Kinetic studies of Gly28:Ser mutant form of Bacillus pumilus lipase: changes in k(cat) and thermal dependence.
Lipases are useful catalysts for a wide variety of industrial purposes. Herein we report the stability and thermal dependence of the activity of wild-type Bacillus pumilus lipase (BplA) and four site-directed mutants designed to improve its thermal stability. The Gly28:Ser mutation produces a dramatic four-fold increase in its k(cat) and a remarkable increase in its stability. While the increase in k(cat) is temperature-independent, the increase in stability shows that the resultant interactions of this mutation have a strong enthalpic component. Thermal dependence of stability, k(cat), K(M) and k(cat)/K(M) were analysed to gain insight on the structural effects of mutations on BplA. Our results are consistent with a gain in enzyme mobility for those mutants displaying enhanced catalytic properties; the analysis of thermal dependence of kinetic parameters indicates that the mutations did not change either the catalytic mechanism or the rate-limiting step of catalysis.
Bustos-Jaimes
Ismael
I
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico. ismaelb@unam.mx
Mora-Lugo
Rodrigo
R
Calcagno
Mario L
ML
Farrés
Amelia
A
D000465:Algorithms; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012694:Serine; C556257:BplA protein, Bacillus pumilus; D008049:Lipase; D005998:Glycine