Enhancement of the aspartame precursor synthetic activity of an organic solvent-stable protease.
The PST-01 protease is highly stable and catalyzes the synthesis of the aspartame precursor with high reaction yields in the presence of organic solvents. However, the synthesis rate using the PST-01 protease was slower than that observed when thermolysin was used. Structural comparison of both enzymes showed particular amino acid differences near the active center. These few residue differences in the PST-01 protease were mutated to match those amino acid types found in thermolysin. The mutated PST-01 proteases at the 114th residue from tyrosine to phenylalanine showed enhancement of synthetic activity. This activity was found to be similar to thermolysin. In addition, mutating the residue in the PST-01 protease with arginine and serine showed more improvement of the activity. The mutant PST-01 protease should be more useful than thermolysin for the synthesis of the aspartame precursor, because this enzyme has higher stability and activity in the presence of organic solvents. The results show the potential of organic solvent-stable enzymes as industrial catalysts.
Ogino
Hiroyasu
H
Department of Chemical Engineering, Osaka Prefecture University, Naka-ku, Sakai, Japan. ogino@chemeng.osakafu-u.ac.jp
Tsuchiyama
Shotaro
S
Yasuda
Masahiro
M
Doukyu
Noriyuki
N
D000595:Amino Acid Sequence; D001218:Aspartame / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D004795:Enzyme Stability / Q000187:drug effects; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009930:Organic Chemicals / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D012997:Solvents / Q000494:pharmacology*; D013379:Substrate Specificity; D013820:Thermolysin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D009930:Organic Chemicals; D012997:Solvents; D010447:Peptide Hydrolases; D013820:Thermolysin; D001218:Aspartame