Cloning and Expression of a Bacillus subtilis Endo-1,3-1,4- -D-Glucanase Gene in Escherichia coli K12

Hinchliffe, Edward

doi:10.1099/00221287-130-5-1285

Cited by 24 publications

(20 citation statements)

References 9 publications

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“…The linkage specificities of these enzymes are varied, the most common being ,-1,3-1,4-glucanases and 13-1,3-glucanases (6,20,32,41,57). There are relatively few reports of members of the family Bacillaceae producing ,B-1,4-glucanases (13,21,41,44).…”

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confidence: 99%

“…Although incapable of degrading crystalline forms of cellulose individually, bacterial P-1,4-glucanases (most likely endo acting) may be able to act synergistically with cellulases of other specificities, such as exo-acting P-1,4-glucanases or P-glucosidases, or both, to achieve the enzymatic bioconversion of cellulose to more commercially useful products. A Bacillus P-1,4-glucanase may also have a role to play in the brewing industry (5,11,20).…”

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confidence: 99%

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Endo-beta-1,4-glucanase gene of Bacillus subtilis DLG

Robson¹,

Chambliss²

1987

J Bacteriol

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The DNA sequence of the BaciUus subtlis DLG endo-,B-1,4-glucanase gene was determined, and the in vivo site of transcription initiation was located. Immediately upstream from the transcription start site were sequences closely resembling those recognized by B. subtilis &43-RNA polymerase. Two possible ribosomebinding sites were observed downstream from the transcription start site. These were followed by a long open reading frame capable of encoding a protein of ca. 55,000 daltons. A signal sequence, typical of those present in gram-positive organisms, was observed at the amino terminus of the open reading frame. Purification of the mature exocellular 0-1,4-glucanase and subsequent amino-terminal protein sequencing defined the site of signal sequence processing to be between two alanine residues following the hydrophobic portion of the signal sequence. The probability of additional carboxy-terminal processing of the 0-1,4-glucanase precursor is discussed. Si nuclease protection studies showed that the amount of ,B-1,4-glucanase mRNA in cells increased significantly as the culture entered the stationary phase. In addition, glucose was found to dramatically stimulate the amount of ,B-1,4-glucanase mRNA in vivo. Finally, the specffic activities of purified B. subtilis DLG endo-0-1,4-glucanase and Trichoderma reesei QM9414 endo-f-1,4-glucanase (EC 3.2.1.4) were compared by using the noncrystalline cellulosic substrate trinitrophenyl-carboxymethyl cellulose.Many members of the family Bacillaceae produce extracellular ,B-glucanases. The linkage specificities of these enzymes are varied, the most common being ,-1,3-1,4-glucanases and 13-1,3-glucanases (6,20,32,41,57). There are relatively few reports of members of the family Bacillaceae producing ,B-1,4-glucanases (13,21,41,44). The two relatively well characterized examples, the alkalophilic Bacillus strain N-4 (21, 48) and Bacillus subtilis DLG (44, 45), produce cellulolytic activity in the form of a carboxymethyl cellulase (CMC) (i.e., P-1,4-glucanase). Although incapable of degrading crystalline forms of cellulose individually, bacterial P-1,4-glucanases (most likely endo acting) may be able to act synergistically with cellulases of other specificities, such as exo-acting P-1,4-glucanases or P-glucosidases, or both, to achieve the enzymatic bioconversion of cellulose to more commercially useful products. A Bacillus P-1,4-glucanase may also have a role to play in the brewing industry (5,11,20). For B. subtilis DLG, ,B-1,4-glucanase production begins at the onset of the stationary phase and is not repressed by either glucose or cellobiose (44), as are many other cellulolytic systems (12,30,53,55). In fact, glucose stimulates enzyme production in some fashion. Additionally, the enzyme is initially translated as a large (ca. 51,500-dalton), enzymatically active intracellular precursor in B. subtilis before undergoing efficient secretion to the exterior of the cell (45). The mature exocellular ,B-1,4-glucanase is 35,200 ± 400 daltons.Cloning the ,3-1,4-glucanase gene ha...

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confidence: 99%

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confidence: 99%

Endo-beta-1,4-glucanase gene of Bacillus subtilis DLG

Robson¹,

Chambliss²

1987

J Bacteriol

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“…Besides the degradation of extracellular proteins by the alkaline protease, the degradation of five other polymeric substrates was increased in the presence of pPR41 ( Fig. 2 (5,9). Degradation of xylan and starch is probably due to xylanase (20) and a-amylase (34), respectively.…”

Section: Resultsmentioning

confidence: 96%

Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis

Amory¹,

Kunst²,

Aubert³

et al. 1987

J Bacteriol

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The sacQ gene from Bacillus An interesting property of Bacillus subtilis is its ability to secrete a great number of enzymes (21), including some industrially useful ones like ax-amylase, proteases, or glticanases. However, the amount of proteins secreted by B. subtilis has been found to be a serious limitation compared to Bacillus strains used in industry, like B. licheniformis, B. amyloliquefaciens, or B. stearothermophilus, which are capable of producing much larger amounts of secreted enzymes.A number of mutations of B. subtilis 168 have been isolated that increase the production of secreted enzymes. sdcU-hyperproducing [sacU(Hy)] mutants (12), which are identical to pap (3, 37) and amyB (25) mutants, were characterized by a pleiotropic phenotype including hyperproduction of levansucrase, protease, and ax-amylase, absence of flagella, and low transformation levels. A similar phenotype was given by the sacQ(Hy) mutation, which was mapped at a different locus (13,14).In this report, we describe the identification of a plasmid containing a DNA fragment from B. licheniformis that confers to the recipient B. subtilis strain a hypersecretion phenotype similar to the one given by the sacQ(Hy) or sacU(Hy) mutation. From subcloning experiments, we obtained evidence that a 46-residue polypeptide is involved in the expression of the phenotype. A series of deletions was camed out in the cloned fragment, and the phenotype was lost when the deletions affected the polypeptide coding sequence. Moreover, hypersecretion was given by a 228-base-pair (bp) Moreover, we clearly show in this report that the sacQ gene isolated from a B. licheniformis high protease producer appears to encode a sacQ protein which is more efficient than the B. subtilis sacQ polypeptide in conferring the hypersecretion phenotype. The natural promoter of the sacQ gene was indeed replaced by an inducible promoter (spac), and the hypersecretion of protease and levansucrase was specifically obtained under conditions of full induction of the promoter. This is strong evidence that the increased expression of both enzymes is due to an increase of sacQ protein synthesis.Nothing, however, is known about the mechanism by which the increased expression of the target genes is produced by sacQ. The six different target genes identified in this study seem to be submitted to different growth phase regulation. For example, levansucrase is only synthesized during exponential growth, whereas alkaline protease is produced during the stationary phase.An attractive hypothesis is to suggest that sacQ regulates the synthesis of an essential component of the secretion machinery, since all of the target genes identified in this report encode a secreted enzyme. However, our results suggest that this hypothesis is unlikely. This is shown from a detailed study of one target of sacQ regulation, the levansucrase gene. We show that the target site is included in a 440-bp fragment located upstream from the coding sequence and ribosome-binding site of levansucrase, which sugges...

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“…The cellobiose metabolic operon from Klebsiella oxytoca has been introduced into E. coli, but the expression level of the cellobiose transporter and metabolic genes was poor, and hence could not support the growth of E. coli on cellobiose (Moniruzzaman et al 1997). Cellulases from several species of Clostridium, Bacillus, Cellulomonas, and Ruminococcus have been expressed and characterized in E. coli (Hinchliffe 1984;Zappe et al 1986;Fierobe et al 1991;ReverbelLeroy et al 1996;Lam et al 1997;ReverbelLeroy et al 1997;Lee et al 2008;Li et al 2009). Co-expression of endoglucanase from B. pumilus and β-glucosidase from Fervidobacterium spp.…”

Section: E Colimentioning

confidence: 99%