1 / 12

Adsorción específica

Purification via lipase-lipase interaction. Immobilized lipase. Low ionic strenght. Esterase. Eg: 5mM buffer. Other proteins. Adsorción específica. Purified Lipase. detergent. J.M. Palomo et al. J.Cromatogr A . 2004 , 1038 , 267-273. Enzyme selection.

michaelmmiller
Download Presentation

Adsorción específica

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Purification via lipase-lipase interaction Immobilized lipase Low ionic strenght Esterase Eg: 5mM buffer Other proteins Adsorción específica Purified Lipase detergent J.M. Palomo et al. J.Cromatogr A.2004, 1038, 267-273.

  2. Enzyme selection Open Conformation Structure of lipase from Pseudomonas fluorescens (PFL) Hydrophobic area Lysines Lid

  3. Purification of lipase from Bacillusthermocatenulatus (BTL) PFL 1. Molecular weight markers 2. Crude extract of BTL 3. Purified BTL Mw (kDa) 5 mM buffer 94 BTL Other proteins 66 45 Specific adsorption SDS,100ºC 30 2 1 3 detergent 20.1 Purified BTL J.M. Palomo et al. J.Cromatogr A.2004, 1038, 267-273.

  4. Preparation of Biocatalysts Conformational Engineering of lipases on solid phase Immobilization strategy …. Lipases …. Etc… Biocatalysts Library

  5. Different orientations of immobilized CAL-B Lid Hydrophobic residues Immobilization on octyl-agarose Carboxylic groups Immobilization on PEI-agarose Ser cat terminal amino group C. antarctica lipase (fracción B) (CAL-B) Lys Immobilization on glyoxyl-agarose Immobilization on BrCN-agarose

  6. Requirements for solid-phase synthesis of lipidated peptides fluorofor Farnesyl tioether (Far) Palmitoyl tioester (Pal) • Linker selection • Incorporation of aa after the palmitoylated cysteine • Access to peptides with different combination of lipidated groups • Introduction of connectors or labeled groups

  7. Lipidated peptides synthesis using sulfonamide linker R-OH R-NH2 Nucleophile attack 2. Solid phase peptide synthesis 1. Acilation Activation ICH2CN 64% J.M. Palomo et al.Angew. Chem. Int Ed. 2006, 45,477-481

  8. Importance of the preparation of semisynthetic Ras proteins Recombinant protein Tailor-made synthesized peptide C-Terminal N-Ras 30% Human cancers • Efect on Ras localization on plasma membrane C.Nicolini , S Schummer, JM Palomo et al. J.Am.Chem.Soc.,2006, 16, 869-874.

  9. Chemoenzymatic processes for the preparation of Neo-glycoconjugates Chemo-enzymatic routes Neo-Glycoconjugates = Carbohydrate oligosaccharides

  10. Regioselective Enzymatic Hydrolysis of peracetylated Monosaccharides χ Lipases hydrolysis - Specificity againts peracetylates molecules - Regioselectivity

  11. Specificity and Regioselectivity of the different immobilized preparations - peracetylated galactose TLL: lipase from T. lanuginosa J.M. Palomo et al. Adv. Synth Cat. 2007, 349,1969-1976

  12. Chemoenzymatic Synthesis of antitumor β-O-Naphtylmetyl-N-peracetylated Lactosamine M. Filice, R.Fernandez-Lafuente, G. Fernandez-Lorente, J.M. Guisan, J.M. Palomo*, M. Terreni*. J Mol. Cat B: Enzym. En prensa.

More Related