Alternative pig liver esterase (APLE) - cloning, identification and functional expression in Pichia pastoris of a versatile new biocatalyst.
Isolated from pig liver as a crude, inhomogeneous enzyme fraction, pig liver esterase (PLE) was found to metabolize a wide range of substrates; often in a highly stereoselective manner. This crude esterase preparation, however, contains several iso-enzymes at proportions varying from batch to batch. Racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate is cleaved enantioselectively by crude PLE, but not by recombinantly expressed gamma-isoform of PLE. Concluding that another PLE iso-enzyme must carry the relevant activity, we cloned and sequenced cDNAs of several PLE isoforms and functionally expressed them in Pichia pastoris. One novel isoform termed alternative pig liver esterase (APLE) was found to hydrolyze methyl-(2R,4E)-5-chloro-2-isopropyl-4-pentenoate in a highly stereoselective manner (E>200). When heterologously expressed and directed for secretion in P. pastoris, APLE was found to be localized in the periplasm. The presence or absence of a putative C-terminal ER retention signal did neither influence functional expression nor cellular localization. The recombinant enzyme, purified by ion exchange chromatography, had a specific activity of 36U (mg protein)(-1) towards racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate.
Hermann
Manuela
M
Applied Biocatalysis Research Centre, Graz, Austria.
Kietzmann
Martin U
MU
Ivancić
Mirela
M
Zenzmaier
Christoph
C
Luiten
Ruud G M
RG
Skranc
Wolfgang
W
Wubbolts
Marcel
M
Winkler
Margit
M
Birner-Gruenberger
Ruth
R
Pichler
Harald
H
Schwab
Helmut
H
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D008110:Liver Extracts; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D019897:Periplasm / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity; D034421:Sus scrofa / Q000378:metabolism*
D005229:Fatty Acids, Monounsaturated; D008110:Liver Extracts; D010446:Peptide Fragments; D011994:Recombinant Proteins; C009785:4-pentenoic acid; D004950:Esterases