Lipase B(from Candida antarctica)

IntroductionLipases 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 224H-atom

Hydrophobic Sidechain Partition (orange) Ile 189, Ile 285

Oxyanion Hole (violet) Gly39, Thr40

Specificity Pocket (yellow) Trp104

Asp 187O-atom

Uppenberg (1994)

Positioning of Substrate

Alcohol side

Acyl sideLipid 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 AlignmentCAL-B 26 SPSSVSKP-ILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMV CAL-A 26 SPSSVSKP-ILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMV Aclav 175 YGKNGKKP-VILVPGTATPAGTTYHFSFAKLGSATNVDVVWLNIPQASLNDIQINAEYVA Aeromic 2 LAAGSQAP-VLFLHGTTSTSKANWSWNWAKAMKSAGRAYCLLDSPNGATGDIQVSAEYVV Strepcin 55 LRDAGDKPTVLFVPGTGLKGEENYAWNYMAELKKKGYQSCWVDSPGRGLRDMQESVEYVV Neofish 174 YGKNGKKP-VILVPGTATPAGTTYYFNFGKLGSAADADVVWLNIPQASLNDVQINSEYVA CAL-B 85 NAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAG-- CAL-A 85 NAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAG-- Aclav 234 YAINYISALTGSN-VAVISWSQGGPDTQWALKYWPTTRDVVDDFIAISPDFHGTVASS-- Aeromic 61 HAIRTMRARAGRP-ISIVGHSQGGMVGRWALKYWPDTRAMVDDYVGLSSSNHGSTSGV-- Strepcin 115 YATRAIQEATGRK-VDLVGHSQGGLLTAWALRFWPDLPGKVDDMVTLGSPFQGTRLAS-- Neofish 233 YAINYISALSESN-VAVLSWSQGGLDTQWALKYWPSTRKVVDDFIAISPDFHGTVVRS-- CAL-B 143 ----PLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPT-TNL-YSATDEIV---QPQ CAL-A 143 ----PLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPT-TNL-YSATDEIV---QPQ Aclav 291 -LACPWLKSLLCSPALWQQAWDSEFISTLRADGGDSAYVPT-TTI-YSSFDEIVQPMSGS Aeromic 118 -GLC--LIQGGCSAANWQQSAGSNFLAALNDGPETFPGIDY-TVI-GTRYDEIV----AP Strepcin 172 -PCRPIAEVAGCPASVLQFARDSNWSKALGADGTPMPAGPSYTTI-YSYADESV--VADG Neofish 290 -LVCPWLAALACTPSLWQQGWNTEFIRTLRGDGGDSAYVPT-TTI-YSTFDEIVQPMSGS CAL-B 194 VSNSPLDSSYLFNGKNVQAQAVCGPLF---VIDHAGSLTSQFSYVVGRSALRSTTGQARS CAL-A 194 VSNSPLDSSYLFNGKNVQAQAVCGPLF---VIDHAGSLTSQFSYVVGRSALRSTTGQARS Aclav 348 QASAILGDARAVGVSNNQVQTVCGSKPAGGIYTHEGVLYNPLAWALAVDAL-THDGPGDP Aeromic 169 PTPSFLEPAP--NVTNTMVQDLCPLQ----IVEHFGMAYDNAAWLIGIDAL-TNPGPAQL Strepcin 228 EAPSLPGAHR-IGV-----QDICPGRPWP---THIAMVVDQVSYDLVADAI-EHPGPADT Neofish 347 QASAILSDSRAVGVSNNHLQTICGGKPAGGVYTHEGVLYNPLAWALAVDAL-THDGPGDP CAL-B 251 ADYG-ITDCNPLPANDLTPEQKVAAAALLA-PAAAAIVAGPKQN-CEPDLMPYARPFAVG CAL-A 251 ADYG-ITDCNPLPANDLTPEQKVAAAALLA-PAAAAIVAGPKQN-CEPDLMPYARPFAVG Aclav 407 SRLNLDDVCGRLLPPQLGLDDFLGTEGLLLVGLAEALAYMPKTL-REPPIAGYAA----- Aeromic 222 DRVS-RATCLRPTMPSVNLLTF-GLDVVSALGVTAKNTIGAETVPAEPELRDYAR----- Strepcin 278 SRID-RAHCAKPVMPLNSQEAVDALPGLLNFPIELLIHSQPWVD-EEPPLRPYAR----- Neofish 406 SRLDLDVVCGRALPPQLGLDDLLGTEGLLLIALAEVLAYRPKTF-GEPAIASYAG-----

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, 293-308• Uppenberg, J.; et al. Biochemistry 1995, 34, 16838-16851• Raza, S.; et al. Protein Science 2001, 10, 329-338• Sanli, H.; Canakci, M. Energy & Fuels 2008, 22, 2713-2719• Dr. Stephen Hughes, ARS-USDA-NCAUR