Product and method

FIELD: medicine.

SUBSTANCE: invention concerns biotechnology and represents a new alpha galactosidase, a DNA molecule coding it. Besides the invention concerns an expression vector containing such DNA molecule, and also a cell transformed by the vector. The invention also concerns the method for making alpha-galaktobiose disaccharides with using new alpha galactosidase.

EFFECT: invention allows for high-efficient alpha-galaktobiose disaccharides.

15 cl, 5 dwg, 1 tbl, 2 ex

 

The invention relates to new α-galactosidase with transglycosylase activity capable of converting melibiose in α-galactosidase disaccharides. In particular, the invention relates to α-galactosidase isolated from a recently discovered strain of Bifidobacterium bifidum.

The invention in particular relates to DNA sequences coding for the selected enzyme α-galactosidase, the enzyme encoded by this DNA sequence, and to the cell host containing a DNA sequence or recombinant vector embedding the DNA sequence. The invention also relates to the use of the enzyme encoded by the DNA sequence, or a cell host containing a DNA sequence or recombinant vector to obtain α-galactosidase.

Bifidobacteria are usually inhabit the lower intestine, and such an environment is poor mono - and disaccharides, as specified sugar, preferably, are consumed by hosts and microbes that are present in the upper intestine. For survival in the lower intestine bifidobacteria produce various kinds of Exo - and endoglycosidase, surface-associated and/or in the form of an extracellular form that is able to utilize various carbohydrates.

In addition hydrolases activity of some enzymes of bifidobacteria possess Tr is nsverse activity. This transglycosylases the field of glycosidase inhibition activity intensively used for enzymatic synthesis of various oligosaccharides, which have been proven to function as the factors stimulating the growth of bifidobacteria.

It is known that some species of bacteria produce β-galactosidase enzymes involved in bacterial metabolism of lactose. So in Mollet, P.L. et al. inAppl & Environ Microbial.,(2001),62,(5), 2276-2283 described the isolation and characterization of three genes of β-galactosidase from Bifidobacterium bifidum. The authors found that all three β-galactosidase capable of catalyzing the formation of beta-linked galactooligosaccharide as a result of transglycosylation.

It is known that some species of bifidobacteria, in contrast to B. bifidum produce α-galactosidase and β-galactosidase. α-Galactosidase belong to the group of glycosylglycerols and can be classified into two groups based on specificity to the substrate, i.e. one such group has specificity to small sugars, such as p-nitrophenyl-α-D-galactopyranoside, melibiose and raffinose, and the other group is able to release galactose from galactomannan such as guar gum, in addition to other small substrates.

It was found that a strain of Bifidobacterium bifidum capable of producing the enzyme with galactosidase activity that converts lactose is a new mixture of galactooligosaccharides, which, as suddenly it was noted, contains up to 35% of the disaccharides, including albizu (Gal (α 1-6)-(Gal). It is known that such a disaccharide (see Paton, J.S. & Paton A.W. (1989),Clin. Microbial. Revs,11, 450-479; Carisson, K.A. (1989),Ann. Revievs Biochem,58, 309-350) has anti-adhesive ability to prevent adhesion of toxins such as Shiga toxin and such pathogens as E. coli, on the walls of the digestive tract.

The strain B. bifidum deposited under number NCIMB 41171 in the National Collection of Industrial &Marine Bacteria, Aberdeen, UK, March 31, 2003 in Addition, he also described in U.S. patent No.2412 380.

It was found that the strain B. bifidum produces α-galactosidase, can turn melibiose in α-galactosidase disaccharides.

The present invention relates to a DNA sequence that encodes a protein with the amino acid sequence shown in SEQ ID NO:2 or hybridities in stringent conditions with the DNA sequence that encodes the considered protein. The DNA sequence shown in SEQ ID NO:1 or may contain fragments or degenerate sequence.

The term "degenerate" refers to DNA sequences which are homologous to the sequence SEQ ID NO:1, at least 50%, more preferably 50-98%, most preferably at 75-95%.

This DNA sequence can contain nucleotide substitutions, insertions or deletions, SP is capable of changing the amino acid sequence of SEQ ID NO:2 is less than 60%, preferably, less than 45%, more preferably less than 25%. Nucleotide substitutions may give conservative substitutions in the amino acid sequences.

In a second aspect the present invention relates to the enzyme encoded by the above DNA sequence. Such an enzyme may contain the amino acid sequence of SEQ ID NO:2 or its fragment.

In the third aspect of the present invention relates to a recombinant vector, preferably expressing the vector containing the above DNA sequence. Such a vector may be introduced into the cell-host such as a bacterium, yeast or fungi. Alternatively, the DNA sequence can be introduced into such a cell is the master. Suitable cell-Khozin may be selected from Bifidobacterium, Lactococcus, Lactobacillus, Bacillus, for example, Bacillus subtilis, or Bacillus circulans, Esherichia and Aspergillus, for example, Aspergillus niger.

When using as a substrate melibiose, the enzyme encoded by the above DNA sequence, produces a mixture of oligosaccharides, mainly α-galactomannan disaccharides.

The above-mentioned enzyme or a host cell can be used to obtain α-galactomannan disaccharides, which may be part of the product, intended to stabilize the activity of the gastrointestinal tract. PR is a product of this type can be selected from the group consisting of dairy products (such as liquid milk, milk powder, for example, whole milk powder, skimmed milk, full-fat dry milk, whey powder, milk for baby food, mix baby food, ice cream, yoghurt, cheese, fermented dairy products), as well as beverages such as fruit juices, baby food, cereal, bread, crackers, pastries, cakes, food additives, dietary supplements, foods with probiotics, foods with prebiotics, animal feed, feed for poultry, and other foods or drinks.

Alternatively, the resulting disaccharides can be used to obtain drugs in the form of tablets or capsules to prevent adhesion to the walls of the intestinal tract pathogens or toxins produced by pathogens. Medicines of this type may be used by the patient, for example, after a course of antibiotics, which often disrupts the normal flora of the digestive tract.

Also the present invention relates to the above method of producing enzymes, which comprises culturing host cells in a suitable culture medium under conditions enabling expression of the enzyme and removing the floor is obtained as a result of enzyme or fermentation products from the culture medium.

Thus, the invention also relates to a method for galactomannan disaccharides, including contacting the above-described enzyme or host cell with a material containing melibiose, under conditions leading to the formation of disaccharides.

Suitable materials containing melibiose, can be selected from commercially available melibiose, raffinose, stachyose or soy extracts.

Brief description of figures

The figure 1 shows the nucleotide sequence (SEQ ID NO:1) α-galactosidase of Bifidobacterium bifidum with the originating or terminating codon indicated in bold italics;

the figure 2 shows the nucleotide sequence according to figure 1 with the amino acid sequence (SEQ ID NO:2) enzyme;

the figure 3 shows the first 540 amino acids amino acid sequence (SEQ.ID. NO:2) according to figure 2;

figure 4 is a graph showing the kinetics of the reaction during the synthesis of galactooligosaccharide α-galactosidase using 40% (wt./wt.) melibiose in 0,1M phosphate buffer as a substrate; and

the figure 5 shows the chromatogram obtained high-performance anion-exchange chromatography of a mixture of α-galactooligosaccharides synthesized using α-galactosidase from B. bifidum NCIMB 41171 using 40% (wt./wt.) melibiose in 0,1M phosphate buffer as substrate at pH 6.0. (Here Glc=glucose; Gal=galactose, Mel=melibiose; SR=degree of polymerization). Arrows, dotted lines, determine the position galactooligosaccharide products.

Genomic DNA was isolated from Bifidobacterium bifidum (NCIMB 41171) using the method of Lawson et al. (1989)Fems Environ Letters,65, (1-2), 41-45. DNA was treated with restriction enzymes, and fragments with a maximum size of 15 TPN ligated vector PBluescript KS(+). Cells of E. coli transformed with vector containing insertions, consisting of > PST processed by enzymes chromosomal DNA of B. bifidum. Clones with α-galactosidase activity were subjected to selection on plates with agar Luria Bertani containing p-nitrophenyl α-D-galactopyranoside and isopropyl-β-D-thiogalactoside (IPTG). Identified two α-galactosidase positive clone (pMeIA1 and pMeIA2).

Two received positive clone treated with enzymes EcroRI, PST and Bam HIshowed similar restriction map, which indicates that they both contain the same DNA insert. Sequencing of the inserted DNA fragment MelA1 was performed using the Sanger dideoxy method of chain termination (Russel P., 2002 iGenetics, Pearson Education Inc., San Francisco, 187-189) using the set to cycle sequencing BigDye Terminator V.3.0. (Applied Biosystems, USA). The DNA sequence MelA1 presented in Figure 1 (SEQ ID NO:1).

Open frame sitiveni the (ORF) was localized using ORF finder from NCBI (National center of Biotechnology information). Used the genetic code of bacteria and determined the length of the reading frame 300 mo. The nucleotide sequence shown in figure 1, was broadcast in six possible frames and was revealed one open reading frame of 759 amino acids encoding the estimated α-galactosidase (translation are shown in Figure 2 SEQ ID NO:2).

The following examples illustrate the present invention.

Example 1

Materials and methods

All chemical reagents and drugs environments used in this work were obtained from Sigma (Dorset, UK), Invitrogen (Paisley, UK), Oxoid (Basingstoke, UK), Qiagen (West Sussex, UK) and Promega (Southmpton, UK).

Bacterial strains

The strain of Bifidobacterium bifidum (NCIMB 41171) kept at cryogenic conditions in Microbank tubes at -70°C. For subsequent experiments, the strain was revived on agar Wilkinson Chalgren (WC) agar (Oxoid, UK) and environment TPY (Wednesday, Trypticase with fitinom and yeast extract) and were grown anaerobically (CO2and N2- 80% and 20%, respectively) at 37°C for 38 hours. The morphology of the colonies and the absence of contamination was checked by staining gram.

Strains E. coli

The strain of Eschericia coli RAllr and DH5a was used in this work, usually incubated in aerobic conditions at 37°C on agar medium Luria-Bertani (LB) or broth (Sambrook J. and Russell W.D. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laborotory Press, New York) and, if necessary, dobavleniyami (100 µg/ml ampicillin and/or 15 µg/ml chloramphenicol) and 40 μl of 2% X-α-galactopyranoside (X-α-Gal), 7 μl of 20% solution of IPTG (isopropyl-β-D-thiogalactoside), which was applied on the surface of a pre-prepared prepared 90 mm agar plates.

In experiments on expression used deficient α-galactosidase strain of E.coli RAllr (Hanatani et al., 1983, J.Biol. Chem,259(3) (genotype: melA-B+, recA-, lacZ-Y-)derived from E. coli. K12. The strain E. coli DH5a (Invitrogen, Paisley, UK) (genotype: F-φ80lacZΔM Δ (lacZYA-argF)U169 recA1 and endA1 hsdR17 (rk-; mk-)phoA supE44 thi-1 gyrA96 relA1λ-used in all other genetic experiments.

Selection of E. coli strains RAllr experiments expression was performed in accordance with genotype. The selected strain does not encode active α-galactosidase due melA mutations in its own DNA. However, this strain contains an active carrier of melibiose, which is needed to transport sugars (melibiose) in the cytoplasm and, consequently, for their metabolism active α-galactosidase. At the present stage of research it is impossible to conclude that occurs if the expression of α-galaxidi Bifidobacterium bifidum intracellular or extracellular. Thus, the presence of an active carrier of melibiose is essential for the identification of α-gal positive clones and, therefore, to highlight the genes encoding α-galactosidase.

It should be noted that in that case the AE, when considering the strain is a mutant recA, which minimizes the recombination of introduced DNA under the influence of the host DNA, increasing the stability of the insertion.

Extraction of Genomic DNA from Bifidobacterium bifidum.

Genomic DNA was isolated from strain Bifidobacterim bifiidum (NCIMB 411171) using the method of Lawson et al. (1989).

According to the described method, the cells were collected from plates in Eppendorf tubes with a capacity of 1.5 ml containing 0.5 ml of TES buffer. Was injected 10 μl of the mixture secrete lysozyme/mutanolysin (4:1, secrete lysozyme 10 mg/ml; mutanolysin 1 mg/ml) and the resulting mixture was stirred and incubated for 30 minutes at 37°C. Next, the cells were treated with 10 µl of proteinase K (concentration 20 mg/ml) and 10 μl of ribonuclease a (10 mg/ml)were mixed and incubated for 1 hour at 65°C. After incubation was added to the system 100 ál of 10% SDS solution and cell lysates were carefully mixed by inversion and incubated for 15 minutes at 65°C, then added to 0.62 ml of a mixture of phenol/chloroform, mixed by inversion before formation of the emulsion. Next, cell lysates were centrifuged with a speed of 6500 rpm for 10 minutes and the upper layer liquid was transferred into a clean Eppendorf tube using for this operation annealed broadband pipette. Extraction (stage deproteinization) was repeated to completely remove the tap fragments. DNA precipitated with addition of 1 ml ice-cold ethanol, and then incubation was performed at least for 30 minutes on ice, or storing overnight in the freezer at -20°C. Genomic DNA was isolated by centrifugation at a speed of 13000 rpm for 5 minutes, and after drying resuspendable in 5 μl of sterile mixture of 1 mm Tris-CI, pH 8.

Extracted DNA was analyzed by gel-electrophoresis and its concentration was measured at 260 nm. Kept at -20°C or -70°C for long period of time, avoiding multiple thawing and freezing to reduce the possibility of destruction.

Vector DNA drug

This study used a pBluescript KS(+) (Stratagen, Norh Torreyv Road). The choice of this clone funds due to the fact that the lac promoter coding plasmid pBluescript KS(+) is required for the initiation of gene transcription, losing its own promoter.

The vector was digested in the presence of the following restriction enzymes: > PST, BamHI and EcoRI in accordance with the manufacturer's instructions, using a ten-fold excess of enzyme relative to the amount of DNA (enzymatic units: μg DNA equivalent to ten units of the enzyme at one µg of plasmid DNA or ten units of enzyme per 0.5 picomoles of plasmid DNA). After thermoinactivation enzyme for 20 minutes at 65°C were analyzed by restriction map using horizontal gel electrophoresis.

Further, the vectors were dephosphorylated using intestinal alkaline phosphatase calf CIAP (Promega, Southampton, UK) according to manufacturer's instructions. The processing efficiency is tested self-legirovaniem vector (Bacteriofag T4 DNA ligase according to the manufacturer's instructions), and then made the transformation of DH5a cells.

A single piece of gel is testimony to the complete digestion of the vector and its single restriction digestion. The degree of digestion of the vector was tested by transformation not legirovannykh molecules competent cells of E. coli DH5a. The number of colonies formed on plates of LB agar, optionally containing ampicillin (100 µl/ml)served as a measure of the number of non-digested molecules and could serve as background signal for subsequent experiments.

Construction of genomic DNA library

Genomic DNA was partially digested in the presence of three restriction enzymes are able to recognize common hexa-nucleotide sequence that include prokaryotic DNA. EcoRI, BamHI and > PST represent restriction endonuclease type II specifically recognizes the sequence type 5'G/AATTC'3, 5'G/GATCC'3 and 5'CTGCA/G'3, respectively, this phenomenon is accompanied by double-strand break, leading to the formation of 5' vystupali the ends of the four nucleotides in these regions, GATS for EcoRI and BamHI, respectively, and the 3' protruding ends using ACGT for > PST.

It was found that all the enzyme had activity and were able to cleave DNA only in the presence of divalent ions of magnesium. Such ions have served only required cofactor.

Restriction digestion of DNA

All restriction pervari samples of genomic DNA were incubated for 2 hours at 37°C and were inactively at 65°C for 20 minutes. Then the reaction mixture was cooled to room temperature and was added the required amount of buffer, subjecting the mixture to mild stirring using a sealed glass capillary. Next, the solutions were loaded into capsules with 0.8% agarose gel (power supply 4-5 V/cm for 14-16 hours) and the size of the digested DNA was determined using 1 TPN standard DNA (Promega, UK) (Sambrook, J, Molecular Cloning: A Laboratory Mannual (2002).

Purification of the fragments generated after restriction digestion

Purification of the fragments from the reaction mixture and the agarose gel was performed using the kit for the extraction of QIAEX gel from Qiagen (West Sussex, UK). Analysis protocols detailed in the manufacturer's instructions.

Ligation and transformation of DNA

After purification of DNA fragments using the kit for gel extraction QIAX, the material was subjected to legirovanie using CIAP-treated pBluescript KC(+) vector. The purpose of ligation certain amount of DNA was transferred into a sterile microcentrifuge tubes with a capacity of 0.5 ml, as shown in table 1.

Table 1
Tube
And
DNA
The number of vector (15 femtomole) (~29,7 ng)
InThe number of vector (15 femtomole ~29,7 ng DNA) plus insert
(foreign matter 15 femtomolar ~69,3 ng)
pUc control (0,056 of femtomole {~100 PG})
In response ligating molar ratio of plasmid DNA vector and embedded DNA fragment was ~1:1. The final concentration of DNA was estimated to be ~10 ng/µl.
Mixture for ligation. Tube And shows the number of self-legirovannoi vector DNA, which should be excluded from the total number of transformants after transformation. The data obtained in vitro To demonstrate ligation of the vector DNA fragments, and the data from column shows the reference value, has allowed the possibility to calculate the efficiency of the transformation process.

Before each operation of ligation of DNA fragments was heated at 45°C for 5 minutes to melt all the "sticky" ends, which was re-annealed in order upon receipt of the fragment. The reaction was carried out according to the instructions Promega for all ligation reactions, the molar ratio of vector:insert DNA was chosen as 1:1.

In tubes a and b were injected at 1.0 μl of 10-fold excess digirolamo buffer and 0.5 Weiss units T4 DNA ligase (Promega, UK), and the entire volume of the reaction mixture was brought to 10 μl with biologically treated water. The tubes were injected with 1.0 μl of 10-fold excess digirolamo buffer, and the entire volume of the reaction mixture was brought to 10 μl with biologically treated water.

The DNA fragments were added in a test tube with water, after which the mixture was heated to 45°C for 5 minutes to melt all the "sticky" ends, which was re-annealed in order upon receipt of the fragment. DNA was cooled to 0°C before adding the remaining ligating reagents and the reaction mixture is incubated over night at 16°C (Sambrook and Rusell, 2001).

After ethanol precipitation and purification legirovannykh fragments (to remove ligiously environment, which may reduce the effectiveness of transformational transformations) further surgeries were performed by following the instructions Hanan. ~50 ng legirovannoi DNA in 5 μl of solution was added to 100 μl of onetenth E. ColiRAllr cells. After heat treatment, and expression in the presence of ampicillin-resistant gene cells distributed across the surface of LB plates containing ampicillin (100 μg/ml), X-α-Gal (40 μl of 2% X-α-Gal) and (7 ál of 20% IPTG).

We measured the number of transformations in each ligation reaction. The number of transformants in vitro was 2×105-1×106CFU/g, while the corresponding number in the sample was between 500 and 600 CFU/mcgil transformed in the sample And reflects the efficiency of processing of vector DNA. The number of transformants in the sample was 2-4×104CFU/µg.

The number of transformants

When legirovanii mixtures in the presence of > PST chromosomal DNA was obtained two α-galactosidase positive clone (pMelA1 and PmelA2) from about 2500 skanirovaniya of transformants, whereas in the presence of the chromosomal DNA, treated with EcoRI and BamHI, was not observed the formation of any positive clone from about 4000 all skanirovaniya transformed.

The digestion of a positive clone

Two positive > PST clone was digested with restriction enzymes EcroRI, PST, BamHI, HindIII, SmaI and kpni restriction sites. Restriction enzymes EcroRI, PST and BamHI showed similar restriction map, one piece ~5 TPN (target gene) and the other ~ about 3 TPN (plasmid DNA), suggesting that these enzymes cut in similar poses the operations. HindIII give a fragment of 6.5 TPN and a fragment of 1.5 TPN, whereas the enzymes SmaI and kpni restriction sites constitute only a fragment size of ~8 TPN, indicating the cleavage of only one position. A similar restriction maps for both plasmids indicate that both contain similar DNA insert.

Sequencing of the DNA sequence

DNA sequencing was performed using set to cycle sequencing BigDye Terminator V.3.0 (Applied Biosystems, USA) and analysis was performed using the ABI Prism 3100 system for fluorescent DNA analysis, including methods capillary electrophoresis.

5'- and 3'-ends embedded DNA fragments sequenced vector-specific primers. Insert additional sequenced using genomic priming (Genome Priming System (GPS-I)) (New England Biolabs, Uk). GPS-1 is an in vitro system based transposon TN7, which uses TnsABC transposase for embedded randomly in the target DNA. Donor: was used to target DNA mass ratio of 1:4 according to the manufacturer's instructions. The number of selected plasmids for sequencing after insertion of transpalmar in the target plasmid was 25. This number was calculated according to the manufacturer's instructions, andbased on 5-fold depth of coverage it assumes a 5-fold depth of coverage.

the unique priming sites at both ends transpiring element allow to sequence both strands of the target DNA at the insertion point.

Mixture for sequencing reactions contained approximately 400-600 ng of plasmid DNA, 3.2 picomole solution of primer and 4 μl of a solution of BigDye terminator.

Identification of open reading frames

Open-reading frames (ORFS) were identified using ORF finder from NCBI. Used the genetic code of bacteria and determined the length of the reading frame 300 mo. The nucleotide sequence was aired in six possible frames and was revealed one open reading frame of 759 amino acids encoding the estimated α-galactosidase (translation are shown in figure 2). Were confirmed by the initiation and termination codons.

Gene α-galactosidase of Bifidobacterium pMelAl the plasmid was expressed in E. coli under conditions of growth that normally inhibit the expression from the inducible E. coli lacZ promoter, located in the flanking region of the cloning vector. These results indicate that endogenous, internal bifidobacterial sequence directed against the gene of α-galactosidase, can serve as a signal of transcription initiation in E. coli.

The start of transcription is indicated in bold italics. The above results indicate that the gene is controlled by its own promoter transcription.

Example 2

Synthesis using cloned enzyme a-galactosidase,

isolated from Bifidobacterium bifidum NCIMB 41171 to glue the ke-host E. coli (strain RA11).

Synthesis, described below, were performed, unless otherwise stated, with whole cells of E. coli RAll as the owner after treatment of E. coli biomass (collected by centrifugation at 10000 g), toluene at a concentration of 2000 ppm, for increasing the permeability of the cells to make it unviable, destroying its cytoplasmic membrane. The biomass of E. coli was prepared as described in p."Strains of E. coli Example 1.

Synthesis using cloned enzyme

Synthesis using α-galactosidase was carried out at the initial concentration of melibiose in the substrate 40% (wt./wt.). Synthesizing the solution was obtained in 0.1 M phosphate buffer with a pH of 6.0. The synthesis was carried out at a temperature of 40°C in water bath shaker at 150 rpm, the Optimum pH for a specific enzyme were chosen based on activity measurements (using as the substrate p-nitrophenyl-α-D-galactopyranoside) specific enzyme preparation at different pH values.

For synthesis using α-galactosidase 2 ml of cell suspension of E. coli RAll (with an activity of 0.3 units/ml) was centrifuged (10000 g) for biomass harvesting, and the supernatant was discharged. This biomass resuspendable 1 g of a 40% (wt./wt.) of the substrate solution, melibiose for carrying out synthesis. The Figure 4 shows the concentration of various sugars present in the mixture during synthesis. The figure 5 shows the chromatogram galactor Goehring mixtures, synthesized α-galactosidase, cloned from .bifidum NCIMB 41171, obtained by high-performance anion-exchange chromatography in conjunction with pulsed amperometric detection (HPAEC-PAD). Table 2 shows the concentrations of sugars galactooligosaccharide mixture at the time optimal synthesis.

Table 2
Carbohydrate composition during the synthesis of α-galactooligosaccharides at 40% (wt./wt.) the initial concentration of melibiose in time, when there is the maximum
the concentration of oligosaccharides
The synthesis. started. the subst.STATE JV ≥ 3STATE SP=2MelGlcGal
% (wt./wt.)Concentration (% of total sugars)
4013,936,6138,0624,117,9
Mel: melibiose, Glc: glucose, Gal: galactose, SP: degree of polymerization.

1. The DNA molecule encoding an α-galactosidase that has a sequence that
a) encodes a protein with the amino acid sequence represented by SEQ ID NO: 2, or
b) hybridized in stringent conditions of hybridization with sequence (a), or
c) is a degenerate sequence of a) or b), which 75-95% homologous to them.

2. The DNA molecule according to claim 1, where the sequence represents SEQ ID NO: 1 or a fragment or degenerate sequence that is at 75-95% homologous her.

3. The DNA molecule according to claim 1 or 2, which is obtained from the sequence degenerate sequence includes nucleotide substitutions, insertions or deletions that result in less than 25% of the modified amino acid sequence of SEQ ID NO: 2 or its fragment.

4. The DNA molecule according to claim 1 or 2, which is obtained from the sequence degenerate sequence contains the nucleotide replacement is a conservative amino acid replacement.

5. α-galactosidase encoded by the DNA sequence according to any one of claims 1 to 4.

6. α-galactosidase containing the amino acid sequence of SEQ ID NO: 2 or a functional fragment.

7. The recombinant expression vector, including the surrounding DNA sequence according to any one of claims 1 to 4.

8. Bacterial cell-host for ekspressirovali α-galactosidase according to claim 5 or 6, containing the DNA sequence according to any one of claims 1 to 4.

9. Bacterial cell-host for ekspressirovali α-galactosidase according to claim 5 or 6, containing the vector according to claim 7.

10. A host cell of claim 8 or 9, where the specified cell is selected from the group consisting of Bifidobacterium, Lactococcus, Lactobacillus, Escherichia, Bacillus and Aspergillus.

11. A host cell of claim 10, where the specified cell is selected from the group consisting of Bifidobacterium bifidum, Bacillus subtilis, Bacillus circulans and Aspergillus niger.

12. The use of α-galactosidase on any of subparagraph 5 or 6 or cells according to any one of p-11 to obtain α-galactomannan disaccharides.

13. The use of α-galactosidase on any of subparagraph 5 or 6 or cells according to any one of p-11 to obtain α-galactomannan disaccharides, which is part of the product selected from the group consisting of dairy products such as liquid milk, milk powder, infant formula, a mixture of baby food, ice cream, yoghurt, cheese, fermented dairy products, beverages such as fruit juices, products for baby food, cereals, bread, biscuits, confectionery, baking, food additives, dietary supplements, foods with probiotics, food products with prebiotics, animal feed, feed for poultry and medicines.

14. How is Holocene α-galactosidase on any of subparagraph 5 or 6, comprising culturing the host cell according to any one of p-11 in a suitable culture medium under conditions allowing to Express the specified enzyme, and removing the resulting α-galactosidase from the culture medium.

15. The method of obtaining α-Galaktionova disaccharide, including the conversion of α-galactosidase on any of subparagraph 5 or 6 or a host cell according to any one of p-11 in contact with the solution melibiose.



 

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10 cl, 26 dwg, 2 tbl, 22 ex

FIELD: pharmacology.

SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.

EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.

35 cl, 13 dwg, 36 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.

EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.

16 cl, 3 tbl, 7 ex

FIELD: food industry.

SUBSTANCE: strain Streptococcus thermophilus which produces lactic acid is described. Sequence of nucleic acids made of the strain producing polysaccharides are also described as well as food or pharmaceutical composition and milk product containing such strain.

EFFECT: strain has strong structural properties.

16 cl, 4 dwg, 6 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of genetic engineering and medicine. Described is animal, non-human, having sequence of nucleic acid encoding presenilin 1, carrying mutations, corresponding to M233T and L235P mutations in PS1 protein of mouse. Animal also contains sequence of nucleic acid, encoding whole gene or part of gene, encoding APP. APP protein represents APP751, originates from human and carries mutations Swedish and London. Animal is intended for application in fight against Alzheimer's disease. Also described are PS1 protein and encoding it nucleic acid.

EFFECT: invention can be used in medicine for discovering compounds intended for Alzheimer's disease treatment.

20 cl, 50 dwg, 1 tbl, 8 ex

FIELD: agriculture.

SUBSTANCE: method is described for improvement of crop capacity in plant parts that provide for crops versus according plants of wild type, including introduction of nucleic acid into plant, which codes cyclin-dependent kinase of D-type (CDKD), containing NXTALRE link, where X stands for any amino acid. Method is disclosed for production of transgenic plant with higher crop capacity, including introduction of CDKD coding nucleic acid into plant or vegetable cell, which contains NXTALRE link, where X stands for any amino acid, and cultivation of vegetable cells in conditions that promote regeneration and growth of mature plant.

EFFECT: invention makes it possible to improve crop capacity of plants.

14 cl, 3 dwg, 5 tbl, 4 ex

Protein // 2380414

FIELD: medicine.

SUBSTANCE: present invention also concerns nucleic acid sequences coding specified fungous lipolytic enzymes. Present invention also concerns foodstuff and method for making foodstuff that involves addition of the fungous lipolytic enzyme under present invention to one or more components of a prepared product. Present invention also includes the method for preparing lysophospholipid and the method for preparing lysoglycolipid wherein said fungous lipolytic enzyme under present invention is used.

EFFECT: possibility to produce advanced foodstuff, including bakery products.

22 cl, 43 dwg, 29 tbl, 16 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to biotechnology and concerns protein of protease NS3/4A HCV or its biologically active fragment, which contains succession, in which residue of amino acid, corresponding to amino acid 156 of protease NS3/4A HCV of wild type, is not the residue of alanine and, not obligatorily, residue of amino acid, which corresponds to aminocacid 168 of protease NS3/4A HCV of wild type, is not asparaginic acid. Invention also relates to polynucleotide, coding said protein, as well as application of said protein in detection of HCV presence in biological sample, as well as in method of determination if the infection, mediated by HCV, in patient is resistant to medication against HCV, in estimation method, if the infected by HCV patient has lower sensitivity or susceptibility to VX-950, as well as in method of candidate examination for inhibitor or potential inhibitor of HCV.

EFFECT: expansion of arsenal of medications for HCV treatment.

68 cl, 17 dwg, 6 tbl, 13 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used in the food industry, as well as in production of detergent materials. A gene of new phospholipase (FvPLA2) is cloned from Fusarium venenatum, which belongs to a group of fungal and bacterial phospholipases XIII PLA2. Analysis has been of physicochemical properties and catalytic activity of the new phospholipase, in accordance with the results of which proposal is made for using FvPLA2 in baking bread, production of vegetable oil, making cheese, as well as in detergent compositions.

EFFECT: nucleotide sequence is defined, which codes FvPLA2 and its active form, through expression of which a recombinant product with phospholipase activity is obtained in a heterologous system.

8 cl, 6 tbl, 11 ex

FIELD: biology.

SUBSTANCE: present invention relates to microbiology and biotechnology. Obtained is a recombinant strain of Yarrowia lipolytica which produces lipase, developed through transformation of the Yarrowia lipolytica Polf strain using integrative plasmid pZ-ura3d4-hp4d-LIP2.

EFFECT: obtained Yarrowia tipolytica strain can be used in obtaining lipase on an industrial scale.

2 dwg, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention concerns biotechnology and claims polynucleotide encoding polypeptide with cysteine protease effect. Also invention concerns coffee tree cell transformed by the indicated polynucleotide.

EFFECT: possibility to modulate coffee aroma precursor level in raw (non-fried) coffee grain.

14 cl, 22 dwg, 7 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: proposed invention concerns biotechnology and represents an improved method of streptokinase production involving genetically engineered strain of Escherichia coli MTCC 5120, which overproduces streptokinase inside a cell and grows in optimised nutrient medium consisting mainly of simple salts and microelements.

EFFECT: efficient and economical method for obtaining streptokinase applicable in thrombolytic therapy.

21 cl, 6 ex

Modified phytases // 2329301

FIELD: biotechnologies.

SUBSTANCE: polypeptide with phytase activity is proposed. The sequences are shown in the description. A polynucleotide coding the said polypeptide is described. The application of the said polypeptide for the use in a feed mix or a feed supplement is described.

EFFECT: allows to increase modified phytase resistance against temperature and humidity.

6 cl, 6 ex, 12 tbl, 10 dwg

FIELD: biotechnology, microbiology, biochemistry, enzymes.

SUBSTANCE: method involves culturing corynebacterium comprising the inserted gene encoding neutral metalloprotease from Streptoverticillium and prostructural moiety of protransglutaminase is split off by neutral metalloprotease produced by this microorganism. Features of neutral metalloprotease are given in the invention text. Also, invention relates to nucleic acid molecule encoding neutral metalloprotease. Invention provides preparing protease that can be used for producing transglutaminase that splits prostructural moiety of protransglutaminase selectively.

EFFECT: improved producing method of enzyme.

8 cl, 12 dwg, 2 ex

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