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.

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.

[Experimental study of therapeutical properties and safety of king crab collagenase-containing ointment]. / Eksperimental'noe izuchenie lechebnykh svoistv mazi, soderzhashchei kollagenazu kamchatskogo kraba.

The effects of ointment containing king crab (Paralithodes camtschatica) collagenase on intact skin, thermal, and pyonecrotic wounds were studied in rats by using hematological, biochemical, immunological, and morphological methods. The ointment for the skin and viscera was shown to be safe. It is highly effective in debriding the infected wounds. Different concentrations of collagenase were tested. The concentration of collagenase was recommended to be 0.2 mg/g ointment for use.

Production of selenomethionine-labeled recombinant human neutrophil collagenase in Escherichia coli.

Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca2+ and Zn2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.

[The immunohistochemical study of suppurative wounds in rats following the application of collagenase from the crab Paralithodes camtschatica]. / Immunogistokhimicheskoe izuchenie gnoinykh ran u krys posle applikatsii kollagenazy kraba Paralithodes camtschatica.

Immunohistochemical study of tissues of purulent wounds in rats after application of the collagenase isolated from the king crab Paralithodes camtschatica has been undertaken. The enzyme therapy resulted in a rapid and efficient removal of necrotic debris. It was accompanied by fibrin elimination from the wound bottom and subsequent formation of new capillaries. Cellular fibronectin with ED-A sequence was identified in the newly formed granulation tissue, which points to its active synthesis in situ. Detection of type I collagen in granulation tissue revealed that wound treatment with crab collagenase had no impact on the development process of the tissue. Polyclonal antibodies against two isozymes of crab collagenolytic protease were obtained. It was shown that after application of both isozymes of the collagenase were accumulated in fibrin deposits at the wound bottom but not penetrated in adherent granulation tissue. These processes underlie the therapeutic effect of the crab collagenase.

[A morphological evaluation of the action of collagenase from the king crab Paralithodes camtschatica on the experimental wound process]. / Morfologicheskaia otsenka vozdeistviia kollagenazy kamchatskogo kraba Paralithodes camtschatica na ranevoi protsess v éksperimente.

The paper presents the results of experimental morphological evaluation of the effect of a new proteolytic enzyme collagenase of crab Paralithodes camtschatica on wound healing in infected and aseptic rabbit wounds. The enzyme was applied on wounds using gauze and gelevin. The findings show that this protease is highly effective for debridement of infected wounds, the effect increasing at gelevin addition. To reach maximal therapeutical effect and diminish the inhibition effect of collagenase on granulation tissue, it is recommended to reduce the dose of protease during debridement process. Clinical application of crab collagenase must be individual as well as duration of wound enzymotherapy.