Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase.
Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.
Fendri
Ahmed
A
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS, BPW, Sfax 3038, Tunisia.
Frikha
Fakher
F
Mosbah
Habib
H
Miled
Nabil
N
Zouari
Nacim
N
Bacha
Abir Ben
AB
Sayari
Adel
A
Mejdoub
Hafedh
H
Gargouri
Youssef
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000494:pharmacology; D002645:Chickens; D003001:Cloning, Molecular; D003089:Colipases / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004655:Emulsions / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008049:Lipase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol / Q000494:pharmacology; D000069463:Olive Oil; D010179:Pancreas / Q000201:enzymology*; D010938:Plant Oils / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D014280:Triglycerides / Q000378:metabolism / Q000494:pharmacology
D001647:Bile Acids and Salts; D003089:Colipases; D003902:Detergents; D004655:Emulsions; D000069463:Olive Oil; D010938:Plant Oils; D014280:Triglycerides; D017830:Octoxynol; D008049:Lipase; C031627:tripropionin; C005830:tributyrin