CARAPAX is a four year project which started in April 2001.
The overall goal of the project is to develop methods for the reproducible generation of bio-active chitosans from shrimp shell and squid pen chitin.

Read more about the progress in Year one, Year two, Year three, Year four and 6-month extension

The first year of the CARAPAX project was mainly devoted to:

1. The production of high quality chitin and chitosans as raw materials:
   The French and Norwegian partners, including the two chitosan producing companies, have optimised the traditional chemical methods of isolating chitin from shrimp shells and squid pen, to ensure minimum depolymerisation and minimum de-N-acetylation. This has yielded the very high molecular weight, very highly N-acetylated chitins to be used as raw materials for the processing into well defined chitosans.
2. The production of the enzymatic tools for chitin and chitosan processing:
   The German and Greek partners have identified and cloned a number of bacterial, fungal, and plant genes coding for chitinases and chitin de-N-acetylases, for the heterologous over-expression and large scale purification of the enzymes. These enzymes will be characterised to be used as diligent tools for the reproducible generation of designer chitosans.
3. The establishment of bio-assays to assess disease protective properties:
   The co-ordinating German partner has set up several plant/pathogen biosystems so that the plant disease protecting bio-activities of the chitosans produced can be assessed for some economically important crop plants and their most devastating fungal pathogens.
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The second year of the CARAPAX project - in addition to continuing the above projects - was mainly devoted to:

The production and characterisation of well defined chitosans:
The Norwegian partner has started to optimise the conditions for the partial de-N-acetylation of chitin followed by partial depolymerisation, while the French partner has invented a novel method for total de-N-acetylation of chitin with minimal depolymerisation (patent pending), followed by controlled partial depolymerisation and then partial re-N-acetylation. Both partners have thus generated chitosans with narrowly and well defined degrees of polymerisation and N-acetylation. These have been characterised in detail concerning their physico-chemical, chemical, and biological properties.
1. The production and characterisation of anti-chitosan antibodies:
   One of the German partners has started generating polyclonal and monoclonal antibodies against some of the well characterised chitosans produced so far. In order to generate anti-chitin antibodies, chitin-mimicking peptides have been identified to be used as antigens.
2. The bio-testing of chitosans to assess their disease protective properties:
   The co-ordinating German partner has continued to set up additional plant/pathogen biosystems for the bio-testing of the chitosans. When the first chitosans became available, they were tested for their ability to elicit or induce plant disease resistance. It became apparent that different chitosans induce different aspects of plant disease resistance, so that the hypothesis of a multiple molecular recognition of the pathogen by an infected host plant was developed.
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The third year of the CARAPAX project was mainly devoted to:

1. The production and characterisation of series of chitosan polymers and oligomers:
   The French and Norwegian partners have continued to generate chitosans with narrowly and well defined degrees of polymerisation and N-acetylation so that series with constant DP and varying DA, or with constant DA and varying DP became available for characterisation and bio-testing. Based on extensive physico-chemical characterisations of these chitosans, the French partner has formulated a general law of behaviour of chitosans in solution.
2. The production and characterisation of chitin and chitosan modifying enzymes:
   The German and Greek partners have heterologously expressed and purified a number of bacterial, fungal, and plant genes coding for chitinases and chitin de-N-acetylases. Together with the Norwegian partner, these enzymes have been characterised for their substrate specificities and the product patterns obtained. These enzymes will now be used to enzymatically generate chitin and chitosan samples to be included in the bio-assays.
3. The establishment of bio-activity matrices for chitosans in plant disease protection:
   The co-ordinating German partner has developed a method for the rapid assessment of antimicrobial activities of chitosans. The introduction of bio-activity matrices to visualise the biological properties of chitosans - elicitation of disease resistance mechanisms and anti-microbial properties - has helped to identify promising candidate chitosans for novel plant protectants. First greenhouse and field trials using these chitosans have been initiated by the German partners.
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The fourth year of the CARAPAX project was mainly devoted to:

The production of chitosan nanoparticles and oligomers with known architecture:
The French and Norwegian partners have generated larger amounts of selected chitosans for the greenhouse and field trials. Concomitantly, the French partner has started to generate series of fully and partially acetylated GlcN- and GlcNAc-oligomers and mixed GlcN/GlcNAc-oligomers with known architecture. The French partner has also started the generation and characterisation of chitin and chitosan nanoparticles to increase their solubility in water.
1. The use and optimisation of chitin and chitosan modifying enzymes and antibodies:
   The German, Greek, and Norwegian partners have collaborated in using the enzymatic tools to generate novel chitosan preparations for bio-testing. The Greek partner continued his efforts in genetically modifying and optimising the substrate specificities and/or product patterns of the enzymes now available. The German partner responsible for antibody production continued trying to identify and characterise clones generating suitable recombinant antibodies against chitin and chitosan.
2. The bio-testing of selected chitosans in plant disease protection:
   The greenhouse and field trials of the German partners have continued to identify promising applications for the most bio-active chitosans identified. Chitosans were used singly or in combination to profit from synergistic effects predicted by the various bio-activity matrices. An attempt was made to enlarge the scope of the general law of behaviour of chitosans to include not only their physico-chemical properties but also their biological activities.
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The 6-month extension of the CARAPAX project was mainly devoted to

Finishing the tasks performed during the first four years:
The French partner has continued the chemical synthesis of partially acetylated, mixed GlcN/GlcNAc-oligomers with fully known architecture. At the end of the project, these were not yet available, but a possible strategy for their synthesis has been identified and validated. This work will be continued after the end of the CARAPAX project. The German, Greek, and Norwegian partners have characterised the novel enzymatic properties of a plant chitinase with unusually strict substrate specificity. The heterologous expression of genes coding for chitosanases and chitin de-N-acetylases in procaryotic and eucaryotic expression systems continued to meet with great problems so that recombinant strains expressing such genes eventually became available, but the yield was yet insufficient for characterising the enzymes. This work will be continued after the end of the CARAPAX project. The generation of chitosan specific antibodies was finally abandoned as even highly sophisticated in vitro techniques did not yield suitable antibodies with convincing binding properties.
1. Identifying strategies for further research into chitosan based plant protection:
   The field trials of the German partners showed that none of the chitosan preparations used had any adverse effects on plant growth and performance. In a number of crops such as strawberry and grapes, promising and reliable results were obtained with selected chitosans at concentrations of 160 g per hectare. Disease protection was generally in the range of ca. 50 % so that the efficacy did not reach that of commercial chemical fungicides. Further research beyond the CARAPAX project will focus on the optimisation of the chitosan used, based on the proposed new general law of behaviour of chitosans which causally links the biological activities of partially acetylated chitosan polymers with their physico-chemical properties. In the future, selected chitosans will be combined with known plant protectants, attempting to identify synergistic effects helping the development of knowledge based, chitosan containing commercial plant protecting or plant strengthening products.
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