Synthesis of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) with thermostabilized low-specific L-threonine aldolase from Streptomyces coelicolor A3(2).
Stability-enhanced mutants, H44, 11-94, 5A2-84, and F8, of L-threonine aldolase (L-TA) from Streptomyces coelicolor A3(2) (SCO1085) were isolated by an error-prone PCR followed by a high-throughput screening. Each of these mutant, had a single amino acid substitution: H177Y in the H44 mutant, A169T in the 11-94 mutant, D104N in the 5A2-84 mutant and Fl81 in the F8 mutant. The residual L-TA activity of the wild-type L-TA after a heat treatment for 20 min at 60 degrees C was only 10.6%. However, those in the stability-enhanced mutants were 85.7% for the H44 mutant, 58.6% for the F8 mutant, 62.1% for the 5A2-84 mutant, and 67.6% for the 11-94 mutant. Although the half-life of the wild-type L-TA at 63 degrees C was 1.3 min, those of the mutant L-TAs were longer: 14.6 min for the H44 mutant, 3.7 min for the 11-94 mutant, 5.8 min for the 5A2-84 mutant, and 5.0 min for the F8 mutant. The specific activity did not change in most of the mutants, but it was decreased by 45% in the case of mutant F8. When the aldol condensation of glycine and 3,4-dihydroxybenzaldehyde was studied by using whole cells of Escherichia coli containing the wild-type L-TA gene, L-threo-3,4-dihydroxyphenylserine (L.-threo-DOPS) was successfully synthesized with a yield of 2.0 mg/ml after 20 repeated batch reactions for 100 h. However, the L-threo-DOPS synthesizing activity of the enzyme decreased with increased cycles of the batch reactions. Compared with the wild-type L-TA, H44 L-TA kept its L-threo-DOPS synthesizing activity almost constant during the 20 repeated batch reactions for 100 h, yielding 4.0 mg/ml of L-threo-DOPS. This result showed that H44 L-TA is more effective than the wild-type L-TA for the mass production of L-threo-DOPS.
NITE Biological Resource Center, National Institute of Technology and Evaluation, 2-5-8 Kazusa-Kamatari, Kisarazu-shi, Chiba 292-0818, Japan. baiksh@kaeri.re.kr
Sang-Ho
SH
Balk
Hideki
H
Yoshioka
Hideaki
H
Yukawa
Shigeaki
S
Harayama
D019943:Amino Acid Substitution / Q000235:genetics; D001547:Benzaldehydes / Q000378:metabolism; D002396:Catechols / Q000378:metabolism; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D015103:Droxidopa / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005998:Glycine / Q000378:metabolism; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006207:Half-Life; D006358:Hot Temperature; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D048372:Streptomyces coelicolor / Q000201:enzymology*
D001547:Benzaldehydes; D002396:Catechols; D004269:DNA, Bacterial; C005581:protocatechualdehyde; D012696:Glycine Hydroxymethyltransferase; D015103:Droxidopa; D005998:Glycine