Characterization of a Novel Subtilisin-like Protease Myroicolsin from Deep Sea Bacterium Myroides profundi D25 and Molecular Insight into Its Collagenolytic Mechanism

Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (<30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal β-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen.     

Autolysis of a novel multidomain subtilase-cold-adapted deseasin MCP-01 is pH-dependent and the surface loops in its catalytic domain, the linker, and the P_proprotein domain are susceptible to proteolytic attack

Cold-adapted deseasin MCP-01 is a novel type subtilase with a multidomain structure containing a catalytic domain, a linker, a P_proprotein domain, and a PKD domain. Its autolysis was pH-dependent due to its flexible structure. N-terminal sequence analysis of the autolytic peptides revealed four autolytic sites in the catalytic domain. Three of these are in the same loops as mesophilic subtilases and one is unlike anything previously reported. Two autolytic sites were deduced in its linker and three in its P_proprotein domain, indicating the linker and the P_proprotein domain are flexible and susceptible to proteolytic attacks. Therefore, during MCP-01 autolysis, the linker and the P_proprotein domain of MCP-01 were easily attacked by proteolysis, resulting in cleavage of the C-terminal region. At the same time, some autolytic sites in the surface loops of the catalytic domain were cleaved. This is the first report describing the autolytic mechanism of a multidomain subtilase.     

Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family

A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP‐01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP‐01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate‐binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP‐01 displayed a non‐strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1’ site in collagen. His211 is a key residue for the P1‐basic‐residue preference of MCP‐01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms.     

Cell surface collagenolysis requires homodimerization of the membrane-bound collagenase MT1-MMP

Pericellular degradation of interstitial collagens is a crucial event for cells to migrate through the dense connective tissue matrices, where collagens exist as insoluble fibers. A key proteinase that participates in this process is considered to be membrane-type 1 matrix metalloproteinase (MT1-MMP or MMP-14), but little is known about the mechanism by which it cleaves the insoluble collagen. Here we report that homodimerization of MT1-MMP through its hemopexin (Hpx) domain is essential for cleaving type I collagen fibers at the cell surface. When dimerization was blocked by coexpressing either a membrane-bound or a soluble form of the Hpx domain, cell surface collagenolytic activity was inhibited in a dose-dependent manner. When MMP-13, a soluble collagenase active as a monomer in solution, was expressed as a membrane-anchored form on the cell surface, homodimerization was also required to cleave collagen. Our results introduce a new concept in that pericellular collagenolysis is regulated by correct molecular assembly of the membrane-anchored collagenase, thereby governing the directionality of the cell to migrate in tissue.     

Elastolytic Mechanism of a Novel M23 Metalloprotease Pseudoalterin from Deep-sea Pseudoalteromonas sp. CF6-2: CLEAVING NOT ONLY GLYCYL BONDS IN THE HYDROPHOBIC REGIONS BUT ALSO PEPTIDE BONDS IN THE HYDROPHILIC REGIONS INVOLVED IN CROSS-LINKING*

Elastin is a common insoluble protein that is abundant in marine vertebrates, and for this reason its degradation is important for the recycling of marine nitrogen. It is still unclear how marine elastin is degraded because of the limited study of marine elastases. Here, a novel protease belonging to the M23A subfamily, secreted by Pseudoalteromonas sp. CF6-2 from deep-sea sediment, was purified and characterized, and its elastolytic mechanism was studied. This protease, named pseudoalterin, has low identities (<40%) to the known M23 proteases. Pseudoalterin has a narrow specificity but high activity toward elastin. Analysis of the cleavage sites of pseudoalterin on elastin showed that pseudoalterin cleaves the glycyl bonds in hydrophobic regions and the peptide bonds Ala–Ala, Ala–Lys, and Lys–Ala involved in cross-linking. Two peptic derivatives of desmosine, desmosine-Ala-Ala and desmosine-Ala-Ala-Ala, were detected in the elastin hydrolysate, indicating that pseudoalterin can dissociate cross-linked elastin. These results reveal a new elastolytic mechanism of the M23 protease pseudoalterin, which is different from the reported mechanism where the M23 proteases only cleave glycyl bonds in elastin. Genome analysis suggests that M23 proteases may be popular in deep-sea sediments, implying their important role in elastin degradation. An elastin degradation model of pseudoalterin was proposed, based on these results and scanning electron microscopic analysis of the degradation by pseudoalterin of bovine elastin and cross-linked recombinant tropoelastin. Our results shed light on the mechanism of elastin degradation in deep-sea sediment.     

Termination of RNA by nucleotides of 9-beta-D-xylofuranosyladenine

The protease susceptibilities of recently identified cartilage collagens HMW, 1α, 2α, and 3α were investigated. Mammlian skin collagenase cleaved the 3α chain under conditions where HMW, 1α and 2α were not degraded. A tumor cell derived type V collagenolytic metalloproteinase degraded HMW, 1α and 2α, but not 3α. Plasmin or leucocyte elastase failed to significantly degrade any of the cartilage collagens when digestion was performed at 25°C (15 hours, enzyme to substrate ratio 1:100). At 36°C but not 33°C α thrombin degraded HMW, 1α and 2α, with little or no degradation of 3α. This pattern of protease susceptibility for HMW, 1α and 2α is therefore similar to type V collagen. The cleavage of 3α by skin collagenase but not by elastase is similar to type II collagen. These results suggest that HMW, 1α and 2α are part of the type V collagen family.     

Novel Use for the Osmolyte Trimethylamine N-oxide: Retaining the Psychrophilic Characters of Cold-Adapted Protease Deseasin MCP-01 and Simultaneously Improving its Thermostability

The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k _cat /K _m) by 290% at 0°C. In the presence of 1 M TMAO, the thermostability (t _1/2) of MCP-01 increased by two- to fivefold at 60∼40°C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40°C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T _m) of MCP-01 by 10.5°C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.     

Identification of a type V collagenolytic enzyme

A neutral metal protease has been identified which cleaves native type V collagen under conditions where pepsinized type IV collagen or the interstitial collagens are not significantly degraded. The enzyme is secreted into the media of cultured M50-76 reticulum cell sarcoma (malignant macrophages) and leiomyosarcoma tumor cells. Biosynthetically labeled type V collagen prepared from organ cultures of human amnion membrane is used for a routine assay of type V collagenolytic activity. The partially purified enzyme a) exists in a latent form requiring trypsin activation for maximum activity; b) has a molecular weight estimated by molecular sieve chromatography of approximately 80,000 daltons; c) is inhibited by EDTA but not phenylmethylsulfonyl fluoride; and d) produces specific cleavage products of both A and B collagen chains.     

Quantitative and metabolic changes of hepatic collagens in rats after carbon tetrachloride poisoning

1. The collagen hydroxyproline in rat liver was composed of 3.5% neutral-soluble collagen, 4.9% acid-soluble collagen and 91.6% insoluble collagen. In labelling studies with [(14)C]proline in vitro, the specific radioactivities of neutral-soluble, acid-soluble and insoluble collagens in rat liver were found to be 233000, 69000 and 830d.p.m./mumol of hydroxyproline respectively after 1h. 2. During subacute carbon tetrachloride poisoning the hepatic content of insoluble collagen markedly increased, whereas those of soluble collagens did not change. During recovery from subacute poisoning hepatic contents of soluble collagens were markedly decreased. 3. After 8 weeks of carbon tetrachloride poisoning the specific radioactivities of hepatic soluble collagens increased, while that of insoluble collagen decreased. During recovery from subacute poisoning, the specific radioactivities of soluble collagens decreased to the normal range and that of insoluble collagen further decreased. 4. Hepatic collagenolytic activity solubilizing insoluble collagen, which differs from mammalian collagenase, decreased under the conditions of the subacute poisoning and also during recovery from subacute poisoning.     

Collagen binding properties of the membrane type-1 matrix metalloproteinase (MT1-MMP) hemopexin C domain. The ectodomain of the 44-kDa autocatalytic product of MT1-MMP inhibits cell invasion by disrupting native type I collagen cleavage

Up-regulation of the collagenolytic membrane type-1 matrix metalloproteinase (MT1-MMP) leads to increased MMP2 (gelatinase A) activation and MT1-MMP autolysis. The autocatalytic degradation product is a cell surface 44-kDa fragment of MT1-MMP (Gly(285)-Val(582)) in which the ectodomain consists of only the linker, hemopexin C domain and the stalk segment found before the transmembrane sequence. In the collagenases, hemopexin C domain exosites bind native collagen, which is required for triple helicase activity during collagen cleavage. Here we investigated the collagen binding properties and the role of the hemopexin C domain of MT1-MMP and of the 44-kDa MT1-MMP ectodomain in collagenolysis. Recombinant proteins, MT1-LCD (Gly(285)-Cys(508)), consisting of the linker and the hemopexin C domain, and MT1-CD (Gly(315)-Cys(508)), which consists of the hemopexin C domain only, were found to bind native type I collagen but not gelatin. Functionally, MT1-LCD inhibited collagen-induced MMP2 activation in fibroblasts, suggesting that interactions between collagen and endogenous MT1-MMP directly stimulate the cellular activation of pro-MMP2. MT1-LCD, but not MT1-CD, also blocked the cleavage of native type I collagen by MT1-MMP in vitro, indicating an important role for the MT1-MMP linker region in triple helicase activity. Similarly, soluble MT1-LCD, but not MT1-CD or peptide analogs of the MT1-MMP linker, reduced the invasion of type I collagen matrices by MDA-MB-231 cells as did the expression of recombinant 44-kDa MT1-MMP on the cell surface. Together, these studies demonstrate that generation of the 44-kDa MT1-MMP autolysis product regulates collagenolytic activity and subsequent invasive potential, suggesting a novel feedback mechanism for the control of pericellular proteolysis.     

Characteristic features in the structure and collagen-binding ability of a thermophilic collagenolytic protease from the thermophile Geobacillus collagenovorans MO-1

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, extracellularly produces a collagenolytic protease with a large molecular mass. Complete nucleotide sequencing of this gene after gene cloning revealed that the collagenolytic protease is a member of the subtilisin family of serine proteases and consists of a signal sequence for secretion, a prosequence for maturation, a catalytic region, 14 direct repeats of 20 amino acids at the C terminus, and a region with unknown function intervening between the catalytic region and the numerous repeats. Since the unusual repeats are most likely to be cleaved in the secreted form of the enzyme, the intervening region was investigated to determine whether it participates in collagen binding to facilitate collagen degradation. It was found that the mature collagenolytic protease containing the intervening region at the C terminus bound collagen but not the other insoluble proteins, elastin and keratin. Furthermore, the intervening region fused with glutathione S-transferase showed a collagen-binding ability comparable to that of the mature collagenolytic protease. The collagen-binding ability was finally attributed to two-thirds of the intervening region which is rich in beta-strands and is approximately 35 kDa in molecular mass. In the collagenolytic protease from strain MO-1, hydrogen bonds most likely predominate over the hydrophobic interaction for collagen binding, since a higher concentration of NaCl released collagen from the enzyme surface but a nonionic detergent could not. To the best of our knowledge, this is the first report of a thermophilic collagenolytic protease containing the collagen-binding segment.     

Cleavage and oligomerization of gliomedin, a transmembrane collagen required for node of ranvier formation

Gliomedin, which has been implicated as a major player in genesis of the nodes of Ranvier, contains two collagenous domains and an olfactomedin-like domain and belongs to the group of type II transmembrane collagens that includes collagens XIII and XVII and ectodysplasin A. One characteristic of this protein family is that constituent proteins can exist in both transmembrane and soluble forms. Recently, gliomedin expressed at the tips of Schwann cell microvilli was found to bind axonal adhesion molecules neurofascin and NrCAM in interactions essential for Na(+)-channel clustering at the nodes of Ranvier in myelinating peripheral nerves. Interestingly, exogenously added olfactomedin domain was found to have the same effect as intact gliomedin. Here we analyze the tissue form of gliomedin and demonstrate that the molecule not only exists as full-length gliomedin but also as a soluble form shed from the cell surface in a furin-dependent manner. In addition, gliomedin can be further proteolytically processed by bone morphogenetic protein 1/Tolloid-like enzymes, resulting in release of the olfactomedin domain from the collagen domains. Interestingly, the later cleavage induces formation of higher order, insoluble molecular aggregates that may play important roles in Na(+)-channel clustering.     

A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1

A collagenolytic protease was purified to homogeneity from thermophilic Bacillus sp. strain MO-1. The protease from strain MO-1 showed high activity toward type I and IV collagens and gelatin. However, peptide substrates (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg and 2-furylacryloyl-Leu-Gly-Pro-Ala) for collagenases were inert as substrates. The collagenolytic protease cleaved oxidized insulin B-chain at 11 sites and degraded type I and IV collagens into anonymous small pieces, suggesting that the protease digests collagens at multiple sites. The collagenolytic protease was far more thermostable than a mesophilic Clostridium histolyticum collagenase. The collagenolytic protease possesses two salient features: (1) it has a very large molecular mass, 210 kDa, and consists of two, identical 105-kDa subunits; (2) it belongs to a serine protease group. The high molecular mass is unique among serine proteases but common for collagenases. The features of the enzyme from strain MO-1 suggest that it is a new collagenolytic protease which is distinct from previously reported collagenases and serine proteases.     

The importance of proline residues in the structure, stability and susceptibility to proteolytic degradation of collagens

Collagens are among proteins that undergo several post-translational modifications, such as prolyl hydroxylation, that occur during elongation of the nascent chains in the endoplasmic reticulum. The major structural collagens, types I, II and III, have large, uninterrupted triple helices, comprising three polyproline II-like chains supercoiled around a common axis. The structure has a requirement for glycine, as every third residue, and is stabilized by the high content of proline and 4-hydroxyproline residues. Action of prolyl hydroxylases is critical. Spontaneous or targeted genetic defects in prolyl hydroxylases can be lethal or result in severe osteogenesis imperfecta. Prolines, as determinants of substrate specificity and susceptibility, also play a role in degradation of collagen by collagenolytic matrix metalloproteinases (MMPs). Targeted mutations in mice in the collagenase cleavage domain have profound effects on collagen turnover and the function of connective tissues. Prolines are thus critical determinants of collagen structure and function.     

Interaction of proteoglycans with the pericellular (1 alpha, 2 alpha, 3 alpha) collagens of cartilage

Interaction between cartilage proteoglycan and the collagen(s) composed of 1 alpha, 2 alpha, and 3 alpha chains was studied in vitro. Most of the collagen was insoluble under the conditions of assay (0.15 M NaCl, 0.008 M phosphate buffer, pH 7.4; 4 degrees C) and was in the form of fibrils 20 nm in diameter or thinner. The larger fibrils had 60-70 nm periodicity, characteristic of native collagens. Proteoglycan monomers which had been labeled by incubating cartilage slices in vitro with Na2 35SO4 were used to assay the interaction. The insoluble collagen fraction bound proteoglycan from solution. At proteoglycan:collagen ratios lower than 1:2, binding was rapid and linear, and the dissociation constant was 1.7 X 10(-9) M. At higher proteoglycan:collagen ratios, more proteoglycan was bound, but at a slower rate. Binding of proteoglycan to collagen did not require fibrils, since soluble 1 alpha, 2 alpha, and 3 alpha containing collagen also bound to proteoglycan and formed an insoluble complex. Denatured collagens did not bind proteoglycan or compete for binding with normal collagen. Optimum binding occurred with intact proteoglycan, but proteoglycan which had been treated with protease was also bound at low levels. Both protease-treated proteoglycan and free chondroitin sulfate competed with intact proteoglycan in the binding assays, but neither chondroitinase ABC-treated proteoglycan nor the oligosaccharides produced by digestion of chondroitin sulfate with testicular hyaluronidase altered the binding of proteoglycan to collagen. Hyaluronic acid did not compete with radioactive proteoglycan, but heparin and dextran sulfate were extremely effective inhibitors of binding. These data suggest a relatively nonspecific interaction between sulfated polyanions and 1 alpha, 2 alpha, and 3 alpha containing collagens. However, given the location of these collagens near the chondrocyte surface, the interaction of fibrillar 1 alpha, 2 alpha, 3 alpha collagen with proteoglycan is likely to occur and to be of biological importance.     

Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and membrane type (MT)1-MMP (MMP-14) are cooperative dynamic components of a cell surface proteolytic axis involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Although MT1-MMP exhibits type I collagenolytic but poor gelatinolytic activities, MMP-2 is a potent gelatinase with weak type I collagenolytic behavior. Recombinant linker/hemopexin C domain (LCD) of MT1-MMP binds native type I collagen, blocks MT1-MMP collagenolytic activity in trans, and by circular dichroism spectroscopy, induces localized structural perturbation in the collagen. These changes were reflected by enhanced cleavage of the MT1-LCD-bound collagen by the collagenases MMP-1 and MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD alone can initiate triple helicase activity. In contrast, the native and denatured collagen binding properties of MMP-2 reside in the fibronectin type II modules, accordingly termed the collagen binding domain (CBD). Recombinant CBD (but not the MMP-2 LCD) also changed the circular dichroism spectra leading to increased MMP-1 and -8 cleavage of native collagen. However, recombinant CBD reduced gelatin and collagen cleavage by MMP-2 in trans as did CBD23, which comprises the second and third fibronectin type II modules, but not the CBD23 mutant W316A/W374A, which neither binds gelatin nor collagen. This indicates that MMP-2 and MT1-MMP bind collagen at a different site than MMP-1 and MMP-8. Thus, MMP-2 utilizes the CBD in cis for collagen binding and triple helicase activity, which compensates for the lack of collagen binding by the MMP-2 LCD. Hence, the MMP family has evolved two distinct mechanisms for collagen triple helicase activity using two structurally distinct domains, with triple helicase activity occurring independent of alpha-chain hydrolysis.     

Collagenolytic subtilisin-like protease from the deep-sea bacterium Alkalimonas collagenimarina AC40T

A new alkaline protease (AcpII) was purified from a culture of the deep-sea bacterium Alkalimonas collagenimarina AC40^T. AcpII degraded collagen three times faster than it degraded casein. The optimal pH was 8.5–9, and the optimal temperature was 45°C for the degradation of collagen. AcpII was completely inhibited by phenylmethylsulfonyl fluoride and partially by EDTA. Cloning and sequencing the gene for AcpII revealed a 2,283-bp open reading frame encoding a protein of 760 amino acids. AcpII comprises a prepropeptide, a catalytic domain that includes a protease-associated domain (PA domain), and tandem repeat prepeptidase C-terminal domains. To elucidate the role of the PA domain of AcpII, we constructed genes for two enzyme derivatives that possessed the catalytic domains with or without the PA domain and expressed them in Escherichia coli. The derivative without the PA domain showed increased specific activities toward all proteinaceous substrates tested, including gelatin, casein, and collagen, compared with those of the derivative with the PA domain.     

Substrate specificity of the collagenolytic serine protease from Uca pugilator: studies with collagenous substrates

The collagenolytic protease from Uca pugilator was studied with respect to its catalytic properties on collagen types I-V. The crab protease degraded all five collagen types, producing multiple cleavages in the triple helix of each native collagen at 25 degrees C. The major early cleavage in the alpha 1 polypeptide chain of collagen types I-III occurred at a 3/4:1/4 locus, resulting in fragments electrophoretically similar to the TCA and TCB products of mammalian collagenase action. Interestingly, a propensity toward this same cleavage was observed even following thermal denaturation of the substrates. The ability of the crab protease to degrade all native collagen types and to catalyze cleavages at multiple loci in the triple helix distinguishes its action from that of mammalian collagenases. The collagenolytic activity of the crab protease was also examined on fibrillar collagen and compared to that of human skin fibroblast collagenase. Enzyme concentrations of fibroblast collagenase which resulted in the saturation of available substrate sites failed to show such an effect in the case of the crab protease. Binding studies of the crab protease to fibrillar collagen likewise indicated substantially reduced levels of enzyme binding in comparison to fibroblast collagenase. These data suggest that the affinity of the crab protease for native collagen is considerably less than the affinity of mammalian collagenase for this substrate.     

Collagenolytic Activity of Some Marine Bacteria

Reconstituted, acid-extracted collagen was used to prepare a medium to screen proteolytic marine bacteria for their ability to elaborate collagenolytic enzymes. The medium was resistant to solubilization by trypsin, hyaluronidase, chondroitinase ABC, and various marine proteinases, but was readily hydrolyzed by commercial Clostridium collagenases. Eighty-seven marine isolates collected in the vicinity of Bermuda, Oahu (Hawaii), and Stone Harbor and Cape May, N. J., were screened. Approximately 44 per cent of the isolates were capable of elaborating enzymes that hydrolyzed reconstituted collagen gels. Several cultures produced collagenolytic enzymes only when grown in the presence of collagen or degradation products of collagen, and with very few exceptions the presence of collagen in the medium greatly enhanced collagenolytic enzyme production. The enzymes from a collagenolytic Bermuda marine isolate were studied in more detail to illustrate that the enzymes capable of hydrolyzing reconstituted collagen were separable from nonspecific proteinases by zone electrophoresis and that these enzymes were true collagenases by virtue of their ability to hydrolyze native bovine Achilles'tendon obtained from three different sources.     

Effects of different buffers on the thermostability and autolysis of a cold-adapted protease MCP-01

A cold-adapted protease MCP-01 was obtained from deep-sea psychrotrophic bacterium Pseudoaltermonas sp. SM9913. The effects of four different buffers, all at 50 mmol/l concentration, on its thermostability and autolysis were studied. The autolysis process of MCP-01 was studied by capillary electrophoresis. The thermostability of MCP-01 increased successively in the following order: carbonate < Tris < phosphate < borate. The optimum temperature for casein hydrolysis also increased in the same order. This suggested that the conformation of MCP-01 was flexible and its autolytic susceptibility was affected by some factors in the buffers such as charge and ionic species. The results also showed that different buffers, in addition to affecting the autolysis speed, gave different patterns of autolysis products. In carbonate buffer, Tris buffer, phosphate buffer and borate buffer, the autolysis patterns of MCP-01 were different. These results suggested that protease MCP-01 probably have different conformations in different buffers, thus exposing different autolysis sites on the enzyme surface. In addition, the loss of activity correlated with the speed of autolysis in the four different buffers, showing that autolysis may be a reason for the low thermostability of the enzyme.