Improvement of the activity of arylmalonate decarboxylase by random mutagenesis

Y. Terao, Kenji Miyamoto, H. Ohta

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Arylmalonate decarboxylase (EC 4.1.1.76) catalyzes enantioselective decarboxylation of α-aryl-α-methylmalonates to give optically pure α-arylpropionates. Recently, we have succeeded in creating a double mutant enzyme that gave opposite enantionmer as the product. Unfortunately, however, the activity of the mutant decreased far lower than that of the native enzyme. Thus, we performed the directed evolution of the mutant via the random mutagenesis method employing the mutator strain Escherichia coli XL1-Red. About 50,000 mutants were screened on color assay plate, and one mutant with higher activity was obtained. Gene analysis of this mutant indicated that the obtained enzyme had an S36N mutation in addition to its original G74C/C188S mutations. The activity of the triple mutant enzyme was tenfold higher than that of the starting doubly mutated enzyme.

Original languageEnglish
Pages (from-to)647-653
Number of pages7
JournalApplied Microbiology and Biotechnology
Volume73
Issue number3
DOIs
Publication statusPublished - 2006 Dec

Fingerprint

Mutagenesis
Enzymes
Mutation
Decarboxylation
Escherichia coli
Assays
Color
Genes
malonate decarboxylase

Keywords

  • Activity
  • Arylmalonate decarboxylase
  • Random-mutagenesis
  • XL1-Red

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Bioengineering
  • Microbiology (medical)

Cite this

Improvement of the activity of arylmalonate decarboxylase by random mutagenesis. / Terao, Y.; Miyamoto, Kenji; Ohta, H.

In: Applied Microbiology and Biotechnology, Vol. 73, No. 3, 12.2006, p. 647-653.

Research output: Contribution to journalArticle

@article{7c137d85d083439d949280506c209d1a,
title = "Improvement of the activity of arylmalonate decarboxylase by random mutagenesis",
abstract = "Arylmalonate decarboxylase (EC 4.1.1.76) catalyzes enantioselective decarboxylation of α-aryl-α-methylmalonates to give optically pure α-arylpropionates. Recently, we have succeeded in creating a double mutant enzyme that gave opposite enantionmer as the product. Unfortunately, however, the activity of the mutant decreased far lower than that of the native enzyme. Thus, we performed the directed evolution of the mutant via the random mutagenesis method employing the mutator strain Escherichia coli XL1-Red. About 50,000 mutants were screened on color assay plate, and one mutant with higher activity was obtained. Gene analysis of this mutant indicated that the obtained enzyme had an S36N mutation in addition to its original G74C/C188S mutations. The activity of the triple mutant enzyme was tenfold higher than that of the starting doubly mutated enzyme.",
keywords = "Activity, Arylmalonate decarboxylase, Random-mutagenesis, XL1-Red",
author = "Y. Terao and Kenji Miyamoto and H. Ohta",
year = "2006",
month = "12",
doi = "10.1007/s00253-006-0518-z",
language = "English",
volume = "73",
pages = "647--653",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
publisher = "Springer Verlag",
number = "3",

}

TY - JOUR

T1 - Improvement of the activity of arylmalonate decarboxylase by random mutagenesis

AU - Terao, Y.

AU - Miyamoto, Kenji

AU - Ohta, H.

PY - 2006/12

Y1 - 2006/12

N2 - Arylmalonate decarboxylase (EC 4.1.1.76) catalyzes enantioselective decarboxylation of α-aryl-α-methylmalonates to give optically pure α-arylpropionates. Recently, we have succeeded in creating a double mutant enzyme that gave opposite enantionmer as the product. Unfortunately, however, the activity of the mutant decreased far lower than that of the native enzyme. Thus, we performed the directed evolution of the mutant via the random mutagenesis method employing the mutator strain Escherichia coli XL1-Red. About 50,000 mutants were screened on color assay plate, and one mutant with higher activity was obtained. Gene analysis of this mutant indicated that the obtained enzyme had an S36N mutation in addition to its original G74C/C188S mutations. The activity of the triple mutant enzyme was tenfold higher than that of the starting doubly mutated enzyme.

AB - Arylmalonate decarboxylase (EC 4.1.1.76) catalyzes enantioselective decarboxylation of α-aryl-α-methylmalonates to give optically pure α-arylpropionates. Recently, we have succeeded in creating a double mutant enzyme that gave opposite enantionmer as the product. Unfortunately, however, the activity of the mutant decreased far lower than that of the native enzyme. Thus, we performed the directed evolution of the mutant via the random mutagenesis method employing the mutator strain Escherichia coli XL1-Red. About 50,000 mutants were screened on color assay plate, and one mutant with higher activity was obtained. Gene analysis of this mutant indicated that the obtained enzyme had an S36N mutation in addition to its original G74C/C188S mutations. The activity of the triple mutant enzyme was tenfold higher than that of the starting doubly mutated enzyme.

KW - Activity

KW - Arylmalonate decarboxylase

KW - Random-mutagenesis

KW - XL1-Red

UR - http://www.scopus.com/inward/record.url?scp=33751021095&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33751021095&partnerID=8YFLogxK

U2 - 10.1007/s00253-006-0518-z

DO - 10.1007/s00253-006-0518-z

M3 - Article

C2 - 16865343

AN - SCOPUS:33751021095

VL - 73

SP - 647

EP - 653

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 3

ER -