Lipase B (from Candida antarctica). Introduction Lipases are a group of enzymes that hydrolyze triglycerides at the lipid- water interface. Triglycerides.

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Presentation transcript:

Lipase B (from Candida antarctica)

Introduction Lipases are a group of enzymes that hydrolyze triglycerides at the lipid- water interface. Triglycerides can be cleaved at all three ester bonds or at one or two specifically. Lipases can show different specificities depending on the lengths of the fatty acids. Lipase B has a specificity towards long chain secondary alcohols, where cleavage at the ester bond forms ethyl esters. Lipase

Sequence 317 Amino Acids - 9 β-sheets - 10 α-helices Uppenberg (1995)

α/β Hydrolase Fold Helices and Sheets only Rotated 90 ˚ (x-axis)

Catalytic Triad Asp 189 His 224 Ser 105

Catalytic Triad within Secondary Structure

Active Site Pocket Ser 105 O-atom His 224 H-atom Hydrophobic Sidechain Partition (orange) Ile 189, Ile 285 Oxyanion Hole (violet) Gly39, Thr40 Specificity Pocket (yellow) Trp104 Asp 187 O-atom Uppenberg (1994)

Positioning of Substrate Alcohol side Acyl side Lipid Molecule Uppenberg (1995)

The order of the catalytic residues is important. Distinguishes which specific type of substrate to be hydrolyzed. Lipase B: Ser-His-Asp Subtilisn Family Proteases: Asp-His-Ser Chymotrypsin Family Proteases: His-Asp-Ser The catalytic serine in lipases is usually identified by the conserved sequence GxSxG, but in Lipase B, the first G is replaced with a T

Sequence Alignment

Serine Hydrolase Mechanism Raza (2001)

Serine Hydrolase Catalysis (enantiomer selective) Raza (2001)

R S Stereoselective towards the R conformation – provides better positioning of the small substituent on the chiral carbon, fitting directly into the oxyanion hole. In the S conformer, the bond between the substituent and chiral carbon would undergo a great degree of strain in order to reach the oxyanion hole. Uppenberg (1995) Raza (2001)

Industrial Applications Attaching Lipase B to an enzyme-carrier resin bead (e.g. Sepabead ® from Resindion Corp.) A solution of alcohol and vegetable oil can be ran through a column packed with these resin beads that contain the Lipase B enzyme, creating a elution of ethyl esters used in biodiesel fuel.

Overall Reaction Lipase B Sanli, Cankci (2008) Enzymatic Process for Biodiesel Production

References Uppenberg, J.; et al. Structure 1994, 2, Uppenberg, J.; et al. Biochemistry 1995, 34, Raza, S.; et al. Protein Science 2001, 10, Sanli, H.; Canakci, M. Energy & Fuels 2008, 22, Dr. Stephen Hughes, ARS-USDA-NCAUR