Thermal stabilization of penicillolysin, a thermolabile 19 kDa Zn2+-protease, obtained by site-directed mutagenesis.
Penicillolysin is a member of the clan MX and the family of M35 proteases. The enzyme is a thermolabile Zn(2+)- protease from Penicillium citrinum with a unique substrate profile. We expressed recombinant penicillolysin in Aspergillus oryzae and generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intention of exploring thermal stabilization of this protein. We based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from A.oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from the edible mushroom Grifora frondsa. The resulting mutant proteins exhibited comparable catalytic efficiency to the wild-type enzyme and some showed a higher tolerance to temperature.
Doi
Yuko
Y
Laboratory of Molecular Enzymology, Graduate School of Bioengineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
Akiyama
Hidetoshi
H
Yamada
Yoshiteru
Y
Ee
Chng Ewe
CE
Lee
Byung Rho
BR
Ikeguchi
Masamichi
M
Ichishima
Eiji
E
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D004220:Disulfides; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D007526:Isoelectric Point; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010407:Penicillium / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013054:Spectrophotometry, Atomic; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*
D004220:Disulfides; D005656:Fungal Proteins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C083042:penicillolysin; D015032:Zinc