Protein Engineering vol. 16 no. 12 pp. 1139-1145, 2003
© 2003 Oxford University Press
Recombinant porcine intestinal carboxylesterase: cloning from the pig liver esterase gene by site-directed mutagenesis, functional expression and characterization
Institute of Chemistry and Biochemistry, Department of Technical Chemistry and Biotechnology, Greifswald University, Soldmannstrasse 16, D-17487 Greifswald, Germany 1Present address: Department of Biochemistry, Lund University, Getingevägen 60, 221 00 Lund, Sweden
2 To whom correspondence should be addressed. e-mail: uwe.bornscheuer{at}uni-greifswald.de
It was shown recently that proline-ß-naphthylamidase from pig liver resembles the 
-subunit of pig liver esterase (PLE), which could be functionally expressed in the yeast Pichia pastoris in recombinant form (rPLE). The gene encoding rPLE shares 97% identity with the published nucleotide sequence of porcine intestinal carboxylesterase (PICE). By site-directed mutagenesis, 22 nucleotides encoding 17 amino acids were exchanged stepwise from the PLE gene yielding the recombinant PICE sequence and eight intermediate mutants. All esterases were successfully produced in P.pastoris as extracellular proteins with specific activities ranging from 4 to 377 U/mg and Vmax/Km values from 12 to 1000 l min–1 x 10–3 using p-nitrophenyl acetate as substrate. Activity-staining of native polyacrylamide gels followed by molecular mass determination suggests that the most active forms of all variants are present as trimers with a molecular mass of 190–210 kDa. All enzymes exhibit the highest activity in the pH range 8–9 and between 60 and 70°C. Almost all esterases show a higher ratio of methyl butyrate hydrolase activity to proline-ß-naphthylamidase activity than rPLE.
Received May 12, 2003; revised October 10, 2003; accepted October 21, 2003
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