Development of an enzyme activity screening system for beta-glucosidase-displaying yeasts using calcium alginate micro-beads and flow sorting.
Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying beta-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls. This system should be a useful method for high-speed screening of yeast cells that display various hydrolyzing enzymes and has potential application to screening randomized libraries of enzyme-displayed yeast cells with higher activities.
Zhou
Yan
Y
Deptartment of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, Japan.
Kajiyama
Shin'ichiro
S
Itoh
Kouichi
K
Tanino
Takanori
T
Fukuda
Nobuo
N
Tanaka
Tsutomu
T
Kondo
Akihiko
A
Fukui
Kiichi
K
D000464:Alginates / Q000737:chemistry*; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D008863:Microspheres; D009795:Octanes / Q000494:pharmacology; D015003:Yeasts / Q000166:cytology / Q000201:enzymology*; D001617:beta-Glucosidase / Q000378:metabolism*
D000464:Alginates; D006603:Hexuronic Acids; D009795:Octanes; D020723:Glucuronic Acid; D001617:beta-Glucosidase; C045798:2,2,4-trimethylpentane