Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system

Abstract

Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30°C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of OPH in the cell surface-expression systems.

Bioadsorption Strategies with Yeast Molecular Display Technology

“…Although this article focused on fundamental applications of adsorption techniques using yeast molecular display systems, environmental solutions could be also achieved with similar systems such as using a phosphorus adsorbent as an antimicrobial agent Ohmoto et al, 2012 . Indeed, we have also investigated a phosphate-dependent molecular display method using the PHO5 gene Shibasaki et al, 2001a and displayed organophosphorus hydrolase Fukuda et al, 2010 . The results suggested that the display of any phosphate-binding proteins could be developed for use with novel phosphorus adsorbents FIG.…”

Estimation of Enzyme Kinetic Parameters of Cell Surface-displayed Organophosphorus Hydrolase and Construction of a Biosensing System for Organophosphorus Compounds

“…9 The yeast strain used was MT8-1 (MATa ade his3 leu2 trp1 ura3). Cells harboring pWIFSSigOPH were grown on a liquid SD-W medium consisting of 0.67% w/v yeast nitrogen base without amino acids, 0.5% w/v glucose, 0.002% w/v adenine sulfate, 0.002% w/v L-histidine-HCl, 0.003% w/v L-leucine, and 0.002% w/v uracil.…”

“…Using this system, the activity of OPH increased by nearly 10-fold compared with that obtained using the α-agglutinin anchor system. 9 Here, we conducted an enzymatic characterization of Flo1p anchor-fused OPH as a biocatalyst for the construction of an OPs biosensor.…”

Bioprospecting thermostable cellulosomes for efficient biofuel production from lignocellulosic biomass

“…Bioadsorption of rare and essential metal ions like tungstate and molybdate have also been reported for adsorption and resource recovery (Nishitani et al 2010;Kuroda et al 2012). Another field of their application is bioremediation, where they are used as pollutant removers (Fukuda et al 2010;Kuroda and Ueda 2010;Kuroda and Ueda 2011). Degradation of many toxic compounds using decontaminating enzymes fused with cellulose-binding CBMs has been reported, which enabled a single-step purification and immobilization of fusion proteins into different cellulosic materials (Xu et al 2002).…”