Molecular cloning of α-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis

Palva, Ilkka

doi:10.1016/0378-1119(82)90191-3

Cited by 236 publications

(97 citation statements)

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“…Since the B. amyloliquefaciens a-amylase gene was previously isolated with very short flanking sequences [3] we decided to test whether the DNA regions surrounding the a-amylase gene in B. amyloliquefaciens would exert any regulatory functions on the a-amylase production. In addition to being of interest to the a-amylase production as such, the potential regulatory elements could also be important when joined to the a-amylase-based expression/secretion vectors constructed in our laboratory [13].…”

Section: Discussionmentioning

confidence: 99%

“…The chromosomal DNA was isolated, purified and then cleaved with restriction endonuclease EcoRI which has a unique cleavage site in the a-amylase gene near the 5' end of the gene [3]. Digested DNA was fractionated by a preparative agarose column gel electrophoresis.…”

Section: Cloning Of the Chromosomal Dna Upstream Of The A-amylase Genementioning

confidence: 99%

“…This allows direct screening of the a-amylase-positive phenotype in an amylasenegative background. The secretion of a-amylase from the positive clones was detected as a clear zone around the colony on starch plates [3]. The resulting hybrid plasmid bearing a functional a-amylase gene could also be used to study directly the possible regulatory functions of the cloned upstream region on the a-amylase gene.…”

Section: Cloning Of the Chromosomal Dna Upstream Of The A-amylase Genementioning

confidence: 99%

“…In our laboratory the a-amylase gene from B. amyloliquefaciens has been cloned in B. subrilis using the plasmid pUBllO as the vector [3]. The gene has been completely sequenced [ 1 I] and the transcription initiation and termination sites determined [12].…”

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Isolation and characterization of a 2.2‐kb operon preceding the α‐amylase gene of Bacillus amyloliquefaciens

Kallio

Ulmanen

Palva

1986

European Journal of Biochemistry

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A DNA region of 2.8 x lo3 base pairs (2.8 kb) upstream of the Bacillus amyloliquefaciens a-amylase gene has been isolated. This DNA gave rise to a 2.2-kb transcript. The 3' end of the transcript was mapped with S1 nuclease and shown to terminate 49 base pairs upstream of the -35 region of the a-amylase promoter. In B. subtilis minicells this 2.2-kb transcript coded for three different polypeptides, thus indicating a polycistronic operon-type structure. The location and the order of the polypeptides were established using DNA deletions. The joining of the 2.2-kb operon to the downstream a-amylase gene in the plasmid PUB110 did not have any significant effect on the level of expression of the a-amylase.Bacilli are known to produce a wide variety of exoenzymes some of which, like a-amylases and proteases, are of commercial importance. At present the a-amylases are the best characterized group of exoenzymes and a-amylase genes have been cloned from various Bacillus species [l -101.In our laboratory the a-amylase gene from B. amyloliquefaciens has been cloned in B. subrilis using the plasmid pUBllO as the vector [3]. The gene has been completely sequenced [ 1 I] and the transcription initiation and termination sites determined [12]. In addition, a set of expression/ secretion vectors have been constructed from the promotersignal sequence region of the a-amylase gene and used to study the expression of foreign genes in B. subtilis [13-151. However, very little is known about the regulation of the expression of the a-amylase genes. Only the a-amylase gene from B. subtilis has been studied at the genetic level. The available data suggest that several loci specifically affect the level of a-amylase production. Some of these loci are closely linked to the structural gene of a-amylase (amyR2, amyR3, tmrA) while others have been mapped elsewhere (amyS [16]). These loci are known to enhance the a-amylase production but the mechanism of this effect at the molecular level is unclear. We have previously tested whether the presence of any of the above loci would effect the expression of the B. amyloliquefaciens a-amylase clone in B. subtilis, but no positive response could be demonstrated [17].In this paper we report the isolation and characterization of a 2.8-kb DNA fragment upstream of the B. amyloliquefaciens a-amylase gene. This 2.8-kb DNA was shown to contain a 2.2-kb operon giving rise to three different polypeptides. However, this operon, when joined to the a-amylase clone, did not affect the production of B. amyloliquefaciens a-amylase in B. subtilis. MATERIALS AND METHODS Bacteria and plasmidsBacillus amyloliquefaciens strain El 8 [ 181 was used for the isolation of total chromosomal DNA to clone the DNA region upstream of the a-amylase gene.B. subtilis BRBl (previously named IH6064) and BRB152 (amy-) were from our collection [9]. A minicell producing B. subtilis strain CUM3 [divIVBl, metB5, thyAl, thyB1, spo(-)] was obtained from Bacillus Genetic Stock Center (Columbus, Ohio, USA; 1A197 in BGSC).Plasmids pKTH10, pKTH2O...

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Section: Discussionmentioning

confidence: 99%

Section: Cloning Of the Chromosomal Dna Upstream Of The A-amylase Genementioning

confidence: 99%

Section: Cloning Of the Chromosomal Dna Upstream Of The A-amylase Genementioning

confidence: 99%

mentioning

confidence: 99%

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Isolation and characterization of a 2.2‐kb operon preceding the α‐amylase gene of Bacillus amyloliquefaciens

Kallio

Ulmanen

Palva

1986

European Journal of Biochemistry

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“…AmyS (Palva, 1982 ;Takkinen et al, 1983 ;Gray et al, 1986 ;Jørgensen et al, 1991a). The segments were chosen so that each swop led to an increase or decrease in the pI, as calculated by the ChargPro programme in the \ software package.…”

Section: Computermentioning

confidence: 99%

Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system

et al. 2000

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A series of chimeric α-amylase genes derived from amyL, which encodes the liquefying α-amylase from Bacillus licheniformis, were constructed in vitro using gene splicing techniques. The gene constructs were cloned in Bacillus subtilis, where their ability to direct the synthesis and secretion of active α-amylase was determined. Detectable α-amylase activity was observed for some, but not all, of the chimeric proteins. Studies on the secretion of wildtype AmyL and its chimeric derivatives revealed that, whilst these proteins were stable in the extracellular milieu, all were subject to some degree of degradation during secretion. The chimeric enzymes were degraded to a greater extent than the native enzyme. These findings suggest that cellassociated proteolysis is a significant problem affecting the use of B. subtilis as host bacterium for the production of heterologous proteins.

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The targeting of bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N‐terminus mature part

Scotti

Præstegaard

Chambert

et al. 1996

Yeast

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Molecular cloning of α-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis

Cited by 236 publications

References 14 publications

Isolation and characterization of a 2.2‐kb operon preceding the α‐amylase gene of Bacillus amyloliquefaciens

Isolation and characterization of a 2.2‐kb operon preceding the α‐amylase gene of Bacillus amyloliquefaciens

Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system

The targeting of bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N‐terminus mature part

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