Thermostable Bacterial Collagenolytic Proteases: A Review.

Collagenolytic proteases are widely used in the food, medical, pharmaceutical, cosmetic, and textile industries. Mesophilic collagenases exhibit collagenolytic activity under physiological conditions, but have limitations in efficiently degrading collagen-rich wastes, such as collagen from fish scales, at high temperatures due to their poor thermostability. Bacterial collagenolytic proteases are members of various proteinase families, including the bacterial collagenolytic metalloproteinase M9 and the bacterial collagenolytic serine proteinase families S1, S8, and S53. Notably, the C-terminal domains of collagenolytic proteases, such as the pre-peptidase C-terminal domain, the polycystic kidney disease-like domain, the collagen-binding domain, the proprotein convertase domain, and the ß-jelly roll domain, exhibit collagen-binding or -swelling activity. These activities can induce conformational changes in collagen or the enzyme active sites, thereby enhancing the collagen-degrading efficiency. In addition, thermostable bacterial collagenolytic proteases can function at high temperatures, which increases their degradation efficiency since heat-denatured collagen is more susceptible to proteolysis and minimizes the risk of microbial contamination. To date, only a few thermophile-derived collagenolytic proteases have been characterized. TSS, a thermostable and halotolerant subtilisin-like serine collagenolytic protease, exhibits high collagenolytic activity at 60°C. In this review, we present and summarize the current research on A) the classification and nomenclature of thermostable and mesophilic collagenolytic proteases derived from diverse microorganisms, and B) the functional roles of their C-terminal domains. Furthermore, we analyze the cleavage specificity of the thermostable collagenolytic proteases within each family and comprehensively discuss the thermostable collagenolytic protease TSS.

The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae.

Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.

Ultrasound-Assisted Enzyme-Catalyzed Hydrolysis of Collagen to Produce Peptides With Biomedical Potential: Collagenase From Aspergillus terreus UCP1276.

Ultrasound has been applied for varied purposes as it provides additional mechanical energy to a system, and is still profitable and straightforward, which are advantages for industrial applications. In this work, ultrasonic treatments were applied to purified collagenase fractions from a fermented extract by Aspergillus terreus UCP 1276 aiming to evaluate the potential effect on collagen hydrolysis. The physical agent was evaluated as an inductor of collagen degradation and consequently as a producer of peptides with anticoagulant activity. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses were also carried out to compare the hydrolysis techniques. The ultrasound (40 kHz, 47.4 W/L) processing was conducted under the same conditions of pH and temperature at different times. The ultrasound-assisted reaction was accelerated in relation to conventional processing. Collagenolytic activity was enhanced and tested in the presence of phenylmethanesulfonyl fluoride inhibitor. Underexposure, the activity was enhanced, reaching more than 72.0% of improvement in relation to the non-exposed enzyme. A period of 30 min of incubation under ultrasound exposure was enough to efficiently produce peptides with biological activity, including anticoagulation and effect on prothrombin time at about 60%. The results indicate that low-frequency ultrasound is an enzymatic inducer with likely commercial applicability accelerating the enzymatic reaction. Bioelectromagnetics. 2020;41:113-120. © 2019 Bioelectromagnetics Society.

Peptide Cross-Linked Poly (Ethylene Glycol) Hydrogel Films as Biosensor Coatings for the Detection of Collagenase.

Peptide cross-linked poly(ethylene glycol) hydrogel has been widely used for drug delivery and tissue engineering. However, the use of this material as a biosensor for the detection of collagenase has not been explored. Proteases play a key role in the pathology of diseases such as rheumatoid arthritis and osteoarthritis. The detection of this class of enzyme using the degradable hydrogel film format is promising as a point-of-care device for disease monitoring. In this study, a protease biosensor was developed based on the degradation of a peptide cross-linked poly(ethylene glycol) hydrogel film and demonstrated for the detection of collagenase. The hydrogel was deposited on gold-coated quartz crystals, and their degradation in the presence of collagenase was monitored using a quartz crystal microbalance (QCM). The biosensor was shown to respond to concentrations between 2 and 2000 nM in less than 10 min with a lower detection limit of 2 nM.

Production and characterization of collagenase from a new Amazonian Bacillus cereus strain.

A new collagenase producing a strain of Bacillus cereus, isolated from the pollen of a bee of Amazon Region (Brazil), had its enzyme characterized and the production medium composition and culture conditions enhanced. A two-level design on three factors, namely initial medium pH, the substrate (gelatin) concentration and agitation intensity, allowed identifying the first two variables as the most significant ones, while a central composite design (CCD) was subsequently used to identify their optimal levels. Statistics highlighted maximized collagenolytic activity when substrate concentration and initial medium pH were selected at their highest levels (positive effects), whereas agitation intensity at the lowest (negative effect). Triplicate runs performed under predicted optimal conditions (pH 7.8 and 1.7% gelatin concentration) yielded a collagenolytic activity (305.39 ± 5.15 U) 4.6- to 15-fold those obtained with the preliminary design. The enzyme displayed optimum activity at 45 °C and pH 7.2, was stable over wide ranges of pH values and temperatures (7.2-11.0 and 25-50 °C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonyl fluoride. The zymogram showed two prominent bands at 50 and 76 kDa. These results are a first attempt to elucidate the features of this new collagenase, its production conditions, and possible scale-up.

Safe and effective subcutaneous adipolysis in minipigs by a collagenase derivative.

Adipocytes attached to the extracellular matrix (ECM) mainly consist of collagen in adipose tissues, while the degradation of ECM by collagenase induces the apoptosis of adipocytes, leading to a decrease in local subcutaneous adipose. To achieve this goal, we are developing a mutant collagenase H (ColH) to remove local subcutaneous fat such as submental fat (SMF). Three vectors were constructed for expressing rColH(FM, mutant for fat melting, with 6xHis tag), rColH(WT, wild-type, with 6xHis tag), and rColH(E451D, E451D mutant, without 6xHis tag) in Escherichia coli. rColH(FM) & rColH(WT) were purified by Ni Sepharose on a laboratory scale, while rColH(E451D) was purified by five chromatography purification steps on a large scale. Then, the stability of rColH(FM) and rColH(WT) was tested by SDS-PAGE to investigate the influence of the E451D mutation on stability. Afterwards, the enzyme kinetics of ColH (mutant or wild-type, with or without His tag) were investigated and compared. Finally, the adipolysis of rColH(E451D) at various doses was tested in vitro and in vivo. The ultrasound results in minipigs suggested that effective adipolysis was induced by rColH(E451D) compared with the negative control, and the histological results suggest dose-dependent fibrosis, necrosis, inflammation and cholesterol cleft formation. These findings indicate the possibility of rColH(E451D) becoming a new injectable drug to safely remove subcutaneous adipose.

U32 collagenase from Pseudoalteromonas agarivorans NW4327: Activity, structure, substrate interactions and molecular dynamics simulations.

A protease of the primary pathogen (Pseudoalteromonas agarivorans NW4327) of the disease affecting the Great Barrier Reef sponge Rhopaloeides odorabile was purified. Zymography demonstrated calcium-dependent collagenase and gelatinase activity of the purified protein. This metalloprotease was identified by matrix assisted laser desorption ionization time-of-flight mass spectrophotometry as a 52,509 Da U32 collagenase. Predicted tertiary structure of U32 collagenase (by Phyre2 fold recognition server) demonstrated 13% identity with known hydrolases establishing novelty of the enzyme. Molecular docking conceived two interacting loops of the collagenase that bound with collagen triple helices and two calcium ions remained centered between the loops. According to ConSurf multiple sequence alignment, the residues of loop1 of the collagenase were mostly conserved while variations among residues of loop2 were comparatively higher than loop1. Asp262, Glu263 of loop1 and Thr363, Lys364, Gln365 of loop2 participated in the interaction with Ca2+ and collagen. Root mean square deviation and root mean square fluctuation values signified higher stability of the collagen-Ca2+-collagenase complex and greater structural stability of the residues of the loops in the complex compared to apocollagenase. Observed properties of NW4327 U32 collagenase and its interaction with collagen were different from similar enzymes of thermophilic bacteria and terrestrial pathogens.

Purification and characterization of a novel extracellular serine-protease with collagenolytic activity from Aspergillus tamarii URM4634.

An extracellular serine-protease from Aspergillus tamarii URM4634 was purified and characterized. The possibility of using Aspergillus tamarii URM4634 protease in detergent formulations and collagenolytic activity was investigated. The protease demonstrated excellent stability at pH range 7.0-11.0, the optimum being at pH 9.0. The enzyme was stable at 40 °C for 180 min, enhanced by Mg++ and Ca++, but inhibited by Zn++, and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggested as serine-protease. The azocasein substrate result showed Km = 0.434 mg/mL and Vmax = 7.739 mg/mL/min. SDS-PAGE and azocasein zymography showed that the purified alkaline protease (2983.8 U/mg) had a molecular mass of 49.3 kDa. The enzyme was purified by column chromatography using Sephadex A50 resin. The proteolytic activity was activated by SDS (sodium dodecyl sulfate), Tween-80, Tween 20 and Triton-100. This study demonstrated that A. tamarii URM4634 protease has potent, stable and compatible collagenolytic activity to the desired level in local laundry detergent brands compared with similar enzymes produced by solid-state fermentation. This protease can thus be chosen as an option in both the food industry to tenderization meat and the detergent industry to washing process.

Purification and characterization of a new thermophilic collagenase from Nocardiopsis dassonvillei NRC2aza and its application in wound healing.

A thermostable metallo-collagenase enzyme (150 kDa), recently identified in a newly isolated actinomycestes strain (Nocardiopsis dassonvillei NRC2aza), has been purified from natural source, characterized to have application in wound healing. A simple 3 step purification procedure gave an increase of purity by 6.23 fold with a specific activity of 387.2 U mg-1. The enzyme activity showed stability across a range of pH (7.0-8.5) and temperature (40-55 °C) with optima at pH 8.0 and 60 °C, respectively. Activators include Mg+2, Ca+2, Zn+2, Na+, K+ and Ba+2, while Mn+2, Co+2, Ni+2and Ag+ ions gave partial inhibition. Full inhibition was given by other tested ions and metalloproteinase inhibitors. Broad substrate specificity was demonstrated including activity against a native collagen. The Km and Vmax of the enzyme using azocollagen were 5.5 mg/ml and 1280 U, respectively. The purified collagenase enhanced wound closure in vitro and in vivo and the repair process was dose dependent. Topical application of the purified collagenase (either of 25 or 50 U) to cutaneous wounds significantly accelerated the rate of wound healing and the formation of granulation tissue. Hence, the purified collagenase has a great potential as a therapeutic agent in wound care and collagen related diseases.

Amplified QCM biosensor for type IV collagenase based on collagenase-cleavage of gold nanoparticles functionalized peptide.

The present study develops a rapid, simple and efficient method for the determination of type IV collagenase by using a specific peptide-modified quartz crystal microbalance (QCM). A small peptide (P1), contains a specific sequence (Pro-Gly) and a terminal cysteine, was synthetized and immobilized to the surface of QCM electrode via the reaction between Au and thiol of the cysteine. The peptide bond between proline and glycine can be specific hydrolyzed cleavage by type IV collagenase, which enabled the modified electrode with a high selectivity toward type IV collagenase. The cleaving process caused a frequency change of QCM to give a signal related to the concentration of type IV collagenase. The morphologies of the modified electrodes were characterized by scanning electron microscope (SEM) and the specific hydrolyzed cleavage process was monitored by QCM. When P1 was modified with gold nanoparticles (P1-Au NPs), the signal could be amplified to further enhance the sensitivity of the designed sensor due to the high-mass of the modified Au NPs. Compared the direct unamplified assay, the values obtained for the limit of detection for type IV collagenase was 0.96 ng mL-1, yielding about 6.5 times of magnitude improvement in sensitivity. This signal enhanced peptide based QCM biosensor for type IV collagenase also showed good selectivity and sensitivity in complex matrix.

A new collagenase enzyme of the marine sponge pathogen Pseudoalteromonas agarivorans NW4327 is uniquely linked with a TonB dependent receptor.

The primary pathogen of the Great Barrier Reef sponge Rhopaloeides odorabile, recently identified as a novel strain (NW4327) of Pseudoalteromonas agarivorans, produced collagenase which degraded R. odorabile skeletal fibers. We now report the collagenase of P. agarivorans as a metalloprotease which required Ca2+ and Zn2+ as cofactors. The collagenase was a TonB dependent receptor (TBDR) having a carboxypeptidase regulatory like domain (CRLD) in the N-terminal along with an outer membrane (OM) channel superfamily domain. The genes for TBDR sub-components and collagenase formed one unified entity in the genome of P. agarivorans NW4327. This association of a collagenase with a TBDR distinguished it from all known functional collagenases till date and for the first time, established the enzymatic capability of TBDRs. Predicted TBDR model demonstrated only 15% identity with ferripyoverdin receptor and the CRLD displayed merely 24% identity with carboxypeptidase catalytic chain. Presence of signal peptide, lack of transmembrane helices, absence of N-terminal in the cytoplasmic side, extracellular localization and recovery from the culture supernatant implicated that the TBDR was secreted. Stronger binding of the collagenase with marine sponge type IV collagen than type I collagen, revealed through molecular docking, indicated higher specificity of the enzyme towards type IV collagen.

Purification and characterization of a collagenase from Penicillium sp. UCP 1286 by polyethylene glycol-phosphate aqueous two-phase system.

Collagenases are proteolytic enzymes capable of degrading both native and denatured collagen, reported to be applied in industrial, medical and biotechnological sectors. Liquid-liquid extraction using aqueous two-phase system (ATPS) is one of the most promising bioseparation techniques, which can substitute difficult solid-liquid separation processes, offering many advantages over conventional methods including low-processing time, low-cost material and low-energy consumption. The collagenase produced by Penicillium sp. UCP 1286 showed a stronger affinity for the bottom salt-rich phase, where the highest levels of collagenolytic activity were observed at the center point runs, using 15.0% (w/w) PEG 3350 g/mol and 12.5% (w/w) phosphate salt at pH 7.0 and concentration. The enzyme was characterized by thermal stability, pH tolerance and effect of inhibitors, showing optimal collagenolytic activity at 37 °C and pH 9.0 and proved to be a serine protease. ATPS showed high efficiency in the collagenase purification, confirmed by a single band in SDS/PAGE, and can in fact be applied as a quick and inexpensive alternative method.

Collagenolytic enzymes produced by fungi: a systematic review

Abstract Specific proteases capable of degrading native triple helical or denatured collagen have been required for many years and have a large spectrum of applications. There are few complete reports that fully uncover production, characterization and purification of fungi collagenases. In this review, authors searched through four scientific on line data bases using the following keywords (collagenolytic OR collagenase) AND (fungi OR fungus OR fungal) AND (production OR synthesis OR synthesize) AND (characterization). Scientific criteria were adopted in this review to classify found articles by score (from 0 to 10). After exclusion criteria, 21 articles were selected. None obtained the maximum of 10 points defined by the methodology, which indicates a deficiency in studies dealing simultaneously with production, characterization and purification of collagenase by fungi. Among microorganisms studied the non-pathogenic fungi Penicillium aurantiogriseum and Rhizoctonia solani stood out in volumetric and specific collagenase activity. The only article found that made sequencing of a true collagenase showed 100% homology with several metalloproteinases fungi. A clear gap in literature about collagenase production by fungi was verified, which prevents further development in the area and increases the need for further studies, particularly full characterization of fungal collagenases with high specificity to collagen.

Collagenase produced from Aspergillus sp. (UCP 1276) using chicken feather industrial residue.

An extracellular collagenolytic serine protease was purified from Aspergillus sp., isolated from the Caatinga biome in northeast Brazil by a two-step chromatographic procedure, using an anion-exchanger and gel filtration. The enzyme was produced by submerged fermentation of feather residue as a substrate. The purified collagenase showed a 2.09-fold increase in specific activity and 22.85% yield. The enzyme was a monomeric protein with a molecular mass of 28.7 kDa, estimated by an SDS-PAGE and AKTA system. The optimum temperature and pH for enzyme activity were around 40°C and pH 8.0, respectively. The enzyme was strongly inhibited by phenyl-methylsulfonyl fluoride, a serine protease inhibitor, and was thermostable until 65°C for 1 h. We then evaluated the enzyme's potential for degradation of Type I and Type V collagens for producing peptides with antifungal activity. Our results revealed that the cleavage of Type V collagen yielded more effective peptides than Type I, inhibiting growth of Aspergillus terreus, Aspergillus japonicus and Aspergillus parasiticus. Both groups of peptides (Type I and Type V) were identified by SDS-PAGE. To conclude, the thermostable collagenase we purified in this study has various potentially useful applications in the fields of biochemistry, biotechnology and biomedical sciences.

Biochemical properties and evaluation of washing performance in commercial detergent compatibility of two collagenolytic serine peptidases secreted by Aspergillus fischeri and Penicillium citrinum.

Filamentous fungi secrete diverse peptidases with different biochemical properties, which is of considerable importance for application in various commercial sectors. In this study, we describe the isolation of two fungal species collected from the soil of decayed organic matter: Aspergillus fischeri and Penicillium citrinum. In a submerged bioprocess, we observed better peptidase production with the fungus P. citrinum, which reached a peak production at 168 h with 760 U/mL, in comparison with the fungus A. fischeri, which reached a peak production at 72 h with 460 U/mL. In both situations, the fermentative medium contained 0.5% crushed feathers as a source of nitrogen. On performing biochemical characterization, we detected two alkaline serine peptidases: The one secreted by P. citrinum had optimal activity at pH 7.0 and at 45°C, while the one secreted by A. fischeri had optimal activity in pH 6.5-8 and at 55-60°C. Metallic ions were effective in modulating these peptidases; in particular, Cu2+ promoted negative modulation of both peptidases. The peptidases were stable and functional under conditions of nonionic surfactants, temperatures up to 45°C for 1 h, and incubation over a wide pH range. In addition, it was observed that both peptidases had the capacity to hydrolyze collagen and performed well in removing an egg protein stain when supplemented into a commercial powder detergent; this was especially true for the peptidase from P. citrinum.

Collagenolytic enzymes produced by fungi: a systematic review.

Specific proteases capable of degrading native triple helical or denatured collagen have been required for many years and have a large spectrum of applications. There are few complete reports that fully uncover production, characterization and purification of fungi collagenases. In this review, authors searched through four scientific on line data bases using the following keywords (collagenolytic OR collagenase) AND (fungi OR fungus OR fungal) AND (production OR synthesis OR synthesize) AND (characterization). Scientific criteria were adopted in this review to classify found articles by score (from 0 to 10). After exclusion criteria, 21 articles were selected. None obtained the maximum of 10 points defined by the methodology, which indicates a deficiency in studies dealing simultaneously with production, characterization and purification of collagenase by fungi. Among microorganisms studied the non-pathogenic fungi Penicillium aurantiogriseum and Rhizoctonia solani stood out in volumetric and specific collagenase activity. The only article found that made sequencing of a true collagenase showed 100% homology with several metalloproteinases fungi. A clear gap in literature about collagenase production by fungi was verified, which prevents further development in the area and increases the need for further studies, particularly full characterization of fungal collagenases with high specificity to collagen.

Filifactor alocis collagenase can modulate apoptosis of normal oral keratinocytes.

To successfully colonize host cells, pathogenic bacteria must circumvent the host's structural barrier such as the collagen-rich extracellular matrix (ECM), as a preliminary step to invasion and colonization of the periodontal tissue. Filifactor alocis possesses a putative Peptidase U32 family protein (HMPREF0389_00504) with collagenase activity that may play a significant role in colonization of host tissue during periodontitis by breaking down collagen into peptides and disruption of the host cell. Domain architecture of the HMPREF0389_00504 protein predicted the presence of a characteristic PrtC-like collagenase domain, and a peptidase domain. Our study demonstrated that the recombinant F. alocis peptidase U32 protein (designated PrtFAC) can interact with, and degrade, type I collagen, heat-denatured collagen and gelatin in a calcium-dependent manner. PrtFAC decreased viability and induced apoptosis of normal oral keratinocytes (NOKs) in a time and dose-dependent manner. Transcriptome analysis of NOK cells treated with PrtFAC showed an upregulation of the genes encoding human pro-apoptotic proteins: Apoptotic peptidase activating factor 1 (Apaf1) cytochrome C, as well as caspase 3 and caspase 9, suggesting the involvement of the mitochondrial apoptotic pathway. There was a significant increase in caspase 3/7 activity in NOK cells treated with PrtFAC. Taken together, these findings suggest that F. alocis PrtFAC protein may play a role in the virulence and pathogenesis of F. alocis.

Proposição de uma nova metodologia para isolamento e cultivo de células-tronco mesenquimais derivadas do tecido adiposo / A new methodology proposition for isolation and culture of adipose-derived mesenchymal stem cells

Introdução: As células-tronco mesenquimais (CTM) têm despertado interesse de vários grupos de pesquisa em função do grande potencial de aplicabilidade em terapia celular e medicina regenerativa. Nesse contexto, o tecido adiposo vem recebendo grande destaque como importante fonte para obtenção de CTM. Os protocolos utilizados atualmente para o isolamento das células-tronco derivadas do tecido adiposo (ADSC) empregam, de forma geral, o método de digestão enzimática com colagenase extraída de bactéria (Clostridium histolyticun), que pode conter contaminantes, como endotoxinas e outros peptídeos que, eventualmente, poderão resultar em reações adversas nos procedimentos de terapia celular em pacientes humanos. Objetivo: Pretendeu-se no presente estudo adequar e propor uma nova abordagem empregando a metodologia de dissociação mecânica para isolamento de CTM derivadas de tecido adiposo de ratos. Métodos: As células cultivadas foram analisadas quanto ao potencial de adesão, proliferação e tempo de duplicação celular, por meio de uma curva de crescimento. As células isoladas e cultivadas a partir do tecido adiposo foram também analisadas quanto ao potencial de diferenciação in vitro nas linhagens adipogênica, condrogênica e osteogênica. Resultados: Os resultados mostraram que o tempo de duplicação (velocidade de crescimento) da população celular isolada por dissociação mecânica é mais expressivo quando comparado com a técnica de digestão enzimática. As células isoladas do tecido adiposo apresentaram potencial de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Conclusão: Os resultados obtidos permitem concluir que a metodologia de dissociação mecânica apresenta-se como uma alternativa viável, de baixo custo e, como tal, extremamente promissora no sentido de permitir que a colagenase de origem bacteriana (Clostridium histolyticun) torne-se um componente prescindível para isolamento e cultivo de células provenientes do tecido adiposo.

Background: Mesenchymal stem cells (MSCs) have attracted interest of several research groups due to the large potential applicability in cell therapy and regenerative medicine. In this context, adipose tissue has received high profile as an important source in order to obtain MSC. The protocols currently suggested for the isolation and culture of adipose- -derived stem cells (ADSC) utilize, in general, the enzymatic digestion method with bacterial collagenase (Clostridium histolyticun) which may contain contaminants such as endotoxin and other peptides that eventually may result in adverse reactions in the cell therapy procedures in human patients. Objective: In this context, it was intended in this study to propose a new methodological approach of mechanical dissociation for isolating and culture of adipose-derived mesenchymal stem cells. Methods: The cultured cells were analyzed for potential adhesion, proliferation and cell doubling time, through a growth curve lineages The cells were also analyzed according to potential for differentiation in adipogenic, chondrogenic and osteogenic lineages. Results: The results showed that the doubling time of the cell population isolated by mechanical dissociation is faster when compared to the enzymatic digestion technique. The isolated cells from adipose tissues howed potential for differentiation in cell lineages osteogenic, adipogenic and chondrogenic. Conclusion: The obtained results allow us to conclude that the methodology of mechanical dissociation, presented in this paper, is a viable, low cost and therefore an extremely promising alternative in order to permit that the bacterial collagenase, from Clostridium histolyticun, become a dispensable component for isolation and cultivation of adipose-derived stem cells.

[ABILITY OF MICROORGANISMS FROM DIFFERENT ECOLOGICAL NICHES TO HYDROLYZE THE INSOLUBLE PROTEINS].

Screening of protease producers with specificity to insoluble and hard soluble protein substrates of animal origin (collagen, fibrin, elastin and keratin) was carried out. It was studied the bacterial cultures (24 strains) isolated from water and periphyton of enclosures with dolphins, and also from exhalations, oral cavity and skin of dolphins. Some bacterial strains isolated from water and periphyton of enclosures hydrolyzed collagen (5-23 U/ml) and elastin (20-32 U/ml). Thus all tested cultures did not possess the property of extracellular keratinases synthesis. The streptomycetes (48 strains) were isolated from the soil of Black Sea coastal strip near Odessa and Saky, from parkland and the shores of freshwater lake in Saky and from the soil of Atlantic Ocean coastal strip near Albufena (Portugal). Several streptomycetes have been found to appeare the perspective producers of extracellular keratinase and collagenase. The strains isolated from the soil of the coastal strip area both sea and freshwater lake in Saky possessed the highest activity (up to 5 U/mg).

Glutenase and collagenase activities of wheat cysteine protease Triticain-α: feasibility for enzymatic therapy assays.

Insufficient and/or improper protein degradation is associated with the development of various human pathologies. Enzymatic therapy with proteolytic enzymes aimed to improve insufficient proteolytic activity was suggested as a treatment of protease deficiency-induced disorders. Since in many cases human degradome is incapable of degrading the entire target protein(s), other organisms can be used as a source of proteases exhibiting activities distinct from human enzymes, and plants are perspective candidates for this source. In this study recombinant wheat cysteine protease Triticain-α was shown to refold in vitro into an autocatalytically activated proteolytic enzyme possessing glutenase and collagenase activities at acidic (or close to neutral) pH levels at the temperature of human body. Mass-spectrometry analysis of the products of Triticain-α-catalyzed gluten hydrolysis revealed multiple cleavage sites within the sequences of gliadin toxic peptides, in particular, in the major toxic 33-mer α-gliadin-derived peptide initiating inflammatory responses to gluten in celiac disease (CD) patients. Triticain-α was found to be relatively stable in the conditions simulating stomach environment. We conclude that Triticain-α can be exploited as a basic compound for development of (i) pharmaceuticals for oral administration aimed at release of the active enzyme into the gastric lumen for CD treatment, and (ii) topically active pharmaceuticals for wound debridement applications.