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WO2016055839A1 - Extraction de complexes enzymatiques à partir de streptomyces gougerotii 101, préparation de biopréparations multienzymatiques, et leur application - Google Patents

Extraction de complexes enzymatiques à partir de streptomyces gougerotii 101, préparation de biopréparations multienzymatiques, et leur application Download PDF

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WO2016055839A1
WO2016055839A1 PCT/IB2014/067226 IB2014067226W WO2016055839A1 WO 2016055839 A1 WO2016055839 A1 WO 2016055839A1 IB 2014067226 W IB2014067226 W IB 2014067226W WO 2016055839 A1 WO2016055839 A1 WO 2016055839A1
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multienzyme
biopreparation
wound
treatment
preparation
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Saulius Grigiskis
Vilma CIPINYTE
Bronislovas TVASKA
Ieva URBANAVICIUTE
Rytis RIMDEIKA
Mykolas MAURICAS
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UAB "BIOCENTRAS"
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/54—Mixtures of enzymes or proenzymes covered by more than a single one of groups A61K38/44 - A61K38/46 or A61K38/51 - A61K38/53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/007—Preparations for dry skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention is attributed to the fields of pharmaceutical biotechnology and biomedicine. It comprises the extraction of enzyme complexes from Streptomyces gougerotii 101, preparation of multienzyme biopreparations and their practical application.
  • Wound healing which is initiated and regulated by central nervous and immune organism systems, has four stages: inflammation, migration (granulation), epithelialization and scar maturation (as seen in picture 1). The same processes happen during the aforementioned wound healing, independently from their origin, tissue wound contamination or other factors.
  • a blood clot forms upon breaking skin and blood-vessels underneath it.
  • Thrombocytes within a clot excrete cytokines and growth factors: PDGF, EGF, IGF-1, FGF, TGF ⁇ .
  • PDGF central nervous system
  • EGF EGF
  • IGF-1 IGF-1
  • FGF FGF
  • TGF ⁇ TGF ⁇
  • Inflammation stage is an important and complicated process. Wound is open, thus creating a high infection risk, as damaged and necrotized tissue are a favourable medium for pathogenic microorganisms.
  • Basic immune response lasts for 25 hours. Even though inflammation is a required process, it also slows down regeneration and can be harmful. Sometimes infection or acute inflammation can complicate the inflammation stage and it can last from 20 days to 2 years. Delayed inflammation stage hinders healing and other processes, thus wounds become chronic and slow healing; hypertrophic scar might form, impeding the functions of surrounding undamaged tissues.
  • Inflammatory wound healing stage starts upon all the required factors and immune cells reaching the wound through a circulatory system.
  • Neutrophils come first and dominate the damaged place during the first 3 days. Their main function is the phagocytosis of microbes and foreign bodies. After phagocytosis is finished, neutrophils experience apoptosis and are removed by macrophages migrating into the wound. Maximal macrophage concentration in a wound is reached after ⁇ 24-36 h. Macrophages, just like neutrophils, phagocytize microbes, other particles, and clean the wound surface. Moreover, macrophages activate NK cells, which start synthesizing interferon gamma (IFN ⁇ ), which in turn activates macrophages themselves.
  • IFN ⁇ interferon gamma
  • macrophages have another important function - they attract to the damaged place and activate adaptive immunity cells, T and B lymphocytes. After arriving to the damaged place, activated T-lymphocytes start synthesizing IFN ⁇ , which, as it was already mentioned, activates macrophages and amplifies their influence. It must be mentioned that immune system cannot fight all microorganisms, thus infection can spread further than the primary place of infection and cause sepsis - acute whole-body inflammation.
  • Patent WO 2013/109004 A1 describes a wound covering of chitosan-silver nanoparticles, which has antibacterial and slow release properties, however slow antibacterial effect also lengthens the fight with an infection.
  • antibacterial compounds whose influence is based on an inhibition of enzymes synthesized by pathogenic microorganisms, e.g.
  • Patent US 2014/0134210 A1 describes an infection treatment method using antibiotics together with immunostimulator lysophosphatidylcholine, which stimulates immune cells: monocytes, macrophages, T-lymphocytes and neutrophils. Research proved that a synergetic action of those two compounds is more effective for treating infections than their effect separately.
  • Patent US 2014/0171358 A1 describes antibacterial compositions of one or more fatty acids: DGLA, 15-OHEPA and/or 15-HETrE, which can be used in combination with antibiotics. Antimicrobial effect of lipases is described in a patent US 2014/0193889 A1 . Lipases degrade microorganism cell walls and cell membrane components, thus impeding their growth and reproduction. There is a known medical preparation Flaminal®, containing enzymes glucose oxidase and lactoperoxidase. Preparation SertaSilTM with an anti-infection effect described in patent WO2010/079209 A2 , contains enzyme serratiopeptidase.
  • TNF- ⁇ stimulates the synthesis of proteases, such as: elastase, myeloperoxidase, acid hydrolase, collagenase and lysosomes, within neurophils and macrophages degrading necrotic tissues. Additionally, the inhibitors of these proteases, suppressing the protease activity on the wound bed, are secreted, so as not to damage live tissue. This inhibition slows down the removal of necrotic tissues from the wound, thus lengthening both the inflammation stage and an overall wound healing duration. Moreover, inflammation cells are an important growth factor and the source of cytokines, initiating wound healing proliferation stage.
  • Patent US 2014/0207050 A1 describes a method of using anaesthetics together with electrostimulation, thus inducing contractions of local and deep muscles, which in turn improve blood circulation and increase oxygen concentration in a wound; wound healing process is quicker and there is lower risk of complications.
  • Tissue removal is a medical removal of dead, damaged or infected tissue in order to improve wound healing and regeneration of a healthy tissue.
  • debridement There are a few methods of debridement: surgical, mechanical, autolytic, enzymatic, etc.
  • Autolytic debridement intensifies natural autolytic processes in a wound, sustains moist environment and regulates the excess of exudate.
  • Autolytic debridement is a long process, which cannot be applied to infected wounds. Mechanical removal of necrotic tissues can damage healthy tissues both within the wound and around it, and isn't selective.
  • Surgical debridement is painful, can elicit bleeding, requires the use of pain suppressants, plus a part of healthy tissues is lost.
  • Enzymatic debridement is especially selective and local method applied for the treatment of slowly healing wounds. Exogenous enzymes, usually proteases, effecting in combination with endogenous enzymes formed within a wound, are used for this method.
  • Enzymes performing debridement can be extracted from plants, microorganisms or animals.
  • Bromelain a protease of vegetative origin, is frequently used as an active component in medical preparations for debridement, and is described in patents: US 2013/0156745 A1, US 8119124 B2 and US 8128589 B2 .
  • Bromelain is a cysteine endopeptidase derived from pineapples.
  • Bromelain preparation 'NexoBrid' is used to treat skin burns.
  • Preparation Accuzyme ® has an active component enzyme papain, which performs debridement.
  • papain is a proteinase derived from papaya fruits. Its advantage against other proteinases is an ability to act in a wide pH range (3 to 12). However papain alone is not so effective, thus it's used together with urea, which denaturates dead proteins and allows them to be degraded by papain. Clinical study showed no significant effect on faster wound healing.
  • Patent RU 2280076 C1 describes enzymes of animal origin. Enzymes, extracted from Kamchatka crab, display collagenasic, proteasic, ribonucleasic, deoxyribonucleasic, phosphodiesterasic, phosphatasic, amylasic, lipasic and glucanasic activities. Due to this reason, complexes of those enzymes or individual enzymatic preparations can be widely used in biotechnology, medicine and cosmetology. Patent authors state that this multipurpose preparation is suitable for the treatment of festering wounds and is more effective than preparations containing only collagenases or proteases; however not any single preparation or its application study are described.
  • Preparation Elase ® uses a mixture of two enzymes: fibrinolysin and deoxyribonuclease.
  • Fibrinolysin degrades fibrin and dissolves blood clots, also inactivates fibrinogen and some clotting factors. This enzyme widens blood vessels on the wound bed. By degrading fibrin and necrotic tissues, it helps the macrophages to enter the wound. Dry fibrinolysin is stable, but dissolved loses its activity after 6-8 hours. Fibrinolysin reaction products are not resolved, thus they must be removed from the wound surface.
  • Deoxyribonuclease is extracted from cattle pancreas. This enzyme degrades nucleic acids and lowers the viscosity of exudate. It is soluble in water and active in a wide pH range, but loses its activity in a room temperature.
  • Endopeptidase trypsin extracted from pancreas, removes necrotic tissues without damaging live tissue. Clinical trials determined that this enzyme can increase re-epithelialization, increase blood circulation and lower the formation of oedemas within wounds. However, more extensive clinical trials are required before the efficacy of this enzyme wound treatment is determined.
  • the most known preparations with trypsin are Xenaderm ® and Granulex ® .
  • Preparation Santyl® designed for wound cleaning and treatment, has microbiological collagenase isolated from Clostridium histoliticum in its composition. This protease selectively degrades distinct collagen within the necrotic tissues, but is useless against keratin, fats and fibrin.
  • the optimal activity pH for this collagenase is 6 to 8.
  • Preparation Varidase ® also contains two enzymes: streptokinase and streptodornase.
  • Streptokinase is produced by ⁇ -hemolytic streptococci. This enzyme transforms plasminogen into plasmin, thus stimulating the fibrolysis of wound exudate.
  • Streptodornase is produced by hemolytic streptococci. This enzyme is a deoxyribonuclease, which performs DNA hydrolysis without any damage to live cells.
  • Patent US 2003/0198631 A1 describes a medical enzymatic preparation for debridement with extracellular metalloendopeptidase 'Thermolysin' , isolated from microorganism Bacillus thermoproteolyticus .
  • This protease is characterized by high specificity for two proteins: collagen and fibrin. Due to this specificity, the preparation can perform only a few functions during a complex wound healing process.
  • Fibroblasts, keratinocytes and endothelium cells start synthesizing growth hormones once the wound is cleaned from microbes and necrotic tissues (table 1).
  • TGF- ⁇ Cell Synthesized growth factor Keratinocytes TGF- ⁇ , TGF- ⁇ , IL-1 Fibroblasts IGF-1, bFGF, TGF- ⁇ , PDGF, KGF, CTGF Endothelium cells bFGF, PDGF, VEGF
  • A, so called, granulation tissue forms in a damaged place from endothelium cells, fibroblasts, keratinocytes, inflammation macrophages, lymphocytes and intercellular matrix.
  • Proteases perform an important function in a cell migration stage. Collagenase, elastase and trypsin degrade desmosomes and hemidesmosomes, and thus help fibroblasts and endothelium cells separating from basement membrane on the wound bed and freely migrate into a wound cavity. Also, protease inhibitors, suppressing protease activity on the wound bed, are secreted into the wound, in order to prevent damaging live tissue.
  • Collagen fibrils are being constantly remodeled by proteases secreted by neutrophils, macrophages, fibroblasts, endothelium and epithelium cells. Re-epithelialization happens simultaneously - wound is covered in a layer of epithelium cells, while new blood vessels are being formed. Fibroblasts synthesize collagen, elastin and proteoglycans, which form the primary scar.
  • Scar maturation can last from a few months to a few years, depending on the origin and size of wounds. Scars of simple cuts or slash wounds mature in 1 to 2 months, while of burns can last 30 or more months. An upset fibroblast function increases collagen synthesis, thus inciting the formation of hypertrophic and keloid scars. During this stage it is prudent to use preparations with collagenase.
  • Patent WO2010079209 A2 describes preparation SertaSil TM for the treatment of various wounds, and whose main active component is proteolytic enzyme serratiopeptidase. This protease has been isolated from a non-pathogenic microorganism Serratia E 15 . Patent describes the functions this preparation performs in a wound: fights an infection, effectively removes necrotic tissue, soothes pain, regulates the amount of exudate, regulates wound moisture balance, decreases wound inflammation and oedemas, reduces bleeding.
  • Patent WO 2011104630 A1 describes an enzyme complex, formed of proteases, carbohydrolases and lipases, and isolated from a fungus Conidiobolus brefeldianus culture. These enzymes can be used separately or in mixtures.
  • Authors present possible application areas: leather (animal fur) treatment, detergents, food, textile, silk production and the utilization of its byproducts, analytic reagents, pharmacy, cosmetics, molecular biology and etc.
  • leather animal fur
  • detergents e.g., aqueous fungus fungus brefeldianus culture
  • analytic reagents e.g., fungus Conidiobolus brefeldianus culture.
  • Patent US 2013/0202581 A1 describes compositions for wound treatment, which use three hydrolases extracted from pancreas: protease, lipase and amylase. Authors say that these enzymes can be used in different concentrations for the creation of wound treatment preparations. It is also noted that these hydrolases stimulate epidermis cells, thus speeding up wound healing and preventing the formation of scars. It is also said that these enzymatic compositions are not effective against infections caused by Staphylococcus aureus and Escherichia coli .
  • Enzymes are also used in cosmetic products; most frequently in skin cleansers. Enzyme based skin cleansers do not contain acids or scrubbing granules, which can irritate skin. The working principle of those cleansers is the dissolving of dead skin cells by enzymes. Skin care product market has products working on a similar principle. Enzymes used in cosmetics can be extracted from different sources. A few examples of such sources are presented further. Patents US 5705166 , US 6416769 B1 and US 8377434 B2 describe enzymes used for the production of skin cleansers and extracted from unripe papaya fruits. Patent CA 2377357 describes enzymes used for cosmetic products and extracted from Atlantic cod ( Gadus morhua) .
  • Patents US 4556554 and US 2010/0080787 A1 describe enzyme complexes used for the production of skin care cosmetics.
  • Patent US 6551606 B1 describes an enzyme complex from coconut ( Cocos nucifera) milk.
  • Patent US 2005/0249720 describes enzyme complexes used for cosmetic products and extracted from pineapple, mango and papaya fruits.
  • the aim of our invention is to create a multienzyme preparation so effective, it wouldn't have the aforementioned drawbacks, would be easily applied either for the treatment of different wounds, for skin care products or for anti-bacterial preparations. Moreover it would functionally act during all the wound healing processes together with immune system, have a wide spectrum anti-bacterial effect, selectively remove necrotic tissues, lower the risk of scar formation, speed up the healing duration and be suitable for the treatment of wounds of various origin and type. Also we seek to adapt the multienzyme preparation for skin care and skin disease treatment.
  • the goal of this invention is to create production technology for the enzyme complexes isolated from Streptomyces gougerotii 101 , and to apply them in the production of multienzyme preparations.
  • the essence of this invention is to create multienzyme preparations, assisting human immune system, from enzyme complexes, isolated from Streptomyces gougerotii 101 , and displaying proteolytic, collagenasic, esterasic, amylolytic, lipasic, glucanasic, deoxyribonucleasic and lytic activities; they would be used for wound treatment, removal of pathogenic microorganisms and skin care.
  • a skin care and wound treatment method, described in this invention is different from other known ones, as multienzyme preparations, composed of enzymes produced by Streptomyces gougerotii 101 with characteristic proteolytic, collagenasic, esterasic, amylolytic, lipasic, glucanasic, deoxyribonucleasic and lytic activities, are used.
  • the second difference is that it is possible to change the contents and activity of enzyme complexes, produced by Streptomyces gougerotii 101 , by using different inductors for growth medium: yeast (A) or collagen (B).
  • the third difference is that it is possible to change the contents and activity of enzymes, produced by Streptomyces gougerotii 101 , by changing inductor concentration in a growth medium.
  • the fourth difference is that enzyme complexes A and B can be used together or separately for the production of multienzyme biopreparation.
  • enzyme complex A and B concentrations in a multienzyme biopreparation can vary from 0 to 100 percent.
  • obtaining multienzyme biopreparation can comprise these steps:
  • a and B enzyme complexes with protein precipitation agents, such as inorganic salts (e.g. NH 4 SO 4 , CaCl 2 ), ketones (e.g. CH 3 COCH 3 ), saturated aliphatic alcohols (e.g. CH 3 CH 2 OH, CH 3 CHOHCH 3 ) and etc.;
  • protein precipitation agents such as inorganic salts (e.g. NH 4 SO 4 , CaCl 2 ), ketones (e.g. CH 3 COCH 3 ), saturated aliphatic alcohols (e.g. CH 3 CH 2 OH, CH 3 CHOHCH 3 ) and etc.;
  • the seventh difference is that multienzyme preparation can be concentrated employing ultrafiltration or vacuum evaporation.
  • the eighth difference is that during the preparation of multienzyme biopreparation, it can be fractioned with ultrafiltration, using 5, 10, 15 and 50 kDa membranes.
  • the ninth difference is that the multienzyme biopreparation lyses a series of microorganisms: Micrococcus lysodeicticus, Staphylococcus albus, Staphylococcus aureus, Streptococcus haemolyticus, Streptococcus paracitrovorum, Pseudomonas aeruginosa, Escherichia coli 078, Escherichia coli 12K, Pseudomonas fluorescens, Saccharomyces cerevisiae, Saccharomyces vini, Candida utilis.
  • the tenth difference is that multienzyme preparation can be used for pharmaceutical and cosmetic compositions together with a suitable additive promoting wound healing and improving skin condition.
  • the eleventh difference is that multienzyme biopreparation can be used for immune system stimulation during the wound treatment, treatment of bacterial diseases and caring for skin.
  • the twelfth difference is that a multienzyme preparation with increased lipasic activity can be used in a composition, which would be suitable for the treatment of chronic and slowly healing wounds.
  • the thirteenth difference is that a multienzyme biopreparation with increased lytic and glucanasic activities can be used in a composition, which would be suitable for the treatment of infected wounds.
  • the fourteenth difference is that a multienzyme biopreparation with increased proteolytic and collagenasic activities can be used in a composition, which would be suitable for the treatment of necrotic wounds.
  • the fifteenth difference is that a multienzyme biopreparation with decreased enzymatic activity can be used in a composition, which would be suitable for the skin care and treatment of skin diseases.
  • the sixteenth difference is that a multienzyme biopreparation can be used in a composition, which would be suitable for the treatment of bacterial diseases.
  • compositions with a multienzyme biopreparation can be in a consistence of liquid, ointment or hydrogel.
  • Fig. 1 Principal scheme of physiological and biochemical processes of wound healing.
  • Fig. 3 Biosynthesis of Streptomyces gougerotii 101 strain enzymes, inductor is yeast (step II);
  • Fig. 5. Obtaining multienzyme complex A from culture liquid (step IV);
  • Fig. 6. Obtaining multienzyme complex B from culture liquid (step V);
  • Fig. 7 Multienzyme complexes' A and B composition after concentration (SDS-PAGE electrophoresis method);
  • Fig. 8 Principal scheme of enzyme complex A preparation (inductor is yeast);
  • Fig. 9 Principal scheme of enzyme complex B preparation (inductor is collagen);
  • M ultienzyme biopreparation obtainment technology is based on the extraction of enzyme complexes synthesized by Streptomyces gougerotii 101 microorganism and their use in pharmacy, production of cosmetic and antibacterial compositions. Enzymes composing a multienzyme biopreparation have following effect:
  • Wound is an open way for pathogenic microorganisms, moreover necrotic tissues and exudates create favourable conditions for their reproduction.
  • General immune response takes about 24 hours; during that time processes in a wound can complicate irreversibly, starting an infection.
  • multienzyme biopreparation compositions have a wide spectrum lytic activity and break down pathogenic microorganisms in a wound. They also hydrolyze denaturated proteins, liquefy necrotized tissue in a wound, stimulate the growth of granulation tissue, speed up the wound cleaning and healing, and lower the swelling and inflammation of nearby tissues.
  • Collagenase in a multienzyme preparation liquefies a hard layer of clotted blood, necrotic tissues and degrades the overabundance of collagen, which lowers the risk of scar formation while treating festering wounds.
  • protein-rich medium without blood circulation favourable for the growth of microorganisms, is removed; bacterial wound contamination is decreased and infectious processes are suppressed. And so, conditions for further wound healing stages, tissue regeneration and improvement in blood circulation, are made.
  • Additives within the multienzyme biopreparation composition like sodium alginate, increase the absorption of exudate.
  • Polyethylene glycol creates polymer grid, which immobilizes enzymes and prevents the loss of enzyme activity, which allows a lower amount of bandaging per day.
  • Glycerol attracts holds and bonds water.
  • Compositions with a higher concentration of glycerol create a longer lasting moist environment in a wound during all the healing stages.
  • Epithelium cells require moisture in order to easier move from the foci of re-epithelialization on the edges of the wound, and fill-in the whole wound. These cells move on the wound bed in dry wounds. In moist conditions, cells can migrate throughout the whole site of the wound and they heal faster.
  • Enzyme complexes with proteolytic, collagenasic, esterasic, amylolytic, lipasic, glucanasic, deoxyribonucleasic and lytic activities are obtained from actinomycete Streptomyces gougerotii 101 strain.
  • Streptomyces gougerotii 101 strain JSC 'Biocentras' microorganism collection registration number K-91 was isolated from soil in south-east Lithuania. Its characteristics are:
  • Culture surface mycelium is white or colourless, average thickness of hyphae is 0.6-0.8 microns. Spores are oval or oblong, surface is smooth.
  • Abundant mycelium (aerial mycelium is white; substrate mycelium is light brown) forms while growing culture Streptomyces gougerotii 101 on a solid maize agar No. 2.
  • White aerial and sand-coloured substrate mycelium form on oat agar.
  • optimal growth temperature is 28-30 °C
  • pH is 7.0-7.5. It hydrolyses starch, casein, collagen, Tween 80, ⁇ -glucans and DNA. Curdles and peptonizes milk, liquefies gelatine, lyses wall cells of yeast and bacteria. Assimilates glucose, saccharose, fructose, xylose, doesn't or only slightly assimilates mannite and raffinose. Proteolytic, collagenasic, DNasic, esterasic, yeast lysing, beta-glucanasic, bacteria lysing and amylolytic activity indexes were measured in order to evaluate the hydrolytic activity of enzymes synthesized by S.
  • gougerotii 101 strain The strain was seeded onto Petri dishes with solid medium with added sodium caseinate, collagen DNA, Tween 80, baking yeast cells, baking yeast beta-glucan, Mycrococcus lysodeikticus cells and starch, in order to determine the activity indexes of these enzymes. Enzyme activity indexes were calculated after 96 h incubation at a temperature of 30 °C, by dividing the hydrolysis zones' diameter of respective substrates by colony diameter. Results are shown in Picture 2.
  • Streptomyces gougerotii 101 strain selection is performed and cell collections are prepared for culture storage.
  • Multienzyme biopreparation production starts with the biosynthesis of Streptomyces gougerotii 101 enzymes, where a culture liquid is obtained.
  • Streptomyces gougerotii 101 produces enzymes with proteolytic, collagenasic, esterasic, amylolytic, lipasic, glucanasic, deoxyribonucleasic and lytic activities.
  • the composition and activity of synthesized enzymes are regulated by changing inductors (yeast A or collagen B) and their amount.
  • Enzymatic activity Multienzyme complex A inctor yeast
  • Multienzyme complex B inctor collagen
  • Proteolytic activity U/cm 3 10-22 5-7.5
  • Collagenasic activity U/cm 3 700-1180 700-930
  • Lipasic activity U/cm 3 100-145 10-20
  • Glucanasic activity U/cm 3 5-7.5 2-3.4
  • DNasic activity U/cm 3 0.21 - 6.
  • Amylasic activity U/cm 3 2-5 1-3 7.
  • Esterasic activity U/cm 3 15000-18000 12000-16000 8. Lytic activity, U/cm 3 20-60 80-100
  • Multienzyme biopreparation Taking a note of multienzyme biopreparation's antibacterial properties, it was applied for the degradation of pathogenic microorganisms isolated from patients' nasopharynx, throat, teeth and festering wounds.
  • Multienzyme biopreparation breaks down cell walls both for pathogenic and non-pathogenic microorganisms; it especially strongly lyses streptococci and staphylococci, which are widely spread as causes of infectious diseases.
  • Multienzyme complex lyses Clostridium perfringens bacterium, which causes gas gangrene (Table 3).
  • Microorganisms Incubation duration 1 h Escherichia coli Pseudomonas fluorescens Bifidobacterium species Saccharomyces cerevisiae Saccharomyces vini Candida utilis Bacillus subtilis Aspergillus niger Aspergillus awamori Penicillium funiculosum 35 10 0 15 0 20 0 0 0 0 0
  • Multienzyme biopreparation has a three-way application. These biopreparations can be used for the production of pharmaceutical, anti-bacterial and cosmetic compositions. The application depends on the activity of the biopreparation and the chosen enzyme complex A or B.
  • multienzyme biopreparation compositions for the treatment of wounds of various origin and type, are created.
  • compositions for the production of anti-bacterial preparations, are created.
  • compositions for skin care and skin disease treatment, are created. Concentrations of enzyme complexes A and B can vary between 0 and 100 % within the multienzyme biopreparation compositions.
  • Multienzyme biopreparation compositions for wound treatment are Multienzyme biopreparation compositions for wound treatment:
  • a randomized, controlled, single-blind clinical trial of parallel groups was carried out for the multienzyme biopreparation compositions - hydrogels - intended for wound treatment.
  • 80 patients with forearm and hand 2B° depth burns were selected. Burns were treated with the applications of multienzyme biopreparation compositions - hydrogels - on the wound surface; re-bandaging was carried out every day until the total epithelialization. Patients were inspected and wounds were evaluated on these days (starting with the burn day): 0-3 (arriving to the hospital patient department), 7 ⁇ 1; 14 ⁇ 1; 21 ⁇ 1.
  • Concentrations of enzyme complexes A and B can vary between 0 and 100 % within the multienzyme biopreparation compositions for skin care and skin disease treatment.
  • Multienzyme biopreparation compositions for skin care and treatment are Multienzyme biopreparation compositions for skin care and treatment:
  • NK cells - transformed lymphocyte lacking determinants inherent to T and B cells.
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • IGF-1 IGF-1 - growth factor similar to insulin.
  • FGF - fibroblast growth factor FGF - fibroblast growth factor
  • TGF- ⁇ - transforming growth factor ⁇ TGF- ⁇ - transforming growth factor ⁇ .
  • TGF- ⁇ - transforming growth factor ⁇ TGF- ⁇ - transforming growth factor ⁇ .
  • IL-1 - interleukin 1 family is a group of 11 cytokines, which has a main role in immune and inflammatory reaction processes.
  • IFN ⁇ - interferon gamma is a dimerized soluble cytokine.
  • KGF - keratinocyte growth factor KGF - keratinocyte growth factor
  • CTGF connective tissue growth factor
  • VEGF vascular endothelial growth factor

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Abstract

La présente invention porte sur les domaines de la biotechnologie et de la biomédecine. L'essence de la présente invention porte sur l'utilisation d'enzymes extraites de Streptomyces gougerotii 101 dans le but de créer des préparations multienzymatiques ayant une activité protéolytique, collagénasique, estérasique, amylolytique, lipasique, glucanasique, désoxyribonucléasique et lytique, et de les utiliser pour la mise au point de produits cosmétiques, de préparations antibactériennes et de préparations pour le parage des plaies. La technologie de mise au point de biopréparations multienzymatiques se distingue par le fait que, au fur et à mesure que vous y modifiez la concentration des enzymes, leurs domaines d'application changent aussi.
PCT/IB2014/067226 2014-10-10 2014-12-22 Extraction de complexes enzymatiques à partir de streptomyces gougerotii 101, préparation de biopréparations multienzymatiques, et leur application Ceased WO2016055839A1 (fr)

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EP14833280.2A EP3204035A1 (fr) 2014-10-10 2014-12-22 Extraction de complexes enzymatiques à partir de streptomyces gougerotii 101, préparation de biopréparations multienzymatiques, et leur application

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LT2014115A LT6177B (lt) 2014-10-10 2014-10-10 Fermentų kompleksų išskyrimas iš steptomyces gougerotii 101, daugiafermentinių biopreparatų ruošimas bei taikymas
LTLT2014-115 2014-10-10

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