Biochemical characterization and high-level production of oxidized polyvinyl alcohol hydrolase from Sphingopyxis sp. 113P3 expressed in methylotrophic Pichia pastoris.
The Sphingopyxis sp. 113P3 gene oph, encoding oxidized polyvinyl alcohol hydrolase (OPH), was optimized with the preferred codons of Pichia pastoris and ligated into the pPIC9K vector behind the α-factor signal sequence. The vector was then transfected into P. pastoris GS115 and genomic integration was confirmed. Large-scale production of recombinant protein was performed by induction with 14.4 g/L methanol at 22 °C in a 3-L bioreactor. The maximal OPH activity obtained was 68.4 U/mL, which is the highest activity reported. The optimal pH and temperature of recombinant OPH were 8.0 and 45 °C, respectively. OPH activity was stable over a pH range of 5.0-8.5, and at a maximal temperature of 45 °C. The K cat /K m of recombinant OPH was 598 mM(-1) s(-1), which was 4.27-fold higher than that of recombinant OPH derived from Escherichia coli. The improved catalytic efficiency of OPH expressed in recombinant P. pastoris makes it favorable for industrial applications.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China, yangyu5352@gmail.com.
Yu
Y
Yang
Long
L
Liu
Jianghua
J
Li
Guocheng
G
Du
Jian
J
Chen
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D042301:Sphingomonadaceae / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases