Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen.

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)10, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2' positions, which may be attributed to the larger space available for substrate binding at the S2 and S2' sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.

Enzymatic vitreolysis using reengineered Vibrio mimicus-derived collagenase.

Purpose: Collagen is a key player contributing to vitreoelasticity and vitreoretinal adhesions. Molecular reorganization causes spontaneous weakening of these adhesions with age, resulting in the separation of the posterior hyaloid membrane (PHM) from the retina in what is called complete posterior vitreous detachment (PVD). Incomplete separation of the posterior hyaloid or tight adherence or both can lead to retinal detachment, vitreomacular traction syndrome, or epiretinal membrane formation, which requires surgical intervention. Pharmacological vitrectomy has the potential of avoiding surgical vitrectomy; it is also useful as an adjunct during retinal surgery to induce PVD. Previously studied enzymatic reagents, such as collagenase derived from Clostridium histolyticum, are nonspecific and potentially toxic. We studied a novel collagenase from Vibrio mimicus (VMC) which remains active (VMA), even after deletion of 51 C-terminal amino acids. To limit the activity of VMA to the vitreous cavity, a fusion construct (inhibitor of hyaluronic acid-VMA [iHA-VMA]) was made in which a 12-mer peptide (iHA, which binds to HA) was fused to the N-terminus of VMA. The construct was evaluated in the context of PVD. Methods: VMA and iHA-VMA were expressed in Escherichia coli, purified, and characterized with gelatin zymography, collagen degradation assay, fluorescamine-based assay, and cell-based assays. Two sets of experiments were performed in New Zealand albino rabbits. Group A (n = 10) received iHA-VMA, while group B (n = 5) received the equivalent dose of VMA. In both groups, saline was injected as a control in the contralateral eyes. Animals were monitored with indirect ophthalmoscopy, optical coherence tomography (OCT), and B-scan ultrasonography. Retinal toxicity was assessed with hematoxylin and eosin (H&E) staining of retinal tissue. Results: The activity of iHA-VMA and VMA was comparable and 65-fold lower than that of C. histolyticum collagenase Type IV. In the iHA-VMA group, all the rabbits (n = 10) developed PVD, with complete PVD seen in six animals. No statistically significant histomorphological changes were seen. In the VMA group, four of the five rabbits developed complete PVD; however, retinal morphological changes were seen in two animals. Conclusions: iHA-VMA displays targeted action confined to the vitreous and shows potential for safe pharmacologic vitreolysis.

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.

Extemporaneous Preparation of Injectable and Enzymatically Degradable 3D Cell Culture Matrices from an Animal-Component-Free Recombinant Protein Based on Human Collagen Type I.

Injectable hydrogels are considered important to realize safe and effective minimally invasive therapy. Although animal-derived natural polymers are well studied, they typically lack injectability and fail to eliminate the potential risks of immunogenic reactions or unknown pathogen contamination. Despite extensive research activities to explore ideal injectable hydrogels, such state-of-the-art technology remains inaccessible to non-specialists. In this article, the design of a new injectable hydrogel platform that can be extemporaneously prepared from commercially available animal-component-free materials is described. The hydrogels can be prepared simply by mixing mutually reactive aqueous solutions without necessitating specialized knowledge or equipment. Their solidification time can be adjusted by choosing proper buffer conditions from immediate to an extended period of time, that is, few or several tens of minutes depending on the concentration of polymeric components, which not only provides injectability, but enables 3D encapsulation of cells. Mesenchymal stromal/stem cells can be encapsulated and cultured in the hydrogels at least for 2 weeks by traditional cell culture techniques, and retrieved by collagenase digestion with cell viability of approximately 80%. This hydrogel platform accelerates future cell-related research activities.

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.

Proteolytic Rafts for Improving Intraparenchymal Migration of Minimally Invasively Administered Hydrogel-Embedded Stem Cells.

The physiological spaces (lateral ventricles, intrathecal space) or pathological cavities (stroke lesion, syringomyelia) may serve as an attractive gateway for minimally invasive deployment of stem cells. Embedding stem cells in injectable scaffolds is essential when transplanting into the body cavities as they secure favorable microenvironment and keep cells localized, thereby preventing sedimentation. However, the limited migration of transplanted cells from scaffold to the host tissue is still a major obstacle, which prevents this approach from wider implementation for the rapidly growing field of regenerative medicine. Hyaluronan, a naturally occurring polymer, is frequently used as a basis of injectable scaffolds. We hypothesized that supplementation of hyaluronan with activated proteolytic enzymes could be a viable approach for dissolving the connective tissue barrier on the interface between the scaffold and the host, such as pia mater or scar tissue, thus demarcating lesion cavity. In a proof-of-concept study, we have found that collagenase and trypsin immobilized in hyaluronan-based hydrogel retain 60% and 28% of their proteolytic activity compared to their non-immobilized forms, respectively. We have also shown that immobilized enzymes do not have a negative effect on the viability of stem cells (glial progenitors and mesenchymal stem cells) in vitro. In conclusion, proteolytic rafts composed of hyaluronan-based hydrogels and immobilized enzymes may be an attractive strategy to facilitate migration of stem cells from injectable scaffolds into the parenchyma of surrounding tissue.

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.

Collagenase-assisted wound bed preparation: An in vitro comparison between Vibrio alginolyticus and Clostridium histolyticum collagenases on substrate specificity.

Bacterial collagenase from the aerobic non-pathogenic Vibrio alginolyticus chemovar iophagus is an extracellular metalloproteinase. This collagenase preparation is obtained through a fermentation process and is purified chromatographically, resulting in a highly purified 82-kDa single-band protein that does not contain non-specific proteases or other microbial impurities. V. alginolyticus collagenase was added to a hyaluronan (HA)-based device to develop a novel debriding agent to improve the treatment of ulcers, necrotic burns, and decubitus in the initial phase of wound bed preparation. In this study, an in vitro biochemical characterisation of V. alginolyticus collagenase versus a commercial preparation from a Clostridium histolyticum strain on various dermal extracellular matrix (ECM) substrates was performed. V. alginolyticus collagenase demonstrated its ability to carry out the enzymatic cleavage of the substrate, allowing a selective removal of necrotic tissues while sparing healthy tissue, as reported in clinical studies and through routine clinical experience. in vitro tests under physiological conditions (pH, presence of Ca++, etc.) have demonstrated that V. alginolyticus collagenase exhibits very poor/limited non-specific proteolytic activity, whereas the collagenase preparation from C. histolyticum is highly active both on collagen and on non-collagenic substrates. This finding implies that while the V. alginolyticus enzyme is fully active on the collagen filaments that anchor the necrotic tissue to the wound bed, it does not degrade other minor, but structurally important, components of the dermal ECM. This feature could explain why collagenase preparation from V. alginolyticus has been reported to be much gentler on perilesional, healthy skin.

Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage.

Inhibitory effects of mycosporine-2-glycine isolated from a halotolerant cyanobacterium on protein glycation and collagenase activity.

Mycosporine-2-glycine (M2G), isolated from the halotolerant cyanobacterium Aphanothece halophytica, was purified and characterized in order to determine its utility as a cosmetic and pharmaceutical ingredient. M2G efficiently inhibited protein crosslinking. The inhibitory activity of M2G was significantly greater than that of the well-known Maillard reaction inhibitor aminoguanidine. In addition, M2G and other known mycosporine-like amino acids inhibited bacterial collagenase activity. To the best of our knowledge, this is the first report describing that M2G specifically inhibits the formation of advanced glycation end-products (AGEs), which play a critical role in ageing process and age-related diseases. These observations indicate that M2G may have potential therapeutic applications by suppressing the formation of AGEs and inhibiting excess collagenase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycosporine-like amino acids (MAAs) are known as multifunctional natural compounds. The MAA mycosporine-2-glycine (M2G), isolated from the halotolerant cyanobacterium Aphanothece halophytica, has potential therapeutic applications for the prevention of skin ageing. Purified M2G was endotoxin-free. M2G had greater inhibitory activity of protein cross-linking compared with well-known inhibitor, aminoguanidine and hindered bacterial collagenase activity. The mechanisms for these inhibitory activities of M2G are discussed in this study.

Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome.

Pseudogymnoascus destructans is the causative agent of white-nose syndrome, a disease that has caused the deaths of millions of bats in North America. This psychrophilic fungus proliferates at low temperatures and targets hibernating bats, resulting in their premature arousal from stupor with catastrophic consequences. Despite the impact of white-nose syndrome, little is known about the fungus itself or how it infects its mammalian host. P. destructans is not amenable to genetic manipulation, and therefore understanding the proteins involved in infection requires alternative approaches. Here, we identify hydrolytic enzymes secreted by P. destructans, and use a novel and unbiased substrate profiling technique to define active peptidases. These experiments revealed that endopeptidases are the major proteolytic activities secreted by P. destructans, and that collagen, the major structural protein in mammals, is actively degraded by the secretome. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans-conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host.

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.

The effect of tissue surface modification with collagenase and addition of TGF-ß3 on the healing potential of meniscal tears repaired with tissue glues in vitro.

The aim of the current in vitro study was to investigate if tissue surface modification with collagenase and addition of the TGF-ß3 can increase the number of cells present in meniscus tears repaired with the use of newly developed tissue adhesives based on isocyanate-terminated block copolymers. Cylindrical explants were harvested from the inner part of bovine menisci. To simulate a full-thickness tear, the central core of the explants was removed and glued back into the defect, with or without incubation in collagenase solution prior to gluing. The repair constructs were then cultured with or without addition of TGF-ß3, and assessed for their histological appearance. The histological staining of the constructs confirmed that both developed adhesives were not cytotoxic. After 28 days, meniscus cells were present in direct contact with the glues. The addition of TGF-ß3 to the culture medium resulted in the presence of cells that formed a sheath inside the simulated tear and in increased cell numbers at the edges of annulus of the explants. In the group in which the tissue was incubated in collagenase and cultured in medium containing TGF-ß3, thicker layers of cells were observed. These results suggest that repairing the torn meniscus with tissue adhesives after pre-treatment of the tissue with collagenase and stimulation with TGF-ß3 is a very promising treatment method, especially when treating the inner avascular part of the meniscus. Nevertheless, longer-term in vitro and in vivo studies are needed to confirm the beneficial effects of this combination therapy.

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.

Immunophenotypic characterization of ovine mesenchymal stem cells.

The clinical potential of multipotent mesenchymal stem cells (MSCs) has led to the essential development of analytical tools such as antibodies against membrane-bound proteins for the immunophenotypic characterization of human and rodent cells. Such tools are frequently lacking for emerging large animal models like the sheep that have greater relevance for the study of human musculoskeletal diseases. The present study identified a set of commercial nonspecies specific monoclonal antibodies for the immunophenotypic characterization of ovine MSCs. A protocol combining the less destructive proteolytic activity of accutase and EDTA was initially developed for the detachment of cells from plastic with minimum loss of cell surface antigens. A range of commercially available antibodies against human or rodent MSC antigens were then tested in single and multistain-based assays for their cross-reactivity to bone marrow derived ovine MSCs. Antibody clones cross-reactive to ovine CD73 (96.9% ± 5.9), CD90 (99.6% ± 0.3), CD105 (99.1 ± 1.5), CD271 (97.7 ± 2.0), and MHC1 (94.0% ± 7.2) antigens were identified using previously reported CD29, CD44, and CD166 as positive controls. Multistaining analysis indicated the colocalization of these antigens on MSCs. Furthermore, antibody clones identified to cross-react against white blood cell antigens exhibited either negative (CD117 (0.1% ± 0.1)) or low (MHCII (10.5% ± 16.0); CD31 (14.6% ± 4.2), and CD45 (39.4% ± 31.8)) cross-reactivity with ovine MSCs. The validation of these antibody clones to sheep MSC antigens is essential for studies utilizing this large animal model for stem cell-based therapies. © 2016 International Society for Advancement of Cytometry.

Bacterial collagenases - A review.

Bacterial collagenases are metalloproteinases involved in the degradation of the extracellular matrices of animal cells, due to their ability to digest native collagen. These enzymes are important virulence factors in a variety of pathogenic bacteria. Nonetheless, there is a lack of scientific consensus for a proper and well-defined classification of these enzymes and a vast controversy regarding the correct identification of collagenases. Clostridial collagenases were the first ones to be identified and characterized and are the reference enzymes for comparison of newly discovered collagenolytic enzymes. In this review we present the most recent data regarding bacterial collagenases and overview the functional and structural diversity of bacterial collagenases. An overall picture of the molecular diversity and distribution of these proteins in nature will also be given. Particular aspects of the different proteolytic activities will be contextualized within relevant areas of application, mainly biotechnological processes and therapeutic uses. At last, we will present a new classification guide for bacterial collagenases that will allow the correct and straightforward classification of these enzymes.

Human glandular organoid formation in murine engineering chambers after collagenase digestion and flow cytometry isolation of normal human breast tissue single cells.

Women with high mammographic density (MD) are at increased risk of breast cancer (BC) after adjustment for age and body mass index. We have developed a murine biochamber model in which both high MD (HMD) and low MD (LMD) tissue can be propagated. Here, we tested whether cells isolated by collagenase digestion and fluorescence-activated cell sorting (FACS) from normal breast can be reconstituted in our biochamber model, which would allow cell-specific manipulations to be tested. Fresh breast tissue was collected from women (n = 7) undergoing prophylactic mastectomy. The tissue underwent collagenase digestion overnight and, in some cases, additional FACS enrichment to obtain mature epithelial, luminal progenitor, mammary stem, and stromal cells. Cells were then transferred bilaterally into biochambers in SCID mice (n = 5-7) and incubated for 6 weeks, before harvesting for histological analyses, and immunohistochemical staining for cytokeratins (CK), vimentin, Ki-67, murine macrophages, and Cleaved Caspase-3. Biochambers inoculated with single cells after collagenase digestion or with flow cytometry contained glandular structures of human origin (human vimentin-positive), which expressed CK-14 and pan-CK, and were proliferating (Ki-67-positive). Glandular structures from the digested tissues were smaller than those in chambers seeded with finely chopped intact mammary tissue. Mouse macrophage infiltration was higher in the chambers arising from digested tissues. Pooled single cells and FACS fractionated cells were viable in the murine biochambers and formed proliferating glandular organoids of human origin. This is among the first report to demonstrate the success of formed human glandular organoids from isolated primary mammary cells in the murine biochamber model.

A metallo-keratinase from a newly isolated Acinetobacter sp. R-1 with low collagenase activity and its biotechnological application potential in leather industry.

Microbial keratinase is a well-recognized enzyme that can specifically degrade insoluble keratins. A keratinase-producing bacterium was isolated from a duck ranch soil and identified as Acinetobacter sp. R-1 based on the biochemical characteristics and 16S rDNA gene sequencing. It showed high keratinase activity and low collagenase activity. The keratinase was purified to electrophoretic homogeneity with 6.69% recovery, 2.68-fold purification and an estimated molecular weight of 25 kDa. Additionally, the keratinase showed optimal activity at 50 °C and pH11. Keratinase activity of Acinetobacter sp. significantly increased in the presence of Li(+), Na(+), and Ca(2+), while it was completely inhibited by EDTA, indicating it was a metallo-keratinase. Moreover, the crude keratinase from Acinetobacter sp. R-1 could thoroughly depilate goat skin and simultaneously modify the wool surface, which indicated its applicable potential in leather and textile industries.

[Screening, identification and fermentation optimization of a collagenase-producing strain and purification of the collagenase].

Objective: The purpose of this study was to isolate novel strains from the soil nearby meat processing factories to produce collagenase. After the yield of collagenase from the strain improved, the collagenase was purified and used for hydrolyzing collagen. Methods: The strain was identified based on morphological features, physiological and biochemical characteristics and 16S rRNA gene phylogenetic tree analysis. The yield of collagenase was increased by optimizing the fermentation condition, and the collagenase isolated from the fermentation supernatant of the strain was finally purified with strong anion exchange resins. Results: The collagenase-producing strain was identified as Bacillus cereus. The optimized fermentation conditions of the strain were: 2.0% glucose as optimum carbon source, 1.5% tryptone as optimum nitrogen source, 0.005% of Ca2+ as optimum metal ion. The optimum temperature and pH were 37 ℃ and 7.5, respectively. Under the optimum conditions, the enzyme activity of collagenase was (65.81±2.06) U/mL, 1.5-fold increased than that before the optimization. After purified with strong anion exchange resins, a collagenase with the purity higher than 90%, the molecular weight about 100 kDa, and the specific activity of 7615.0±78.7 U/mg was obtained. Conclusion: The activity of Bacillus cereus collagenase was higher than the reported collagenases. Using this novel collagenase, collagen could be degraded into short biological peptides in a short time. Hence, this collagenase has application prospects in many fields, such as food, medical, health care products and cosmetics.

Structures of three polycystic kidney disease-like domains from Clostridium histolyticum collagenases ColG and ColH.

Clostridium histolyticum collagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca(2+)-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca(2+)-bound holo s2b (1.4 Šresolution, R = 15.0%, Rfree = 19.1%) and holo s2a (1.9 Šresolution, R = 16.3%, Rfree = 20.7%), as well as of Ca(2+)-free apo s2a (1.8 Šresolution, R = 20.7%, Rfree = 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 Šresolution, R = 16.9%, Rfree = 21.2%; 1.6 Šresolution, R = 16.2%, Rfree = 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved ß-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent ß-strand. This ß-bulge and the genesis of a Ca(2+) pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3. B-factor analyses suggest that in the presence of Ca(2+) the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca(2+) binding. Conserved residues not only interact with Ca(2+), but also position the Ca(2+)-interacting water molecule. Ca(2+) aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca(2+) binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.