High-level expression of Myrothecium verrucaria bilirubin oxidase in Pichia pastoris, and its facile purification and characterization.
Bilirubin oxidase (BO) from Myrothecium verrucaria (authentic BO) catalyzing the oxidation of bilirubin to biliverdine was overexpressed in the methylotrophic yeast, Pichia pastoris. The cDNA encoding BO was cloned into the P. pastoris expression vector pPIC9K under the control of the alcohol oxidase 1 promoter and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. The productivity of recombinant BO (rBO) in P. pastoris was approximately 5000 U/L of culture broth, being about 2.5- and 250-fold higher than rBO expressed in Aspergillus oryzae and S. cerevisiae, respectively. The calculated molecular mass of rBO consisting of 538 amino acids was 60,493 kDa, however, that of SDS-PAGE was 66 kDa because of non-native type N-linked sugar chains. The spectroscopic properties of rBO were typical of multicopper oxidase containing four Cu ions per protein molecule. The specific activity to oxidize bilirubin was 57 U/mg, having a value about twice that of authentic BO and rBO expressed in A. oryzae. Moreover, the thermostability of rBO expressed in P. pastoris was significantly high compared to the authentic BO previously reported. Accordingly, a heterologous expression system of rBO to meet clinical and industrial needs was constructed.
Kataoka
Kunishige
K
Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan. kataoka@cacheibm.s.kanazawa-u.ac.jp
Tanaka
Kazuhiro
K
Sakai
Yoko
Y
Sakurai
Takeshi
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D015870:Gene Expression; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D006999:Hypocreales / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008970:Molecular Weight; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014170:Transformation, Genetic
D004271:DNA, Fungal; D011994:Recombinant Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase