The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono- and diglyceride specific lipase.
The extracellular lipase from Penicillium camembertii has unique substrate specificity restricted to mono- and diglycerides. The enzyme is a member of a homologous family of lipases from filamentous fungi. Four of these proteins, from the fungi Rhizomucor miehei, Humicola lanuginosa, Rhizopus delemar and P. camembertii, have had their structures elucidated by X-ray crystallography. In spite of pronounced sequence similarities the enzymes exhibit significant differences. For example, the thermostability of the P. camembertii lipase is considerably lower than that of the H. lanuginosa enzyme. Since only the P. camembertii enzyme lacks the characteristic long disulfide bridge, corresponding to Cys22-Cys268 in the H. lanuginosa lipase, we have engineered this disulfide into the former enzyme in the hope of obtaining a significantly more stable fold. The properties of the double mutant (Y22C and G269C) were assessed by a variety of biophysical techniques. The extra disulfide link was found to increase the melting temperature of the protein from 51 to 63 degrees C. However, no difference is observed under reducing conditions, indicating an intrinsic instability of the new disulfide. The optimal temperature for catalytic activity decreased by 10 degrees C and the optimum pH was shifted by 0.7 units to more acidic.
Yamaguchi
S
S
Tsukuba Research Laboratories, Amano Pharmaceutical Co. Ltd., Japan.
Takeuchi
K
K
Mase
T
T
Oikawa
K
K
McMullen
T
T
Derewenda
U
U
McElhaney
R N
RN
Kay
C M
CM
Derewenda
Z S
ZS
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004075:Diglycerides; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D005989:Glycerides; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D013379:Substrate Specificity; D013696:Temperature
D017931:DNA Primers; D004075:Diglycerides; D004220:Disulfides; D005989:Glycerides; D008049:Lipase