Biodiversity and biocatalyst activity of culturable hydrocarbonoclastic fungi isolated from Marac–Moruga mud volcano in South Trinidad

AbstractMud volcanoes (MVs) are visible signs of oil and gas reserves present deep beneath land and sea. The Marac MV in Trinidad is the only MV associated with natural hydrocarbon seeps. Petrogenic polyaromatic hydrocarbons (PAHs) in its sediments must undergo biogeochemical cycles of detoxification as they can enter the water table and aquifers threatening ecosystems and biota. Recurrent hydrocarbon seep activity of MVs consolidates the growth of hydrocarbonoclastic fungal communities. Fungi possess advantageous metabolic and ecophysiological features for remediation but are underexplored compared to bacteria. Additionally, indigenous fungi are more efficient at PAH detoxification than commercial/foreign counterparts and remediation strategies remain site-specific. Few studies have focused on hydrocarbonoclastic fungal incidence and potential in MVs, an aspect that has not been explored in Trinidad. This study determined the unique biodiversity of culturable fungi from the Marac MV capable of metabolizing PAHs in vitro and investigated their extracellular peroxidase activity to utilize different substrates ergo their extracellular oxidoreductase activity (> 50% of the strains decolourized of methylene blue dye). Dothideomycetes and Eurotiomycetes (89% combined incidence) were predominantly isolated. ITS rDNA sequence cluster analysis confirmed strain identities. 18 indigenous hydrocarbonoclastic strains not previously reported in the literature and some of which were biosurfactant-producing, were identified. Intra-strain variability was apparent for PAH utilization, oil-tolerance and hydroxylase substrate specificity. Comparatively high levels of extracellular protein were detected for strains that demonstrated low substrate specificity. Halotolerant strains were also recovered which indicated marine-mixed substrata of the MV as a result of deep sea conduits. This work highlighted novel MV fungal strains as potential bioremediators and biocatalysts with a broad industrial applications.

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Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects

Protein and Peptide Letters ◽

10.2174/0929866527666191223141205 ◽

2020 ◽

Vol 27 (5) ◽

pp. 400-410

Author(s):

Valentina De Luca ◽

Luigi Mandrich

Keyword(s):

Gene Evolution ◽

Sequence Divergence ◽

High Specificity ◽

Industrial Applications ◽

Point Of View ◽

Enzyme Promiscuity ◽

Primary Activity ◽

Starting Point ◽

Main Activity

: Enzymes are among the most studied biological molecules because better understanding enzymes structure and activity will shed more light on their biological processes and regulation; from a biotechnological point of view there are many examples of enzymes used with the aim to obtain new products and/or to make industrial processes less invasive towards the environment. Enzymes are known for their high specificity in the recognition of a substrate but considering the particular features of an increasing number of enzymes this is not completely true, in fact, many enzymes are active on different substrates: this ability is called enzyme promiscuity. Usually, promiscuous activities have significantly lower kinetic parameters than to that of primary activity, but they have a crucial role in gene evolution. It is accepted that gene duplication followed by sequence divergence is considered a key evolutionary mechanism to generate new enzyme functions. In this way, promiscuous activities are the starting point to increase a secondary activity in the main activity and then get a new enzyme. The primary activity can be lost or reduced to a promiscuous activity. In this review we describe the differences between substrate and enzyme promiscuity, and its rule in gene evolution. From a practical point of view the knowledge of promiscuity can facilitate the in vitro progress of proteins engineering, both for biomedical and industrial applications. In particular, we report cases regarding esterases, phosphotriesterases and cytochrome P450.

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Investigation of the effect of loratadine and calcium ions on oxidoreductase activity of catalase enzyme

Current Enzyme Inhibition ◽

10.2174/1573408016999200817173859 ◽

2020 ◽

Vol 16 ◽

Author(s):

Edhem Hasković ◽

Safija Herenda ◽

Zehra Halilović ◽

Snežana Unčanin ◽

Denis Hasković ◽

...

Keyword(s):

Calcium Ions ◽

Enzyme Reaction ◽

Analytical Techniques ◽

Competitive Inhibitor ◽

Cellular Functions ◽

Oxidoreductase Activity ◽

H1 Antihistamines ◽

Activity Of Enzymes ◽

The Given

Background: The Spectrophotometric method is one of the most suitable analytical techniques for testing the activity of enzymes under the influence of various factors. Methods: The effect of H1-antihistamines of loratadine and calcium ions on enzyme catalase under in vitro conditions was investigated in this paper. Results and Discussion: It has been shown that loratadine isa partial inhibitor of catalase, but this effect is diminished in the presence of calcium ions. Calcium as well as other cations are important for many biological and cellular functions. The kidneys play a central role in the homeostasis of these ions. The activity of the catalase enzyme under the given conditions, the type of inhibition,and the kinetic parameters of the enzyme reaction were determined. Conclusion: We concluded that loratadine is a partially competitive inhibitor.

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An In Vitro Cytotoxicity Assay Using Rat Hepatocytes and MTT and Coomassie Blue Dye as Indicators

Alternatives to Laboratory Animals ◽

10.1177/026119299101900309 ◽

1991 ◽

Vol 19 (3) ◽

pp. 352-360

Author(s):

Kazuhiko Otoguro ◽

Kanki Komiyama ◽

Satoshi Ωmura ◽

Charles A. Tyson

Keyword(s):

Mtt Assay ◽

Membrane Integrity ◽

Cell Number ◽

Cellular Protein ◽

Blue Dye ◽

Tetrazolium Salt ◽

Sprague Dawley ◽

Lactate Dehydrogenase Activity ◽

Coomassie Blue

Isolated hepatocytes from male Sprague-Dawley rats suspended in culture medium supplemented with either 0.2 or 2% bovine serum albumin (BSA) were allowed to attach to collagen coated 96-well dishes. Ten test chemicals from the MEIC list and salicylic acid were added individually to the dishes, and at the end of 24 and 48 hours, cytotoxicity was determined by measuring MTT (tetrazolium salt) reduction (mitochondrial integrity) and total cellular protein using Coomassie blue dye (reflecting cell number). Total cellular lactate dehydrogenase activity was also determined in some experiments, as an indicator of plasma membrane integrity. The relative toxicities of the test chemicals were quantified by the estimation of EC10, EC20 and EC50 values for each parameter. Except for one chemical, digoxin, in the MTT assay, cytotoxic potency increased with incubation time. The hepatocytes tended to be more sensitive to the chemicals in medium containing 0.2% BSA than in medium containing 2% BSA. Simple linear regression analyses of the log transformed data from the MTT assay versus log oral LD50 in rats for the test chemicals gave the best results using EC10 at 24 hours (r2 = 0.86). With protein as the cytotoxic indicator, the best results were obtained with EC values in the medium containing 2% BSA, again at 24 hours (r2 = 0.83). These results suggest that the MTT and Coomassie blue dye assays could be useful indicators for testing the cytotoxic potential of chemicals in rat hepatocyte cultures.

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Characterization of Phospholipase Activity of the Pseudomonas aeruginosa Type III Cytotoxin, ExoU

Journal of Bacteriology ◽

10.1128/jb.187.3.1192-1195.2005 ◽

2005 ◽

Vol 187 (3) ◽

pp. 1192-1195 ◽

Cited By ~ 42

Author(s):

Hiromi Sato ◽

Jimmy B. Feix ◽

Cecilia J. Hillard ◽

Dara W. Frank

Keyword(s):

Pseudomonas Aeruginosa ◽

Substrate Specificity ◽

Yeast Extract ◽

Enzyme Activation ◽

Phospholipase Activity ◽

Eukaryotic Protein ◽

Type Iii

ABSTRACT Recombinant ExoU (rExoU) and yeast extract were used to optimize an in vitro phospholipase assay as a basis for identifying the mechanism for enzyme activation and substrate specificity. Our results support a model in which a eukaryotic protein cofactor or complex facilitates the interaction of rExoU with phospholipid substrates.

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Substrate specificity in vivo and in vitro in the formation of stilbenes. Biosynthesis of rhaponticin

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(80)90332-x ◽

1980 ◽

Vol 200 (1) ◽

pp. 72-78 ◽

Cited By ~ 40

Author(s):

Norbert Rupprich ◽

Heinz Hildebrand ◽

Helmut Kindl

Keyword(s):

Substrate Specificity

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The in vivo and in vitro substrate specificity of quinoprotein glucose dehydrogenase ofAcinetobacter calcoaceticusLMD79.41

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1987.tb02122.x ◽

1987 ◽

Vol 43 (2) ◽

pp. 195-200 ◽

Cited By ~ 22

Author(s):

P. Dokter ◽

J.T. Pronk ◽

B.J. Schie ◽

J.P. Dijken ◽

J.A. Duine

Keyword(s):

Substrate Specificity ◽

Glucose Dehydrogenase

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LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

Biochemical Journal ◽

10.1042/bj20060379 ◽

2006 ◽

Vol 398 (3) ◽

pp. 531-538 ◽

Cited By ~ 116

Author(s):

Yukiko Mizutani ◽

Akio Kihara ◽

Yasuyuki Igarashi

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Family Members ◽

Fatty Acyl ◽

Ceramide Synthase ◽

Broad Substrate Specificity ◽

Acid Protein ◽

Amino Acid Protein

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

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Differential susceptibilities of isolated hamster lung cell types to amiodarone toxicity

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y98-084 ◽

1998 ◽

Vol 76 (7-8) ◽

pp. 721-727 ◽

Cited By ~ 3

Author(s):

M W Bolt ◽

W J Racz ◽

J F Brien ◽

T M Bray ◽

T E Massey

Keyword(s):

Alveolar Macrophages ◽

Gradient Centrifugation ◽

Cell Types ◽

Clara Cell ◽

Blue Dye ◽

Alveolar Type ◽

Specific Cell ◽

Type Ii ◽

Clara Cells

Treatment of cardiac dysrhythmias with the iodinated benzofuran derivative amiodarone (AM) is limited by pulmonary toxicity. The susceptibilities of different lung cell types of male Golden Syrian hamsters to AM-induced cytotoxicity were investigated in vitro. Bronchoalveolar lavage and protease digestion to release cells, followed by centrifugal elutriation and density gradient centrifugation, resulted in preparations enriched with alveolar macrophages (98%), alveolar type II cells (75-85%), and nonciliated bronchiolar epithelial (Clara) cells (35-50%). Alveolar type II cell and Clara cell preparations demonstrated decreased viability (by 0.5% trypan blue dye exclusion) when incubated with 50 µM AM for 36 h, and all AM-treated cell preparations demonstrated decreased viability when incubated with 100 or 200 µM AM. Based on a viability index ((viability of AM-treated cells ÷ viability of controls) × 100%), the Clara cell fraction was significantly (p < 0.05) more susceptible than all of the other cell types to 50 µM AM. However, AM cytotoxicity was greatest (p < 0.05) in alveolar macrophages following incubation with 100 or 200 µM AM. There was no difference between any of the enriched cell preparations in the amount of drug accumulated following 24 h of incubation with 50 µM AM, whereas alveolar macrophages accumulated the most drug during incubation with 100 µM AM. Thus, the most susceptible cell type was dependent on AM concentration. AM-induced cytotoxicity in specific cell types may initiate processes leading to inflammation and pulmonary fibrosis.Key words: amiodarone, susceptibility, alveolar macrophage, accumulation.

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