Biosynthesis, Isolation and Magnetization of gellan spheres for biorecognition of therapeutic His-tag proteins

• Autor(a) principal: Coelho, Joana Filipa da Silva
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/6687
• topic: Biossíntese; Catechol-O-Methyltransferase; Goma de Gelana; Magnetização; Sphingomonas Paucimobilis; Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas

The bacterium Sphingomonas paucimobilis ATCC 31461 has the ability to produce a low cost anionic polysaccharide gellan gum, composed of a tetrasaccharide structure of glucose, glucuronic acid and rhamnose units. This biopolymer has many applications in the food, pharmaceutical and cosmetic industries. Typically, for gellan aggregation, the presence of divalent ions is required to decrease the electrostatic repulsion between helices, allowing the cross-link. Curiously, the affinity interaction between transition metal ions and histidine has been used as a site-specific, noncovalent method for the purification and immobilization of recombinant proteins bearing three to ten consecutive histidine amino acids at their amino- or carboxyl-terminus. Thus, the present project intends to formulate and magnetize biosynthetic gellan spheres to extract COMT protein from a lysate sample. Gellan gum production was improved by using two different media, N medium and S medium. The fermentation procedure was performed with a stirring of 250 rpm at 30 ºC for 48 hours, and a higher production of gellan gum was achieved through the S medium. Then, in order to recover the gellan gum produced by fermentation, different processes, like filtration, dialysis, washes with acetone and ether and dissolution in distilled water, were tested. The best method to recover gellan gum with a high degree of purity, comparatively with the commercial gellan gum was the filtration with acetone and ether and dissolution with distilled water. All the samples obtained from the different procedures of recovery were analyzed by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The gellan spheres were prepared by water-in-oil emulsion technique. For that, the gellan gum was dissolved in distilled water with a stirring of 300 rpm at 90 ºC during 30 minutes and this solution was transferred to a syringe and trickled in individual drops into a 100 % vegetable cooking oil solution with a 750 rpm stirring, at 100 ºC. The mixture was transferred to different solutions of 200 mM BaCl2, CaCl2, CoCl2, CuCl2 and NiCl2 with a stirring of 750 rpm for 30 minutes, at room temperature. The spheres with nickel as a cross-linker were magnetized by the chemical co-precipitation method. All the spheres with different cross-linkers and magnetized spheres were characterized morphologically and chemically by scanning electron microscopy and energy-dispersive X-Ray spectroscopy. Finally, the gellan spheres were used to capture model proteins, BSA and lysozyme, and a more complex protein, SCOMT, through the batch method. Thus, different conditions to bind and elute the protein were study, through the variation of the amount of spheres, the presence of urea in the lysate, the pH and ionic strength of the binding and elution solutions. So, the best conditions for the capture of the total protein from the complex lysate are the equilibrium of 10 mL of spheres (with or without magnetization) at acidic pH, binding step with urea in the SCOMT lysate and elution step with the increase of pH and then the ionic strength. However, to selectively isolate the SCOMT, it could be more adequate to explore the equilibrium and binding conditions with a pH around 7.5 and the elution condition by decreasing the pH to the protein isoelectric point with magnetic spheres.