Production method of keratinase from penicillium citrinum

FIELD: biotechnologies.

SUBSTANCE: production method of keratinase provides for directed adaptation of strain Penicillium citrinum PC-54-91"ВИЛАР" by three-time subcultivation on Czapek agar medium containing 2% of hair keratin as a carbon source. Cultivation under deep conditions on Czapek medium containing 10% of hair keratin and 0.5% of saccharose during 6 days. Separation of biomass from culture fluid with further extraction of target product by two-stage chromatographic cleaning involving gel filtration on TSK-Gel TOYOPEARL HW-40 and affine chromatography on protein A to sepharose CL-4B with further lyophilisation.

EFFECT: invention allows increasing ferment yield, obtaining keratinase preparation decomposing the hair α-keratin, and improving cleaning degree of ferment preparation.

2 tbl, 5 ex

 

The invention relates to the field of biotechnology, in particular, to the production of enzymes using microbial synthesis and can be used to produce potent drugs keratins used in medicine, cosmetics, light industry and agriculture in cases where the hydrolysis of keratin.

The aim of this work is to obtain high-purity drug keratinase with high yield and enzymatic activity able to hydrolyze α-keratin.

In the complex of proteolytic enzymes produced by microorganisms, an important place is occupied by the enzymes that break down keratin - keratinase (Y.S. 3.4.21-24, 99). Keratins are proteins, consisting of a long polypeptide chains, stabilized by disulfide bonds, hydrophobic interactions, and hydrogen bonds, and containing a high percentage of hydrophobic amino acid residue, insoluble in water and resistant to conventional proteoliticheskikh enzymes. On the secondary structure of the protein family of keratins are divided into two groups: α-keratins, organized in the form of α-helix, characterized by a high content of cysteine and many disulfide bonds that are included in hair, wool, nails, horns and hooves of mammals (hard α-keratin), and epidermis (soft α-keratin), and β-keratins shaped bore the zigzag channels at polypeptide chains (β-sheets), with a lower content of cysteine, included in the feathers, beak, claws of birds, scales of reptiles and exoskeletons of arthropods, in addition to α-keratins.

The range of application of microbial keratinase is extremely wide: in the agro-industry for the conversion of keratin-containing wastes in the production of feed hydrolysates, additives and nitrogen fertilizers; in the biomedical, pharmaceutical and cosmetic industry for the hydrolysis of prions, preparation of vaccines in the treatment of dermatophytosis, production of bioactive peptides, removal of keratin in the treatment of psoriasis and acne, degradation of keratinized skin and hair; in the leather industry for the process of dehairing alternatively traditional, polluting the environment, the method of tanning leather using sulfides; in the textile industry for modification of the fibers; in the manufacture of detergents and biopolymers (biodegradable films, adhesives, coatings); for wastewater treatment, as well as the conversion process of keratin-containing waste into biofuel [Brandelli, 2010].

Producers microbial keratinase are bacteria, mainly Bacillis sp., actinomyces and some deuteromycete. Currently, the commercial drug keratinase produced by the company "Merck" from the culture fluid actinomycete Streptomyces fradiae I.M. No. 3739[Nickerson, 1961]. Deep cultivation of produce in a nutrient medium, containing, along with mineral salts keratin substrate (sterilized Undenatured wool, feathers or flour from hooves) within 7 days. For deposition of keratinase the cultural filtrate liquid is treated with ammonium sulfate, followed by desalting the resulting sludge by dialysis and freeze drying of the drug. Despite the high enzymatic activity of this keratinase used for dehairing in the leather industry, the purification degree is small (activity compared with culture fluid increases 14-fold), which limits the field of application of this drug.

Also known is a method of obtaining keratinase using Streptomyces chromogenus S.graecus LEAH 0832, increasing output keratinase 11 times by changing the pH of culture medium [Stallions, 1990].

Keratinase most studied microorganisms capable of destroying β-keratins and only a small number of produce enzymes with pronounced activity against α-keratins, especially to keratin of hair and wool. Described is a method of obtaining various bacterial keratinase, consisting in the selection of strain on agar medium, the culturing in a nutrient medium containing 0.5% of flour out of her hair, 5 soy flour, 1% corn flour and 1% fish meal, and how to obtain a filtrate of the culture fluid [Yang, 2005]

Of particular interest is the obtaining of proteolytic enzymes with high keratinase activity against α-keratins, but not with collagenase activity, since their use due to the lack of destruction of collagen allows to obtain high-quality leather, as well as pharmaceutical and cosmetic preparations. Discovered the Bacillus subtilis strain S14, secreting keratinase with the specified properties under cultivation in a medium containing whey and bullish wool (40 g/l). However, the activity of this subtilisin-like keratinase, purified using ionoobmennoi chromatography, was not investigated, and was okharakterizovana only the culture fluid [Macedo, 2005]. Recently reported on a strain of Bacillus subtilis AMR, is able to hydrolyze human hair [Mazotto, 2009]. Pre hair was degreased with a mixture of chloroform/methanol (1:1), washed, chopped and added in 0.06 M phosphate buffer (pH 7,2) in an amount of 10 g/l, containing 0.01% of yeast extract. Maximum keratinolytic activity of the culture liquid after 8 days amounted to 163 units/ml, however, the activity determination was performed using a "soluble" keratin, which can lead to a substantial increase of the obtained results is tov.

Currently, much interest has grown as producers of proteolytic enzymes with keratinolytic effect of micromycetes, non-dermatophytes. Reported method for the keratinase from Myceliophtora thermophilia GZUIFR-H94 by cultivation of a producer on a medium containing as a source of carbon and nitrogen, 10 g/l pen flour and 0,98 g/l of urea at 38°C [Yongquan, 2009].

To the producers-deuteromycetes capable of high speed to hydrolyze α-keratin is one of the strains of Aspergillus flavus, which reportedly can be used as dailywage agent at surface cultivation on the environment with breast serum added as inducer of the synthesis of protease [Malathi, 1991]. Lack of work is the definition does not actually keratinase activity, but only of enzyme activity towards casein. Purification of the enzyme and determination of substrate specificity were not performed. It should also be noted that deuteromycete is a potential producer of aflatoxins.

A method of obtaining keratinase from the culture fluid Doratomyces microsporus and Paecilomyces marguandii under cultivation in liquid medium containing soybean flour as an inductor keratinase, which is able to hydrolyze α-keratins EP is of the dermis and nail, but to a much lesser extent, keratin hair, wool and feathers, and with low collagenolytic activity [Gradizar, 2005].

Deuteromycetes, non-dermatophytes that can form keratinase, gidrolizuemye nails, human hair or feathers are some species of the genus Penicillium [Marcondes, 2008], including Penicililum citrinum [Anby, 2006; Gordonov, 2009]. It should be noted that Penicillium citrinum is not pathogenic and is of great practical importance in biotechnology and medicine as producer of statins [Li, 2011], plant growth regulators [Kuramata, 2007] and various enzymes, such as used in the food industry nuclease P1required to obtain 5-Yasinovka acid [Ichishima, 1991], and lipase [Pimentel, 1996]. Among the enzymes synthesized by this micromycetes hold an important place and proteolytic enzymes, which include keratinase.

As the prototype was chosen as the method of obtaining keratinase from Penicillium citrinum [Anby, 2006]. The technique of culturing Penicillium citrinum allowed the authors to consider the studied strain potential producer of keratinase. The maintenance of the strain was carried out in the environment Saburo. Deep cultivation was carried out in a nutrient medium containing 1% keratin substrate in the form of a powder of low-fat with a mixture of chloroform-methanol (1:1), washed distilleria the Noi water and dried feathers as the sole source of carbon and nitrogen, and mineral salts (g/l): 1.5 K2HPO4, 0,05 MgSO4×7N2Oh, 0,025 CaCL2, 0,015 FeSO4×7H2O and 0.005 ZnSO4×7H2O,pH 7.5, seeded spore suspension of 106spores in 1 ml of medium at 30°C, agitation of 100 rpm for 3 weeks. Certinatly activity of the culture liquid, filtered and subjected to zentrifugenbau (8000 g, 10 min), amounted to 1.48 keratinizing units/ml was higher than that of many other studied species of deuteromycetes-determination (genera: Penicillium, Aspergillus, Cladosporium, Paecilomyces) and dermatophytes (Microsporium gypseum, Chrisosporium keratinophillum, Myceliopthora vellerea). The allocation of keratinase was not carried out.

Because of the isolation and purification of keratinase from the culture fluid Penicillium citrinum not yet developed, we have considered ways of getting other proteolytic enzymes specified producer.

There is a method of allocating penicillamine - metalloproteinases from Penicillium citrinum [Yamaguchi, 1993] by chromatography of the enzyme on CM-sepharose, rechromatography on CM-sepharose and final purification using FPLC-Superose 12. The resulting electrophoretic homogeneous product with a molecular weight of 18 kDa, however, the degree of purification and the yield of the enzyme has not been described. Certinatly activity penicillamine not been studied.

The literature describes a method of separation of Surinov the th protease, belonging to the family of subtilisin, Sekretareva Penicillium citrinum 52-5, under cultivation on mineral medium containing 0.5% glucose and 1.5% beef extract for 4 days [Su, 2001]. Clean filtered from biomass extract included: salting out with ammonium sulfate to 80% on ice, centrifugation, dissolving the precipitate in 10 mm phosphate buffer (pH 7,8), dialyzed against this buffer, separating undissolved proteins by centrifugation and final purification using ion-exchange chromatography on CM-sepharose. In the result, it was possible to purify the enzyme 3.4 times, the yield of protease was 14.1 per cent.

Describes a method for serine protease, inactivating the lactate dehydrogenase from Penicillium citrinum KE-1 [Watazu, 1994]. Deuteromycete were cultured in liquid medium containing 1% glucose, 1% meat extract, 1% polypeptide and 0.3% NaCl, pH 7.0 at 30°C for 6 days. Cleaning procedure of the supernatant obtained after centrifugation of the culture liquid, included the following stages: deposition of protein with ammonium sulfate, centrifugation, dissolving the precipitate in 10 mm phosphate buffer (pH 6.0), dialysis against the same buffer, ion-exchange chromatography on CM-sepharose, the concentration of the active fractions by ultrafiltration UF-10 PS membrane, dialysis against phosphate buffer (pH 6.0)containing 0.1 M NaCl, chromatographia Sephadex G-100, dialysis against distilled water and freeze drying. As a result, the enzyme was purified 124 times, the yield was 81%.

The problem to which the invention is directed, is increasing keratinase activity culture Penicillium citrinum PC-54-91 VILAR, reducing the time to conduct the process of cultivation and development of procedures for the isolation and purification of keratinase to obtain a purified enzyme preparation with high output, is able to hydrolyze α-keratin.

These goals are achieved by using as the source of keratinase deuteromycete Penicillium citrinum PC-5 4-91 VILAR with pre-directed adaptation of inoculum by three reseeding on Chapek's medium containing as the sole source of carbon and nitrogen 2% keratin hair; deep cultivation producer on the modified Chapek's medium containing 10% keratin hair; applying a two-step chromatographic purification method, including gel-filtration on TSK-Gel TOYOPEARL HW-40 ("TOYO SODA") is a gel-based spherical hydrophilic granules (diameter 45 μm) of polyethylene oxide with a pore size of 50 And the range of molecular weights of the partial protein from 15 000 to 100, Yes, no sorbing substances of protein nature, using as eluent 0.15 M NaCL solution, and affinity chromatography fraction mole is Blarney weight exceeding 15,000 Yes using protein And sepharose CL-4B (Pharmacia") - sorbent with a diameter of granules 60-140 μm on the basis of agarose, a polysaccharide, a simple link which is agrobios, and as a ligand is used, the protein And possessing the ability to absorb keratinase, which elute 1 M solution of acetic acid (pH 2,4), and further freeze-drying of the drug.

Definition keratinase activity carried out by the modified method of Anbu et al (2006). Per unit keratinase activity (KE/ml) accept such quantity of enzyme, which as a result of proteolysis 20 mg keratin hair at 37°C for 1 h in the presence of 3.8 ml of 100 mm Tris-HCL buffer (pH 7,8) and 0.2 ml of the filtrate of the culture fluid obtained using a membrane filter with a pore size of 0.23 μm, or 0.2 ml of eluent when measured on a spectrophotometer (λ=280 nm) after filtration of the incubation mixture increases the optical density of the experimental samples over the control, equal to 0.1. Specific keratinase activity calculated in terms of mg of extracellular protein identified by the method of Lowry.

Example 1. To obtain a seed culture of Penicillium citrinum PC-54-91 VILAR) were grown for 7 days in an incubator at 26°C on a beveled surface of the agar medium of čapek, of the following composition (%): NaNO3- 0,2; KH2PO4- 0,1; MgSO4×7H2O - 0,05; KCl - 0,05; FeSO ×7H2O - 0,001; caso3to 0.3; saccharose - 2; agar - 2, pH 6.8 (the method of obtaining the No. 1 - table 1). Seeds served as a spore suspension of deuteromycete (108spores per 100 ml medium). Cultivation in deep conditions was carried out in flasks with a volume of 300 ml with 100 ml of nutrient medium on a shaker at a speed of 220 rpm at 26°C. the Cultivation was carried out on a modified environment of čapek with partial replacement of sucrose on the keratin substrate of hair - fat-free mixture of chloroform: methanol (1:1), shredded and mindelsohn human hair [Gordonov, 2007] (0,5% sucrose and 1.5% keratin). In the culture filtrates of the fluid deuteromycete obtained by filtering through a membrane filter with a pore size of 0.23 μm at various stages of cultivation, defined the General and specific keratinolytic activity as described above.

Maximum certinatly activity of the culture liquid was 1,08 KE/ml after 10 days of cultivation.

Primer. The culture of Penicillium citrinum PC-54-91 VILAR, grown on agar medium of čapek as in example 1, was perseval one, two or three times (the methods of obtaining the No. 2, 3, 4, respectively - table 1) on agar medium of čapek with the replacement of sucrose on keratin hair for directed adaptation of culture. Conditions of planting and cultivation analogous to example No. 1. Max what I certinatly activity of the culture liquid was 1.47 KE/ml after 8 days of cultivation in the way of getting No. 2, 2,35 KE/ml after 7 days of cultivation in the method of obtaining the No. 3 and 2,60 KE/ml after 6 days in the way of getting No. 4.

Example 3. Cultivation of Penicillium citrinum PC-54-91 VILAR was performed as described in method get No. 4, increasing the concentration of keratin hair in a liquid nutrient medium up to 5 and 10% (ways to get # 5 and # 6, respectively - table 1). Maximum certinatly activity of the culture liquid after 6 days of cultivation amounted to 4.73 KE/ml for method of obtaining the No. 5 and 6.26 KE/ml for method No. 6.

Table 1 presents the results showing the advantages of the proposed method in comparison with the known.

Example 4. Cultivation of Penicillium citrinum PC-54-91 VILAR was performed as described in method get No. 6 (table 1). For isolation and purification of keratinase from the filtrate of the culture fluid was consistently applied the gel filtration and affinity chromatography. For gel filtration were used column "LKB" (of 2.6×40 cm) with gel TOYOPEARL HW-40. The column was applied to 10 ml of the filtrate of the culture fluid. The eluent served 0.15 M NaCl solution. Detection was carried out at a wavelength of 274 nm using flow densitometer Uvicord 2", the rate of elution was 3.3 ml/min Optimization of the chromatography conditions such as the amount of deposited sample and the rate of elution, have enabled us to achieve a very clear distinction pointed to by the x fractions. When this fraction, corresponding to the external volume of the column and a material with a molecular weight higher than 15 kDa, possessed considerable keratinase activity exceeding keratinase activity of the original drug 3.03 times, and was 11.8 KE/mg. However, carried out using a disc-electrophoresis analysis of the material fraction 1 showed that it is not homogeneous and in addition to the basic protein with a molecular weight of about 21 kDa and contains other proteins with higher and lower molecular weights, which required the development of additional cleaning methods enzyme preparation.

Material with keratinase activity, after gel filtration was collected and subjected to purification by the method of affinity chromatography. For these purposes, LKB column volume 9×150 mm was filled with protein And separate in 0.01 M phosphate buffer (pH 7.4) and applied the material fractions obtained after gel filtration with keratinase activity. The elution was performed using sequentially 0.01 M phosphate buffer (same buffer with 1 M NaCl and 1 M solution of acetic acid until complete removal of the adsorbed material. The rate of elution was 10 ml/hour. The optical density of the eluate was monitored at a wavelength of 274 nm using flow densitometer Uvicord 2". It is shown that when carrying out affinity chromatography material, possessing to latinosnow activity was elyuirovaniya with a column under the action of 1 M acetic acid. Certinatly the activity of the other fractions was practically equal to zero. Electrophoretic analysis using disc electrophoresis in SDS page [Sheludko, 1975] identified only one homogeneous protein fraction with a molecular weight of about 21 kDa. After carrying out affinity chromatography enzymatic activity of the obtained preparation was increased to 231,1 KE/mg, i.e. increased by 59.3 times. Table 2 presents the results of a chromatographic purification of keratinase produced by Penicillium citrinum RS-54-91 VILLARS.

The resulting solution of the enzyme preparation was poured in sterile penicillin vials of 5 ml, froze them in the freezer "Sanyo Ultra Low" at -60°C and freeze-dried on a freeze dryer "Edwards (England) at 40°C and 0.1-0.2 ATM.

The activity of enzymes in various fields pH was determined using 0.01 M phosphate buffer solution with pH value: 5.8; 6.4; 7.0; 7.4; 8.0. To study thermal stability of the enzyme preparations were incubated with M phosphate buffer (pH 7.4) for 60 min at temperatures: 20°; 30°; 40°; 50°; 60°C, and then spent the determination of enzyme activity, as described above. To conduct inhibitory analysis used EDTA - metalloproteinase inhibitor and PMSF (phenylmethanesulfonyl), an inhibitor of serine-FR is C, and dithiothreitol (DTT). The effect of inhibitors on keratinase activity was determined by the residual proteolytic activity after pre-incubation of the enzyme (15 min at 37°C) with inhibitors contributed to the final concentration in the sample with 1 µg of the enzyme: EDTA to 1 mm PMSF and DTT to 3 mm. The substrate specificity of the enzyme was determined by measuring the amount of the formed α-amino groups in the process of protein hydrolysis and dipeptide as described previously [Nikitin, 2011]. The reaction was carried out under the following conditions: initial substrate concentration of 20 mm; the weight ratio of enzyme: substrate ratio = 1:50; pH 7.4; 37°C.

Received keratinase active in the pH range from 6.4 to 8.0, with an optimum pH 7.0; in the range from 20 to 40°C, with an optimum at 30°C. the Enzyme loses its activity under the effect of PMSF and practically does not react to the processing EDTA, that is, a serine protease type. DTT reduces the activity keratinase less than PMSF, indicating that the effect of disulfide bonds on the stability of the active centre of the enzyme. Enzymatic activity was very low when used as substrates of dipeptides Ley-Gly and Gly-Gly and collagen, indicating the absence of collagenases the activity of the protease.

Thus, keratinase produced by Penicillium citrinum PC-54-91 VILAR and widely the may according to the invention, belongs to the class of neutral thermolabile protease serine-type enzyme α-keratin and almost no hydrolyzes collagen.

Example 5. To obtain a seed culture of Penicillium citrinum PC-54-91 VILAR) were grown for 7 days in an incubator at 26°C on a beveled surface of the agar medium of čapek, of the following composition (%): NaNO3- 0,2; KH2PO4- 0,1; MgSO4×7H2O - 0,05; KCl - 0,05; FeSO4×7H2O - 0,001; caso3to 0.3; saccharose - 2; agar - 2, pH 6.8. Culture deuteromycete grown on agar medium of čapek, perseval three times on agar medium of čapek with the replacement of sucrose on keratin hair for directed adaptation of culture. Seeds served as a spore suspension of deuteromycete (108spores per 100 ml medium). Cultivation in deep conditions was carried out in flasks with a volume of 300 ml with 100 ml of nutrient medium on a shaker at a speed of 220 rpm at 26°C for 6 days. Cultivation was carried out on a modified environment of čapek with partial replacement of sucrose on the keratin of the hair (with 0.5% sucrose and 10% keratin). In the culture filtrates of the fluid deuteromycete obtained by filtering through a membrane filter with a pore size of 0.23 μm at various stages of cultivation, was determined keratinolytic activity. Maximum keratinolytic actively the TB culture fluid was 6,26±0.38 KE/ml

For isolation and purification keratinase from the filtrate of the culture fluid was consistently applied the gel filtration and affinity chromatography. For gel filtration were used column "LKB" (of 2.6×40 cm) with gel TOYOPEARL HW-40. The column was applied to 10 ml of the filtrate of the culture fluid. The eluent served 0.15 M NaCl solution. Detection was carried out at a wavelength of 274 nm using flow densitometer Uvicord 2", the rate of elution was 3.3 ml/min Optimization of the chromatography conditions such as the amount of deposited sample and the rate of elution, have resulted in a fairly clear separation of these fractions. When this fraction, corresponding to the external volume of the column and a material with a molecular weight higher than 15 kDa, possessed considerable keratinase activity exceeding keratinase activity of the original drug 3.03 times, and was 11.8 KE/mg. However, carried out using a disc-electrophoresis analysis of the material fraction 1 showed that it is not homogeneous and in addition to the basic protein with a molecular weight of about 21 kDa and contains other proteins with higher and lower molecular weights, which required the development of additional cleaning methods enzyme preparation.

Material with keratinase activity, after gel filtration was collected and subjected to purification by the method of Finney chromatography. For these purposes, LKB column volume 9×150 mm was filled with protein And separate in 0.01 M phosphate buffer (pH 7.4) and applied the material fractions obtained after gel filtration with keratinase activity. The elution was performed using sequentially 0.01 M phosphate buffer (same buffer with 1 M NaCl and 1 M solution of acetic acid until complete removal of the adsorbed material. The rate of elution was 10 ml/hour. The optical density of the eluate was monitored at a wavelength of 274 nm using flow densitometer Uvicord 2". It is shown that when carrying out affinity chromatography material with keratinase activity, elyuirovaniya with a column under the action of 1 M acetic acid. Certinatly the activity of the other fractions was practically equal to zero. Electrophoretic analysis using disc electrophoresis in SDS page [Sheludko, 1975] identified only one homogeneous protein fraction with a molecular weight of about 21 kDa. After carrying out affinity chromatography enzymatic activity of the obtained preparation was increased to 231,1 KE/mg, i.e. increased by 59.3 times. Table 2 presents the results of a chromatographic purification of keratinase produced by Penicillium citrinum PC-54-91 VILLARS.

The resulting solution of the enzyme preparation was poured in sterile penicillin vials of 5 ml, froze them in the freezer "Sanyo ltra Low" at -60°C and freeze-dried on a freeze dryer "Edwards (England) 40°C and 0.1-0.2 ATM.

The positive effect of the invention is to increase the production of keratinase culture Penicillium citrinum PC-5 4-91 VILAR 4.2 times, and reduce the time of cultivation from 21 to 6 days compared to the prototype and in obtaining drug keratinase that cleave α-keratin hair in two-step chromatographic process, with the productivity of 19 mg/l of culture liquid and activity 231 KE./mg, yield 70%, and the purification efficiency of 59.3 times.

LITERATURE

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9. Gradizar H., J. Fridrich, I. Krizaj, Jerala R. 200. Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marguandii and Doratomyces microsporus to some known proteases. Appl. Environ. Environ. 71(7)3420-3426

10. Marcondes N.R., Taira C.L., Vandresen D.C., Svindzinski T.I.E., Kadowaki M.K., Peralta M.R. 2008. New feather-degrading filamentous fungi. Microb. Ecol. 56:13-17

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Secretion of keratinolytic enzymes ang keratinolysis by Scopulariopsis brevicaulis and Trichophyton mentagrophytes: regresson analysis. Can. J. Environ 52:1060-1069

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Table 1
Certinatly activity of Penicillium citrinum PC-54-91 VILAR and strain of Penicillium citrinum in the prototype
The method of obtaining, No.No. passage on the modified environmentThe number of keratin (deep cultivation), %Time of cultivation, daysCertinatly activity, KE/ml
101,5101,08±0,08
211,581,47±0,10
321,572,35±0,15
4 31,562,60±0,16
5356to 4.73±0,32
631066,26±0,38
The placeholder01211,48±0,095

Table 2
The scheme of purification of keratinase from the culture fluid Penicillium citrinum PC-54-91 VILAR (per 100 ml of culture medium)
ProcedureTotal protein mgGeneral certinatly activity, KESpecific certinatly activity, KE/mlSpecific certinatly activity, COC mgThe degree of purificationOutput by activity, %
The filtrate RY1525956,26 a 3.91100
Gel filtration414821,6911,83,0381
Affinity chromatography1,84161,30231,159,370

The method of obtaining keratinase, providing directed adaptation of a strain of Penicillium citrinum PC-54-WILER by three reseeding culture on agar medium of čapek, containing as a carbon source, 2% keratin hair cultivation in deep conditions on the Chapek's medium containing 10% keratin hair and 0.5% sucrose for 6 days, followed by separation of the enzyme from the culture filtrate of the fluid by gel filtration on TSK-Gel TOYOPEARL HW-40, elution 0.15 M NaCl, followed by implementation of affinity chromatography on protein And sepharose CL-4B with subsequent elution of 1 M solution acetic acid and lyophilization.



 

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FIELD: biotechnologies.

SUBSTANCE: liquid culture of strain Corynebacterium glutamicum All-Russian collection of industrial microorganisms B-1959 - lysine producer by means of a deep method and liquid cultures of strains Bacillus subtilis All-Russian collection of industrial microorganisms B-8130, Bacillus subtilis All-Russian collection of industrial microorganisms B-2984, Bacillus subtilis All-Russian collection of industrial microorganisms B-4099 and Bacillus licheniformis All-Russian collection of industrial microorganisms B-4162 are prepared. To liquid culture of strain Corynebacterium glutamicum All-Russian collection of industrial microorganisms B-1959 there added in quantity of 30 l is 35 l of the mixture consisting of liquid cultures Bacillus subtilis All-Russian collection of industrial microorganisms B-8130, Bacillus subtilis All-Russian collection of industrial microorganisms B-2984 and Bacillus subtilis All-Russian collection of industrial microorganisms B-4099, which have been taken in the ratio of 6:6:1 respectively. Common mixture of liquid cultures is applied onto the carrier prepared in advance - sterile exhausted beet pulp treated with cellulolytic ferment and enriched with fermentolysate of Saccharomyces cerevisiae yeast. Then, it is mixed and exposed; after that, solid-phase fermentation is carried out under the specified conditions of restricted oxygen access. Liquid culture Bacillus licheniformis All-Russian collection of industrial microorganisms B-4162 containing at least 5.6×108 CFU/g is added in the amount of 65 l. The mixture is mixed and dried to the humidity of 8-10%. Dry powders of purple echinacea and fruits of holy thistle are added in terms of 20-50 g per 1 kg of the end product. The obtained mixture is stirred and subject to crushing till indiscrete mass is obtained.

EFFECT: invention allows improving feedstuff quality.

3 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: strain Aspergillus oryzae RCAM01135 is a producer of proteolytic and amylolytic ferments, which has ability to produce proteolytic and amylolytic ferments. It was deposited at GNU VNIISKHM with the following registration number: RCAM01135. It can be used at production of different food products (fermented spices, additives, and drinks).

EFFECT: invention allows increasing the growth speed and intensity of spore formation.

4 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: strain of unicellular algae Dunaliella salina IPPAS D-295-producer of biologically active substances having antioxidant activity was deposited in the culture collection of microalgae of the Institute of Plant Physiology n.a. K.A.Timiryazev RAS (IPP RAS (IPPAS)) under registration number IPPAS D-295 and can be used to produce biologically active substances having antioxidant activity and antagonistic activity against opportunistic pathogenic bacteria.

EFFECT: invention enables to increases the yield of antioxidant substances.

3 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to a strain of the bacteriophage Escherichia coli ECD4. The strain of the bacteriophage Escherichia coli ECD4 is isolated from faeces of broiler chickens on the culture of the bacteria of the strain Escherichia coli O104.H4 RK1No.112027 and is deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" under the number Ph63. The proposed strain of the bacteriophage has lytic activity in respect to the bacteria of the strain Escherichia coli O104:H4 RKINo.112027, lyses Escherichia of the serotype 0157:H7, does not suppress growth of cells Escherichia coli M-17, has lytic activity in respect to several other clinically significant serotypes of Escherichia, and also to several types of Shigella. The strain of the bacteriophage Escherichia coli ECD4 propagates on the laboratory non-pathogenic strain Escherichia coli K-12 C600F.

EFFECT: invention may be used in creation of new preparations for treatment of a disease caused by bacteria Escherichia coli and for elimination of this pathogen from food products.

4 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed for biodegradation of drotaverine hydrochloride (spasmolytic NOSHPA). The method provides for the process of reaction of drotaverine hydrochloride with cells of the strain Rhodococcus rhodochrous, Institute of Ecology and Genetics of Microorganisms 608. The process is performed under aerobic conditions in the medium containing sources of carbon, nitrogen, phosphorus, mineral salts. At the same time the additional source of carbon in the nutrient medium may be glucose. The cells of the strain Rhodococcus rhodochrous, Institute of Ecology and Genetics of Microorganisms, 608, may be used in the immobilised form. The strain is stored in the Regional Profiled Collection of Alkanotroph Microorganisms (acronym of the collection of the Institute of Ecology and Genetics of Microorganisms). Duration of biodegradation of drotaverine hydrochloride makes at least 80% for 25 days.

EFFECT: invention makes it possible to reduce time of biodestruction of drotaverine hydrochloride and to consider the used strain as a potential agent for cleaning of environment.

4 cl, 4 dwg, 3 ex

FIELD: biotechnology.

SUBSTANCE: method includes preparation of the inoculum of cells Streptomyces sp. MT246 (All-Russian Collection of Microorganisms Ac-2618D). Preparation of the nutrient medium containing one or more sources of carbon, nitrogen, metal ions in the form of soluble salts with fractional feeding of the carbon source during the productive stage. At that, as an additional carbon source the nutrient medium contains rhamnose, and a necessary component of the nutrient medium - MnS04 and sorbent from the group of amberlites XAD. Adding of the inoculate of cells Streptomyces sp. MT246 (All-Russian Collection of Microorganisms Ac-2618D) to the nutrient medium together with rhamnose and MnSO4 in a predetermined ratio and incubating of the medium at pH 7.0-7.5, temperature 25-35°C for 7-10 days. Separation of the residue is carried by centrifugation. The resulting residue is added to ethanol at a predetermined ratio, followed by extraction of tacrolimus at 30 for 2 hours and centrifuging to obtain the extract comprising tacrolimus.

EFFECT: invention enables to increase the yield of tacrolimus.

9 cl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, and represents a method of producing a recombinant mutein [C112S] of human enterokinase light chain, using a bacterium belonging to the genus Escherichia, transformed with an expression plasmid containing a DNA fragment encoding a mutein [C112S] inactive precursor of human enterokinase light chain, comprising a sequence encoding a cleavable N-terminal peptide comprising a hexahistidine cluster and the enterokinase recognition sequence, and a sequence fused with it in frame encoding a precursor of mutein [C112S] of human enterokinase light chain with non-cleavable C-terminal hexahistidine cluster under control of a promoter operating in the bacterial cell.

EFFECT: invention enables to produce the recombinant mutein of human enterokinase light chain with a high yield.

11 cl, 5 dwg, 1 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention represents a method of production of precursor of a recombinant fragment of human tissue plasminogen activator (tPA) (reteplase) using a bacterium belonging to the genus Escherichia, transformed with an expression plasmid containing a DNA fragment encoding a precursor of the recombinant fragment of human tissue plasminogen activator (tPA) (reteplase), comprising a sequence encoding a cleavable N-terminal peptide comprising decahistidine cluster and sequence of enterokinase recognition fused in frame, or a fragment of DNA encoding the [-1] methionyl of fragment of human tPA (reteplase), under the control of a promoter operating in the said bacterial cell.

EFFECT: invention enables to obtain a recombinant fragment of human tissue plasminogen activator with a high yield.

10 cl, 6 dwg, 1 tbl, 8 ex

FIELD: biotechnology.

SUBSTANCE: fermentation of nutritional medium is carried out based on corn starch hydrolysates by actinomycete. Then ultrafiltration of the obtained filtrate of the culture liquid is carried out with an activity of 4250±250 IU/cm3 through membranes with cut-off of 5 kDa. The filtrate obtained is concentrated under vacuum of 0.082 mPa at a temperature of 25-30°C. It is clarified at the temperature of 30-40°C. Its ultrafiltration is carried out again through the membrane with cut-off of 1 kDa. The obtained glycosidase inhibitor is dried and ground.

EFFECT: invention enables to obtain the glycosidase inhibitor with increased activity.

1 tbl, 4 ex

FIELD: food industry.

SUBSTANCE: invention relates to biotechnology, agriculture and zootechnics and may be used for industrial-scale breeding of broiler chickens of highly productive crosses. Prolyser symbiotic preparation based on Escherichia coli VL-613 is activated by way of resuspension with cooled boiled water or sterile physical solution at a temperature of 25°C - 37°C. The preparation in a dissolved form is given to drink to broiler chicken during 1 hour after the preparation activation in accordance with the following scheme. From the 8th day since the chicken birth and to the 22nd day the preparation is given in an amount of 100 mln microbial cells per day (per 1 chicken of "Кобб-500" and "Авиан-48" crosses) or 50 mln microbial cells per day (per 1 chicken of "Смена-7" cross). From the 22nd day and up to broiler chickens growing completion the preparation is given in an amount of 75 mln microbial cells per day (per 1 chicken of "Кобб-500" and "Авиан-48" crosses) or 50 mln microbial cells per day (per 1 chicken of "Смена-7" cross). The preparation is administered orally, preferably - during the morning feeding. The concentration of Escherichia coli VL-613 is equal to 1.75-3.05 billion microbial cells of the preparation per 1 broiler chicken throughout the whole growing cycle.

EFFECT: invention allows to fully replace synthetic lisyn in liquid given to drink to broilers, to increase poultry average daily weight gain, to increase the yield of Category 1 meat and to reduce the concentration of Escherichia coli VL-613 cells.

4 tbl

FIELD: biotechnologies.

SUBSTANCE: strain Aspergillus oryzae RCAM01135 is a producer of proteolytic and amylolytic ferments, which has ability to produce proteolytic and amylolytic ferments. It was deposited at GNU VNIISKHM with the following registration number: RCAM01135. It can be used at production of different food products (fermented spices, additives, and drinks).

EFFECT: invention allows increasing the growth speed and intensity of spore formation.

4 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: protease is presented which has enhanced milk clotting activity, containing amino acid sequence at least 80 % identical to SEQ ID NO: 3, where the said protease has at least one mutation selected from the group consisting of: (a) substitution of glutamine, corresponding to glutamine at a position of 265 in SEQ ID NO: 3, with amino acid; and (b) replacement of glutamine, corresponding to glutamine at a position of 266 in SEQ ID NO: 3, with amino acid. DNA is described which encodes the said protease, the expression vector containing the said DNA, and the cell transformed with the said vector, designed for expression of the said protease. The method of production of protease having enhanced milk clotting activity is proposed, comprising culturing the said transformed cell in the cultural medium and isolation of protease from the cultural medium.

EFFECT: invention enables to obtain the protease with enhanced milk clotting activity.

16 cl, 2 dwg, 4 tbl

FIELD: medicine.

SUBSTANCE: preparation of thrombolytic and fibrinolytic action is produced by submerged cultivation on a liquid nutrient medium, of a strain of basidium fungus of Coprinus lagopides TI-12, or Coprinus lagopides TI-32, or Coprinus lagopides TI-33 stored in the high basidium fungi culture collection of the microbiological synthesis technology department of the St.-Petersburg State Technical University, separation of a mycelial biomass from a native solution. The prepared solution is concentrated by ultrafiltration with using a semipermeable membrane with a nominal molecular-mass rejection limit up to 20 kDa followed by cool dehumidification in the mode suitable for protein compounds. The invention has allowed detecting Coprinus lagopides from basidium fungi of Coprinus exhibiting high thrombolytic and fibrinolytic activity.

EFFECT: preparation in the low concentration shows a high level of thrombolytic and fibrinolytic activity in vivo.

2 dwg, 4 tbl, 5 ex

FIELD: food industry.

SUBSTANCE: oyster mushroom fruit bodies are homogenised, the obtained homogenate is frozen followed by defrosting and dividing into extract and residue. Residue is mixed with distilled water in mass ratio of 1:2, held at +4-+8°C, the secondary extract is separated and mixed with previously obtained extract. Food acid is added into the obtained mixture to achieve pH 3.8 and it is precipitated by saturation of the solution with sodium chloride at +4-+8°C. The obtained residue is separated and dried by freezing at -18°C. The dried residue is kept in a refrigerator. Before using, it is dissolved in a minimum amount of water and centrifuged.

EFFECT: high milk-clotting activity and increased ratio of milk-clotting and general proteolytic activity.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method of obtaining milk-turning ferment includes deep cultivation of agaric macromycetes Coprinus lagopides on liquid medium, which contains sources of carbon, nitrogen and mineral salts.

EFFECT: ferment with high level of milk-turning activity and low level of general proteolytic activity.

3 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: strain of basidium fungus Trametes hirsuta (Wulfen) Pilat, deposited with collections of the OOO PKF "BIGOR" enumerated as CF-28, is extracted by repeated passages from monoglobular isolates of deep culture of the strain TsNIIMOD 56. The said strain features an active synthesis of extracellular laccase. The laccase activity makes some 20 to 26 ME/ml.

EFFECT: production of the basidium fungus strain with active extracellular laccase synthesis.

2 tbl, 2 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.

2 ex

FIELD: medicine; pharmacology.

SUBSTANCE: substance for dermatological medical products on the basis of a collagenase of a microbic parentage represents an ultrafiltrate allocated from a cultural liquid of Streptomyces lavendulae strain VKPM S-910 with collagenolytic activity of 1800-2500 KEA/ml and proteolytic activity of 120-200 PE/ml.

EFFECT: obtaining of biologically active ultrafiltrate with high collagenolytic and proteolytic activity for an effective utilisation as a part of various wound-healthing preparations and dermatology.

3 ex

FIELD: biotechnology.

SUBSTANCE: disclosed is isolated polypeptide being acid-proof metalloprotease isolated from Thermoascus aurantiacus. Described are strain Thermoascus aurantiacus CGMCC No 0670 for polypeptide production and method for polypeptide production using said strain. Disclosed is method for plant protein treatment to increase digestion value thereof by using said polypeptide.

EFFECT: protease of good acid resistance useful in feed production.

6 cl, 7 ex, 4 tbl

The invention relates to biotechnology and concerning the processing of leather and fur raw materials with the help of a new complex enzyme preparation

FIELD: biotechnologies.

SUBSTANCE: proposed method provides for preparation of inoculating mycelium of basidiomycete, which has been chosen from the following groups: Flammulina velutipes (Curtis) Singer and/or Hericium erinaceus (Bull.) Pers. Preparation of culture medium containing pulverised cold-pressed sunflower cake, soya flour, potassium dihydrogen phosphate, magnesium sulphate and water in the specified ratios. Inoculation of the obtained culture medium with the inoculating mycelium of basidiomycete, cultivation of basidiomycete with further separation of mycelium biomass and extraction of an antitumour agent from the biomass.

EFFECT: invention allows improving an environmental situation due to utilisation of production wastes of food industry.

3 cl, 1 tbl, 4 ex

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