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Lipase B
(from Candida antarctica)
Introduction
Lipases are a group of enzymes that hydrolyze triglycerides at the lipidwater 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
Hydrophobic Sidechain Partition
(orange) Ile 189, Ile 285
Asp 187
O-atom
Ser 105
O-atom
His 224
H-atom
Oxyanion Hole (violet)
Gly39, Thr40
Uppenberg (1994)
Specificity Pocket
(yellow) Trp104
Positioning of Substrate
Lipid Molecule
Alcohol side
Uppenberg (1995)
Acyl side
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
CAL-B
CAL-A
Aclav
Aeromic
Strepcin
Neofish
26
26
175
2
55
174
SPSSVSKP-ILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMV
SPSSVSKP-ILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMV
YGKNGKKP-VILVPGTATPAGTTYHFSFAKLGSATNVDVVWLNIPQASLNDIQINAEYVA
LAAGSQAP-VLFLHGTTSTSKANWSWNWAKAMKSAGRAYCLLDSPNGATGDIQVSAEYVV
LRDAGDKPTVLFVPGTGLKGEENYAWNYMAELKKKGYQSCWVDSPGRGLRDMQESVEYVV
YGKNGKKP-VILVPGTATPAGTTYYFNFGKLGSAADADVVWLNIPQASLNDVQINSEYVA
CAL-B
CAL-A
Aclav
Aeromic
Strepcin
Neofish
85
85
234
61
115
233
NAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAG-NAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAG-YAINYISALTGSN-VAVISWSQGGPDTQWALKYWPTTRDVVDDFIAISPDFHGTVASS-HAIRTMRARAGRP-ISIVGHSQGGMVGRWALKYWPDTRAMVDDYVGLSSSNHGSTSGV-YATRAIQEATGRK-VDLVGHSQGGLLTAWALRFWPDLPGKVDDMVTLGSPFQGTRLAS-YAINYISALSESN-VAVLSWSQGGLDTQWALKYWPSTRKVVDDFIAISPDFHGTVVRS--
CAL-B
CAL-A
Aclav
Aeromic
Strepcin
Neofish
143
143
291
118
172
290
----PLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPT-TNL-YSATDEIV---QPQ
----PLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPT-TNL-YSATDEIV---QPQ
-LACPWLKSLLCSPALWQQAWDSEFISTLRADGGDSAYVPT-TTI-YSSFDEIVQPMSGS
-GLC--LIQGGCSAANWQQSAGSNFLAALNDGPETFPGIDY-TVI-GTRYDEIV----AP
-PCRPIAEVAGCPASVLQFARDSNWSKALGADGTPMPAGPSYTTI-YSYADESV--VADG
-LVCPWLAALACTPSLWQQGWNTEFIRTLRGDGGDSAYVPT-TTI-YSTFDEIVQPMSGS
CAL-B
CAL-A
Aclav
Aeromic
Strepcin
Neofish
194
194
348
169
228
347
VSNSPLDSSYLFNGKNVQAQAVCGPLF---VIDHAGSLTSQFSYVVGRSALRSTTGQARS
VSNSPLDSSYLFNGKNVQAQAVCGPLF---VIDHAGSLTSQFSYVVGRSALRSTTGQARS
QASAILGDARAVGVSNNQVQTVCGSKPAGGIYTHEGVLYNPLAWALAVDAL-THDGPGDP
PTPSFLEPAP--NVTNTMVQDLCPLQ----IVEHFGMAYDNAAWLIGIDAL-TNPGPAQL
EAPSLPGAHR-IGV-----QDICPGRPWP---THIAMVVDQVSYDLVADAI-EHPGPADT
QASAILSDSRAVGVSNNHLQTICGGKPAGGVYTHEGVLYNPLAWALAVDAL-THDGPGDP
CAL-B
CAL-A
Aclav
Aeromic
Strepcin
Neofish
251
251
407
222
278
406
ADYG-ITDCNPLPANDLTPEQKVAAAALLA-PAAAAIVAGPKQN-CEPDLMPYARPFAVG
ADYG-ITDCNPLPANDLTPEQKVAAAALLA-PAAAAIVAGPKQN-CEPDLMPYARPFAVG
SRLNLDDVCGRLLPPQLGLDDFLGTEGLLLVGLAEALAYMPKTL-REPPIAGYAA----DRVS-RATCLRPTMPSVNLLTF-GLDVVSALGVTAKNTIGAETVPAEPELRDYAR----SRID-RAHCAKPVMPLNSQEAVDALPGLLNFPIELLIHSQPWVD-EEPPLRPYAR----SRLDLDVVCGRALPPQLGLDDLLGTEGLLLIALAEVLAYRPKTF-GEPAIASYAG-----
Serine Hydrolase Mechanism
Raza (2001)
Serine Hydrolase Catalysis
(enantiomer selective)
Raza (2001)
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.
R
S
Raza (2001)
Uppenberg (1995)
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.
Enzymatic Process for
Biodiesel Production
Overall Reaction
Lipase B
Sanli, Cankci (2008)
References
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•
•
•
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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