Enhancement of the organic solvent-stability of the LST-03 lipase by directed evolution.
LST-03 lipase from an organic solvent-tolerant Pseudomonas aeruginosa LST-03 has high stability and activity in the presence of various organic solvents. In this research, enhancement of organic solvent-stability of LST-03 lipase was attempted by directed evolution. The structural gene of the LST-03 lipase was amplified by the error prone-PCR method. Organic solvent-stability of the mutated lipases was assayed by formation of a clear zone of agar which contained dimethyl sulfoxide (DMSO) and tri-n-butyrin and which overlaid a plate medium. And the organic solvent-stability was also confirmed by measuring the half-life of activity in the presence of DMSO. Four mutated enzymes were selected on the basis of their high organic solvent-stability in the presence of DMSO. The organic solvent-stabilities of mutated LST-03 lipase in the presence of various organic solvents were measured and their mutated amino acid residues were identified. The half-lives of the LST-03-R65 lipase in the presence of cyclohexane and n-decane were about 9 to 11-fold longer than those of the wild-type lipase, respectively. Some substituted amino acid residues of mutated LST-03 lipases have been located at the surface of the enzyme molecules, while some other amino acid residues have been changed from neutral to basic residues.
Kawata
Takuya
T
Department of Chemical Engineering, Osaka Prefecture University, Naka-ku, Sakai, Osaka 599-8531, Japan.
Ogino
Hiroyasu
H
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009930:Organic Chemicals / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D012997:Solvents / Q000737:chemistry
D009930:Organic Chemicals; D012997:Solvents; D008049:Lipase