Your browser doesn't support javascript.
loading
Synthesis of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) with thermostabilized low-specific L-threonine aldolase from Streptomyces coelicolor A3(2).
Balk, Sang-Ho; Yoshioka, Hideki; Yukawa, Hideaki; Harayama, Shigeaki.
Affiliation
  • Balk SH; 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
J Microbiol Biotechnol ; 17(5): 721-7, 2007 May.
Article in En | MEDLINE | ID: mdl-18051291
ABSTRACT
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.
Subject(s)
Search on Google
Collection: 01-internacional Database: MEDLINE Main subject: Glycine Hydroxymethyltransferase / Enzyme Stability / Mutation, Missense / Streptomyces coelicolor / Droxidopa / Hot Temperature Language: En Journal: J Microbiol Biotechnol Year: 2007 Document type: Article Affiliation country: Japan
Search on Google
Collection: 01-internacional Database: MEDLINE Main subject: Glycine Hydroxymethyltransferase / Enzyme Stability / Mutation, Missense / Streptomyces coelicolor / Droxidopa / Hot Temperature Language: En Journal: J Microbiol Biotechnol Year: 2007 Document type: Article Affiliation country: Japan