Molecular biology of a cryptic dehalogenase from Burkholderia cepacia MBA4

Molecular biology of a cryptic dehalogenase from Burkholderia cepacia MBA4

Laiju Sam(Laiju Sam)

香港大學/Doctor of Philosophy/Botany/博士(2000年)

並列摘要

(Uncorrected OCR)
Abstract of the thesis entitled

MOLECULAR BIOLOGY OF A CRYPTIC DEHALOGENASE FROM Burkholderia cepacia MBA4

Submitted by

Laiju Sam

For the degree of Doctor of Philosophy at The University of Hong Kong

in July 2000

Burkholderia cepacia MBA4 has been previously shown to produce a single dehalogenase in batch culture condition. Other cryptic dehalogenases were detected in cells grown in continuous culture. In this study, one of the cryptic dehalogenases was cloned and characterised. This cryptic haloacid dehalogenase was designated Chd 1 and expressed constitutively in Escherichia coli. The structural gene, chdJ, was isolated from a 1.7-kb PstI fragment. This fragment. contains a functional promoter since the expression of chdl in E. coli is orientation independent. The nucleotide sequence of the fragment was determined and analysed and an open reading frame for 840 amino acids was identified. The nucleotide sequence of chdl did not show any homology with those of other dehalogenase genes. Comparison of the deduced amino acid sequence, however, showed significant homology, ranging from 42-50%, with the amino acid sequences of several other dehalogenases. Phylogenetic analysis indicated that Chd 1 is closely related to dehalogenase CI and dehalogenase IVa of Pseudomonas putida CBS3 and B. cepacia MBA4 respectively.

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The recombinant Chdl had a native molecular weight of 58,000 daltons

whereas the denatured molecular weight was found to be 27,000 daltons. Thus,

the enzyme exists predominantly as a dimer. When the activity towards the two

stereoisomers of 2-monochloropropionic acid were considered individually, the

enzyme was found to be active towards the L-isomer only. The purified enzyme

was most active towards monobromoacetic acid with specific activities of 5.44,

1.5, 4.56, and 1.95 J!mole of halide released/min/mg protein for

monobromoacetic acid, 2-monobromopropionic acid, monochloroacetic acid and

2-monochloropropionic acid respectively. Maximum activity of the enzyme was

observed at pH 6.5. The enzyme was found to be thermolabile.

chdl was cloned in the T7 RNA polymerase driven pRSET A and Irc promoter

based pPROEXHT expression vectors. However, no significant expression of

chdl was obtained in these systems.

Chd 1 differed from other dehalogenases in having a long leader sequence.

This leader sequence contains a potential signal peptidase cleavage site. This is a

property of periplasmic enzymes. In order to detect the unprocessed molecules

of Chd 1, chdl was expressed in the temperature-sensitive E. coli strain IT 41

(lep9-mutant). This strain contains a mutation in the leader peptidase gene. The

leader peptidase is inactive in this strain at the non-permissive temperature of

o

42 C. Precursor molecules of Chd 1 was detected in cultures shifted to this non-

permissive temperature. Periplasmic fractions isolated from E. coli harboring

chdl were found to contain the active dehalogenase.

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並列關鍵字

Pseudomonas - Molecular aspects.