Identification and characterisation of a novel heptapeptide mackerel by-product hydrolysate, and its potential as a functional fertiliser component.

Functional fertilisers for hydroponics are in great demand. Herein, we isolated peptides from mackerel by-products, a valuable source of bioactive peptides. The pellet-phase fraction obtained after cold-acetone extraction exhibited plant growth-promoting activity in wheat hydroponics, and the presumed peptides were determined to be ≤ 1 kDa based on molecular weight cut-off and tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Size exclusion chromatography and matrix-assisted laser desorption ionisation time of flight mass spectrometry analysis were employed for peptide purification and identification. Finally, two peptides were identified, both with linear structures, consisting of amino acid sequences TCGGQGR and KEAGAFIDR. At 1 mg/mL, the heptapeptide performed better than the nonapeptide in terms of wheat growth and health, but neither peptide exhibited antimicrobial activity. Only the heptapeptide displayed significant antioxidant activity, and this activity bioaccumulated in wheat leaves after 7 days of hydroponic growth. The heptapeptide did not match any known metabolites in PepBank, BIOPEP, UniProt or METLIN databases, and is therefore a novel peptide with potential as a functional fertiliser component.

Recovery of antioxidant and antimicrobial peptides through the reutilization of Nile perch wastewater by biodegradation using two Bacillus species.

Nile perch wastewater was biodegraded using two Bacillus species to recover bioactive substances to enhance its reutilization value. The two Bacillus species successfully produced low-molecular-weight substances with a 47.8% degree of hydrolysis. The antioxidant activities of the Nile perch wastewater increased as the biodegradation proceeded, and the culture supernatant exhibited the highest DPPH (80.1%), ABTS (93.1%) and Fe2+ chelating (88.5%) antioxidant activities at 60 h. The antioxidant potential of the biodegraded Nile perch wastewater was found to be higher than those of other fish hydrolysates. Moreover, the biodegraded Nile perch wastewater exhibited effective antimicrobial activity against Vibrio vulnificus, exhibiting a minimal inhibitory concentration of 585 µg mL-1. Two-dimensional thin layer chromatography analysis revealed the specific amino acids responsible for the antioxidant activity, and molecular-weight cut-off ultrafiltration revealed that the <2-kDa fraction exhibited the highest antioxidant activity with the lowest IC50 values (0.43 and 0.22 mg mL-1 for DPPH and ABTS antioxidant activities, respectively). This is the first report of the reutilization of Nile perch wastewater as a natural antioxidant and antimicrobial ingredient for nutraceuticals.

Biodegraded mackerel wastewater selectively inhibits harmful algal blooms.

Functional substances from mackerel wastewater were biodegraded and tested for inhibitory activity against harmful algal blooms (HABs) that are detrimental to aquaculture. The supernatant from a 48 h culture of mackerel wastewater had a half-maximal inhibitory concentration of 0.54-0.68 mg/ml for the three tested HAB organisms (Heterocapsa triquetra, Alexandrium fundyense and Prorocentrum minimum). This inhibitory effect was not observed form 48-h biodegraded nutrient broth culture supernatant, indicating inhibitory substances did not originate from bacterial metabolites. Rough estimation of molecular weight using ultrafiltration indicated that the inhibitory substance was less than 2 kDa, and it did not inhibit the non-HAB organism (Skeletonema costatum), which is essential for preserving marine ecosystems in real-world applications. Furthermore, this inhibitory effect against HABs was not observed for biodegraded culture supernatants of other protein sources (skim milk and okara), indicating that the inhibitory substances were derived from a particular source present only in raw mackerel wastewater. To the best of our knowledge, this is the first report describing the selective inhibitory effects of the culture supernatant of mackerel wastewater against HABs.

Isolation and identification of a novel algicidal peptide from mackerel muscle hydrolysate.

To help remedy damage from harmful algal blooms, an attempt was made to isolate an algicidal substance previously observed to be present in mackerel muscle hydrolysate. Crude extract was obtained by cold acetone precipitation, and it dissolved best in water. Through molecular weight cut-off determination and tricine-SDS PAGE, the algicidal substance was determined to be a peptide of <1 kDa. Based on this result, purification was first performed using size exclusion chromatography and preparative reverse phase high-performance liquid chromatography. Then, the active algicidal fraction was applied to an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry system, followed by MS/MS analysis. The algicidal peptide had linear structure consisting of amino acids with sequence NH-KMNF-COOH. Its calculated properties were: molecular weight 538.66 g/mol; isoelectric point 9.91; net charge +1 at pH 7.0; and 50% hydrophobicity. Algicidal ability of the identified peptide was confirmed using synthesized peptide. The LC50 values toward four harmful algal blooming species were 0.69, 0.83, 0.85 and 1.24 mg/ml for Alexandrium fundyense, A. catenella, Heterocapsa triquetra, and Prorocentrum minimum, respectively. There was no coincidence in the sequence of the identified peptide with those of known metabolites in the APD, Norine, CAMP, UniProt and METLIN databases. Consequently, this algicidal substance originating from mackerel protein was deduced to be a novel peptide that can usefully be applied to relieve harmful algal blooms.

Analysis of proinflammatory gene expression by RBIV infection in rock bream, Oplegnathus faciatus.

Early induction of proinflammatory cytokines is known to regulate the later immune responses to inhibit the progress of infectious diseases. In this study, proinflammatory cytokine gene expression has been studied in immune tissues to understand the early immune response induced by megalocytivirus in rock bream (Oplegnathus faciatus). For this, we have cloned interleukin (IL)-1ß and IL-8 gene and performed the phylogenetic and structural analysis. Also the constitutive gene expressions of IL-1ß and IL-8 were assessed in 12 organs and found to be the highest expression in tail fin and liver, respectively. The expressions of proinflammatory cytokine genes including IL-1ß, IL-8, TNFα and Cox-2, and antiviral genes like Mx and IFN1 were analysed by stimulation with PAMPs and RBIV infection. In vitro study showed the highly up-regulated proinflammatory gene expressions in head kidney and the moderate up-regulation in spleen by LPS. Same concentration of polyI:C moderately upregulated IL-1ß gene expression in head kidney but down-regulated IL-8 and TNFα gene expression in head kidney and spleen at 8 h. Mx and IFN1 gene expressions were highly upregulated by polyI:C in head kidney and spleen cells in vitro. By RBIV infection, proinflammatory gene expressions were initially up-regulated and later down-regulated in head kidney. In spleen, although mostly not significant, proinflammatory cytokine gene expressions were down-regulated by RBIV infection except up-regulation of Cox-2 gene expression by low concentration of RBIV at 24 h. Mx and IFN1 gene expressions were down-regulated by high dose of RBIV infection in vitro. In vivo study revealed that IL-8, TNFα, and IFN1 gene expressions were down-regulated in brain, head kidney, spleen, and gill while up-regulated in heart and liver, indicating differential proinflammatory and antiviral responses in the organs. It is supposed that down-regulation of proinflammatory gene expression in the immune organs may result in the failure of antiviral immune responses, causing high mortalities by megalocytivirus infection in rock bream.

The high reutilization value potential of high-salinity anchovy fishmeal wastewater through microbial degradation.

To provide an option for the reutilization of high-salinity anchovy fishmeal wastewater (FMW), generated during the anchovy fishmeal manufacturing processes, its potential for biodegradation was assessed in 1-l five-neck flasks using a halotolerant and proteolytic microbial consortium. During the first 41 h of biodegradation, the pH, DO, ORP, and dry-sludge weight decreased as the total cell number of the microbial consortium increased steadily; the COD(Cr)/TN ratios remained between 4.0 and 5.5, respectively, indicating the stable metabolic degradation of organic matter. The ORP tended to increase after 41 h, and the unpleasant fishy smell disappeared once positive ORP values were achieved. The removal percentages of COD(Cr) and TN were 59.0 and 54.4%, respectively, and the dry-sludge weight decreased from 115.5 to 68.0 g, with a degradation rate of 0.59 g h(-1), during the 80 h experiment. The supernatant from the culture of the anchovy FMW at 70 h (culture supernatant) was phytotoxin-free, and the level of total amino acids was 8.04 g 100 g(-1), comparable to that of commercial fertilizers. In hydroponic cultures containing red bean and barley, the culture supernatant demonstrated a good fertilizing ability. The culture supernatant also exhibited a high degree of antioxidant activity, with a 52.3% hydroxyl radical-scavenging activity and 0.16 reducing power (at OD 700 nm). Moreover, the culture supernatant inhibited DNA damage from hydroxyl radicals, enhancing the reutilization value of anchovy FMW. This report presents the first description of high-salinity anchovy FMW possessing a high reutilization value potential both for agriculture and medicine.

Characterization of salt-tolerant ß-glucosidase with increased thermostability under high salinity conditions from Bacillus sp. SJ-10 isolated from jeotgal, a traditional Korean fermented seafood.

The ß-glucosidase gene, bglC, was cloned from Bacillus sp. SJ-10 isolated from the squid jeotgal. Recombinant BglC protein overexpression was induced in Escherichia coli. The optimal pH and temperature of the enzyme, using p-nitrophenyl-ß-D-glucopyranoside (pNPßGlc) as a substrate, were pH 6 and 40 °C, respectively. Enzymatic activity increased by 3.3- and 3.5-fold in the presence of 15% NaCl and KCl, respectively. Furthermore, enzyme thermostability improved in the presence of NaCl or KCl. At 45 °C in the presence of salts, the enzyme was stable for 2 h and maintained 80% activity. In the absence of salts, BglC completely lost activity after 110 min at 45 °C. Comparison of the kinetic parameters at various salt concentrations revealed that BglC had approximately 1.5- and 1.2-fold higher affinity and hydrolyzed pNPßGlc 1.9- and 2.1-fold faster in the presence of 15% NaCl and KCl, respectively. Additionally, the Gibb's free energy for denaturation was higher in the presence of 15% salt than in the absence of salt at 45 and 50 °C. Since enzymatic activity and thermostability were enhanced under high salinity conditions, BglC is an ideal salt-tolerant enzyme for further research and industrial applications.

Reuse of red seaweed waste by a novel bacterium, Bacillus sp. SYR4 isolated from a sandbar.

A potent bacterial strain was isolated from a sandbar and identified as Bacillus sp. SYR4 for the reuse of red seaweed waste. The isolate possessed both agarase and carrageenase activities. The optimal pH and temperature for the degradation of both agar and carrageenan by the isolate were found to be pH 7.5 and 30 °C, respectively. The effects of cations on cell growth and degradation ability of the isolate were significant in comparison with controls. The isolate produced 0.27 and 0.29 g l(-1) of reducing sugars from 1 g l(-1) of agar and carrageenan, respectively. When the isolate was cultivated in red seaweed powder medium for 10 days, the yield of reducing sugars was 24 %. As a result, the eco-friendly reuse of red seaweed waste by this isolate appears to be feasible for the production of reducing sugars and could be a valuable resource. To the best of our knowledge, this is the first study to directly demonstrate the ability of Bacillus sp. SYR4 to degrade both agar and carrageenan.

Conversion of red-algae Gracilaria verrucosa to sugars, levulinic acid and 5-hydroxymethylfurfural.

This study employed a statistical methodology to investigate the optimization of conversion conditions and evaluate the reciprocal interaction of reaction factors related to the process of red-algae Gracilaria verrucosa conversion to sugars (glucose, galactose), levulinic acid and 5-hydroxymethylfurfural (5-HMF) by acidic hydrolysis. Overall, the conditions optimized for glucose formation included a higher catalyst concentration than did those for galactose, and these conditions for galactose were similar to those for 5-HMF. Levulinic acid production, meanwhile, was optimized at a higher reaction temperature, a higher catalyst concentration, and a longer reaction time than was glucose, galactose or 5-HMF production. By this approach, the optimal yields (and reaction conditions) for glucose, galactose, levulinic acid, and 5-HMF were as follows: glucose 5.29 g/L (8.46 wt%) (reaction temperature 160 °C, catalyst concentration 1.92%, reaction time 20 min), galactose 18.38 g/L (29.4 wt%) (160 °C, 1.03%, 20 min), levulinic acid 14.65 g/L (18.64 wt%) (180.9 °C, 2.85%, 50 min), and 5-HMF 3.74 g/L (5.98 wt%) (160.5 °C, 1%, 20 min).

High antioxidant and DNA protection activities of N-acetylglucosamine (GlcNAc) and chitobiose produced by exolytic chitinase from Bacillus cereus EW5.

Chitin-degrading bacterial strains were screened and tested for their ability to degrade shrimp-shell waste (SSW). Among the potential strains, B. cereus EW5 exhibited the highest chitin-degrading ability compared with other strains and produced 24 mg of reducing sugar per gram of dry SSW after 4 days of incubation. A TLC analysis of SSW biodegradation revealed that the chitosaccharides produced in the culture supernatant were mainly N-acetylglucosamine (GlcNAc) and chitobiose due to the isolate's exolytic chitinase activity. The culture supernatant exhibited a high degree of antioxidant activity, as indicated by 83% DPPH, 99.6% ABTS, 51% hydroxyl radical scavenging activity and 0.34 reducing power. The formation of GlcNAc and chitobiose during biodegradation of SSW is considered to be the major contributor to the antioxidant activity. The EW5 culture supernatant also displayed inhibition of DNA damage, enhancing the reutilization value of SSW. This report presents the first description of fermented production of GlcNAc and DNA protective activity of culture supernatant from SSW by B. cereus.

Recovery of algal oil from marine green macro-algae Enteromorpha intestinalis by acidic-hydrothermal process.

In this study, the recovery of algal oil from Enteromorpha intestinalis based on an acidic-hydrothermal reaction was investigated. Overall, the algal oil yield after the acidic-hydrothermal reaction was increased under the conditions of high reaction temperature, high catalyst concentration, and long reaction time within the tested ranges. Significantly, catalyst concentration, compared with reaction temperature and time, less affected algal oil recovery. The optimal acidic-hydrothermal reaction conditions for production of algal oil from E. intestinalis were as follows-200 °C reaction temperature, 2.92 % catalyst concentration, 54 min reaction time. Under these conditions, an 18.6 % algal oil yield was obtained. By increasing the combined severity factor, the algae oil recovery yield linearly increased.

Microbacterium oxydans, a novel alginate- and laminarin-degrading bacterium for the reutilization of brown-seaweed waste.

There is a growing demand for the efficient treatment of seaweed waste. We identified six bacterial strains from the marine environment for the reutilization of brown-seaweed waste, and the most potentially useful strain, Microbacterium oxydans, was chosen and further investigated. Plate assays indicated that this bacterial isolate possessed both alginate lyase and laminarinase activities. The optimal inoculum size, pH, temperature and substrate concentration for the degradation of brown-seaweed polysaccharides by the isolate were as follows: 20% (v v(-1)), pH 6.0, 37 °C, and 5 g L(-1) for alginate and 20% (v v(-1)), pH 6.0, 30 °C, and 10 g L(-1) for laminarin, respectively. During 6 d in culture under the optimal conditions, the isolate produced 0.17 g L(-1) of reducing sugars from alginate with 11.0 U mL(-1) of maximal alginate lyase activity, and 5.11 and 2.88 g L(-1) of reducing sugars and glucose from laminarin, respectively. In particular, a fair amount of laminarin was degraded to glucose (28.8%) due to the isolate's exolytic laminarinase activity. As a result, the reutilization of brown-seaweed waste by this isolate appears to be possible for the production of reducing sugars as a valuable resource. This is the first study to directly demonstrate the ability of M. oxydans to degrade both alginate and laminarin.

Characterisation of a novel Bacillus sp. SJ-10 ß-1,3-1,4-glucanase isolated from jeotgal, a traditional Korean fermented fish.

A novel ß-1,3-1,4-glucanase gene was identified in Bacillus sp. SJ-10 (KCCM 90078) isolated from jeotgal, a traditional Korean fermented fish. We analysed the ß-1,3-1,4-glucanase gene sequence and examined the recombinant enzyme. The open reading frame of the gene encoded 244 amino acids. The sequence was not identical to any ß-glucanases deposited in GenBank. The gene was cloned into pET22b(+) and expressed in Escherichia coli BL21. Purification of recombinant ß-1,3-1,4-glucanase was conducted by affinity chromatography using a Ni-NTA column. Enzyme specificity of ß-1,3-1,4-glucanase was confirmed based on substrate specificity. The optimal temperature and pH of the purified enzyme towards barley ß-glucan were 50 °C and pH 6, respectively. More than 80 % of activity was retained at temperatures of 30-70 °C and pH values of 4-9, which differed from all other bacterial ß-1,3-1,4-glucanases. The degradation products of barley ß-glucan by ß-1,3-1,4-glucanase were analysed using thin-layer chromatography, and ultimately glucose was produced by treatment with cellobiase.

Scaled-up bioconversion of fish waste to liquid fertilizer using a 5 L ribbon-type reactor.

A scaled-up conversion process of fish waste to liquid fertilizer was performed in a 5 L ribbon-type reactor. Biodegradation was performed by inoculation of autoclaved fish waste with 5.84 × 10(5) CFU mL(-1) of mixed microorganisms for 96 h. As a result, the pH changed from 6.92 to 5.72, the cell number reached 7.28 × 10(5) CFU mL(-1), and approximately 430 g (28.3%) of fish waste was degraded. Analyses indicated that the 96 h culture of inoculated fish waste possessed comparable fertilizing ability to commercial fertilizers in hydroponic culture with amino acid contents of 6.91 g 100 g(-1). Therefore, the scaled-up production achieved a more satisfactory fish waste degradation rate (3.61 g h(-1)) than the flask-scale production (0.24 g h(-1)). The biodegraded broth of fish waste at room temperature did not undergo putrefaction for 6 months due to the addition of 1% lactate.

Cloning, expression analysis and enzymatic characterization of cathepsin S from olive flounder (Paralichthys olivaceus).

Cathepsin S is a critical protease for the regulation of MHC class II immune responses, and thus is a potential target for developing immunosuppressive drugs in the pathogenesis of degenerative and autoimmune diseases. In this study, we cloned a cDNA encoding for cathepsin S (PoCtS) from the olive flounder, Paralichthys olivaceus. The 1170 bp PoCtS cDNA contained an open reading frame of 1014 bp, which consisted of a 25-residue putative signal peptide, a 96-residue propeptide and the 216-residue mature enzyme. The tissue-specific expression pattern of PoCtS, determined via RT-PCR and real-time PCR analysis, revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however IL-1beta, IL-6, IL-8 and PoCtS expression increased significantly in muscle 6h post-injection of bacterial lipopolysaccharide (LPS). The cDNA encoding proenzyme of PoCtS was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4T-1 vector. Also, the recombinant proPoCtS protein was overexpressed in E. coli BL21(DE3) as a 60 kDa fusion protein. Cathepsin S activity was detected through the cleavage of synthetic fluorogenic peptide substrates, such as Z-Val-Val-Arg-AMC and Z-Phe-Arg-AMC. The optimum pH for the protease activity was determined to be 8. This is the first report that characterized the enzymatic properties and analyzed the expression of piscine cathepsin S.

Molecular cloning, expression, and characterization of cathepsin L from mud loach (Misgurnus mizolepis).

Cathepsin L is an important protease in the initiation of protein degradation and one of the most powerful endopeptidases. In this study, we cloned mud loach (Misgurnus mizolepis) cathepsin L (MlCtL) cDNA, and the pro-mature enzyme of MlCtL (proMlCtL) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4 T-1 vector. The recombinant proMlCtL was overexpressed in E. coli DH5αMCR as a 62-kDa protein. Its activity was quantified by measuring the cleavage of synthetic fluorogenic peptide substrates, and the protease activity of proMlCtL was also demonstrated by gelatin zymography. Antipain and leupeptin were shown to inhibit the protease activity of proMlCtL. Our results suggest that the structural features and evolutionary relationship of the mud loach cathepsin L gene were similar to that of the other mammalian cathepsin Ls; however, the proMlCtL protein was more stable at neutral and alkaline pH. The optimum temperature for the proMlCtL enzyme was found to be 40 °C. In addition, proMlCtL activity was dependent upon the presence of several metal ions and detergents.

Coenzyme Q10 production in a 150-l reactor by a mutant strain of Rhodobacter sphaeroides.

For the commercial production of CoQ(10), batch-type fermentations were attempted in a 150-l fermenter using a mutant strain of R. sphaeroides. Optimum temperature and initial aeration rate were found to be 30 degrees C and 2 vvm, respectively. Under optimum fermentation conditions, the maximum value of specific CoQ(10) content was achieved reproducibly as 6.34 mg/g DCW after 24 h, with 3.02 g/l of DCW. During the fermentation, aeration shift (from the adequate aeration at the early growth phase to the limited aeration in active cellular metabolism) was a key factor in CoQ(10) production for scale-up. A higher value of the specific CoQ(10) content (8.12 mg/g DCW) was achieved in fed-batch fermentation and comparable to those produced by the pilot-scale fed-batch fermentations of A. tumefaciens, which indicated that the mutant strain of R. sphaeroides used in this study was a potential high CoQ(10) producer. This is the first detailed study to demonstrate a pilot-scale production of CoQ(10) using a mutant strain of R. sphaeroides.

Identification and characterization of microorganisms from earthworm viscera for the conversion of fish wastes into liquid fertilizer.

Five bacteria isolated from earthworm viscera and identified as Brevibacillus agri, Bacillus cereus, Bacillus licheniformis, and Brevibacillus parabrevis by 16S rRNA sequencing were employed in the conversion of fish wastes generated from a restaurant specializing in sliced raw fish into fertilizer. Within 120h after inoculation of autoclaved fish waste with 5.15 x 10(5) CFU ml(-1) mixed isolates, the amount of dry sludge decreased from 29.4 to 0.2g, the pH changed from 7.05 to 5.70, and the cell number reached 6.45 x 10(5) CFU ml(-1). Analyses of an 84-h culture of inoculated fish waste indicated low phytotoxicity in a seed germination test, an amino acid content of 5.71 g 100 g(-1), a low concentration of heavy metals (Pb, As, Cd, Hg, Cr, Cu, Ni and Zn), and a N/P/K level of 2.33%. Therefore the converted fish waste has the potential for use as liquid fertilizer, although the low NPK level is a concern. This is the first demonstration of the reutilization of fish wastes as a liquid fertilizer.

Purification, molecular cloning, and biochemical characterization of subtilisin JB1 from a newly isolated Bacillus subtilis JB1.

An extracellular gelatinolytic enzyme obtained from the newly isolated Bacillus subtilis JB1, a thermophilic microorganism relevant to the aerobic biodegradation process of fish-meal production, was purified via ammonium sulfate precipitation, Sephadex G-200 Gel filtration chromatography, and one-dimensional gel electrophoresis separation and subsequently identified via peptide mass fingerprinting and chemically assisted fragmentation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The subtilisin JB1 gene was sequenced and its recombinant protein prosubtilisin JB1 was expressed in Escherichia coli, and the purified prosubtilisin JB1 (62 kDa) protein was digested with gelatin, bovine serum albumin, azocasein, fibrinogen, and the fluorogenic peptide substrate Ala-Ala-Phe-7-amido-4-methylcoumarin hydrochloride, whereas the serine protease inhibitors phenylmethylsulfonyl fluoride and chymostatin completely inhibited its enzyme activity at an optimal pH of 7.5. Thus, our results show that subtilisin JB1 may serve as a potential source material for use in industrial applications of proteolytic enzymes and microorganisms for fishery waste degradation and fish by-product processing.

Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus).

We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3-24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme.