Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris.
The glucan 1,3-beta-glucosidase A gene (exgA) from Aspergillus oryzae and fused to the Saccharomyces cerevisiae signal peptide (α-factor) was expressed under the control of either a constitutive (GAP) or an inducible (AOX1) promoter in Pichia pastoris. A 1.4-fold higher extracellular enzyme activity (2 U/ml) was obtained using the AOX1 inducible expression system than with the GAP constitutive promoter (1.4 U/ml). The purified recombinant ExgA enzyme, with a yield of 10 mg protein/l culture supernatant, was about 40 kDa by SDS-PAGE analysis with a specific activity of 289 U/mg protein. The enzyme was optimally active at 35 °C and pH 5.0 and displayed a K(M) and V(max) of 0.56 mM and 10,042 μmol/(min mg protein), respectively, with p-nitrophenyl-β-D-glucopyranoside as the substrate. Moreover, it was tolerant to glucose inhibition with a K(i) of 365 mM.
Biological Science Program, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok 10330, Thailand. nassapat.boonvitthya@hotmail.com
Nassapat
N
Boonvitthya
Phatrapan
P
Tanapong
Patcharaporn
P
Kanngan
Vorakan
V
Burapatana
Warawut
W
Chulalaksananukul
D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D043326:Glucan 1,3-beta-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013696:Temperature
D004271:DNA, Fungal; D005656:Fungal Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D043326:Glucan 1,3-beta-Glucosidase; D005947:Glucose