A study on the diversity and production of microbial extracellular nucleases :potential anti-biofilm enzymes

Abstract

Microorganisms have been viewed as planktonic, free living single cells but predominantly they exist as sessile multispecies communities in the natural environment forming ‘biofilms’. Biofilms are beneficial for organisms to survive in natural environment as well as for biotechnological applications such as microbial fuel cells and bioremediation. However, biofilms are associated with disease persistence and biofouling and are comprised of adhered microbes within a hydrated matrix rich in polysaccharides, proteins and extracellular DNA (eDNA). eDNA is an important structural component and its degradation by deoxyribonucleases may be a novel approach to eradicate biofilm related problems. The present work was undertaken in this context to discover and produce microbial nucleolytic enzymes for applications for the control of harmful biofilms. Eighty six out of 260 bacterial isolates which included thermophilic and psychrophilic strains, showed deoxyribonuclease activity. The diversity and function of extracellular nucleases was also investigated throughout the microbial world using bioinformatics tools. Sequence driven analysis suggested that major bacterial lineages contain diverse extracellular nucleases with biological function related to nitrogen, phosphate and carbohydrate metabolism, protection, survival and virulence. Production optimisation for one specific extracellular nuclease, NucB, from Bacillus licheniformis EI-34-6 was carried out. This enzyme was previously known to cleave eDNA causing biofilm dispersal, and may therefore be used commercially to remove biofilms. The understanding of B. licheniformis physiology was applied in order to enhance NucB production 10-fold. For further characterisation of the enzyme and to Abstract iv understand its biological mechanism in breaking down biofilms, NucB was expressed in the SURE expression host B. subtilis NZ8900. This allowed a 68-fold increase in protein yield. NucB protein has been purified to high degree purity with specific nuclease activity of 15000 U/mg of protein. Biophysical characterisation showed that the protein was thermally stable and could reversibly refold. Statistical optimisation of extracellular nucleases production in diverse bacteria grown at different temperatures was demonstrated as a promising methodology for enhancing key enzyme secretion. The effectiveness of biofilm disruption by NucB was successfully tested with different single species biofilms grown on polystyrene, glass, and stainless steel surfaces. Biofilm dispersal efficiency of other microbial nucleases ranged between 60 – 95 % of removal after 1 h. The results presented in this thesis demonstrate that bacteria were able to produce nucleases across broad temperature range. In context to biofilm dispersal, bioinformatic analysis speculates the ecological implication of secreted diverse microbial extracellular nuclease-like genes were to decide the fate of eDNA and play pivotal role in nutrient cycling of the eco-system. Bioprocess development confirmed process optimisation can reliably produce functional and well-folded recombinant NucB at levels suitable for applications where biofilm removal is needed. Production optimisation of extracellular nucleases from diverse bacteria expanded the availability of different nucleases with wide range of anti-biofilm properties. Evidence is also presented to show that extracellular nucleases can disperse preformed microbial biofilms on different substrata. Microbial extracellular nucleases therefore appear to be a rich and unexplored source of anti-biofilm enzymes.