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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Microsporum canis is the main zoophylic dermatophyte in dogs and cats, and it is also an important zoonotic agent. The literature showed that cats are asymptomatic carriers of M. canis. This is apparently due to host resistance and/or the presence of strains with lower virulence. This study was aimed to evaluate the keratinolytic, elastinolytic and collagenolytic activities of M. canis strains and their relationship with symptomatic and asymptomatic cats. In addition, these strains were analysed by RFLP. The strains isolated from cats with clinical dermatophytosis had higher keratinase and elastase activity than those isolated from asymptomatic animals (p minus than 0.05). There were not differences in RFLP patterns based on Hind III digestion.

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