Supplementary

postgraduate thesis: Molecular biology of a cryptic dehalogenase from Burkholderia cepacia MBA4

TitleMolecular biology of a cryptic dehalogenase from Burkholderia cepacia MBA4
Authors
Issue Date2000
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Sam, L.. (2000). Molecular biology of a cryptic dehalogenase from Burkholderia cepacia MBA4. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
Abstract(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. 1 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. 11
DegreeDoctor of Philosophy
SubjectPseudomonas - Molecular aspects.
Dept/ProgramBotany

 

DC FieldValueLanguage
dc.contributor.authorSam, Laiju-
dc.date.issued2000-
dc.identifier.citationSam, L.. (2000). Molecular biology of a cryptic dehalogenase from Burkholderia cepacia MBA4. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.description.abstract(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. 1 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. 11-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.source.urihttp://hub.hku.hk/bib/B21827187-
dc.subject.lcshPseudomonas - Molecular aspects.-
dc.titleMolecular biology of a cryptic dehalogenase from Burkholderia cepacia MBA4-
dc.typePG_Thesis-
dc.identifier.hkulb2182718-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBotany-
dc.description.natureabstract-
dc.description.naturetoc-

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