Development of bioreactor system for L-tyrosine synthesis using thermostable tyrosine phenol-lyase.
An efficient enzyme system for the synthesis of L-tyrosine was developed using a fed-batch reactor with continuous feeding of phenol, pyruvate, and ammonia. A thermo- and chemostable tyrosine phenol-lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM phenol at pH 8.5 and 40 degrees C. The fed-batch system (working volume, 0.5 1) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4 M ammonium chloride (pH 8.5). The system produced 130 g/l of L-tyrosine within 30 h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-tyrosine was 94% on the basis of the supplied phenol.
Kim
Do Young
DY
Systems Microbiology Research Center, KRIBB, Daejeon 305-806, Korea.
Rha
Eugene
E
Choi
Su-Lim
SL
Song
Jae Jun
JJ
Hong
Seung-Pyo
SP
Sung
Moon-Hee
MH
Lee
Seung-Goo
SG
D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D019149:Bioreactors / Q000382:microbiology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014443:Tyrosine / Q000096:biosynthesis*; D014447:Tyrosine Phenol-Lyase / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014443:Tyrosine; D014447:Tyrosine Phenol-Lyase