Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

Honjo, Masaru; Manabe, Kazuaki; Shimada, Hiroaki; Mita, I.; Nakayama, Akira; Furutani, Yoshio

doi:10.1016/0168-1656(84)90018-x

Cited by 22 publications

(5 citation statements)

References 16 publications

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“…This sequence (Fig. 3) revealed an open reading frame (ORF) of (8,12,44) and is, therefore, probably also synthesized as a pre-pro protein. The putative pre structure of the B. caldolyticus Npr shows characteristics of a typical signal sequence (43); the NH2-terminal part (n region, amino acids 1 to 4) contains two positively charged amino acids (Lys-3 and Arg-4) and is followed by the h Fig.…”

Section: Methodsmentioning

confidence: 99%

“…These enzymes contain one zinc atom per molecule and may be stabilized by calcium binding (17). Several npr genes have been cloned and expressed in Bacillus subtilis, including the genes from B. subtilis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, and their nucleotide sequences have been determined (8,12,18,33,38,40,44). Recently, the npr gene from Bacillus brevis was also cloned and sequenced (la).…”

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confidence: 99%

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A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product

Burg¹,

Enequist²,

Haar³

et al. 1991

J Bacteriol

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By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolyticaily active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtUis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb Hinfl-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. stearothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8°C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostabiity was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B.caldolyticus.Bacterial species belonging to the genus Bacillus secrete a variety of enzymes, some of which are of economical importance, in particular the ax-amylases and proteinases (5). The various Bacillus species show large variations in optimum growth temperatures, which are often, but not invariably, reflected in the thermostabilities of their extracellular enzymes (23). By comparing thermostable and thermolabile variants of homologous enzymes, information on the mechanisms involved in thermostability of enzymes can be obtained (22,31,37).We selected the neutral proteases from bacilli as a model system in such an approach. Neutral proteases (Npr) are metalloendoproteinases which show optimum activity at neutral pH. These enzymes contain one zinc atom per molecule and may be stabilized by calcium binding (17). Several npr genes have been cloned and expressed in Bacillus subtilis, including the genes from B. subtilis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, and their nucleotide sequences have been determined (8,12,18,33,38,40,44). Recently, the npr gene from Bacillus brevis was also cloned and sequenced (la). Additionally, the primary and tertiary structures of the neutral proteases from Bacillus cereus and Bacillus thermoproteolyticus have been determined (21,28,34,35). The neutral proteases from B. subtilis and B. amyloliquefaciens are rather thermolabile, whereas the enzymes from B. stearothermophilus and ...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product

Burg¹,

Enequist²,

Haar³

et al. 1991

J Bacteriol

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“…subtilis was reported [ 7 , 8 ], the period of gene modification on neutral protease improvement has begun. Up to now, numerous Bacillus neutral proteases have been characterized and genes encoding for these enzymes have been cloned and sequenced from Bacillus subtilis [ 9 – 11 ], Bacillus amyloliquefaciens [ 12 , 13 ], Bacillus cereus [ 14 ], Bacillus stearothermophilus [ 15 , 16 ], Bacillus thermoproteolyticus [ 17 , 18 ], Bacillus nematocida [ 19 ], Bacillus caldolyticus [ 20 ], etc. A number of these studies have undertaken expression of neutral proteases by multicopy plasmids or by modifying regulatory elements in heterologous hosts like Escherichia coli or B .…”

Section: Introductionmentioning

confidence: 99%

Engineering of a Bacillus amyloliquefaciens Strain with High Neutral Protease Producing Capacity and Optimization of Its Fermentation Conditions

et al. 2016

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The neutral protease has high potential for industrial applications, and attempts to improve enzyme expression level have important application values. In the present study, a neutral protease-encoding gene, Banpr, was cloned from Bacillus amyloliquefaciens strain K11, and a genetic manipulation method specific for this difficult-to-transform strain was developed for the high-level expression of neutral protease. The recombinant plasmid pUB110-Banpr was constructed in Bacillus subtilis strain WB600 and then transformed into strain K11 under optimized conditions. A positive transformant 110N-6 with the highest protease secreting capacity on skim milk plates and great genetic stability for more than 100 generations was selected for further study. Optimization of the fermentation conditions increased the enzyme activity of strain 110N-6 to 8995 ± 250 U/ml in flask culture and 28084 ± 1282 U/ml in 15-l fermentor, which are significantly higher than that of the native strain K11 and industrial strain B. subtilis AS.1398, respectively. The high expression level and extreme genetic stability make B. amyloliquefaciens strain 110N-6 more favorable for mass production of neutral protease for industrial uses.

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“…Genes from various species of the genus Bacillus have been cloned into B. subtilis and found to be functional, for example leuA and ilvC from B. amyloliquefaciens (Yoshimura et al, 1983), neutral protease genes from B. amyloliquefaciens (Honjo et al, 1984) and B. stearothermophilus (Fujii et al, 1983), hisH from B. Iicheniformis (Weinrauch & Dubnau, 1983), and a-amylase genes from B. amyloliquefaciens (Palva, 1982; Lehtovaara et al, 1984) and B. lichenformis (Joyet et al, 1984). for the presence of spores.…”

Section: Introductionmentioning

confidence: 99%

Cloning and Sequencing of a Gene from Bacillus amyloliquefaciens that Complements Mutations of the Sporulation Gene spoIID in Bacillus subtilis

Turner

Mandelstam

1986

Microbiology

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A segment of DNA from Bacillus amyloliquefaciens, which complemented a mutant sporulation gene, spoIID68, in Bacillus subtilis, was cloned into a derivative of the temperate bacteriophage phi 105. The segment of DNA included an entire structural gene and complemented the mutation spoIID298, in addition to spoIID68, in B. subtilis. The nucleotide sequence of the gene from B. amyloliquefaciens was determined and compared with that of the B. subtilis gene; 74% homology was found in the coding region. Amino acid primary sequences derived from the nucleotide sequences of the two genes were also compared. The gene from B. amyloliquefaciens coded for a protein of 344 amino acid residues, one more than the protein coded by the corresponding gene from B. subtilis. Comparison of the primary amino acid sequences of the two genes showed that 78% of the residues were completely conserved and 8% were semi-conserved. Variation, however, was not random, i.e. some segments were much more highly conserved than others. Both proteins had a hydrophobic region at the N-terminus.

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Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

Cited by 22 publications

References 16 publications

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product

Engineering of a Bacillus amyloliquefaciens Strain with High Neutral Protease Producing Capacity and Optimization of Its Fermentation Conditions

Cloning and Sequencing of a Gene from Bacillus amyloliquefaciens that Complements Mutations of the Sporulation Gene spoIID in Bacillus subtilis

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