Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity

AbstractAn antibacterial ∼11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5–6.2) and at low temperature (4–35°C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70°C for 15 min, suggesting relatively high protein structure stability. Sequence-analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme

The Enzyme and the cDNA Sequence of a Thermolabile and Double-Strand Specific DNase from Northern Shrimps (Pandalus borealis)

Inge W. Nilsen et al...Background We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis) and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies. Methodology/Principal Findings A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis). The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65°C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN). Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases. Conclusions/Significance The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature, strengthens its potential for in vitro applications.This work was supported by the Research council of Norway, project number 138822/130, Nofima Marin and Biotec Marine Biochemicals. Since IWN and KØ are employees at Nofima Marin and LJH, ME, DRG and OL are employees at Marine Biochemicals the funders played a role in the design, data analysis and the decision to publish.Peer reviewe

Efficient Screening of Marine Extracts for Protease Inhibitors by Combining FRET Based Activity Assays and Surface Plasmon Resonance Spectroscopy Based Binding Assays

The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose

HerrZyme - Enzymkarakterisering og teknologiutvikling for kommersialisering av enzym fra restråstoff av sild

Forprosjektet HerrZyme fokuserer på undersøkelser av en delvis renset esterase fra silderestråstoff med kommersielt formål. Enzymet bryter ned kortkjedede lipider og har så langt vist seg å ha attraktive egenskaper for bl.a. detergentindustrien, herunder høy katalytisk aktivitet ved lave temperaturer. I dette prosjektet skulle avklaringer av enzymets relevante egenskaper for ulike bruksbetingelser og – formål gjennomføres. Esteraseaktivitet er målt med substratet para-nitrofenyl butyrat med mål å påvise pH, temperatur og løsemiddel tåleevne. Undersøkelsene viser på god aktivitet i pH mellom pH 4.8 og pH 12.0. Aktiviteten er også god etter inkubasjon ved 37 °C men ikke ved 45 °C, hvilket viser på kuldeadaptert enzym. Enzymet tåler ikke inkubasjon i 50 % av forskjellige løsemidler, men har 100 % gjenstående aktivitet etter to timers inkubasjon natrium polyfosfat. Innen prosjektet har esteraseaktiviteten blitt fullstendig renset og analysert med LC-MSMS på Q-TOF, men fullstendig indentifisering var ikke mulig. En vellykket pilot skale rensingsforsøk ble gjennomført. Mye tid ble brukt til å implementere assayer for å finne nye aktiviteter med andre substrater, endo- og eksogene, men ingen ny aktivitet ble funnet under prosjektet hvilket forhindret substrat-, regio-, og enantio-selektivitets¬karakterisering. I summering, til tross for mange lovende egenskaper gjenstår identifisering av enzymet og at finne dess naturlige aktivitet

Efficient Screening of Marine Extracts for Protease Inhibitors by Combining FRET Based Activity Assays and Surface Plasmon Resonance Spectroscopy Based Binding Assays

The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose

Rapport/Report 23/2014 English Summary

-Forprosjektet HerrZyme fokuserer på undersøkelser av en delvis renset esterase fra silderestråstoff med kommersielt formål. Enzymet bryter ned kortkjedede lipider og har så langt vist seg å ha attraktive egenskaper for bl.a. detergentindustrien, herunder høy katalytisk aktivitet ved lave temperaturer. I dette prosjektet skulle avklaringer av enzymets relevante egenskaper for ulike bruksbetingelser og – formål gjennomføres. Esteraseaktivitet er målt med substratet para-nitrofenyl butyrat med mål å påvise pH, temperatur og løsemiddel tåleevne. Undersøkelsene viser på god aktivitet i pH mellom pH 4.8 og pH 12.0. Aktiviteten er også god etter inkubasjon ved 37 °C men ikke ved 45 °C, hvilket viser på kuldeadaptert enzym. Enzymet tåler ikke inkubasjon i 50 % av forskjellige løsemidler, men har 100 % gjenstående aktivitet etter to timers inkubasjon natrium polyfosfat. Innen prosjektet har esteraseaktiviteten blitt fullstendig renset og analysert med LC-MSMS på Q-TOF, men fullstendig indentifisering var ikke mulig. En vellykket pilot skale rensingsforsøk ble gjennomført. Mye tid ble brukt til å implementere assayer for å finne nye aktiviteter med andre substrater, endo- og eksogene, men ingen ny aktivitet ble funnet under prosjektet hvilket forhindret substrat-, regio-, og enantio-selektivitets¬karakterisering. I summering, til tross for mange lovende egenskaper gjenstår identifisering av enzymet og at finne dess naturlige aktivitet.Within the project HerrZyme, a partly purified esterase activity stemming from herring by-products has been investigated from a commercial perspective. The activity shows good durability to prolonged incubations between pH 4.8 and pH 12.0, but not to prolonged incubations at temperatures over 45 °C. 30% of maximum activity is retained at 10°C, suggesting a cold-adapted enzyme. In spite of considerable effort being put into purifying and sequencing the enzyme, together with the establishing of a number of enzyme assays to find activity with natural esterase compounds, the true nature of this esterase activity remains to be solved

Rapport/Report 23/2014 English Summary

-Forprosjektet HerrZyme fokuserer på undersøkelser av en delvis renset esterase fra silderestråstoff med kommersielt formål. Enzymet bryter ned kortkjedede lipider og har så langt vist seg å ha attraktive egenskaper for bl.a. detergentindustrien, herunder høy katalytisk aktivitet ved lave temperaturer. I dette prosjektet skulle avklaringer av enzymets relevante egenskaper for ulike bruksbetingelser og – formål gjennomføres. Esteraseaktivitet er målt med substratet para-nitrofenyl butyrat med mål å påvise pH, temperatur og løsemiddel tåleevne. Undersøkelsene viser på god aktivitet i pH mellom pH 4.8 og pH 12.0. Aktiviteten er også god etter inkubasjon ved 37 °C men ikke ved 45 °C, hvilket viser på kuldeadaptert enzym. Enzymet tåler ikke inkubasjon i 50 % av forskjellige løsemidler, men har 100 % gjenstående aktivitet etter to timers inkubasjon natrium polyfosfat. Innen prosjektet har esteraseaktiviteten blitt fullstendig renset og analysert med LC-MSMS på Q-TOF, men fullstendig indentifisering var ikke mulig. En vellykket pilot skale rensingsforsøk ble gjennomført. Mye tid ble brukt til å implementere assayer for å finne nye aktiviteter med andre substrater, endo- og eksogene, men ingen ny aktivitet ble funnet under prosjektet hvilket forhindret substrat-, regio-, og enantio-selektivitets¬karakterisering. I summering, til tross for mange lovende egenskaper gjenstår identifisering av enzymet og at finne dess naturlige aktivitet.Within the project HerrZyme, a partly purified esterase activity stemming from herring by-products has been investigated from a commercial perspective. The activity shows good durability to prolonged incubations between pH 4.8 and pH 12.0, but not to prolonged incubations at temperatures over 45 °C. 30% of maximum activity is retained at 10°C, suggesting a cold-adapted enzyme. In spite of considerable effort being put into purifying and sequencing the enzyme, together with the establishing of a number of enzyme assays to find activity with natural esterase compounds, the true nature of this esterase activity remains to be solved

Exploring the effect of inhibitors, cooking and freezing on melanosis in snow crab (Chionoecetes opilio) clusters

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