Differences in the degradation of native collagen by two microbial collagenases.

The early stages of degradation of native collagen by two bacterial collagenases were studied by electron microscopy and by automatic Edman degradation. The purified collagenase from Clostridium histolyticum was shown to cleave native collagen at several sites, but not progressively from the N-terminus, as had been previously suggested. The homogeneous collagenase from Achromobacter iophagus cleaves native collagen preferentially at two sites corresponding to the interbands 33-34 and 41-42. The latter lies within the region cleaved by the eukaryotic collagenases.     

Polycystic kidney disease-like domains of clostridial collagenases and their role in collagen recruitment

Bacterial collagenases exhibit a multimodular domain organization. While the N-terminal collagenase unit harbors the catalytic zinc and suffices to degrade peptidic substrates, collagen substrates come in different types, explaining the requirement for accessory domains such as polycystic kidney disease (PKD)-like domains for efficient catalysis. How the recognition and unfolding of (micro-)fibrillar or triple-helical collagen is accomplished are only poorly understood. Here, we present the crystal structure of the PKD-like domain of collagenase G from Clostridium histolyticum. The β-barrel structure reveals a two-tier architecture, connected by kinked hinge segments. Together with sheet extension as a generic oligomerization mechanism, this explains the cooperativity among accessory domains as well as their adaptivity to varying substrates.     

SLRP interaction can protect collagen fibrils from cleavage by collagenases

Decorin, fibromodulin and lumican are small leucine-rich repeat proteoglycans (SLRPs) which interact with the surface of collagen fibrils. Together with other molecules they form a coat on the fibril surface which could impede the access to collagenolytic proteinases. To address this hypothesis, fibrils of type I or type II collagen were formed in vitro and treated with either collagenase-1 (MMP1) or collagenase-3 (MMP13). The fibrils were either treated directly or following incubation in the presence of the recombinant SLRPs. The susceptibility of the uncoated and SLRP-coated fibrils to collagenase cleavage was assessed by SDS/PAGE. Interaction with either recombinant decorin, fibromodulin or lumican results in decreased collagenase cleavage of both fibril types. Thus SLRP interaction can help protect collagen fibrils from cleavage by collagenases.     

A model for interstitial collagen catabolism by mammalian collagenases.

Mammalian collagenases cleave all three alpha chains of native, triple-helical types I, II, and III collagens after the Gly residue of the partial sequence Gly-[Ile or Leu]-[Ala or Leu] at a single locus approximately three-fourths from the amino terminus. There are an additional 31 sites in the triple-helical regions of types I, II, III, and IV collagens that contain the same partial sequence but are not hydrolyzed. A model has been developed to explain this remarkable specificity. The mammalian collagenase cleavage site in interstitial collagens is distinguished by: (a) a low side-chain molal volume-, high imino acid (greater than 33%)-containing region that is tightly triple-helical, consisting of four Gly-X-Y triplets preceding the cleavage site, (b) a low imino acid-containing (less than 17%), loosely triple-helical region consisting of four Gly-X-Y triplets following the cleavage site, and (c) a maximum of one charged residue for the entire 25 residue cleavage site region, which is always an Arg that follows the cleavage site in subsite P'5 or P'8. In addition, the high imino acid-containing region cannot have an imino acid adjacent to the cleaved Gly-[Ile or Leu] bond (i.e. in subsite P2). Careful scrutiny of the 31 non-cleaved sequences reveals that none of those sites shares all of the characteristics of the cleavage site. The criterion of this model thus explain both cleaved and non-cleaved sequences in the triple-helical regions of types I, II, III, and IV collagen, and are supported by all known experimental and theoretical results on collagen catabolism and structure.     

Nereis cuticle collagen. Proteolysis by marine vibrial and clostridial collagenases

Proteolysis of Nereis cuticle collagen by two bacterial collagenases was investigated using viscosimetry, enzyme kinetics, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and ion exchange chromatography of collagenolytic peptides. Collagenase of the marine Vibrio B-30 completely degrades native cuticle collagen at 7 degress C with a turnover number 50 times greater than that of the clostridial collagenase. Although turnover numbers for the two enzymes are comparable when using denatured cuticle collagen as substrate, the vibrial collagenase appears to cleave twice as many peptide bonds per mg of cuticle collagen as does the clostridial enzyme. Sodium dodecyl sulfate gel electrophoresis of collagenase-digested native cuticle collagen reflects the resistance of the collagen to clostridial collagenase; however, the vibrial enzyme completely degrades the cuticle collagen with the formation of one transient intermediate (Mr 400,000). Peptide analysis of fully digested denatured cuticle collagen reveals that the two enzymes have a number of qualitative and quantitative similarities. Despite these, however, only the vibrial collagenase seems capable of extensively degrading native cuticle collagen.     

Differential susceptibility of type X collagen to cleavage by two mammalian interstitial collagenases and 72-kDa type IV collagenase

We have studied the degradation of type X collagen by human skin fibroblast and rat uterus interstitial collagenases and human 72-kDa type IV collagenase. The interstitial collagenases attacked the native type X helix at two loci, cleaving residues Gly92-Leu93 and Gly420-Ile421, both scissions involving Gly-X bonds of Gly-X-Y-Z-A sequences. However, the human and rat interstitial enzymes displayed an opposite and substantial selectivity for each of these potential sites, with the uterine enzyme catalyzing the Gly420-Ile421 cleavage almost 20-fold faster than the Gly92-Leu93 locus. Values for enzyme-substrate affinity were approximately 1 microM indistinguishable from the corresponding Km values against type I collagen. Interestingly, in attacking type X collagen, both enzymes manifested kinetic properties intermediate between those characterizing the degradation of native and denatured collagen substrates. Thus, energy dependence of reaction velocity revealed a value of EA of 45 kcal, typical of native interstitial collagen substrates. However, the substitution of D2O for H2O in solvent buffer failed to slow type X collagenolysis significantly (kH/kD = 1.1), in contrast to the 50-70% slowing (kH/kD = 2-3) observed with native interstitial collagens. Since this lack of deuterium isotope effect is characteristic of interstitial collagenase cleavage of denatured collagens, we investigated the capacity of another metalloproteinase with substantial gelatinolytic activity, 72-kDa type IV collagenase, to degrade type X collagen. The 72-kDa type IV collagenase cleaved type X collagen at both 25 and 37 degrees C, and at loci in close proximity to those attacked by the interstitial enzymes. No further cleavages were observed at either temperature with type IV collagenase, and although values for kcat were not determined (due to associated tissue inhibitor of metalloproteinases-2), catalytic rates appeared to be substantial in comparison to the interstitial enzymes. In contrast, type X collagen was completely resistant to proteolysis by stromelysin. Type X collagen thus appears to be highly unusual in its susceptibility to degradation by both interstitial collagenase and another member of the metalloproteinase gene family.     

Magnetotactic bacteria,magnetosomes and their application

Magnetotactic bacteria (MTB) are a diverse group of microorganisms with the ability to orient and migrate along geomagnetic field lines. This unique feat is based on specific intracellular organelles, the magnetosomes, which, in most MTB, comprise nanometer-sized, membrane bound crystals of magnetic iron minerals and organized into chains via a dedicated cytoskeleton. Because of the special properties of the magnetosomes, MTB are of great interest for paleomagnetism, environmental magnetism, biomarkers in rocks, magnetic materials and biomineralization in organisms, and bacterial magnetites have been exploited for a variety of applications in modern biological and medical sciences. In this paper, we describe general characteristics of MTB and their magnetic mineral inclusions, but focus mainly on the magnetosome formation and the magnetisms of MTB and bacterial magnetosomes, as well as on the significances and applications of MTB and their intracellular magnetic mineral crystals.     

Evaluation of methods for extraction of bacteria from soil

Abstract Several methods for dispersion of soil were tested for possible use in procedures for extraction of bacteria. Physical cell damage on cells and efficiency in extraction of indigenous cells from soil, were investigated. Cell damage by the dispersion methods was investigated by measuring the physical cell integrity and viability of pure cultures of Escherichia coli and Bacillus subtilis , as well as soil bacteria extracted from soil, when dispersed in slurries of γ-sterilized soil. Separation of bacteria and soil particles on the basis of buoyant density was conducted with the nonionic density gradient medium Nycodenz. When slurries of γ-sterilized soil with added pure cultured cells were centrifuged (10000 × g ) over cushions of Nycodenz (1.3 g ml⁻¹), practically all the added cells were recovered in a layer on top of the cushion. This proves that a reversible attachment and cosedimentation is not an important phenomenon in this procedure. The efficiency of the different dispersion methods for the extraction of indigenous soil bacteria, was assessed after separation of dislodged and attached soil bacteria. This separation was done either on the basis of sedimentation rate by low speed centrifugation, or buoyant density by Nycodenz density gradient centrifugation. The physical dispersion by ultrasonic treatment and chemical dispersion by the use of a chelating agent together with a detergent, were inferior to physical dispersion either by Waring blender (for large volumes) or a rotating rubber pestle treatment (for smaller volumes). The physical dispersion did not appear to be destructive to the cells tested.     

Biological properties of L-forms and their parent bacteria

L-forms of Staphylococcus aureus, Streptococcus faecalis, Listeria monocytogenes and Salmonella typhy and their parent bacteria were examined for biological properties and compared with their parental forms. Some L-forms differed from their parent bacteria and required a longer incubation period.     

Halotolerant, alkaliphilic urease-producing bacteria from different climate zones and their application for biocementation of sand

Microbially induced calcium carbonate precipitation (MICP) is a phenomenon based on urease activity of halotolerant and alkaliphilic microorganisms that can be used for the soil bioclogging and biocementation in geotechnical engineering. However, enrichment cultures produced from indigenous soil bacteria cannot be used for large-scale MICP because their urease activity decreased with the rate about 5 % per one generation. To ensure stability of urease activity in biocement, halotolerant and alkaliphilic strains of urease-producing bacteria for soil biocementation were isolated from either sandy soil or high salinity water in different climate zones. The strain Bacillus sp. VUK5, isolated from soil in Ukraine (continental climate), was phylogenetically close in identity (99 % of 16S rRNA gene sequence) to the strain of Bacillus sp. VS1 isolated from beach sand in Singapore (tropical rainforest climate), as well as to the strains of Bacillus sp. isolated by other researchers in Ghent, Belgium (maritime temperate climate) and Yogyakarta, Indonesia (tropical rainforest climate). Both strains Bacillus sp. VS1 and VUK5 had maximum specific growth rate of 0.09/h and maximum urease activities of 6.2 and 8.8 mM of hydrolysed urea/min, respectively. The halotolerant and alkaliphilic strain of urease-producing bacteria isolated from water of the saline lake Dead Sea in Jordan was presented by Gram-positive cocci close to the species Staphylococcus succinus. However, the strains of this species could be hemolytic and toxigenic, therefore only representatives of alkaliphilic Bacillus sp. were used for the biocementation studies. Unconfined compressive strengths for dry biocemented sand samples after six batch treatments with strains VS1and VUK5 were 765 and 845 kPa, respectively. The content of precipitated calcium and the strength of dry biocemented sand at permeability equals to 1 % of initial value were 12.4 g Ca/kg of dry sand and 454 kPa, respectively, in case of biocementation by the strain VS1. So, halotolerant, alkaliphilic, urease-producing bacteria isolated from different climate zones have similar properties and can be used for biocementation of soil.     

Purification of two alcohol dehydrogenases from Zymomonas mobilis and their properties

Summary Two alcohol dehydrogenases (ADHI and ADHII, EC 1.1.1.1) were purified to homogeneity from the cell extract of Zymomonas mobilis. The subunit molecular weights of ADHI and ADHII were 40,000 and 38,000, respectively, and both enzymes were homologous dimers. The optimal pHs of ADHI in ethanol oxidation and acetaldehyde reduction reactions were 9.5 and 4.5, and those of ADHII were 9.5 and 6.5, respectively. The optimal temperatures of ADHI and ADHII were 55° C and 45° C, respectively. ADHI was heat-inactivated at 65° C at a 10-fold higher rate than ADHII. ADHI and ADHII were inhibited by 4 M and 1 mM p-chloromercuribenzoate, respectively, and the inhibitions were reversed by the addition of 70 mM 2-mercaptoethanol. ADHII activity was enhanced by 0.02 to 2 mM CoCl₂ and inhibited by 0.4 mM o-phenanthroline; and the activity of inactivated ADHII was restored by addition of 1 mM CoCl₂ or ZnCl₂.ADHI was active on most primary alcohols but not secondary alcohols. ADHII was active on only ethanol, n-propanol, allylalcohol, and furfuryl alcohol.In the anaerobic culture of Z. mobilis, ADHII activity accounted for more than 80% of total alcohol dehydrogenase activity. In aerobic culture, ADHII was the main enzyme but was produced only in the early growth phase.     

Biodegradable collagen from Scomberomorus lineolatus skin for wound healing dressings and its application on antibiofilm properties

The major resolution of the study was to develop a dynamic form of natural biopolymer material to improve the wound healing by inhibition of biofilm formation on the surface. The extraction of collagen was effectively prepared from Scomberomorus lineolatus fish skin. Lyophilized collagen sheet was liquefied in 0.5M acetic acid to form acidic solubilized collagen (ASC) for further analysis. Physicochemical characterization of ASC was performed by various techniques using a standard protocol. The yield of ASC form S.lineolatus is higher (21.5%) than the previous reported studies. The effect of collagen solubility is gradually decreases with increasing concentration of NaCl and collagen is mostly soluble in acidic pH conditions. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of ASC contains α chain composition of α₁ and α₂ subunits and was characterized as type I collagen. Ultraviolet absorption was regulated as the appropriate wavelength to optimize the collagen. Fourier-transform infrared spectroscopy and X-ray diffraction confirmed that the isolated collagen is a triple-helical structure. The biofilm formation of Pseudomonas aeruginosa was significantly reduced by collagen incorporated with isolated 3,5,7-trihydroxyflavone (collagen-TF) sheet up to 70%. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay executed on fibroblast cell lines (L929) shows that the collagen-TF sheet was 100% compatible to enrich the cell adhesion and proliferation. The current study was the first report to extract, purify, and characterize ASC from S. lineolatus fish skin and characterize as type I collagen. Based on the result, we design the natural biodegradable collagen loaded with TF compound (collagen-TF) for antibiofilm properties. Compared with different sources of polymer, fish skin collagen is more effective and can be used as a biopolymer sheet for wound healing, food, drug delivery, tissue engineering, and pharmaceutical application.     

Differences in the physical properties of collagenases isolated from rheumatoid synovium and human skin

During purification of human collagenase from normal skin and rheumatoid synovium differences have been observed with regard to the size and charge properties of the two enzymes. Gel filtration experiments in Sephadex G-100 superfine have allowed molecular weights of approximately 63,000 and 32,000 daltons to be calculated for the skin and rheumatoid synovial enzyme respectively. Ion exchange chromatography using Sephadex QAE, A-50 has shown the rheumatoid synovial enzyme to possess charge properties more basic than that of the skin enzyme. Elution of collagenase activity from $\text{7}\text{1}\text{2}\text{\%}$ polyacrylamide gels has produced no evidence for a ‘fast-moving’ component of either enzyme.     

Production and properties of keratinolytic proteases from three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils

The keratinolytic potential and protease properties of three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils was described. Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 were able to degrade feather meal, producing high amounts of soluble proteins and forming thiol groups. The proteases of strains K12, A22 and P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; this last is an uncommon feature for bacterial keratinases. The optimal temperature was in the range 45–55°C. All three proteases were active towards azokeratin and were inhibited by EDTA, suggesting that they are keratinolytic metalloproteases. The proteolytic activity of K12 was stimulated by organic solvents and the detergent SDS, suggesting its potential application for detergent formulations and peptide synthesis. Strains A22, K12 and P3 have great potential for use in biotechnological processes involving hydrolysis of keratinous byproducts.