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J.M. Palomo et al. J.Cromatogr A.2004, 1038,

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Presentation on theme: "J.M. Palomo et al. J.Cromatogr A.2004, 1038,"— Presentation transcript:

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

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

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

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

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

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

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

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

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

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

11 J.M. Palomo et al. Adv. Synth Cat. 2007, 349,1969-1976
Specificity and Regioselectivity of the different immobilized preparations - peracetylated galactose TLL: lipase from T. lanuginosa Enzime Support Activity [mol/h. mgprot] conversion [%] Yield 1-OH 6-OH others TLL Octyl 0.48 100 99 BrCN 0.01 95 85 10 PEI 0.02 25 14.5 7.5 3 J.M. Palomo et al. Adv. Synth Cat. 2007, 349,

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.

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