EP3204035A1

EP3204035A1 - Extraction of enzyme complexes from streptomyces gougerotii 101, preparation of multienzyme biopreparations and their application

Info

Publication number: EP3204035A1
Application number: EP14833280.2A
Authority: EP
Inventors: Saulius Grigiskis; Vilma CIPINYTE; Bronislovas TVASKA; Ieva URBANAVICIUTE; Rytis RIMDEIKA; Mykolas MAURICAS
Original assignee: Uab "biocentras"
Current assignee: Uab "biocentras"
Priority date: 2014-10-10
Filing date: 2014-12-22
Publication date: 2017-08-16
Also published as: LT2014115A; LT6177B; WO2016055839A1

Abstract

This invention is attributed to the fields of biotechnology and biomedicine. The essence of this invention is to use enzymes extracted from Streptomyces gougerotii 101 in order to create multienzyme preparations with proteolytic, collagenasic, esterasic, amylolytic, lipasic, glucanasic, deoxyribunocleasic and lytic activities, and to employ them in the development of cosmetics, antibacterial preparations and preparations for wound care. Multienzyme biopreparation development technology is distinguishable by the fact that, as you change enzyme concentrations within, their application field changes too.

Description

Extraction of enzyme complexes from Streptomyces gougerotii 101, preparation of multienzyme biopreparations and their application

Technical Field

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.

Background Art

Wound - mechanical, chemical, thermal or other damage of skin, mucous membrane or deeper tissue. 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.

Pic 1. Wound healing principal scheme. Based on T. Hunt's 'Basic principles of wound healing'

Inflammation stage

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‑β. These compounds have a dual effect: activate nearby immune cells and transmit a signal about the inflammation to central nervous system (shown in picture 2). 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.

Pic 2. Inflammation regulation scheme

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 (IFNγ), which in turn activates macrophages themselves. Additionally, 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 IFNγ, 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.

There is a series of preparations for fighting infection in wounds. That can be iodine solutions, like BETADINE®, described in a patent US 5986162 A, antiseptic preparations like SSD (which includes an antibacterial compound silver sulfadiazine), described in a patent US5514657 A or antibacterial medical honey Medihoney®, described in a patent EP2040721 B1. USA Food and Drug Administration (FDA) confirmed that silver is a natural antibiotic that fight microorganisms. There are silver infused sterile antibacterial bandages described in patents US 7704523 B2, WO 2004/002384 A1, US 7005556 B1, US 2007/0275043 A1. Silver is also used in other products: colloid solutions, hydrogels and etc., described in patents WO 2005/018543 A2, US 6706279 B1. Antibacterial compound used in a patent WO 2002/100448 A1 is triclosan, however laboratory tests have proven a negative impact this compound has on a human endocrine system. 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. There are antibacterial compounds, whose influence is based on an inhibition of enzymes synthesized by pathogenic microorganisms, e.g. inhibitor of beta-lactamase, which degrades penicillin, described in a patent US 2014/0194386 A1. Another antibacterial compound described in a patent US 2014/0163038 A1 inhibits Fab enzyme, which participates in a bacteria fatty acid synthesis cycle, thus suppressing microorganism reproduction and development. 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 SertaSil™ with an anti-infection effect described in patent WO2010/079209 A2, contains enzyme serratiopeptidase.

The main drawback of all those preparations is that they only perform a single function - in order to achieve an effective wound healing it is important to remove all the causes slowing the wound healing. Fat-based lotions and antibiotic preparations shouldn't be used, since the former stimulate inflammation and festering, while the latter cannot enter necrotic tissue and wound doesn't receive a needed antibiotic concentration. Antibiotics are of narrow spectrum and do not affect fungi and viruses, plus microbes quickly gain a resistance to antibiotics. The main causes of slow wound healing are dead necrotic tissues in a wound and pathogenic bacteria. According to the scientific literature, early and full removal of dead tissues affects quicker wound epithelialization, lowers the risk of scar formation and ensures faster restoration of functions.

During the healing stage of an inflamed wound, macrophages and neurophils clean wound surface from microbes and cell debris, and initiate tissue repair stage. Growth factor TNF-α 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.

There are various ways of healing the wound. 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.

In order for a wound to heal and avoid complications, it is important to encourage the removal of dead tissue during the inflammation stage. Tissue removal (debridement) - is a medical removal of dead, damaged or infected tissue in order to improve wound healing and regeneration of a healthy tissue. 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.

Theoretically these enzymes should improve the wound healing, as exudate is mostly comprised of fibrin and nucleoproteins. The efficacy of this preparation to remove necrotic tissues and increase granulation has been studied during clinical trials, using physiological solution as a control. Treatment results only showed the superiority of this preparation for 19 patients out of 34.

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^®.

Not a small amount of microbiological enzymatic preparations, used for wound treatments, is also known.

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 β-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.

Due to the enzyme selectivity towards a substrate in current market products for wound cleaning and healing, it can end in a slow and only partial necrotic tissue removal. Additionally, their effect can be limited according to all the processes happening during the wound healing.

Migration (granulation) and epithelialization stages

Fibroblasts, keratinocytes and endothelium cells start synthesizing growth hormones once the wound is cleaned from microbes and necrotic tissues (table 1).

Table 1. Growth factors synthesized by cells

Cell	Synthesized growth factor
Keratinocytes	TGF-β, TGF-α, IL-1
Fibroblasts	IGF-1, bFGF, TGF-β, PDGF, KGF, CTGF
Endothelium cells	bFGF, PDGF, VEGF

These synthesized compounds stimulate cell migration, proliferation, formation of new capillaries and synthesis of extracellular proteins.

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. This suppression extends the necrotic tissue removal from the wound, thus lengthening not only the inflammation stage, but the whole wound healing duration too. 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

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^™ 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. However, we currently don't know of any wound treatment preparation with the aforementioned enzymes and their mixtures.

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.

Aforementioned wound treatment and skin care methods do not solve all the therapeutic problems:

there are no universal complex preparations with an anti-bacterial effect, selectively removing necrotic tissues, stimulating organism immune system, lowering the risk of scar formation and acting throughout all the healing stages;

there are no complex biopreparations, whose application depends on enzyme concentration;

there are no biopreparations capable of managing wound healing speed;

there are no complex biopreparations for skin care and skin disease treatment.

That's why 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.

Technical Solution

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.

The fifth difference is that enzyme complex A and B concentrations in a multienzyme biopreparation can vary from 0 to 100 percent.

The sixth difference is that obtaining multienzyme biopreparation can comprise these steps:

a) preparation of A and B enzyme complexes;

b) concentration and fractionation of A and B enzyme complexes;

c) treatment of A and B enzyme complexes with protein precipitation agents, such as inorganic salts (e.g. NH₄SO₄, CaCl₂), ketones (e.g. CH₃COCH₃), saturated aliphatic alcohols (e.g. CH₃CH₂OH, CH₃CHOHCH₃) and etc.;

d) chromatographic separation of A and B complexes;

e) A and B complex stabilization with saturated aliphatic polyhydroxy alcohols, whose carbon chain length is no shorter than C₃ (e.g. glycerol, sorbitol, polyvinyl alcohol);

f) optionally mixing of complexes A and B.

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.

The seventeenth difference is that compositions with a multienzyme biopreparation can be in a consistence of liquid, ointment or hydrogel.

Description of Drawings

Fig. 1. Principal scheme of physiological and biochemical processes of wound healing.

Fig. 2. Preparation of Master and Working cell collections of Streptomyces gougerotii 101 culture (step I);

Fig. 3. Biosynthesis of Streptomyces gougerotii 101 strain enzymes, inductor is yeast (step II);

Fig. 4. Biosynthesis of Streptomyces gougerotii 101 strain enzymes, inductor is collagen (step III);

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);

Fig. 10. Principal scheme for multienzyme biopreparation application;

Fig. 11. Principal scheme for multienzyme biopreparation use.

Best Mode

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:

a) proteases start working in an early wound healing stage and participate in all the subsequent stages. Firstly, they perform debridement, degrade a complex clot structure. Proteases also lyse necrotic tissues, exudates and proteins within it, thus shortening the duration of inflammation stage. They block pain stimulators histamine and bradykinin, speed up the degradation of inflammation stimulators and toxic compounds, stimulate and regulate the functional activity of macrophages. These enzymes participate in a migration stage, degrade desmosomes and hemidesmosomes. It helps for fibroblasts and endothelium cells to separate from the basement membrane on the wound bed and freely migrate within a wound cavity.

b) glucanase lyses the microbe cell walls, thus stops their development in a wound and prevents the infection. Moreover, β-glucans formed due to the effect of glucanase within multienzyme biopreparations, activate phagocytes, promote the synthesis of γ-interferon and so stimulate organism immune system and speed up wound healing processes.

c) DNase hydrolyses the DNA of broken cells - it speeds up the wound cleaning from necrotic tissues. This enzyme selectively degrades nucleic acids and decreases the viscosity of exudates, thus facilitating the movement and migration for the cells participating in wound healing processes.

d) lipase increases the amount of growth factors in a wound; they in turn speed up angiogenesis, proliferation and migration of keratinocytes and fibroblasts, activate the endothelial cells and promote collagen synthesis. Due to the effect of lipases, the synthesis of prostaglandins decreases, thus the swelling around the wound goes down.

e) amylase regulates EGF secretion, which activates fibroblast and keratinocyte proliferation and migration in a wound. Speeds up the closure of the wound.

f) elastase stimulates innate immune system during the early stages, and during the later stages participates in tissue regeneration.

g) collagenase selectively degrades collagen into smaller molecules, which are later hydrolyzed by other proteases. Collagenase is effective in cleaning a damaged place from necrotic tissue, speeds up the formation of granulation tissue. Collagenase improves the migration of keratinocytes and epithelium cells into a damaged place and their proliferation, which speeds up the wound layering in epithelium and formation of new tissues. Collagenase decreases collagen amount synthesized by fibroblasts during the scar maturation, and thus prevents the formation of protuberant and uneven scar, i.e. the scar gets softer and flatter with a normal collagen amount.

h) enzymes with a lytic activity participate in the breakdown of microorganism cell walls, thus suppressing their development within a wound and prevent infection and complications.

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. It is prudent to treat wounds with multienzyme biopreparation compositions immediately after the damage received, in order to remove all the factors stopping or complicating the wound healing. First of all, 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. With the removal of necrotized tissues using multienzyme biopreparation compositions, 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.

Mode for Invention

The description of producent Streptomyces gougerotii 101

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:

Ø Morphological

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.

Ø Cultural properties

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.

Ø Physiological and biochemical

It's an aerobe, optimal growth temperature is 28-30 °C, pH is 7.0-7.5. It hydrolyses starch, casein, collagen, Tween 80, β-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.

Picture 2. Activity indexes of hydrolytic enzymes synthesized by S. gougerotii 101 strain

STEPS FOR THE PRODUCTION OF ENZYME COMPLEXES OBTAINED FROM Streptomyces gougerotii 101

Step I

Firstly, in order to obtain enzyme complexes with optimal proteolytic, collagenasic, esterasic, amylolytic, lipasic, glucanasic, deoxyribonucleasic and lytic activities, Streptomyces gougerotii 101 strain selection is performed and cell collections are prepared for culture storage.

Steps II and III

Multienzyme biopreparation production starts with the biosynthesis of Streptomyces gougerotii 101 enzymes, where a culture liquid is obtained. During biosynthesis, 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.

Steps IV and V

Cultural liquids A and B obtained after Streptomyces gougerotii 101 enzyme biosynthesis are centrifuged, filtrated and concentrated using either vacuum evaporation or ultrafiltration. Obtained concentrates A and B are precipitated using main protein precipitation agents. After an additional centrifugation, precipitate is dissolved, and acquired suspension is centrifuged once more.

Table 2 . Enzymatic activities of multienzyme complexes A and B

No.	Enzymatic activity	Multienzyme complex A (inductor yeast)	Multienzyme complex B (inductor collagen)
1.	Proteolytic activity, U/cm³	10-22	5-7.5
2.	Collagenasic activity, U/cm³	700-1180	700-930
3.	Lipasic activity, U/cm³	100-145	10-20
4.	Glucanasic activity, U/cm³	5-7.5	2-3.4
5.	DNasic activity, U/cm³	0.21	-
6.	Amylasic activity, U/cm³	2-5	1-3
7.	Esterasic activity, U/cm³	15000-18000	12000-16000
8.	Lytic activity, U/cm³	20-60	80-100

1. Proteolytic activity unit - the amount of enzyme preparation, which excretes 1 micromole of tyrosine per minute, while hydrolyzing casein or sodium caseinate at a temperature of 37 °C and pH 8.0.

2. Collagenasic activity unit - the amount of enzyme, which excretes 1 micromole of α‑leucine per 5 hours, while hydrolyzing collagen at a temperature of 37 °C and pH 7.5.

3. Lipasic activity unit - the amount of enzyme, which releases 1 nanomole of p-nitrophenol per minute from substrate p-nitrophenyl, at a temperature of 40 °C and pH 8.0.

4. Glucanasic activity unit - an activity, which breaks down 0.1 μmol of insoluble β‑1,3‑gliukan per minute at optimal conditions (pH is 7.0; temperature is 50 ° C).

5. DNAsic activity unit - the amount of enzyme, which breaks down 1μg of DNA per 10 minutes, at a temperature of 37 °C.

6. Amylasic activity unit - bacterial α-amylase (S.A. Rapidaz) unit is the amount of enzyme, which breaks down 1 mg of starch to erythrodextrin per minute, at a temperature of 30±0.2 °C and pH 5.6-5.8.

7. Esterasic activity unit - the amount of enzyme, which releases 1 nanomole of p‑nitrophenol per minute from substrate p-nitrophenyl butyrate, at a temperature of 40 °C and pH 8.0.

8. Lytic activity unit - the amount of enzyme, which lowers reaction mixture optical density by 0.1 in 30 minutes, at a temperature of 37 °C and pH 8.0 (substrate is Micrococcus lysodeikticus culture).

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 perfringensbacterium, which causes gas gangrene (Table 3).

Table 3 . Antibacterial effect of multienzyme biopreparation.

Microorganisms	Lysed percentage of cells, %
	Incubation duration, 15 min.
Micrococcus lysodeicticus Staphylococcus albus Staphylococcus aureus Streptococcus haemolyticus Streptococcus agalacitae Streptococcus paracitrovorum Pseudomonas aeruginosa Clostridium perfringens	32 50 60 40 45 30 40 30
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

Preclinical study of multienzyme biopreparation has been performed. Toxicological, pharmacological and preparation efficacy analyses (State Food and Veterinary Service authorization to perform laboratory trials with animals No. 0141) were performed during preclinical study stage. All the studies were performed according to the plans prepared and signed in advance. Mice, rabbits and guinea pigs were used for the studies. All the toxicological tests used (as it is specified in EU directives) the concentration of the studied multienzyme preparation, whose proteolytic and collagenasic activity 6 times exceeded the activities of medical preparation form. To sum up, it can be stated that the extent and results of performed toxicological studies show that the preparation is non-toxic and safe to use.

Conclusions:

Ø Skin sensitization study on guinea pigs. Conclusion: no reddening or swelling noticed, negative reaction to a provocative sample - preparation doesn't sensitize the skin;

Ø Accute skin irritation test on rabbits. Conclusion: average skin irritation mark is equal to zero - preparation does not irritate skin;

Ø Accute eye irritation test on rabbits. Conclusion: rabbit eye irritation was evaluated as 0.25, so it is recommended to take care so that preparation does not enter the eyes - mildly burns eyes;

Ø Repeated dose (28 days) toxicity through skin test. While studying repeated preparation dose toxicity on C57W mice, it was determined that preparation is non-toxic at a concentration of 0.8 U/cm.

Ø Accute toxicity through the digestive tract on C57W mice. Preparation is a compound of low danger.

Ø Burn treatment efficacy, using different preparation concentrations (0.5 U/cm³, 0.8 U/cm³, 1.0 U/cm³, 1.6 U/cm³) by proteolytic activity, was studied. Healthy adult Wistar population rat males were used for the study. Study results showed that the most optimal proteolytic activity for the treatment of thermal skin burns is 0.8 U/cm³. The use of the studied complex enzyme preparation with a proteolytic activity of 0.8 U/cm³ for I-III degree thermal skin burn treatment, allows shortening the healing duration by an average of 20 %. The treatment with this preparation leaves no scars, which is very relevant treating face, hand and leg burns.

The span and results of preclinical studies show that the multienzyme preparation is safe, non-toxic, and can be used for the development of pharmaceutical, cosmetic and anti-bacterial compositions.

The application of multienzyme complexes

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. In the first case, multienzyme biopreparation compositions, for the treatment of wounds of various origin and type, are created. In the second case, compositions, for the production of anti-bacterial preparations, are created. In the third case, 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:

1. Hydrogel for necrotic wounds

Ingredients	Concentration, %
Enzyme complex A (70 %) + B (30 %)	20
Sodium alginate	3
Polyethylene glycol	20
Glycerol	7
Water	50

2. Hydrogel for III degree burns

Ingredients	Concentration, %
Enzyme complex A (50 %) + B (50 %)	30
Sodium alginate	3
Polyethylene glycol	20
Glycerol	6
Water	40
Sea-buckthorn oil	1

3. Hydrogel for I and II degree burns

Ingredients	Concentration, %
Enzyme complex A (60 %) + B (40 %)	20
Sodium alginate	3
Polyethylene glycol	20
Glycerol	6
Water	50
Sea-buckthorn oil	1

4. Hydrogel for sores and bedsores

Ingredients	Concentration, %
Enzyme complex A (20 %) + B (80 %)	20
Sodium alginate	3
Polyethylene glycol	20
Glycerol	5
Water	50
Sea-buckthorn oil	1
Pot marigold tincture	1

5. Hydrogel for infected wounds

Ingredients	Concentration, %
Enzyme complex A (90 %) + B (10 %)	30
Sodium alginate	3
Polyethylene glycol	20
Glycerol	7
Water	40

THE EFFICACY OF MULTIENZYME BIOPREPARATION COMPOSITIONS FOR HEALING WOUNDS

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. Clinical expressions in a wound (wound length, width, depth, exudates, erythema, fluctuation, local fever in a wound, pain/sensitivity during palpation, swelling/induration, necrosis, fibrin, granulations, epithelialization) were evaluated on these days and the wound was photographed. Result evaluation was performed six months after the burn, once an active scar remodeling process has ended. Conclusions regarding clinical trials of multienzyme biopreparation compositions - hydrogels - for wound treatment:

Ø Effectively absorbs the excess exudate;

Ø Effectively cleans wound surface from dead tissue;

Ø Effectively suppresses the infection;

Ø Maintains moist wound environment;

Ø Creates optimal microclimate;

Ø Prevents wounds from drying;

Ø No reddened wound edges;

Ø No pain in between bandaging;

Ø Promotes faster wound closure;

Ø No side effects;

Ø No scars after treatment;

Ø Wounds of any type and size epithelialize in average 18 days.

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:

1. Lotion for frostbites/burns

Ingredients	Concentration, %
Enzyme complex A (50 %) + B (50 %)	10
Glycerol	60
Sea-buckthorn oil	5
Water	25

2. Lotion for calluses

Ingredients	Concentration, %
Enzyme complex A (50 %) + B (50 %)	10
Glycerol	60
Water	30

3. Lotion for scars

Ingredients	Concentration, %
Enzyme complex A (10 %) + B (90 %)	10
Glycerol	60
Pot marigold tincture	5
Water	25

Creams:

5. Skin cleanser

Ingredients	Concentration, %
Enzyme complex A (20 %) + B (80 %)	5
Glycerol	4
Water	70
Polyethylene glycol	1
Sorbitan monostearate	5
Sorbitan oleate	5
Coconut oil	10

6. For dry skin

Ingredients	Concentration, %
Enzyme complex B (100 %)	10
Glycerol	20
Water	40
Polyethylene glycol	2
Sorbitan monostearate	6
Sorbitan oleate	6
Coconut oil	10
Sea-buckthorn oil	6

7. Foot care

Ingredients	Concentration, %
Enzyme complex A (20 %) + B (80 %)	20
Glycerol	20
Water	30
Polyethylene glycol	1,5
Sorbitn monostearate	10
Sorbitan oleate	10
Coconut oil	5
Mint oil	3,5

8. Scar removal

Ingredients	Concentration, %
Enzyme complex B (100 %)	30
Glycerol	20
Water	20
Polyethylene glycol	1
Sorbitan monostearate	10
Sorbitan oleate	10
Coconut oil	8
Pot marigold tincture	1

Multienzyme biopreparation anti-bacterial composition:

1. Anti-bacterial oral mucosa spray

Ingredients	Concentration, %
Enzyme complex A (100 %)	10
Glycerol	15
Water	70
Sea-buckthorn oil	0,5
Mint oil	0,5
Pot marigold tincture	4

ABBREVIATIONS

B cells - white blood cells, determining organism's humoral response.

T cells - blood cells belonging to an agranulocyte (mononuclear leucocyte) group; one of lymphocyte populations.

NK cells - transformed lymphocyte, lacking determinants inherent to T and B cells.

PDGF - platelet-derived growth factor.

EGF - epidermal growth factor.

IGF-1 - growth factor similar to insulin.

FGF - fibroblast growth factor.

TGF-β - transforming growth factor β.

TGF-α - transforming growth factor α.

IL-1 - interleukin 1 family (IL-1 family) is a group of 11 cytokines, which has a main role in immune and inflammatory reaction processes.

IFNγ - interferon gamma is a dimerized soluble cytokine.

bFGF - basic fibroblast growth factor.

KGF - keratinocyte growth factor.

CTGF - connective tissue growth factor.

VEGF - vascular endothelial growth factor.

REFERENCES:

1. Schultz, Gregory S., et al. 'Wound bed preparation: a systematic approach to wound management.' Wound repair and regeneration 11.s1 (2003): S1-S28.

2. Falabella, Anna F. 'Debridement and wound bed preparation.' Dermatologic Therapy 19.6 (2006): 317-325.

3. Werner, Sabine, and Richard Grose. 'Regulation of wound healing by growth factors and cytokines.' Physiological reviews 83.3 (2003): 835-870.

4. Püllen, Rupert, et al. 'Prospective randomized double‐blind study of the wound‐debriding effects of collagenase and fibrinolysin/deoxyribonuclease in pressure ulcers.' Age and ageing 31.2 (2002): 126-130.

5. Ramundo, Janet, and Mikel Gray. 'Enzymatic wound debridement.' Journal of Wound Ostomy & Continence Nursing 35.3 (2008): 273-280.

6. Wang, Xiao-Jing, et al. 'Role of TGFβ-mediated inflammation in cutaneous wound healing.' Journal of Investigative Dermatology Symposium Proceedings. Vol. 11. No. 1. Nature Publishing Group, 2006.

7. Tsirogianni K. A., Moutsopoulos N. M, Moutsopoulos H. M. Wound healing: Immunological aspects, Injury, Int. J. Care Injured, 2006, 37S:S5 - S12.

8. Martin, Paul. 'Wound healing-aiming for perfect skin regeneration.' Science 276.5309 (1997): 75-81.

9. Curtis, R., et al. 'Accuzyme (R) Papain-Urea Ointment versus Collagenase Santyl (R) Ointment in the Treatment of Partial Thickness Burn Wounds: 191.' Journal of Burn Care & Research 23 (2002): S136.

10. Shi, Lei, and Dennis Carson. 'Collagenase Santyl ointment: a selective agent for wound debridement.' Journal of Wound Ostomy & Continence Nursing 36.6S (2009): S12-S16.

11. Kang, Jeehoon, et al. 'Activated platelet supernatant can augment the angiogenic potential of human peripheral blood stem cells mobilized from bone marrow by G-CSF.' Journal of molecular and cellular cardiology (2014).

12. Van Meijer, M., and H. Pannekoek. 'Structure of plasminogen activator inhibitor 1 (PAI-1) and its function in fibrinolysis: an update.' Fibrinolysis 9.5 (1995): 263-276.

Claims

1. Multienzyme biopreparation, characterized in that it comprises the enzymes and their complexes produced by Streptomyces gougerotii 101 and having characteristic proteolytic, collagenasic, esterasic, amylolytic, lipasic, glucanasic, deoxyribonucleasic and lytic activities.

2. Multienzyme biopreparation according to claim 1, wherein the growth medium determines the enzymes produced by Streptomyces gougerotii 101 due to one of inductors consisting of yeast (enzyme complex A) or collagen (enzyme complex B).

3. Multienzyme biopreparation according to claim 2, wherein growth medium is composed of different inductor concentrations.

4. Multienzyme biopreparation according to any of claims 1-3, wherein the preparation encompasses complexes A and B together and separately.

5. Multienzyme biopreparation according to any of claims 1-4, wherein the concentrations of complexes A and B are from 0 to 100 %.

6. Method for the preparation of multienzyme biopreparation according to any of claims 1-5, wherein it comprises these steps:

a) preparation of A and B enzyme complexes;

b) concentration and fractionation of A and B enzyme complexes;

d) chromatographic separation of A and B complexes;

f) a selective mixing of complexes A and B.

7. Method for the preparation of multienzyme biopreparation according to claim 6, wherein A and B enzyme complexes are concentrated using ultrafiltration or vacuum evaporation during step b).

8. Method for the preparation of multienzyme biopreparation according to claim 6, wherein A and B enzyme complexes are fractionated using ultrafiltration and employing 5, 10, 15 and 50 kDa membranes.

9. Multienzyme biopreparation according to any of claims 1-5 , wherein it lyses microorganisms: Micrococcus lysodeicticus, Staphylococcus albus, Staphylococcus aureus, Streptococcus haemolyticus, Streptococcus paracitrovorum, Pseudomonas aeruginosa, Escherichia coli 078, Escherichia coli 12K, Pseudomonas fluorescens, Saccharomyces cerevisiae, Candida utilis.

10. Cosmetic or pharmaceutical composition comprising multienzyme biopreparation according to any of claims 1-5 and 9 and pharmaceutically acceptable excipient, used for promoting wound healing and improving skin conditions.

11. Cosmetic or pharmaceutical composition according to claim 10, used for immune system stimulation and treatment of different wounds, skin care and treatment, treatment of bacterial infections.

12. Composition according to claims 10 and 11, wherein the composition with increased lipasic activity is intended for the treatment of chronic and slowly healing wounds.

13. Composition according to claims 10 and 11, wherein the composition with increased lytic and glucanasic activities is intended for the treatment of infected wounds.

14. Composition according to claims 10 and 11, wherein the composition with increased proteolytic and collagenasic activities is intended for the treatment of necrotic wounds.

15. Composition according to claims 10 and 11, wherein the composition with decreased enzymatic activity is intended for skin care and treatment of skin diseases.

16. Composition according to claims 10 and 11, wherein the composition is intended for the production of anti-bacterial preparations.

17. Application according to any of claims 10-16, wherein the compositions can be of liquid, ointment or hydrogel consistence.