In vitro evolution of lipase B from Candida antarctica using surface display in hansenula polymorpha.
ALipase B from Candida antarctica (CalB) displayed on the cell surface of H. polymorpha has been functionally improved for catalytic activity by molecular evolution. CalB was displayed on the cell surface by fusing to a cell-wall anchor motif (CwpF). A library of CalB mutants was constructed by in vivo recombination in H. polymorpha. Several mutants with increased whole-cell CalB activity were acquired from screening seven thousand transformants. The two independent mutants CalB10 and CalB14 showed an approximately 5 times greater whole-cell activity than the wild-type. When these mutants were made as a soluble form, CalB 10 showed 6 times greater activity and CalB14 showed an 11 times greater activity compared with the wild-type. Sequence analyses of mutant CALB genes revealed amino acid substitutions of Leu278Pro in CalB10 and Leu278Pro/Leu219Gln in CalB14. The substituted Pro278 in both mutants was located near the proline site of the alpha10 helix. This mutation was assumed to induce a conformational change in the alpha10 helix and increased the k(cat) value of mutant CalB approximately 6 times. Site-directed mutagenized CalB, LQ (Leu219Gln) was secreted into the culture supernatant at an amount of approximately 3 times more without an increase in the CalB transcript level, compared with the wild-type.
Kim
So-Young
SY
Systems Microbiology Research Center Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejon 305-333, Korea.
Sohn
Jung-Hoon
JH
Pyun
Yu-Ryang
YR
Yang
In Seok
IS
Kim
Kyung-Hyun
KH
Choi
Eui-Sung
ES
D019943:Amino Acid Substitution / Q000235:genetics; D004252:DNA Mutational Analysis; D019020:Directed Molecular Evolution / Q000379:methods*; D005656:Fungal Proteins; D007700:Kinetics; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica