ENZYMES FROM MARINE MICROORGANISMS FOR THE PREPARATION OF BIOLOGICALLY ACTIVE MOLECULES

This PhD project focuses on the identification, isolation and characterization of new biocatalysts able to generate biologically active molecules with significant enantioselectivity. Through screening, we identified marine strains, from MaCuMBA (Marine Culturable Microorganism for Biotechnological Applications) and BIODEEP (Biotechnologies form the deep) European project collections, which show a marked enantioselectivity on intermediates of molecules of biological interest. Biotransformation substrate range included pramipexole, as main target, but it also embraces other common building blocks for synthetic industrial preparation. The stereoselective reduction of structurally different ketones using halotolerant marine yeasts (Meyerozyma guilliermondii and Rhodotorula mucilaginosa) was studied using cells grown and bio-converted in seawater. The preparation of valuable chemicals through water-saving (bio)processes based on the direct exploitation of seawater is a significant step towards sustainable biocatalysis. By choosing a suitable strain, high yields and stereoselectivity could be achieved in most cases. Notably, high chemoselectivity and enantioselectivity were observed using R. mucilaginosa in the reduction of aromatic β-ketonitriles, which allowed the recovery of the optically pure corresponding alcohols; notably, reduction with whole cells of yeasts generally give a mixture of undesired products, as observed with M. guilliermondii. Keto-reduction potential of thirty-three marine bacterium species was checked and afterwards the possibility to convert this substrate directly into the optically pure amine was investigated: marine bacteria were screened to identify transaminase activity. Based on the previous results in terms of halotolerance and transaminase activity, the marine bacterium strain Virgibacillus pantothenticus 21D was selected for the genome sequencing in order to clone and express an ω-transaminase enzyme. A recombinant non-marine ketoreductase from Pichia glucozyma (KRED1-Pglu) was used for the enantioselective reduction of various cyclic ketones including pramipexole ketone intermediate. Thanks to co-factor recycling system, the purified enzyme showed very promising results. The soluble expression of a novel omega transaminase from a newly isolated halotolerant marine bacterium Virgibacillus pantothenticus was attained. Despite of several standard methodologies applied, the marine wild-type enzyme was total insoluble in E. coli host and it was satisfactorily solubilized by one single-point mutation, allowing the characterization of the new omega transaminase. The enzyme shows an interesting salt and solvent tolerance, in accordance to its origin and it results particularly active on some interesting building blocks molecules.