Cloning and expression of a Clostridium acetobutylicum α-amylase gene in Escherichia coli

Verhasselt, P

doi:10.1016/0378-1097(89)90473-4

Cited by 13 publications

(13 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, because of the truncated sequence, this assumption still is preliminary. An a-amylase of C. acetobutylicum has recently been cloned (34), but sequence data are not yet available. From the published restriction map, no direct homology to our sequence could be detected.…”

Section: Discussionmentioning

confidence: 99%

Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum

Gerischer

Dürre

1990

J Bacteriol

View full text Add to dashboard Cite

Acetoacetate decarboxylase (ADC) (EC4.1.1.4) of Clostridium acetobutylicum DSM 792 was purified to homogeneity, and its first 25 N-terminal amino acids were determined. Oligonucleotide probes deduced from this sequence were used to detect positive clones in partial gene banks derived from Sau3A and HaeIII digests with following ligation into the vector pUC9. In Escherichia coli, the 2.1-kbp HaeHII clones expressed high levels of ADC activity. The expression was independent of the orientation of the insert with respect to the lac promoter of the vector and also of the addition of isopropyl-l-D-thiogalactopyranoside, thus indicating that sequences located on the clostridial DNA controlled transcription and translation. From the E. coli clone with the recombinant plasmid pUG93 containing the 2.1-kbp HaeIII fragment, the ADC protein was purified and compared with the native enzyme. Both were indistinguishable with respect to the molecular mass of subunits and native protein as well as to activity stain. The 2.9-kbp Sau3A fragment could be shown to contain the amino terminus of the acetoacetate decarboxylase (adc) gene but did not express enzyme activity. It partially overlapped with the HaeHI fragment, spanning together 4,053 bp of the clostridial genome that were completely sequenced. Four open reading frames (ORFs) could be detected, one of which was unambiguously assigned to the acetoacetate decarboxylase (adc) gene. Amino acid sequences of the N terminus and the catalytic center as deduced from the nucleotide sequence were identical to sequences obtained from direct analysis of the protein. Typical procaryotic transcriptional and translational start and stop signals could be found in the DNA sequence. Together with these regulatory sequences, the adc gene formed a single operon. The carboxyl terminus of the enzyme proved to be rather hydrophobic. In vitro transcription-translation assays resulted in formation of ADC and ORF3 gene product; the other two ORFs were not expressed. Whereas no homology of the adc gene and ORF2 could be detected with sequences available in the EMBL or GenBank data bases, the obviously truncated ORF1 showed significant similarity to a-amylase of Bacillus subtilis. The restriction pattern and N-terminal amino acid sequence (as deduced from the nucleotide sequence) of ORF3 proved to be identical to those of the large subunit of acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase.

show abstract

Section: Discussionmentioning

confidence: 99%

Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum

Gerischer

Dürre

1990

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…L. reuteri total DNA was isolated according to Verhasselt et al (1989) as modified by Nagy et al (1995). General procedures for cloning, transformation, DNA manipulations and agarose gel electrophoresis were as described by van Hijum et al (2002).…”

Section: Methodsmentioning

confidence: 99%

Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri

et al. 2004

View full text Add to dashboard Cite

show abstract

“…The membranes were then washed twice at room temperature with 300 mM NaCl-30 mM sodium citrate containing 0.1% SDS and twice with 150 mM NaCl-15 mM sodium citrate containing 0.1% SDS at 45 and 53ЊC, respectively. Total DNA of Rhodococcus species was extracted by the method of Verhasselt et al (84), modified by including an ampicillin and lysozyme pretreatment of cells, as carried out for the protein isolation. DNA fragments of interest were purified from agarose gels and cloned in pUC18 or LambdaGEM-12.…”

Section: Methodsmentioning

confidence: 99%

Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase

et al. 1995

View full text Add to dashboard Cite

Determination of the N-terminal sequences of two EPTC (S-ethyl dipropylcarbamothioate)-induced proteins from thiocarbamate-degradingEvidence for the involvement of this cytochrome P-450 system in EPTC degradation and herbicide biosafening for maize was obtained by complementation experiments using EPTC-negative Rhodococcus erythropolis SQ1 and mutant FAJ2027 as acceptor strains. N dealkylation by cytochrome P-450 and conversion of the released aldehyde into the corresponding carboxylic acid by aldehyde dehydrogenase are proposed as the reactions initiating thiocarbamate catabolism in Rhodococcus sp. strain NI86/21. In addition to the major metabolite N-depropyl EPTC, another degradation product was identified, EPTC-sulfoxide.

show abstract

Cloning and expression of a Clostridium acetobutylicum α-amylase gene in Escherichia coli

Cited by 13 publications

References 11 publications

Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum

Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum

Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri

Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase

Contact Info

Product

Resources

About