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 β-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℃. 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.
There were many reports that elevations in the levels of active and latent collagenase in gingival crevicular fluid(GCF) have been correlated positively with periodontal disease activity. To provide a simple diagnostic approach for testing GCF collagenolytic activity, the detection limit of enzyme activity was compared using radiofibril assay(Sodek et.al.1981) and spectrophotometric collagenolytic assay(Nethery et al. 1986). The detection limits of both assay for standard bacterial enzyme were similar and the radiofibril assay showed a little (1/2) lower detection limit for tad pole collagenase. To evaluate the relationship between periodontal tissue destruction and the collagenolytic activity, GCF was collected, and latent and active enzyme activities were measured by a spectrophotometric collagenolytic assay. Twelve subjects showing progressive lesions were selected according to the presence of immediate tissue destruction, frequent abscess formation, and increasing need for tooth extraction, and the absence of underlying systemic disease and previous antibiotic medication history within 6 months. Comparisons were made between sites with either: 1) inflammation with a previous history of progressive loss of periodontal tissue and bone support(2l progressive sites): 2) previous history of bone loss and periodontal destruction but now clinically stable(12 comparably stable sites); or 3) no loss of periodontal tissue and bone support(11 control sites including 5 gingivitis sites and 6 healthy sites). Active collagenase activity was the highest in the progressive sites and decreased in the order of the gingivitis sites, the stable sites, and the healthy sites. The total enzyme activity was $2{\sim}3$ fold higher in the progressive sites and the gingivitis sites, compared to the stable and the healthy sites. The ratio of active to total collagenolytic activity was twice in the progressive sites. Analysis of active collagenase level(5mU) and the ratio of active to total collagenolytic activity(0.8) as a diagnositic test indicates that these measurements have the sensitivity of 0.81 and 0.86, the specificity of 0.70 and 0.65, and the overall agreement of 0.75 and 0.73, respectively. Thus, this method has significant merits as a diagnostic tool to determine wherher the site is in a state of remission or progression.
True collagenolytic enzymes in animal tissues were first demonstrated by Gross and Lapiere (1962), who showed the ability of such an enzyme in the culture medium of living explants of tadpole tissue to degrade a specific substrate of undenatured collagen under physiological conditions. Recently, tumor-associated collagenolytic activity has been demonstrated in human neoplasm and in ascites V Carcinoma. This investigation have been peforme to determine whether or not a collagen lytic enzyme could e found in isolated solid Tawa sarcoma of Donryu female rat obtained the culture medium. The results were as follows.
1. 11.5mg% of hydroxyproline contained in Donryu rat skin collagen, which was extracted by 0.5M acetic acid.
2. Cultivation of solid Tawa sarcoma tissues on reconstituted rat skin collagen gels showed lysis of adjacent gel after 18 hours, and much more extensive lysis after 5 days.
3. Collagen substrate was not attacked by the common proteolytic enzymes, trypsin, pepsin, and pronase.
A marine bacterium for producing an collagenolytic protease was isolated from the southern sea of Korea and identified as Vibrio vulnificus and named as Vibrio vulnificus CYK279H. This strain producing an collagenolytic protease was showed high activity toward collagen and gelatin as substrate. The optimum initial pH, NaCl, and temperature for cell growth and protease production was 7.5, 2.0% and 25$^{\circ}C$, respectively. Optimization for collagenolytic protease production was composed of 0.3% D-galactose, 0.6% yeast extract, 4.0% gelatin, 0.2% (NH$_4$)$_2$SO$_4$, and 0.2 mM ferric citrate in artificial sea water. The maximum protease production was required gelatin and yeast extract. The collagenolytic protease production by Vibrio vulnificus CYK279H reached a maximum of 73 unit/l after the cultivation for 18 h under the optimized medium.
Final goal of periodontal treatment is to reconstruct the destructed periodontal tissue as well as to remove the necrotic pathologic elements. The purpose of this study is to investigate on the effect of Zizyphi extract to the inhibitory ability on collagenolytic activity of P gingivalis, biologic activity of gingival fibroblasts, and on the collagen and protein synthesis of gingival fibroblasts. Gingival fibroblast from giniva of first bicuspids from patient for orthodontic treatment were used and cultured. For the measurement of inhibitory ability of collagenolytic activity, crude enzyme was extracted and used on the basis of modified Ono's method. On the inhibition of collagenolytic enzyme from herbal extracts, collagenokit CLN-100 were used. The cellular activity of gingival fibroblast, were studied using MTT solution and measured optical density on 570mm by ELISA reader. To measure the effects on the ability of whole protein and collagen synthesis, cell membrane was destructed with ultrasonic grinder after culturing, centrifuged and counted by liquid scintilation counter. The inhibitory effects on producing of $IL-l{\beta}$ by monocyte, after promotion of producing $IL-l{\beta}$ by LPS, were compared with the mixture of herbal extracts and other drugs using thymocyte stimulation assay. About inhibitory effects of $PGF_2$. by gingival fibroblasts, herbal extract was compared with the addition of the other control groups using enzyme imunoassay. On the inhibition of collagenolytic activity by P. gingivalis, benzene extracts showed the most efficient inhibitory effects among the $19{\mu}g/ml$ of the compared extracts and 40.5% by Tetracycline. On the cellular activity promoting effects, compared extracts showed a bit of more effects than PDGF of $100{\mu}g/ml$ concentration and IGF of $20{\mu}g/ml$ concentration. All of the PDGF, IGF, Zizyphi Fructus extract should increase in collagen synthesis, but especially 70% ethylalcohol extracts of Zizyphi Fructus showed comparably high effects among the compared extracts. Effects on whole protein synthesis were slightly increased on every extract but especially 70% ethylalcohol extract showed significantly effective than any other estract. On the inhibitory effects of Zizyphi Fructus $IL-l{\beta}$ production by monocyte, compared extracts showed 70% of highly inhibitory effect than that of 60% inhibition effects on controlled group and each extracts showed no significant difference. In $PGF_2$ production inhibitroy effect of Zizyphi Fructus gingival fibroblasts, Herbal extracts showed 70% of inhibition comparing with tat of 90.2% of controlled group, but each extracts showed similar effects excluding the $H_2O$ extracts. These results suggested that Zizyphi Fructus might be useful medicine for inhibition of inflammatory mediator including $IL-l{\beta}$ and $PGF_2$.
This research aimed to assess the effect of collagenolytic proteases from Bacillus subtilis B13 and Bacillus siamensis S6 for tenderizing goat meat during wet aging. Collagenolytic proteases B13 and S6 were prepared at 5 U/mL of collagenolytic activity before injecting into goat meat with 10% (v/w) of initial weight. The control sample was injected with distilled water and used as a negative control. The injected meats were placed in vacuum-sealed bags and wet aged at 4℃ for 0, 3, 5, 7, 14, and 21 days. Thereafter, total aerobic count and physicochemical quality were elucidated. Both enzyme-treated samples from B13 and S6 aged for 5 days showed an acceptable microbial quality with lower than 5.7 Log CFU/g. These conditions produced the tender meats by the reduction in shear force accounting for 30% for B13 and 26% for S6 as compared to the control. Moreover, the enzyme-treated samples showed lower values of hardness, gumminess, and chewiness, with higher springiness and trichloroacetic acid-soluble peptides than the control (p<0.05). The detrimental impact on cooking loss and lipid oxidation was not found. Enzyme-injected meat had a lower cooking loss than the control (p<0.05) with no significant difference in lipid oxidation (p>0.05). Notably, meats treated with B13 and S6 were lower in CIE L* value as compared to the control (p<0.05) with no significant impact on CIE a* and CIE b* (p>0.05). These results suggested that these two collagenolytic proteases could enhance the quality of goat meat in terms of tenderness and reduce the aging time for meat tenderization.
Porpilyromonas endodontalis is specifically involved in endodontic infections. The bacterium can be isolated almost exclusively only from infected rool canals. P. gingivalis also has been implicated in endodontic infection. Pathogemcity of P. gingival is is attributed to a variety of virulence factors, especially proteases, produced by the bacterium. Importance of P. endodontalis in endodontic infection has been revealed. However, the pathogenic property of P. endodontalis has not been extensively studied. The present study was undertaken to characterize the proteolytic activity of P. endodontalis and compare the activity with that of P. gingivalis which has the most potent and diverse proteases among oral bacteria. For this purpose, culture supematants(SUP) and cell extracts(CE) were obtained from these two bacteria and were subjected to zymography using 15% polyacrylamide gel copolymerized with gelatin, type I, IV collagens or albumin. Hydrolysis of the collagens was further investigated by the cleavage assay using native type I and IV collagens in solution-phase. The results were as follows: 1. P. endodontalis apparently has a proteolytic activity that is comparable with that of P. gingivalis. 2. SUP and CE obtained from P. endodontalis and P. gingival is showed the strongest activity for gelatin, followed by type I and IV collagens, and albumin. 3. In the zymography, no noticeable difference in proteolytic activity for gelatin and albumin between the SUP and CE was observed, but in the cleavage assay using native collagens, the SUP showed a stronger collagenolytic activity than the CE. 4. The gelatinolytic activity of both the SUP and CE from these two bacteria was diminished in the presence of $CaCl_2$ or reducing agents such as ${\beta}$-mercaptoethanol and dithiothreitol(DTT). 5. Type I(calf skin and human placenta) collagenolytic activity of P. endodontalis and P. gingivalis was reduced by DTT but not affected by $CaCl_2$. The inhibitory effect of DTT, however, was reduced to some extent by $CaCl_2$. 6. Type IV collagenolytic activity of these two bacteria was not affected by $CaCl_2$ but increased to some extent in association with the reducing agents. 7. Hydrolysis of albumin by P. endodontalis and P. gingivalis was demonstrated only in the presence of the reducing agents. The overall results indicate that with respect to proteolytic activity, P. endodontalis appears to be as potent as P. gingivalis, or maybe more, and its proteolytic characteristic is similar to that of P. gingivalis. This suggests that P. endodontalis has so potent proteolytic activity that can participate by itself in endodontic infections and apical periodontitis, causing tissue destruction.
A collagenolytic enzyme, produced by Vibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Ser-Asn. The optimum temperature and pH for the enzyme activity were $35^{\circ}C$ and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8-8.0 and $20{\sim}35^{\circ}C$, respectively. The purified enzyme was strongly activated by $Zn^{2+},\;Li^{2+},\;and\;Ca^{2+}$, but inhibited by $Cu^{2+}$. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.
The oral microbiota such as P. gingivalis, P. intermedia and A. actinomycetemcomitans play a primary role in the initiation and progression of the periodontal disease. The purpose of this study was to evaluate the antimicrobial effects and inhibitory effects of honokiol and magnolol on the bacterial collagenase activity, cytotoxicity and cytokine production of periodontopathic microorganisms. The antimicrobial activities of honokiol and magnolol was evaluted with minimum inhibition concentration. Honokiol was more active than magnolol, but less than chlorhexidine on antimicrobial activity. The inhibitory effects of magnolol and honokiol on the collagenolytic activity and cytotoxicity were evaluated using a Collagenokit CLN-100 and rapid colorimetric assay (MTT method) for cellular growth and survival of gingival fibroblast and periodontalligament cell and $[^3H]-thymidine$ incorporation for the gingival epithelial cell. The inhibitory effects on the collagenolytic activity was the highest in chlorhexidine, and the lowest in magnolol. Magnolol had the lowest cytotoxic effect and chlorhexidine had the highest. The inhibitory effects on cytokine production was evaluated using $interleukin-1{\beta}$ ELISA kit (Cistron Biotech.), IL-6, $TNF-{\alpha}$ ELISA kit (Genzyme) and inhibitory effects were higher than bacterial LPS and there is no difference among the honokiol, magnolol and chlorhexidine. From these results, the antimicrobial and antienzymatic activities of honokiol and magnolol were seemed to inhibit bacterial growth and enzyme activities with lesser cytotoxic activities. Therefore, it was suggested that honokiol and magnolol are very effective antimicrobial agents on periodontal pathogens.
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