Biological Sciences

Dr Claire Hellio

Marine Biochemistry

I work on marine biochemistry studies since 1995. My work has been performed in collaboration with Prof Yves Le Gal (Marine station of Concarneau) , Dr Benoit Veron (Algobank, University of Caen, France), Dr Helene Thomas-Guyon (University of La Rochelle, France) and Dr Tristan Renault (IFREMER, France) and focused on two main enzymes: Histidase and Phenoloxidase.

1- Phenoloxidase study in 2 marine invertebrates: Mytilus edulis & Crassostrea gigas.

Research performed by Dr Claire Hellio, Prof Yves Le Gal, Dr Helene Thomas-Guyon and Dr Tristan Renault, from 1998 to date.

1a - Mytilus edulis: development of a new antifouling test

Mussels are among the major fouling macroorganisms which cause serious problems by settling on man-made surfaces. The blue mussel, Mytilus edulis, attaches to the substratum by means of adhesive plaques connected to a stem of byssus. These plaques are thought to be produced by the action of a phenoloxidase on a protein precursor by oxidising phenols to catechols and catechols to o-quinones from covalent bonding with amines. The plaque adheres to a variety of substrata including rocky surfaces, slates, iron or polystyrene and even on Teflon plates in sea water.

The antifouling effect on mussel often is determined by recording the attachment ability of adults or larvae of mussels or barnacles. Two different methods presently are used : the first is based on the process of adhesion of mussels to solid substrata. The second concerns the effect of the sample upon direct application on the foot of the mussel. However, both of these methods are inconvenient in that they require a large quantity of material for testing and are time consuming. To develop new antifouling paints, it is very important to test and quantify the efficiency of the new materials. However all the methods used are characterised by a lack of precision in the study of the binder evolution and in the assessment of paint performances. Our present study presents a bioassay procedure for antifouling substances and is based on the measurement of activity of the phenoloxidase purified from the foot of M. edulis .

This program has led to 1 paper and to the development of new antifouling test.

  • As a first step in developing a related bioassay, pure phenoloxidase was obtained with a yield of 35%. The enzyme has a molecular mass close to 34 kDa, as estimated by gel filtration and by SDS PAGE, indicating that the enzyme is a monomer.
  • The second step in developing a related bioassay was to establish a standard protocol for the utilisation of the purified phenoloxidase. Vm and Km values were determined as respectively 0.058 Δ DO/min and 1.6 mM. The phenoloxidase has an optimum of activity at pH 6.8 (phosphate buffer) with a maximum of activity at 35 °C. Approximately 80% of the activity was retained after incubation of the enzyme for 30 min at 35°C and a concentrated solution of the enzyme was stable at 4°C. No inactivation occurred upon freeze-dying of a dilute aqueous solution of the enzyme. This characteristic allowed advance preparation of large quantities of purified phenoloxidase.
  • The last step in developing a related bioassay was to validate this new assay technique as an antifouling bioassay. In order to realise it, we have tested two compounds often utilised in the composition of the antifouling coatings : CuSO 4 and TBTO. With the phenoloxidase test, we obtain a level of inhibition around 80% of the enzyme activity, these results show that an antifouling activity can be correlated with a high level of inhibition of the phenoloxidase activity. This assay technique leads to the same result that the previous methods used (plate assay and foot retraction) with the advantage of being rapid, repetitive and only few consuming products. These characteristic make it suitable for screening naturally occurring antifouling compounds.

For more information, see

C H ellio , N Bourgougnon, Y Le Gal (2000) Phenoloxidase (E. C. 1.14.18.1) from Mytilus edulis byssus gland : purification, partial characterization and application for screening products with potential antifouling activities. Biofouling. 16: 235-244.

1b - Crassostrea gigas: development of a new indicator of oyster’s health

In invertebrates, haemocytes and some proteolytic stunts (coagulation, melanin synthesis and opsonisation) are involved in defence reactions toward pathogens. The prophenoloxidase (ProPO) system, as the origin of melanin production, has been suggested as an innate defence mechanism in invertebrates. In arthropods and some bivalve molluscs, it has been demonstrated that phenoloxidase (PO) is present in the haemolymph as an inactivated form of Pro-phenoloxidase. ProPO is cleaved by gentle proteolyse via an endogen activator system or exogen agents, to PO, the enzymatic active form. Non-enzymatic polymerisation of o-diphenols leads to the production of melanin, a common response of invertebrates to the entry of a infections agent. The activation of the enzymatic system by bacterial or fungal components is an additional clue of the involvement of this enzyme in invertebrate immune defence mechanisms. Conversion of pro-PO to PO induced by small amounts of exogen molecules from: bacteria (lipopolysaccharides (LPS)), algae (laminarin) or yeast (zymosan) and suggests that the pro-PO system is a non-self recognition system.

Understanding the activation of the pro-PO system in Crassostrea gigas could be used as a probe for the evaluation of their health and could provide rapid inexpensive tests for environmental stress. This of particular importance as the aquaculture of oysters contributes significantly to the economic development of many tropical, subtropical and temperate countries. But aquaculture is threatened by the repeated appearance of new diseases, non-infectious and infectious. Difficulties in controlling these diseases come mainly from the differences in susceptibility of the animals according to their developmental stage (from larvae to adults) and from the diversity of pathogens (parasites, viruses, fungi and bacteria) that affect them. Antibiotics have been used intensively as preventive and curative measures but such practices are now questioned due to the appearance of drug-resistant bacteria and their harmful effects on the environment. As an outcome, alternative treatments as well as animal health monitoring have to be established as preventing measures. Investigation of the innate immune systems may give new insights into the management and control of diseases in aquaculture.

One paper has just been submitted, our main results were:

  • We confirmed that a PO-like activity can be detected in vitro in the acellular fraction of the haemolymph from adult Pacific oysters, C. gigas.
  • PO is present in the acellular hemolymph in the form of a pro-enzyme and required a proteolytic cleavage for activation.
  • The PO like activity in C. gigas is sensitive to in vitro activation by LPS, zymosan or laminarin. Increase of the enzymatic activity does not appear to be dependent of the concentrations of exogen molecules used.
  • Increase of the enzymatic PO-like activity by exogen molecules in the study suggests that the pro-PO system takes place in non-self recognition and defence reactions in the adult C. gigas.
  • The present study establishes the effect of various factors on the pro-PO. Some molecules (tropolone, DETC, b -mercaptoethanol and PTU) inhibit the enzymatic phenoloxidase like response. On the other hand, some natural molecules (laminarin, zymosan, TPCK and LPS) stimulated this enzymatic system in C. gigas.
  • This study unambiguously demonstrated its presence as a proenzyme (proPO) in acellular fraction of haemolymph of C. gigas. Microbial substances enhance enzyme activity in vitro suggesting its potential role in host defence. The activation responses of this proenzyme in C. gigas to exogenous proteases, microbial cell wall components, and its susceptibility to protease inhibitors in vitro was similar to the proPO activation system of arthropods. The similarities in activation responses of haemolymph proPO system in animals belonging to two different phyla tend to imply a unifying biochemical mechanism for immune recognition among invertebrates.
  • In aquaculture, animals are submitted to stressful environmental and ecological conditions and this have been linked to high-density animal populations, pollution and nutritional imbalances. As ProPO and PO are involved in the immune response, a biochemical test on PO or Pro-Po activities could be used as a probe to measure health conditions of animals.

2 - Utilization of amino acid (histidine) by two microalgae: Chlamydomonas reinhardtii & Dunaliella tertiolecta

Research performed by Dr Claire Hellio, Dr Benoit Veron, Prof Yves Le Gal, from 1995 to date.

One of the important macronutrients controlling phytoplanktonic growth is nitrogen which is mainly available as inorganic nitrogen. During the spring, most of the nitrogen is present as nitrate. Autotrophic and heterotrophic organism using nitrate for growth convert it into ammonium and then into the different forms of organic nitrogen. In summer, as a consequence of the phytoplankton bloom, nitrate is depleted in large parts of the surface waters and the concentration of dissolved organic nitrogen compounds are then often higher than that of inorganic nitrogen. Under such conditions, it would be advantageous for phytoplanktonic species to have the ability to use organic nitrogen also, a large portion of which consists of dissolved free amino acids and dissolved combined amino acids. Understanding of the cellular and molecular mechanisms underlying nitrogen utilization in planktonic macroalgae is one of the obligatory keys to the development of ecological models. With this objective, we investigated the ability of Dunaliella tertiolecta & Chlamydomonas reinhardtii to grow with histidine as nitrogenous nutrient and examined the conditions of expression of histidase and related enzyme systems as a function of the combined nitrogen of carbon supplies.

This program has led to 3 publications (and 1 in preparation).

Our main results were:

  • The utilisation of His operated only when the ammonium source in the culture was nearly exhausted and the growth curve exhibited a profile characterising a phenomenon of diauxy, which can be interpreted as a manifestation of a catabolic repression mechanism. The most rapidly and available metabolised nitrogen sources, NH 4 +, inhibited or/and repressed the utilisation of the secondary N source, His. These results describe more precisely that D. tertiolecta & C. reihnardtii are able to use His for supporting their growth. However it can be seen from these experiments that the degradation of histidine is not complete. One interpretation could be that, under a given level, His is spared from degradation in ammonia and glutamate and could directly refuel the metabolic pathways.
  • When the NH 4 + concentration falls (below 4 mM for D. tertiolecta & 0.5 mM for C. reihnardtii), the 2 enzymes of histidine degradation pathway are expressed and showed a linear relationship between both activities. A similar observation was made when simultaneously measuring histidase and histidine permease in the same conditions. These results indicate that histidase, urocanase and histidine permease are submitted to a common system of regulation.
  • Histidase of D. tertiolecta & C. reihnardtii were purified and characterised. The molecular mass of enzyme was close to 155 kDa, corresponding to 4 identical sub-units of 38.2 kDa. These results are analogous to those discover in Pseudomonas aeruginosa (198 kDa), Bacillus subtilis (220 kDa), and Streptomyces griseus.
  • The substrate saturation curves of histidase at room temperature display a biphasic behaviour, and two sets of Hill numbers and Vm and Km values were determined when [His] were higher or lower than 20.5 mM. This is an indication that histidase may be allosterically regulated and show a cooperative response with respect to substrate concentration. This observation suggests that histidase, in addition to being regulated at the expression level is also subject to a more direct regulation operating at the level of enzyme activity. In the absence of a well structured separation between degradative and biosynthetic activity in the cell, histidase may be allosterically regulated and display a cooperative response with respect to histidine concentration.
  • We obtained a good indication that histidase may be allosterically regulated by some nucleotides, most notably purine nucleotides but possibly by cytosine nucleotides. Preliminary indications suggest that the inhibition by a mixture of effectors is simply additive. Thus, control of histidase would be achieved by the total concentration of all inhibitory compounds, each influenced by its binding constant. From a physiological viewpoint, however, the most likely regulators would be ATP and GTP, since these are in higher concentrations than any other nucleotides. Reversal of inhibition would require a drop in the pools of all inhibitory nucleotides and not simply a change in the degree of phosphorylation. The pattern of inhibition presently observed may reflect a biosynthetic aspect of the enzyme's function rather than its more acknowledged role in degradation for energy production via glutamate formation. One might reason that the histidine degradation pathway is an important source for C1 units essential for purine formation, since formyl glutamate, an intermediate in histidine degradation, can donate a formyl group to tetrahydrofolate. Under the conditions where histidine serves as a major carbon source, histidase may be regulated by the size of the total nucleotide pool, reflecting the need for the additional synthesis of purine nucleotides.

For more information, see:

C H ellio , Y Le Gal (1998) Histidine utilization by the unicellular alga Dunaliella tertiolecta. Comp. Biochem. Physiol. 119A, 4: 753-758.

C H ellio , Y Le Gal (1999) Histidase from the unicellular alga Dunaliella tertiolecta : purification and partial characterization. Eur. J. Phycol. 34: 71-78

C Hellio , B Veron, Y Le Gal (2004). Amino acid utilization by the unicellular alga Chlamydomonas reinhardtii (CCAP 11/32A): specific study of histidine. PPB. 42: 257-264