Enzymatic production of soluble bioactive β-1,3-glucans from paramylon

Paramylon is a high-molecular weight unbranched β-1,3-glucan polymer produced by the flagellated microalga Euglena gracilis. Paramylon and other β-1,3-glucans have been reported to have antitumor and immunostimulating activities, making them ideal candidates as nutraceuticals or adjuvants. Their bioactivity can be increased by breaking them down to shorter-chain soluble saccharides by chemical or enzymatic methods. Here, chemical, physical and enzymatic approaches were examined as individual or combined strategies to facilitate the production of soluble bioactive compounds from paramylon. A microwave method was developed for the pretreatment of paramylon granules, which otherwise are resistant to enzymatic hydrolysis. The granules were sourced commercially after attempts to produce paramylon in an open pond cultivation proved unsuccessful. The paramylon granules were hydrolysed with a selection of commercially-sourced enzymes and a dye-based assay was devised to assess the efficacy of the hydrolysis. The new microwave pretreatment resulted in improved enzyme accessibility and thereby substantially enhanced the hydrolysis of paramylon granules. The soluble β-1,3-glucans produced with this method were characterised by highperformance liquid chromatography (HPLC) and an immunological assay on mouse macrophages showed that they were bioactive. The pathway for enzymatic degradation of paramylon in E. gracilis was also explored with Escherichia coli cells transformed with an E. gracilis-derived cDNA library. Recombinants were screened for the production of E. gracilis β-1,3-glucanases using an assay which combines fluorescence-activated cell sorting (FACS) with enzyme activity tests in microtiter plates. Both methods were based on the use of the fluorogenic β-glucan analogue fluorescein di-β-D glucopyranoside. However, the identification of new enzymes was prevented probably due to the suboptimal quality of the original cDNA library. In a parallel approach, protein fractions obtained from E. gracilis were analysed using mass spectrometry and a recently published transcriptomic database. Four putative enzymes with a high amino acid sequence similarity to known β-1,3-glucanase sequences were identified based on their translated DNA sequences. One of the corresponding genes was introduced into E. coli and into Saccharomyces cerevisiae to produce a recombinant enzyme. Unfortunately, expression levels were too low for reliable enzyme activity assays and further characterisation. Finally, various commercially-available and in-house enzyme preparations were shown to degrade paramylon on a dye-based plate assay. This new source of paramylon-degrading enzymes deserves further characterisation, which unfortunately was outside the time-frame of this project but could form the basis for promising future work