Gene cloning and biochemical characterization of 4-N-trimethylaminobutyraldehyde dehydrogenase II from Pseudomonas sp. 13CM

Md Rezaul Bari, Maizom Hassan, Naoki Akai, Jiro Arima, Nobuhiro Mori

Research output: Contribution to journalArticle

Abstract

The gene encoding 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABaldehyde-DH) from Pseudomonas sp. 13CM, responsible for the conversion of 4-N-trimethylaminobutyraldehyde (TMABaldehyde) to γ-butyrobetaine in the carnitine biosynthesis pathway, isolated by shotgun cloning and expressed in Escherichia coli DH5α. The recombinant TMABaldehyde-DH was purified 19. 5 fold to apparent homogeneity by hydrophobic and affinity chromatography and biochemically characterized. The enzyme was found to be a trimer with identical 52 kDa subunits. The isoelectric point was found to be 4. 5. Optimum pH and temperature were found respectively as pH 9. 5 and 40 °C. The Km values for TMABaldehyde, 4-dimethylaminobutyraldehyde, and NAD+ were respectively, 0. 31, 0. 62, and 1. 16 mM. The molecular and catalytic properties differed from those of TMABaldehyde-DH I, which was discovered initially in Pseudomonas sp. 13CM. The new enzyme, designated TMABaldehyde-DH II, structural gene was inserted into an expression vector pET24b (+) and over-expressed in E. coli BL21 (DE3) under the control of a T7 promoter. The recombinant TMABaldehyde-DH from Pseudomonas sp. 13CM can now be obtained in large quantity necessary for further biochemical characterization and applications.

Original languageEnglish
Pages (from-to)683-692
Number of pages10
JournalWorld Journal of Microbiology and Biotechnology
Volume29
Issue number4
DOIs
Publication statusPublished - 2013

Fingerprint

Pseudomonas
Organism Cloning
Genes
Escherichia coli
Carnitine
Isoelectric Point
Firearms
Enzymes
Affinity Chromatography
NAD
4-N-trimethylaminobutyraldehyde dehydrogenase
Temperature
gamma-trimethylaminobutyraldehyde

Keywords

  • 4-N-trimethylaminobutyraldehyde
  • Gene cloning
  • L-carnitine biosynthetic pathway
  • Pseudomonas sp. 13CM
  • Quaternary ammonium compound

ASJC Scopus subject areas

  • Biotechnology
  • Physiology
  • Applied Microbiology and Biotechnology

Cite this

Gene cloning and biochemical characterization of 4-N-trimethylaminobutyraldehyde dehydrogenase II from Pseudomonas sp. 13CM. / Bari, Md Rezaul; Hassan, Maizom; Akai, Naoki; Arima, Jiro; Mori, Nobuhiro.

In: World Journal of Microbiology and Biotechnology, Vol. 29, No. 4, 2013, p. 683-692.

Research output: Contribution to journalArticle

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