Microalgal Enzymes with Biotechnological Application

Vingiani, Giorgio Maria (2022). Microalgal Enzymes with Biotechnological Application. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00015444

Abstract

The aim of this thesis was to address the current state of art of microalgal enzymes with biotechnological application, and to expand the current knowledge of enzymes with the potential to find proper application in industry or novel market niches, via their production and biochemical characterization. Available transcriptomic datasets were used to identify possible enzymes of interest. Specifically, after a review of the available scientific literature (Chapter 1) my experimental activities focused on different enzymes identified in the transcriptome of the marine diatom Cylindrotheca closterium.

Studied enzymes were two di-n-butyl phthalate hydrolases (CcDBPH1-2), putatively involved in phthalate degradation (Chapter 2), two platelet activating factor- acetylhydrolase (CcPAF-AH1-2), involved in lipid metabolism and inflammation modulation, which role in diatom is still unknown (Chapter 3), a L-asparaginase (CcASNase), that is an enzyme with a well-supported market niche in cancer treatment and acrylamide mitigation (Chapter 4).

The experimental pipeline of CcDBPH1-2, starting from transcript identification till protein functional activity studies, was completed and reached the stage of protein purification and activity assay. In addition, different bioinformatics analyses and cc_dbph1-2 expression by RT-qPCR in C. closterium grown with different concentrations of DBP were also performed confirming evidences of the protein bioremediation activity.

The expression of CcPAF-AH1-2 was hampered by the strong toxic effect that recombinant proteins production induced in E. coli cells. However, the mutation of the catalytic serine 243 of CcPAF-AH2 allowed enzyme accumulation in 1L cultures.

Finally, the expression of CcASNase was successful in BL21 (DE3) and Rosetta 2 (DE3) E. coli strains. However, the low amount of produced protein will need further optimization of the growth conditions.

Overall, this PhD thesis represents a contribution to the growing number of studies aimed to unravel the application of microalgae as an eco-friendly and eco-sustainable source of novel enzymes for human applications.

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