Extracellular complex of chitinolytic enzymes of <Emphasis Type="Italic">Clostridium paraputrificum</Emphasis> strain J4 separated by membrane ultrafiltration

Membrane diafiltration was used for separation of the extracellular complex of chitinolytic enzymes of C. paraputrificum J4 free from contaminants with molar mass higher than 100 kDa and lower than 30 kDa. The enzyme complex containing β-N-acetylglucosaminidase (NAGase) and six endochitinases was concentrated on a membrane with cut-off 30 kDa. In this retentate, the NAGase/endochitinase specific activity was 13.5/6.5-times higher than in the initial culture filtrate. The proportion (in %) of endochitinases: 23 (90 kDa), 42 (86 kDa), 8 (72 kDa), 16 (68 kDa) and 8 (60 kDa) was calculated from their peak areas (determined by densitometry) in images of zymograms. NAGase (38 kDa) was less active and stable at pH lower than 4 and higher than 8 but it was more temperature-stable than endochitinases, especially at 40–60 °C. In contrast to endochitinases, the pH optimum of NAGase activity was shifted by ca. 0.7 pH units to the alkaline region. Extracellular NAGase together with six endochitinases secreted by C. paraputrificum J4 were separated by membrane diafiltration and characterized by molar mass, stability and activity in dependence on pH and temperature. The knowledge of composition of chitinolytic enzymes, their pH and temperature stability is useful for optimization of the separation process.     

Chitinolytic Enterobacter agglomerans Antagonistic to Fungal Plant Pathogens

Three Enterobacter agglomerans strains which produce and excrete proteins with chitinolytic activity were found while screening soil-borne bacteria antagonistic to fungal plant pathogens. The chitinolytic activity was induced when the strains were grown in the presence of colloidal chitin as the sole carbon source. It was quantitated by using assays with chromogenic p-nitrophenyl analogs of disaccharide, trisaccharide, and tetrasaccharide derivatives of N-acetylglucosamine. A set of three fluorescent substrates with a 4-methylumbelliferyl group linked by (beta)-1,4 linkage to N-acetylglucosamine mono- or oligosaccharides were used to identify the chitinolytic activities of proteins which had been renatured following their separation by electrophoresis. This study provides the most complete evidence for the presence of a complex of chitinolytic enzymes in Enterobacter strains. Four enzymes were detected: two N-acetyl-(beta)-d-glucosaminidases of 89 and 67 kDa, an endochitinase with an apparent molecular mass of 59 kDa, and a chitobiosidase of 50 kDa. The biocontrol ability of the chitinolytic strains was demonstrated under greenhouse conditions. The bacteria decreased the incidence of disease caused by Rhizoctonia solani in cotton by 64 to 86%. Two Tn5 mutants of one of the isolates, which were deficient in chitinolytic activity, were unable to protect plants against the disease.     

Actinobacterial chitinase-like enzymes: profiles of rhizosphere versus non-rhizosphere isolates

The objective of this study was to determine if antifungal actinomycetes isolated from rhizosphere and non-rhizosphere soils exhibit different chitinase-like production and (or) induction patterns. Selected isolates from both habitats were compared. Chitinase-like levels and isoform characteristic patterns were evaluated over time in culture fluids of isolates grown on media containing different combinations of colloidal chitin and fungal cell wall (FCW) preparation. Supernatants were also subjected to native and non-native polyacrylamide gel electrophoresis (PAGE), using glycol chitin amended gels. For non-native PAGE, protein samples were denatured by two different approaches. Multiple active bands, ranging from 20 to 53 kDa and present in varying amounts, were detected in gels for most strains. Different substrate preferences were observed among strains, and different chitinase-like enzymes were produced, depending upon the substrate combinations used. The presence of FCW in the medium induced specific chitinase-like enzymes not observed otherwise. Enzymatic activities and profiles of the isolates, however, were strain and substrate specific rather than habitat specific. However, a sagebrush rhizosphere soil had a larger actinomycete community with higher chitinolytic activities than the nearby bulk soil. The use of PAGE to compare chitinase-like proteins induced in media with and without FCW was useful for identifying chitinase-like enzymes potentially involved in antifungal activity.     

Chitinolytic enzymes from bacterium inhabiting human gastrointestinal tract -- critical parameters of protein isolation from anaerobic culture

The object of this study are chitinolytic enzymes produced by bacterium Clostridium paraputrificum J4 isolated from the gastrointestinal tract of a healthy human. In particular, we focus on the development of purification protocols, determination of properties of the enzymes and their activity profiles. The process of bacteria cultivation and isolation of chitinolytic complex of enzymes showing specific activities of endo-, exo-chitinase and N-acetyl-β-glucosaminidase was optimized. A range of various purification procedures were used such as ultrafiltration, precipitation, chromatographic separations (ion-exchange, size exclusion, chromatofocusing) in altered combinations. The optimal purification protocol comprises two or three steps. Individual samples were analyzed by SDS/PAGE electrophoresis and after renaturation their activity could be detected using zymograms. Mass spectroscopy peptide fragment analysis and MALDI analysis of the purest samples indicate presence of endochitinase B (molecular mass about 85 kDa) and of 60-kDa endo- and exochitinases.     

Synthesis of Extracellular Chitinase by Wild-Type B-10 and Mutant M-1 Strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, whereas M-1 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused oversynthesis of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the synthesis of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Microbial reclamation of shellfish wastes for the production of chitinases

Shrimp and crab shell powder (SCSP), prepared by treating shellfish processing waste with boiling and crushing, was used as a substrate for isolating chitinolytic microorganisms. Three potential strains (E1, J1, and J1-1) were isolated and identified as Bacillus cereus, B. alvei, and B. sphaericus, respectively. Three extracellular chitinases (FB1, FB2, and FB3) were purified from the culture supernatants of Bacillus cereus E1, B. alvei J1, and B. sphaericus J1-1, respectively. The molecular weights of FB1, FB2, and FB3 were 71,000, 71,000, and 65,000, respectively, by SDS-PAGE. The pIs for FB1, FB2, and FB3 were 7.1, 7.2, and 7.4, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FB1 were pH 9, 50 degrees C, pH 7 to 10, and 70 degrees C; those of FB2 were pH 9, 60 degrees C, pH 5 to 9, and 70 degrees C; and those of FB3 were pH 7, 50 degrees C, pH 5 to 9, and 60 degrees C. The activities of all enzymes were strongly inhibited by Hg(2+) and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol.     

Extracellular chitinase production by wild-type B-10 and mutant M-1 strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, while M-10 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused overproduction of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the production of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Genomic analysis and initial characterization of the chitinolytic system of Microbulbifer degradans strain 2-40

The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP). The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40. The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in N-acetyl-D-glucosamine (GlcNAc) metabolism. Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin. The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside (MUF-triNAG). The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains. ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues. In addition, ChiB and CbpA contained glutamic acid-rich domains. At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date. A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified. The activities of two apparent periplasmic (HexA and HexB) N-acetyl-beta-D-glucosaminidases and one cytoplasmic (HexC) N-acetyl-beta-D-glucosaminidase were demonstrated. Genes involved in GlcNAc metabolism, similar to those of the Escherichia coli K-12 NAG utilization operon, were identified. NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a nagA mutation in E. coli K-12. Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome. Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40.     

Interactions of bacteria with different mechanisms for chitin degradation result in the formation of a mixed-species biofilm

In this study, interactions between bacteria possessing either released or cell-associated enzymes for polymer degradation were investigated. For this, a co-culture of Aeromonas hydrophila strain AH-1N as an enzyme-releasing bacterium and of Flavobacterium sp. strain 4D9 as a bacterium with cell-associated enzymes was set up with chitin embedded into agarose beads to account for natural conditions, under which polymers are usually embedded in organic aggregates. In single cultures, strain AH-1N grew with embedded chitin, while strain 4D9 did not. In co-cultures, strain 4D9 grew and outcompeted strain AH-1N in the biofilm fraction. Experiments with cell-free culture supernatants containing the chitinolytic enzymes of strain AH-1N revealed that growth of strain 4D9 in the co-culture was based on intercepting N-acetylglucosamine from chitin degradation. For this, strain 4D9 had to actively integrate into the biofilm of strain AH-1N. This study shows that bacteria using different chitin degradation mechanisms can coexist by formation of a mixed-species biofilm.     

Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus chitonophagus</Emphasis>

Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80°C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.     

Excretome of the chitinolytic bacterium <Emphasis Type="Italic">Clostridium paraputrificum</Emphasis> J4

A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-d-glucosaminide, N,N′-diacetyl-β-d-chitobiose, or N,N′,N˝-triacetyl-β-d-chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.     

Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae

A novel chitinase was detected in extracellular culture fluids of the entomopathogenic fungus Metarhizium anisopliae (ATCC 20500) grown in liquid medium containing chitin as a sole carbon source. A chitinase was purified to near homogeneity from culture broth of M. anisopliae by DEAE-Sephacel, CM-Sepharose CL-6B ion-exchange chromatography, and gel filtration with Superose 12HR. The molecular mass of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 60 kDa and the optimum pH of the enzyme was 5.0. This molecular mass is different from values of 33, 43.5, and 45 kDa for endochitinases and 110 kDa for an exochitinase (N-acetylglucosaminidase) from M. anisopliae ME-1 published previously. In addition, N-terminal sequences of 60-kDa chitinase are different from those of 43.4- and 45-kDa endochitinases. The purified enzyme showed high chitinolytic activity against colloidal, crystalline chitin of crab shells as well as against p-nitrophenyl-beta-d-N-acetylglucosamide, p-nitrophenyl-beta-d-N, N'-diacetylchitobiose, and p-nitrophenyl-N, N'-N"-triacetylchitotriose, indicating that this enzyme has both endo- and exochitinase activity.     

Partial characterization of chitinolytic enzymes from Streptomyces albidoflavus

Streptomyces albidoflavus NRRL B-16746 secreted three types of chitinolytic enzymes: N -acetyl-glucosaminidase, chitobiosidase and endochitinase. Optimal activity for all three types of enzymes occurred at pH 4–6; however 55–74% of the chitobiosidase and endochitinase activity was detectable at pH 8–10. Chitobiosidase activity originated from two strongly acidic (pI < 3.0) proteins with molecular mass of 27 kDa and 34 kDa, while endochitinase activity originated from five major acidic proteins (pI 5.1, 5.3, 5.75, 5.8–5.9 and 6.4) with molecular mass of 59, 45, 38.5, 27 and 25.5 kDa. Purified chitobiosidases significantly reduced spore germination and germ tube elongation of Botrytis cinerea and Fusarium oxysporum. Chitinolytic enzymes with significant activity at pH 4–10 may be used, transgenically, to reduce the growth and/or development of a broad spectrum of insects and fungi that are major economic pests.     

Extractionless method for the determination of urinary caffeine metabolites using high-performance liquid chromatography coupled with tandem mass spectrometry

Caffeine is metabolised in humans primarily by cytochromes P450 1A2 and 2A6, xanthine dehydrogenase/oxidase, and N-acetyltransferase 2. The activities of these enzymes show a large variation due to genetic polymorphisms and/or induction by xenobiotics. Ratios of different caffeine metabolites in urine or other body fluids are frequently used to characterise the individual/actual activity of these enzymes. The common analytical method involves extensive sample preparation, followed by HPLC-UV. The presence of numerous other UV-absorbing chemicals in body fluids affects the sensitivity and selectivity of this method. We have developed an HPLC-electrospray-MS-MS method for the determination of 11 caffeine metabolites and two internal standards after a simple, extractionless preparation. Blank urine, obtained after 5 days on a methylxanthine-free diet, contained small amounts of some caffeine metabolites, but no other components producing any confounding signals. Eleven metabolites and internal standards were recovered at 90 to 110% after addition to the blank urine (0.1 to 2.5 micro M in the final sample involving a 20-fold dilution of urine) in the 0.1-2.5 micro M concentration range. Other metabolites, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU), were detected with similar recovery and precision, but required higher concentrations (3 to 30 micro M). AFMU was completely converted into AAMU by a short alkalisation of urine. The method was explored in six healthy individuals after consuming coffee (4 mg caffeine per kg body mass). These experiments demonstrated the simplicity, high sensitivity and selectivity of the method under conditions used for phenotyping.     

An improved LC-MS/MS method for the quantification of prostaglandins E(2) and D(2) production in biological fluids

We report an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that accurately measures prostaglandins D(2) (PGD(2)) and E(2) (PGE(2)) in cell culture supernatants and other biological fluids. The limit of detection for each prostaglandin was 20 pg/ml (0.20 pg, 0.55 fmol on-column), and the interday and intraday coefficients of variation were less than 5%. Both d(4)-PGE(2) and d(4)-PGD(2) were used as surrogate standards to control for differential loss and degradation of the analytes. Stability studies indicated that sample preparation time should be less than 8h to measure PGD(2) accurately, whereas preparation time did not affect PGE(2) measurement due to its greater stability in biological samples. As an application of the method, PGD(2) and PGE(2) were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE(2) but no PGD(2), whereas the murine macrophage cell line RAW 264.7 produced PGD(2) and only trace amounts of PGE(2). This direct comparison showed that COX-2 gene expression can lead to differential production of PGD(2) and PGE(2) by epithelial cells and macrophages. Because PGE(2) is antiasthmatic and PGD(2) is proasthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macrophages in the lung contributes to the pathogenesis of chronic obstructive pulmonary disease (COPD), bronchiectasis, asthma, and lung cancer.     

A soluble form of the interleukin 4 receptor in biological fluids

Murine biological fluids and murine cell culture supernatants were analyzed for the presence of soluble murine interleukin 4 receptor (sIL4R) with the use of two monoclonal antibodies directed against the receptor. Mouse urine, serum, ascitic fluid, and cell culture supernatants contained varying levels of immunoreactive protein. All of the immunoreactive protein possessed interleukin 4 (IL 4) binding activity. Following partial purification of ascitic fluid a protein was isolated that binds IL 4 with high affinity. This data is consistent with the fact that murine biological fluids contain a soluble version of the murine IL 4 receptor that arises via secretion of the soluble receptor and/or via shedding of the extracellular portion of the full-length receptor from the cell surface.     

Enzymatic depolymerization of N-succinylchitosan

A complex of chitinolytic enzymes of Streptomyces kurssanovii and also lysozyme and Celloviridin, an industrial cellulase preparation, were demonstrated to provide for an enzymatic hydrolysis of N-succinylchitosan. Our studies were carried out on a high-molecular N-succinylchitosan with M of 390 kDa and a substitution degree of 0.8 as a substrate. All the enzymatic preparations were shown to be suitable for the obtaining of low-molecular derivatives of N-succinylchitosan. The complex of enzymes from S. kurssanovii showed the greatest activity: they reduced the characteristic viscosity of initial solution of the substrate by 78% for 30 min. A biodegradation of N-succinylchitosan of various molecular masses was shown to proceed under the action of lysozyme, and the cleavage reaction was revealed to decelerate at a decrease in the polymer molecular mass. A use of N-succinylchitosan in a complex with drugs for a prolongation of their action in a live organism was presumed.     

Generic method for quantification of FLAG-tagged fusion proteins by a real time biosensor

Availability of rapid quantitative protein-expression analysis is often the bottleneck in high throughput screening applications. A real time biosensor was employed to establish a quantitative assay for FLAG fusion proteins using FLAG-tagged bacterial alkaline phosphatase as standard. A range of FLAG-tagged bacterial alkaline phosphatase concentrations were injected over the anti-FLAG M2 antibody surface of the biosensor and used as standards to determine the concentration of different FLAG-tagged proteins with a molecular mass of 18.1 kDa respectively 49.3 kDa from yeast culture supernatants. The M2 immobilized chip was found to retain binding capacity following regeneration for at least 120 cycles. This real time biosensor method allows the quantitation of proteins from culture supernatants using a calibration curve obtained with a different protein. Further benefits include the short assay time of approximately 5 min, the small amount of sample required (35 microl per injection) and the ability to monitor the binding event in real time.     

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.