Efficient production of active form of recombinant cassava hydroxynitrile lyase using Escherichia coli in low-temperature culture.
Overexpression and production of the high concentration of hydroxynitrile lyase from cassava (Manihot esculenta (MeHNL, EC 4.1.2.39)) were investigated. Hydroxynitrile lyase is a useful enzyme for the production of optically active cyanohydrin compounds. The production of MeHNL was increased by changing the rare codons of the original sequence of cassava MeHNL. However, most of the produced MeHNL was in the insoluble form. In order to increase the solubility of MeHNL, the effects of the cultivation temperature were investigated. When the cultivation temperature was reduced, the cell yield and the ratio of soluble MeHNL increased significantly. The enzyme activity and yield at low-temperature cultures (17 degrees C) were 850 times higher than those obtained at the optimum growth temperature of 37 degrees C. The rate of MeHNL production in the present study was calculated as 3,000 unit/h. Low-temperature cultivation was very effective in improving the productivity of the active form of MeHNL. Unlike the temperature-shift method, low-temperature cultivation has more potential for the large-scale production of MeHNL for the optically active cyanohydrin production.
Semba
Hisashi
H
Strategic Technology Research Center, Nippon Shokubai Co Ltd, Tsukuba, Ibaraki, Japan. hisashi_semba@shokubai.co.jp
Ichige
Eita
E
Imanaka
Tadayuki
T
Atomi
Haruyuki
H
Aoyagi
Hideki
H
D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D002365:Manihot / Q000201:enzymology*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D003062:Codon; D010940:Plant Proteins; D011994:Recombinant Proteins; D000446:Aldehyde-Lyases; C079359:hydroxymandelonitrile lyase