Filamentous Marine Fungi Research Articles

Antitumor activity of urease-free l-asparaginase with low glutaminase coactivity produced by marine-derived Aspergillus flavus

l-asparaginase, which has a high therapeutic effect in various cancer diseases, hydrolyzes l-asparagine to aspartate and ammonia and is mainly used in treating Acute Lymphoblastic Leukaemia. This study aims to investigate the l-asparaginase production potential of marine filamentous fungi and the partial purification and characterization of the l-asparaginase. The rapid plate screening method was used to measure the l-asparaginase activity of 94 marine fungi. l-asparaginase producers were then screened for glutaminase and urease activity. The selected extracellular l-asparaginase producer, Aspergillus flavus MF10, showed 2.82 U/mL l-asparaginase activity in small-scale fermentation. l-asparaginase was concentrated by ultrafiltration followed by acetone precipitation. The specific activity of the enzyme, purified 4,8 fold by partial purification, was determined as 259 U/mg. There were no urease and glutaminase activities in the fermentation broth, but low glutaminase activity (1.27 U/mL) was detected in the concentrated fermentation broth. The optimum pH and temperature of the l-asparaginase were 8.0 and 40 °C, respectively. l-asparaginase was stable at pH 7.0–8.0 and can maintain its activity at 70% at 40 °C after 1 h. While the enzyme decreased the viability of SH-SY5Y (neuroblastoma) to 43% and LNCap (prostate cancer) to 61%, it did not show cytotoxic effects in healthy cell line L929 (fibroblast). It was also determined that l-asparaginase showed low cytotoxicity in HaCaT (keratinocyte) cells. In conclusion, it was shown that urease-free l-asparaginase with low glutaminase coactivity, produced from marine-derived A. flavus MF10, has the potential for medical use.

Ecological insights and potential application of marine filamentous fungi in environmental restoration

Ecological insights and potential application of marine filamentous fungi in environmental restoration

Species Composition and Hydrolase Enzyme (EC.3) Activity of Fungi Isolated from Thasala Mangroves, Nakhon Si Thammarat Province, Southern Thailand

In the present study, we aimed to investigate diversity of manglicolous and endobiotic fungi associated with mangrove plants from Thasala mangroves, Nakhon Si Thammarat province, southern Thailand. Extracellular hydrolase enzymes (EC.3) including amylase, cellulase, lipase, protease, and pectinase from isolated fungi were evaluated. A total of 31 obligate marine fungal species was recorded from various Thasala mangroves sites. These included 24 ascomycetes (77.4 %), 1 mucoromycete (3.2 %), 3 basidiomycetes (9.7 %), 4 asexual morphs (12.9 %), and tentatively identified fungi (9.7 %). The average percentage occurrence of the fungi recorded from each site ranged from 0.4 to 16%. At all sites, the common filamentous marine fungi were Cumulospora sp., Halorosellinia oceanica, Kallichroma tethys, Leptosphaeria sp., Rimora mangrovei, Lulworthia sp., Phoma sp., Halenospora varia, Halocyphina villosa, Nia vibrissa, and Sclerococcum haliotrephum. Also, the most frequent marine fungi include Eutypella naqsii, Sammeyersia grandispora, Marinosphaera mangrovei, Nemania maritima, and Verruculina enalia. Seven mangrove plants were selected for endophytic fungal isolation including Acanthus ebracteatus, Acrostichum aureum, Avicennia alba, Bruguiera cylindrica, Rhizophora apiculata, R. mucronata, and Sonneratia alba. The isolation rate (IR) and colonization rate (CR) of fungal endobiota varied for all plant studied. The isolation rate ranged from 24 - 53.3 %, while colonization rate varied from 29.3 - 61.3 %. The fungal isolates were screened for the production of hydrolase enzymes (EC.3), of which 37.2 % exhibited enzyme activities. The 16 out of 28 obligate marine fungi (57.1 %) and 13 out of 50 fungal endophytes (26 %) had enzyme production efficiency toward specific substrates or an enzymatic index (EI) higher than 1.4. None of the fungal isolates depicted lipase activity. Additionally, the environmental stressors (temperature, pH, salinity) affecting enzyme production were evaluated in order to discover potential candidates for industrial application.
 HIGHLIGHTS
 
 Diversity of obligate marine fungal species was estimated from Thasala mangroves
 The obligate marine fungi and fungal endophytes had hydrolase enzyme (EC.3) production efficiency toward specific substrates or an enzymatic index (EI) higher than 1.4
 The temperature, pH, and salinity affecting enzyme production were evaluated in order to discover potential candidates for industrial application
 
 GRAPHICAL ABSTRACT

A Review on Bioactive Compounds from Marine-Derived Chaetomium Species.

Filamentous marine fungi have proven to be a plentiful source of new natural products. Chaetomium, a widely distributed fungal genus in the marine environment, has gained much interest within the scientific community. In the last 20 years, many potential secondary metabolites have been detected from marine-derived Chaetomium. In this review, we attempt to provide a comprehensive summary of the natural products produced by marine-derived Chaetomium species. A total of 122 secondary metabolites that were described from 2001 to 2021 are covered. The structural diversity of the compounds, along with details of the sources and relevant biological properties are also provided, while the relationships between structures and their bioactivities are discussed. It is our expectation that this review will be of benefit to drug development and innovation.

Fungi Can Be More Effective than Bacteria for the Bioremediation of Marine Sediments Highly Contaminated with Heavy Metals.

The contamination of coastal marine sediments with heavy metals (HMs) is a widespread phenomenon that requires effective remediation actions. Bioremediation based on the use of bacteria is an economically and environmentally sustainable effective strategy for reducing HM contamination and/or toxicity in marine sediments. However, information on the efficiency of marine-derived fungi for HM decontamination of marine sediments is still largely lacking, despite evidence of the performance of terrestrial fungal strains on other contaminated matrixes (e.g., soils, freshwater sediments, industrial wastes). Here, we carried out for the first time an array of parallel laboratory experiments by using different combinations of chemical and microbial amendments (including acidophilic autotrophic and heterotrophic bacteria, as well as filamentous marine fungi) for the bioremediation of highly HM-contaminated sediments of the Portman Bay (NW Mediterranean Sea), an area largely affected by long-term historical discharges of mine tailings. Our results indicate that the bioleaching performance of metals from the sediment is based on the addition of fungi (Aspergillus niger and Trichoderma sp.), either alone or in combination with autotrophic bacteria, was higher when compared to other treatments. In particular, fungal addition allowed obtaining bioleaching yields for As eight times higher than those by chemical treatments and double compared with the addition of bacteria alone. Moreover, in our study, the fungal addition was the only treatment allowing effective bioleaching of otherwise not mobile fractions of Zn and Cd, thus overtaking bacterial treatments. We found that the lower the sediment pH reached by the experimental conditions, as in the case of fungal addition, the higher the solubilization yield of metals, suggesting that the specific metabolic features of A. niger and Trichoderma sp. enable lowering sediment pH and enhance HM bioleaching. Overall, our findings indicate that fungi can be more effective than acidophilic autotrophic and heterotrophic bacteria in HM bioleaching, and as such, their use can represent a promising and efficient strategy for the bioremediation of marine sediments highly contaminated with heavy metals.

The Presence of Marine Filamentous Fungi on a Copper-Based Antifouling Paint

Marine biofouling is undesirable growth on submerged substances, which causes a major problem for maritime industries. Antifouling paints containing toxic compounds such as copper are used to prevent marine biofouling. However, bacteria and diatoms are usually found in biofilms developed on such paints. In this study, plastic panels painted with a copper-based self-polishing antifouling paint were exposed to biofouling for 6 months in the Marina Bandar Rowdha, Sea of Oman. Clean panels were used as a control substratum. Marine filamentous fungi from protected and unprotected substrate were isolated on a potato dextrose agar. Pure isolates were identified using sequences of the ITS region of rDNA. Six fungal isolates (Alternaria sp., Aspergillus niger, A. terreus, A. tubingensis, Cladosporium halotolerans, and C. omanense) were obtained from the antifouling paint. Four isolates (Aspergillus pseudodeflectus, C. omanense, and Parengyodontium album) were isolated from clean panels and nylon ropes. This is the first evidence of the presence of marine fungi on antifouling paints. In comparison with isolates from the unprotected substrate, fungi from the antifouling paint were highly resistant to copper, which suggests that filamentous fungi can grow on marine antifouling paints.

High throughput, small scale methods to characterise the growth of marine fungi.

Various marine fungi have been shown to produce interesting, bioactive compounds, but scaling up the production of these compounds can be challenging, particularly because little is generally known about how the producing organisms grow. Here we assessed the suitability of using 100-well BioScreen plates or 96-well plates incubated in a robot hotel to cultivate eight filamentous marine fungi, six sporulating and two non-sporulating, to obtain data on growth and substrate (glucose, xylose, galactose or glycerol) utilisation in a high throughput manner. All eight fungi grew in both cultivation systems, but growth was more variable and with more noise in the data in the Cytomat plate hotel than in the BioScreen. Specific growth rates between 0.01 (no added substrate) and 0.07 h-1 were measured for strains growing in the BioScreen and between 0.01 and 0.27 h-1 for strains in the plate hotel. Three strains, Dendryphiella salina LF304, Penicillium chrysogenum KF657 and Penicillium pinophilum LF458, consistently had higher specific growth rates on glucose and xylose in the plate hotel than in the BioScreen, but otherwise results were similar in the two systems. However, because of the noise in data from the plate hotel, the data obtained from it could only be used to distinguish between substrates which did or did not support growth, whereas data from BioScreen also provided information on substrate preference. Glucose was the preferred substrate for all strains, followed by xylose and galactose. Five strains also grew on glycerol. Therefore it was important to minimise the amount of glycerol introduced with the inoculum to avoid misinterpreting the results for growth on poor substrates. We concluded that both systems could provide physiological data with filamentous fungi, provided sufficient replicates are included in the measurements.

Characterization of Amylolytic Activity by a Marine-Derived Yeast Sporidiobolus pararoseus PH-Gra1

Marine yeasts have tremendous potential in industrial applications but have received less attention than terrestrial yeasts and marine filamentous fungi. In this study, we have screened marine yeasts for amylolytic activity and identified an amylase-producing strain PH-Gra1 isolated from sea algae. PH-Gra1 formed as a coral-red colony on yeast–peptone–dextrose (YPD) agar; the maximum radial growth was observed at 22 °C, pH 6.5 without addition of NaCl to the media. Based on the morphology and phylogenetic analyses derived from sequences of internal transcribed spacer (ITS) and a D1/D2 domain of large subunit of ribosomal DNA, PH-Gra1 was designated Sporidiobolus pararoseus. S. pararoseus is frequently isolated from marine environments and known to produce lipids, carotenoids, and several enzymes. However, its amylolytic activity, particularly the optimum conditions for enzyme activity and stability, has not been previously characterized in detail. The extracellular crude enzyme of PH-Gra1 displayed its maximum amylolytic activity at 55 °C, pH 6.5, and 0%–3.0% (w/v) NaCl under the tested conditions, and the activity increased with time over the 180-min incubation period. In addition, the crude enzyme hydrolyzed potato starch more actively than corn and wheat starch, and was stable at temperatures ranging from 15 °C to 45 °C for 2 h. This report provides a basis for additional studies of marine yeasts that will facilitate industrial applications.

A Novel High-Throughput Screening Platform Identifies Itaconate Derivatives from Marine Penicillium antarcticum as Inhibitors of Mesenchymal Stem Cell Differentiation.

Worldwide diffused diseases such as osteoarthritis, atherosclerosis or chronic kidney disease are associated with a tissue calcification process which may involve unexpected local stem cell differentiation. Current pharmacological treatments for such musculoskeletal conditions are weakly effective, sometimes extremely expensive and often absent. The potential to develop new therapies is represented by the discovery of small molecules modulating resident progenitor cell differentiation to prevent aberrant tissue calcification. The marine environment is a rich reserve of compounds with pharmaceutical potential and many novel molecules are isolated from macro and microorganisms annually. The potential of small molecules synthetized by marine filamentous fungi to influence the osteogenic and chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) was investigated using a novel, high-throughput automated screening platform. Metabolites synthetized by the marine-derived fungus Penicillium antarcticum were evaluated on the platform. Itaconic acid derivatives were identified as inhibitors of calcium elaboration into the matrix of osteogenically differentiated hMSCs and also inhibited hMSC chondrogenic differentiation, highlighting their capacity to impair ectopic calcification. Bioactive small molecule discovery is critical to address ectopic tissue calcification and the use of biologically relevant assays to identify naturally occurring metabolites from marine sources represents a strategy that can contribute to this effort.

A conspectus of the filamentous marine fungi of Sweden

Abstract Marine filamentous fungi have been little studied in Sweden, which is remarkable given the depth and width of mycological studies in the country since the time of Elias Fries. Seventy-four marine fungi are listed for Sweden based on historical records and recent collections, of which 16 are new records for the country. New records for the country are based on morphological identification of species mainly from marine wood, and most of them from the Swedish West Coast. In some instances, the identifications have been made by comparisons of sequences obtained from cultures with reference sequences in GenBank. Corollospora angusta, Corollospora filiformis, and Corollospora pulchella, previously known from tropical/subtropical areas, are recorded for the first time for Sweden. The arctic Havispora longyearbyensis was also found. Kalmusia longispora and Neocamarosporium calvescens were reported for the first time from marine habitats.

Abstract 1864: Verticillin A causes DNA damage and apoptosis in high grade serous ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy affecting women worldwide and the fifth most common cause of cancer related deaths among U.S. women. New targeted therapies are needed to prevent HGSOC progression and metastasis related lethality from the disease. The goal of this study was to test the novel natural compound, Verticillin A, for its anticancer properties and mode of action in HGSOC cells. Verticillin A is an epipolythiodioxopiperazine (ETP) alkaloid that is isolated from several terrestrial and marine filamentous fungi and has been shown to be cytotoxic in several cancer cell lines including OVCAR8, OVCAR4 and Kuramochi. Our data indicated that Verticillin A treatment caused cytotoxicity in HGSOC cell lines in a dose-dependent manner. Furthermore, treatment with Verticillin A in HGSOC cell line OVCAR8 and OVCAR4 enhanced apoptosis, which was demonstrated by PARP cleavage and Annexin V/ Propidium iodide staining. To determine whether Verticillin A caused in vivo tumor growth inhibition, OVCAR8-RFP cells were xenografted in mice to form tumors and the mice were treated with Verticillin A. Encapsulated nanoparticles of Verticillin A decreased tumor growth in vivo and had low cytotoxicity compared to the naked drug. RNA-Seq analysis was performed with OVCAR8 cells treated with Verticillin A and the data found an upregulation of apoptosis signaling pathway and oxidative stress response and downregulation of cancer stemness signaling pathways. A proteomic histone profiling performed in OVCAR8 cells indicated that Verticillin A caused epigenetic modifications with global changes in histone methylation and acetylation marks. Thus, our study identifies Verticillin A as a novel epigenetic modifier in ovarian cancer cells and indicates therapeutic potential for treatment of HGSOC. Note: This abstract was not presented at the meeting. Citation Format: Amrita Salvi, Julia Austin, Daniel Lantvit, Joanna Burdette. Verticillin A causes DNA damage and apoptosis in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1864.

Induced effect of Ca2+ on curvulamine synthesis by marine-derived fungus Curvularia sp. IFB-Z10 under submerged fermentation

Abstract Curvulamine is a novel framework alkaloid with prominent antibacterial and acetylcholinesterase inhibitory activities produced by marine fungus, Curvularia sp. IFB-Z10. However, the low yield severely restricts its further pharmaceutical researches and applications. The study was aimed to enhance curvulamine production based on CaCl2 addition strategy and further clarify the underlying regulation mechanism of Ca2+. In this work, the optimal addition conditions of CaCl2 were firstly obtained, and curvulamine production achieved 42.96 mg/L under supplying 5 mM CaCl2 at 0 h, which was 3.33-fold that of control in shake flask. It was further found that cytosolic Ca2+ level was markedly increased through calcium channel under CaCl2 stimulation. Relatively high intracellular Ca2+ level could induce fungus to principally form pellets with small diameter and compact structure (morphology), and significantly up-regulate the transcriptional and expression levels of key biosynthetic genes, which collectively promoted curvulamine synthesis. Finally, CaCl2 addition strategy was successfully applied in 5-L bioreactor. This work provided an effective regulation strategy to improve curvulamine production in submerged fermentation of Curvularia sp. and it would facilitate further research on the synthesis regulation of secondary metabolites from other marine filamentous fungi.

Carbon utilization profile of the filamentous fungal species Fusarium fujikuroi, Penicillium decumbens, and Sarocladium strictum isolated from marine coastal environments

Facultative marine filamentous fungi have recently emerged as a functional component in coastal marine systems. However, little is known about their ecological role and functions in biogeochemical cycles. Penicillium decumbens, S. strictum, and F. fujikuroi were isolated from the coastal upwelling zone off south-central Chile. Their carbon profiles were characterized using Biolog FF MicroPlates. These species used a wide range of carbon sources, mainly carbohydrates, but also amino acids, suggesting the use of metabolic routes that include glycolysis/gluconeogenesis. Substrate richness revealed a great capacity for the utilization of nutritional sources, reflected by the following Shannon Indices of utilization of specific substrates: 4.02 for S. strictum, 4.01 for P. decumbes, and 3.91 for F. fujikuroi, which reveals a high physiological capacity for oxidizing different substrates. Significant differences were found between 18 substrates utilized by all three species. Results suggest that filamentous fungi should be considered an integral part of the marine microbial community and included in biogeochemical cycling models of upwelling ecosystems.

Growth of marine fungi on polymeric substrates.

BackgroundMarine fungi are a diverse group of opportunistic and obligate organisms isolated from marine environments. These fungi are now often included in screens for novel metabolites, while less attention has been given to their production of hydrolytic enzymes. Most enzymes derived from marine microorganisms have been obtained from marine bacteria. The enzymes produced by marine fungi may have different properties than those derived from bacteria or from terrestrial fungi. Here we assess the growth of six filamentous marine fungi on a wide range of polymeric substrates as an indication of their general capacity to produce hydrolytic enzymes.ResultsCalcarisporium sp. KF525, Tritirachium sp. LF562, Bartalinia robillardoides LF550, Penicillium pinophilum LF458, Scopulariopsis brevicaulis LF580 and Pestalotiopsis sp. KF079 all grew on both casein and gelatin as N-source, indicating secretion of proteases. All species also grew on starch, laminarin, xylan, pectin and oil, indicating production of amylases, glucanases, xylanases, pectinases and lipases. Growth on cellulose occurred but was weaker than on xylan. All strains also grew to some extent on sulphated arabinogalactan, although only LF562 could utilise arabinose. Four strains grew on the sulphated ulvans, whereas only KF525 grew on agar or carrageenan. KF525 and LF562 showed limited growth on alginate. Although fucose was used as carbon source by several species, fucoidan did not support biomass production.ConclusionsMarine fungi could be excellent sources of a wide range of hydrolytic enzymes, including those able to hydrolyse various seaweed polymers. Although the native hosts may secrete only small amounts of these enzymes, the genes may provide a rich source of novel enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0233-5) contains supplementary material, which is available to authorized users.

Major bioactive metabolites from marine fungi: A Review.

Biologists and chemists of the world have been attracted towards marine natural products for the last five decades. Approximately 16,000 marine natural products have been isolated from marine organisms which have been reported in approximately 6,800 publications, proving marine microorganisms to be a invaluable source for the production of novel antibiotic, anti tumor, and anti inflammatory agents. The marine fungi particularly those associated with marine alga, sponge, invertebrates, and sediments appear to be a rich source for secondary metabolites, possessing Antibiotic, antiviral, antifungal and antiyeast activities. Besides, a few growth stimulant properties which may be useful in studies on wound healing, carcinogenic properties, and in the study of cancers are reported. Recent investigations on marine filamentous fungi looking for biologically active secondary metabolites indicate the tremendous potential of them as a source of new medicines. The present study reviews about some important bioactive metabolites reported from marine fungal strains which are anti bacterial, anti tumour and anti inflammatory in action. It highlights the chemistry and biological activity of the major bioactive alkaloids, polyketides, terpenoids, isoprenoid and non-isoprenoid compounds, quinones, isolated from marine fungi.

Diversity of culturable marine filamentous fungi from deep‐sea hydrothermal vents

As now very few studies have been carried out on deep-sea marine fungi, this field remains relatively unknown. However, their presence inside benthic microbial eukaryotes at deep-sea vents was recently pointed out from molecular microbial ecology studies. We report here an attempt to describe the culturable part of mycological communities in deep-sea vent ecosystems that is an important step in understanding their diversity, abundance and function. Physiological characterization revealed strains that are more or less adapted to deep-sea conditions. Those results suggest the presence of true marine organisms and other more ubiquitous. Phylogenetical characterization highly correlated to physiological data revealed the presence of fungi that have been previously described and unknown ones until now, belonging to new taxonomic groups. This survey encourages for further work in order to complete descriptions and also to describe the ecological role of these organisms in such extreme environments.

Filamentous fungal population and species diversity from the continental slope of Bay of Bengal, India

Filamentous fungal diversity from the sediments of the continental slope of Bay of Bengal was studied. Sediment samples were collected during two voyages in 2004 and 2005. Filamentous fungal population from both the cruises showed a range of 5.17–59.51 CFU/g and 3.47–29.68 CFU/g, respectively. Totally 16 fungal genera were recorded from both the cruises. Aspergillus was found to be the dominant genus and the overall percentage occurrence was as follows: Deuteromycotina 74%, Ascomycotina 17%, Basidiomycotina 4% and non-sporulating 5%. Diversity indices were calculated and during both the cruises species richness ( d) varied from 0.912 to 3.622 and 1.443 to 4.588; evenness ( J′) varied from 0.9183 to 1.000 and 0.8322 to 1.000 and Shannon–Wiener index ( H′ log 2) varied from 0.9183 to 1.000 and 1.000 to 3.690. The higher diversity was found in Divipoint transect (northern Bay of Bengal). 95% confidence interval and ellipse showed that the stations were well lying within the funnel. Cluster analysis and MDS grouped the northern transects which showed higher diversity. BVSTEP resulted in isolation of 23 species which were most influential in the marine filamentous fungal diversity of the continental slope of Bay of Bengal. Thus, a lower population range and higher diversity of marine filamentous marine fungi in the sediments of the continental slope of Bay of Bengal was recorded.

The Current Chemical Utility of Marine and Terrestrial Filamentous Fungi in Side-Chain Chemistry

This review focuses on three aspects of the application of marine and terrestrial filamentous fungi to side-chain chemistry from 2000 to early 2006. The first section covers the generation of novel biologically active side-chain containing metabolites. This includes a range of antibiotic, cytotoxic and novel bioactive compounds. The second section discusses the use of these fungi both whole cell and lipases of fungal origin in the chemical modification of side-chains. Examples include reductive desymmetrization of pro-chiral ketones, dynamic kinetic resolution and unique reactions. The final section details the degradation of side-chains by fungi and includes lignin and steroid transformations for determination metabolism and the generation of new skeletal structure.

Comparative Characterization of Laminarinases from the Filamentous Marine Fungi Chaetomium Indicum Corda and Trichoderma Aureviride Rifai

Marine filamentous fungi (103 strains) isolated from various marine habitats were studied for their ability to produce extracellular O-glycosylhydrolases. Cultural filtrates of these strains were shown to contain a series of glycanases (laminarinases, amylases, cellulases, pustulanases) and glycosidases (β-glucosidases, N-acetyl-β-glucosaminidases, β-galactosidases, α-mannosidases). Two species of marine fungi from different habitats were chosen for isolation of laminarinases and detailed study on enzyme properties. The fungus Chaetomium indicum associated with the alga Fucus evanescens C. Agardh was collected near the Kuril Islands, and Trichoderma aureviride was sampled from bottom deposits of South China Sea. Properties of extracellular laminarinases were similar: temperature optimums (40–45 °C), molecular masses (54–56 kDa), Km (0.1–0.3 mg mL−1). Temperature stability of laminarinase of C. indicum was significantly higher than those from Trichoderma aureveride. It is shown that these enzymes are specific to β-1,3-bonds in glucans, release predominantly glucose from laminaran and do not catalyze reaction of transglycosylation. Accoding to these data enzymes are exo-1,3-β-D-glucan-glucanohydrolases (EC 3.2.1.58). Inhibitor analysis demonstrated the significant role of tryptophan and tyrosine residues in the catalytic activity of enzymes. Molecules of T. aureviride laminarinase contained the functionally important thiol group.

Molecular approaches for assessing fungal diversity in marine substrata

A range of molecular techniques is available to assess fungal diversity in natural environments. These include denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), single-strand conformation polymorphism (SSCP), terminal restriction fragment length polymorphism (T-RFLP), amplified rDNA restriction analysis (ARDRA), amplified ribosomal intergenic spacer analysis (ARISA) and cloning. The techniques, polymerase chain reaction (PCR) primers for environmental work and their potential limitations are discussed in this review, together with applications for describing the diversity of filamentous marine fungi associated with their substrata. It is unlikely that a single technique can be used to assess all aspects of diversity, however the limited number of studies with a molecular approach has demonstrated that marine substrata contain a greater fungal diversity than previously recognised. These studies have also shown that it is important to combine mycological and molecular approaches to accurately assess diversity.