Efficient in vitro synthesis of cis-polyisoprenes using a thermostable cis-prenyltransferase from a hyperthermophilic archaeon Thermococcus kodakaraensis.
The Tk-idsB encoding cis-prenyltransferase which catalyzes consecutive cis-condensation of isopentenyl diphosphate to allylic diphosphate was isolated from a hyperthermophilic archaeon Thermococcus kodakaraensis, and enzymatic characteristics of the recombinant Tk-IdsB were examined. Tk-IdsB was not fully denatured even at 90 degrees C and preferably utilizes both C(10) and C(15) allylic diphosphates to yield mainly the C(60)-C(65) products. Based on structural models, single alanine-substitution mutants at Glu68, Lys109, or Leu113 were constructed, showing that all the three produced longer chains (C(65)-C(70)) than the wild-type and the substitution at 109 (K109A) was the most effective. Tk-IdsB was applied to an organic-aqueous dual-phase system and more than 90% of the products were recovered from the organic phase when 1-butanol or 1-pentanol was overlaid. When 1-octanol was overlaid, 70% of the products were obtained from the upper organic phase. The product distributions were changed depending on the hydrophobicity of organic solvents used. Tk-IdsB was then immobilized onto silica beads to make Tk-IdsB more tolerant, showing that half-life of enzyme at 80 degrees C was prolonged by immobilization. When the immobilized Tk-IdsB was applied in the organic-aqueous dual-phase system, immobilized Tk-IdsB catalyzed consecutive condensation more efficiently than the unimmobilized one.
Yamada
Yosuke
Y
Department of Bioscience, Nanobiotechnology Research Center, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337, Hyogo, Japan.
Fukuda
Wakao
W
Hirooka
Kazutake
K
Hiromoto
Takeshi
T
Nakayama
Jun-Ichi
J
Imanaka
Tadayuki
T
Fukusaki
Ei-Ichiro
E
Fujiwara
Shinsuke
S
D020003:1-Octanol / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045782:Hemiterpenes / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000378:metabolism*; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004800:Enzymes, Immobilized; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C004809:isopentenyl pyrophosphate; D014166:Transferases; C106821:cis-prenyl transferase; D020003:1-Octanol