Production and characterization of recombinant Phanerochaete chrysosporium beta-glucosidase in the methylotrophic yeast Pichia pastoris.
The extracellular beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium was expressed heterologously in the methylotrophic yeast Pichia pastoris. After 7 days' cultivation in an induction medium containing 1% (v/v) methanol, the expression level of the recombinant enzyme was 28,500 U/l, 38 times that of the wild-type enzyme. The specific activity of the crude recombinant enzyme for p-nitrophenyl-beta-D-glucoside was 52 U/mg, 37 times that of the wild-type enzyme; this difference made the purification of the enzyme simple. On a SDS-PAGE, the molecular mass of the recombinant enzyme was 133 kDa, and that of the wild-type enzyme was 116 kDa, but the difference had no effect on the hydrolysis of cellobiose or p-nitrophenyl-beta-D-glucoside. We concluded that the recombinant enzyme produced by Pichia pastoris retains the catalytic properties of the wild-type enzyme from Phanerochaete chrysosporium.
Kawai
Rie
R
Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.
Yoshida
Makoto
M
Tani
Tomomi
T
Igarashi
Kiyohiko
K
Ohira
Tsuyoshi
T
Nagasawa
Hiromichi
H
Samejima
Masahiro
M
D002482:Cellulose / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D007700:Kinetics; D020075:Phanerochaete / Q000201:enzymology* / Q000254:growth & development; D010843:Pichia / Q000201:enzymology*; D011485:Protein Binding; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D001617:beta-Glucosidase / Q000096:biosynthesis*
D012331:RNA, Fungal; D011994:Recombinant Proteins; D002482:Cellulose; D001617:beta-Glucosidase