is increased to 27-29°C and uncompensated pH range is narrowed to 4.0-5.0. At reduction of dissolved oxygen concentration to minimum level and its further increase, temperature of 30-33°C and pH of 4.0-6.0 are maintained.
EFFECT: enhanced activity of culture fluid containing laccase.
1 tbl, 3 ex
FIELD: chemistry, biochemistry.
SUBSTANCE: proposed method includes deep cultivation of the strain of fungi Trametes hirsuta (Wulfen) Pilat, OOO PKF "BIGOR" CF-28 on a nutrient medium, separation of the fungi biomass, membrane concentration of the filtrate. The nutrient medium composition incorporates growth factors in concentration of 0.1-1.02 wt % and wheat flour in concentration of 2.0-4.0 wt % is used as a source of carbon. The cultivation is carried out at 28 to 34°C, pH 4.0 to 4.5, the concentration of dissolved oxygen making at least 1.5 to 2.0 mg·dm-3. The membrane concentration is effected with the membrane element pore sizes varying from 0.1 to 0.5 microns, at a pressure difference of 1.0 to 4.0 MPa and temperature of 20 to 40°C. Laccase activity makes some 140 to 200 ME.
EFFECT: production of fermented laccase preparation with high activity.
SUBSTANCE: claimed method includes production of homogenate from wheat Triticum aestiivum L. coarse grinding flour by extraction of protein structure with aqueous solution containing EDTA and DTT/ Extraction is carried out at continuous agitation of flour and aqueous solution in ratio of 1:3. Obtained extract is separated from solid phase by centrifugation at 3000-5000 rmp for 15-30 min/ Obtained supernatant is twice fractionated. The first fractionation is carried out at 20 % saturation of solution with ammonium sulfate; the second one is carried out at 70 % saturation followed by deposit separation using centrifugation after the first and second centrifugation. Obtained deposit id is resuspended in 0.1 M tris-HCl buffer containing EDTA and separated by centrifugation at 10000-14000 rpm. Isolated target crude product is salted out on column with Sefadex. Extract is purified by ion-exchange chromatography on column with Sefadex DEAE A50 in 0.1 M tris-HCl buffer followed by rechromatography of target product on column with Sefadex DEAE A50 in 0.1 M tris-HCl buffer, containing EDTA. Purified extract is freeze-dried.
EFFECT: enzyme with various purification ratio.
11 cl, 2 dwg, 1 tbl, 2 ex
FIELD: biotechnology, gene engineering, medicine.
SUBSTANCE: new chimerical enzyme proteins, in particular proteins comprising fragments of porcine uricase and baboon uricase (PBC and PKS) are obtained by increasing of number of accessible PEG-binding sites in mammalian uricase by substitution of at least one fragment of mammalian lysine-free native enzyme amino acid sequence with homological lysine-containing uricase fragment of mammalian belonging to another specie and screening of uricase variants based on activity and immunogenicity after conjunction with PEG. Disclosed uricase variants maintain after PEGylation sufficiently the same uricolytic activity that non-modified enzyme and are sufficiently non-immunogenic ones. Also describes are methods for production of new chimerical uricase proteins by recombinant DNA technologies and vector constructs and expression systems used therefore.
EFFECT: intermediates for medicines with low immunogenicity and enhanced bioavalibility.
16 cl, 14 dwg, 4 tbl, 7 ex
FIELD: biotechnology, biochemistry, enzymes, microbiology.
SUBSTANCE: invention proposes a method for microbiological oxidation of N-, O- or S-heterocyclic mono- or multinuclear aromatic compounds. The method involves culturing the recombinant microorganism that expresses cytochrome P-450-dependent monooxygenase BM-3 from Bacillus megaterium with amino acid sequence represented in SEQ ID NO:2 that comprises at least one functional mutation in region 86-88 and, if necessary, at least one functional mutation in one of regions 73-82, 172-224, 39-43, 48-52, 67-170, 300-335 and 352-356. The prepared oxidation product is isolated from medium, Invention provides carrying out oxidation of organic compounds with enhanced degree of effectiveness.
EFFECT: enhanced effectiveness of enzymes.
17 cl, 1 tbl, 7 ex
FIELD: genetic engineering, biochemistry.
SUBSTANCE: method involves insertion of functional or nonfunctional sequence of nucleic acid encoding polypeptide that shows activity of enzyme desaturase into genome that encodes the plant oil or microorganism. After culturing the transformed organism in suitable conditions oil or triglyceride is isolated from this organism. Enhanced or diminished production of polypeptide in organism with activity of the corresponding desaturase results to change of the amount of fatty acid in it and with the new content of calendulic acid in it. Invention can be used in production of saturated or unsaturated fatty acids or triglycerides with the enhanced content of such acids.
EFFECT: improved and valuable properties of gene.
8 cl, 7 dwg, 6 ex
FIELD: biotechnology, biochemistry.
SUBSTANCE: invention proposes a sequence of nucleic acid that encodes a polypeptide possessing activity of ▵6-acetylenase/▵6-desaturase or ▵6-desaturase and a polypeptide itself. Indicated sequence of nucleic acid is contacted with oil-producing organism followed by its culturing for isolation of triglycerides comprising in organism. Invention provides preparing triglycerides with the enhanced content of unsaturated fatty acids.
EFFECT: improved preparing method.
6 cl, 1 dwg, 1 tbl, 11 ex
FIELD: biotechnology, in particular gene engineering, pharmaceutical and food processing industry.
SUBSTANCE: DNA sequence (1341 n.p.) encoding fatty acid -desaturase (447 amino acid residue, 57 kD) of nematode Caenorhabditis elegants is isolated and characterized. Obtained DNA-sequence is expressed in bacterium and yeast cells to produce Biologically active enzyme recombinant form. Said recombinant form is capable to catalyze conversion of dihomo-γ-linolenic acid to arachidonic acid and eicosatetraenoate to eicosapentaenoate.
EFFECT: method for large-scale production of polyunsaturated fatty acid.
15 cl, 4 dwg, 1 ex
SUBSTANCE: present invention relates to biotechnology and immunology. An antibody against angiopoietin-2 is proposed. Versions of the antibody are disclosed, which are produced by hybridome ATCC PTA-7258, ATCC PTA-7259, ATCC PTA-7260. The corresponding coding nucleic acid and expression vector are disclosed. A host cell which produces the antibody based on the said vector is described. The disclosed antibodies have Kd of the order of 10-10-10-12 M, for the antibody 3.19.3 (from ATCC PTA-7260) IC50=99 nM. The said antibody properties can be used in treating human tumours.
EFFECT: design of a method of treating pathological angiogenesis based on an antibody and use of the antibody to prepare a medicinal agent for treating pathological angiogenesis.
33 cl, 18 dwg, 18 tbl, 24 ex
SUBSTANCE: trans-sialydase enzyme has been recovered from a unicell Trypanosoma congolense. Trans-sialydase is characterised by one of the following amivo acid sequences: SEQ ID NO:2, SEQ ID NO:4 or a sequence being 75% identical with one of said sequences.
EFFECT: extended range of enzymes with trans-sialydase activity.
6 cl, 3 dwg, 1 tbl, 6 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention pertains to biotechnology, animal husbandry and medicine. Proposed is a transgenic animal from the family of bulls which produces in its milk a recombinant human growth hormone in amount of at least 92 ng/ml per month on average. Also disclosed is a method of creating such a transgenic animal which involves cloning the genetic make-up which codes the hGH gene and a beta-casein promoter in an expression vector. Further, there is transfection into fetal somatic cells of cattle, usually fibroblasts, and nuclear transfer into enucleated oocytes of cattle, that way forming transgenic embryos. The invention can be used in animal husbandry and medicine.
EFFECT: invention enables to obtain a large amount of the human growth hormone.
43 cl, 5 dwg, 3 tbl, 9 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: present invention relates to biochemistry and specifically to a modified polypeptide of a HIV-1 gp41 envelope glycoprotein, a polynucleotide which codes the modified polypeptide and an expression vector which contains a coding modified polypeptide of the HIV-1 gp41 envelope glycoprotein. The modified polypeptide of the HIV-1 gp41 envelope glycoprotein contains an amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 14, where the sequence between positions 603 and 615 or 598 and 622 SEQ ID NO: 1 or the sequence between positions 530 and 542 or 525 and 549 SEQ ID NO: 14 is replaced by a linker fragment which is an oligopeptide SEQ ID NO: 2.
EFFECT: improved solubility of a modified polypeptide of the HIV-1 gp41 envelope glycoprotein without changing its immunogenic reactivity.
10 cl, 8 dwg, 1 tbl, 3 ex
SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.
EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.
17 cl, 1 dwg, 7 tbl, 7 ex
SUBSTANCE: integrative plasmid vector contains selective marker for selection of transformants in cells E.coli, site of replication start, site of integration and expression cassette, which consists of promotor, transcription terminator and selective marker, and integration site is represented by sequence of DNA that codes area 18S of messenger RNA.
EFFECT: improved productivity of strain.
3 cl, 4 dwg, 10 ex
SUBSTANCE: invention represents polypeptide, having α-L-arabinofuranosidase activity selected from the following polypeptides: polypeptide with SEQ ID No. 2, polypeptide, amino-acid sequence of which is located between positions 28 and 400 SEQ ID No. 2, fragment of polypeptide with SEQ ID No. 2, having activity of α-L-arabinofuranosidase, polypeptide having activity of α-L-arabinofuranosidase B and expressing 80% identity with polypeptide SEQ ID No. 2. Invention also relates to polynucleotide, which codes this polypeptide, expression cassette and vector, containing polynucleotide, and master organism that contains this polypeptide.
EFFECT: expanded arsenal of mediums for hydrolysis of α-L-arabinofuranosyl links in arabinofuranosyl-oligosaccharide compounds.
9 cl, 6 dwg, 2 tbl, 1 ex
SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.
EFFECT: applicable in medicine for preparing a vaccine.
52 cl, 32 dwg, 7 tbl, 29 ex
SUBSTANCE: invention relates to immunology and biotechnology. Described are versions of the humanised antibody CD45RO/RB which carry a light and a heavy strand. Versions of the following are disclosed: isolated polynucleotide, coding antibody, expression vector containing a polynucleotide and host cells containing the expression vector. Described also is use of the antibody to treat and/or prevent various diseases, including as a component of a pharmaceutical composition.
EFFECT: invention provides antibodies identified as CD45RO and CD45RB, which can find use in medicine.
9 cl, 14 dwg, 2 tbl, 13 ex
SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.
EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.
57 cl, 45 dwg, 4 tbl, 8 ex
SUBSTANCE: invention relates to biotechnology and represents method of obtaining L-amino acid using bacteria of genus Escherichia, bacterium being modified in such way that activity of alkoholdehydrogenase, coded by gene adhE, is enhanced in said bacterium.
EFFECT: invention allows to obtain L-amino acids with high degree of efficiency.
19 cl, 5 dwg, 4 tbl, 20 ex