Bacillus cereus neutral protease

Feder, Joseph; Keay, Leonard; Garrett, L.R.; Cirulis, N.; Moseley, Martha H.; Wildi, Bernard S.

doi:10.1016/0005-2795(71)90061-4

Cited by 43 publications

(20 citation statements)

References 16 publications

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“…These proteases contain the typical HEXXH amino acid motif, require Zn 2ϩ ions for their activity, and contain multiple Ca 2ϩ ions (up to four) for stability. All enzymes are optimally active at neutral pH (1,5).…”

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confidence: 99%

“…The availability of an impressive amount of sequence, structural, and kinetic data renders this group of proteases an ideal subject for rational design strategies. Although some of the family members have been characterized individually (5,(21)(22)(23)(24), a consistent comparison with an identical substrate set and a uniform set of assay conditions has never been conducted. Previously it was suggested that TLPs exhibit a preference for large hydrophobic P 1 Ј residues (Leu or Phe) (1,17,21,22).…”

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confidence: 99%

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Substrate Specificity in the Highly Heterogeneous M4 Peptidase Family Is Determined by a Small Subset of Amino Acids

Kreij

Venema

Burg³

2000

Journal of Biological Chemistry

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The members of the M4 peptidase family are involved in processes as diverse as pathogenicity and industrial applications. For the first time a number of M4 family members, also known as thermolysin-like proteases, has been characterized with an identical substrate set and a uniform set of assay conditions. Characterization with peptide substrates as well as high performance liquid chromatography analysis of ␤-casein digests shows that the M4 family is a homogeneous family in terms of catalysis, even though there is a significant degree of amino acid sequence variation. The results of this study show that differences in substrate specificity within the M4 family do not correlate with overall sequence differences but depend on a small number of identifiable amino acids. Indeed, molecular modeling followed by site-directed mutagenesis of one of the substrate binding pocket residues of the thermolysin-like proteases of Bacillus stearothermophilus converted the catalytic characteristics of this variant into that of thermolysin.

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confidence: 99%

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confidence: 99%

Substrate Specificity in the Highly Heterogeneous M4 Peptidase Family Is Determined by a Small Subset of Amino Acids

Kreij

Venema

Burg³

2000

Journal of Biological Chemistry

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“…Neutral protease from Baciflus cereus (NP) is a metalloprotease which hydrolyses polypeptide chains at the imino side of hydrophobic and aromatic residues (Feder et al, 1971 ;Sidler et al, 1986a). The amino acid sequence, comprising 317 residues, was determined by protein sequencing (Sidler et al, 1986b).…”

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The structure of neutral protease from Bacillus cereus at 0.2‐nm resolution

Stark

Pauptit

Wilson

et al. 1992

European Journal of Biochemistry

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The crystal structure of the neutral protease from Bacillus cereus has been refined to an R factor of 17.5% at 0.2-nm resolution. The enzyme, an extracellular metalloendopeptidase, consists of two domains and binds one zinc and four calcium ions. The structure is very similar to that of thermolysin, with which the enzyme shares 73% amino-acid sequence identity. The active-site cleft between the two domains is wider in neutral protease than in thermolysin. This suggests the presence of a flexible hinge region between the two domains, which may assist enzyme action. The high-resolution analysis allows detailed examination of possible causes for the difference in thermostability between neutral protease and thermolysin.

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“…The B. subtilis neutral protease A (mature form) shows 56% homology with thermolysin. Two other thermolysin-related neutral proteases have also have been purified from B. stearothermophilus (6,23) and B. cereus (5,29). They share 92 and 84% homology with thermolysin, respectively.…”

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Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis

Tran

Wong

1991

J Bacteriol

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We have cloned from Bacillus subtilis a novel protease gene (nprB) encoding a neutral protease by using a shotgun cloning approach. The gene product was determined to have a molecular mass of 60 kDa. It has a typical signal peptide-like sequence at the N-terminal region. The expression of nprB can be stimulated by using a B. subtilis strain, WB30, carrying a sacUh mutation. Expression of this protease gene results in production of a 37-kDa protease in the culture medium. The first five amino acid residues from the N terminus of the mature protease were determined to be Ala-Ala-Gly-Thr-Gly. This indicates that the protease is synthesized in a preproenzyme form. The purified protease has a pH optimum of around 6.6, and its activity can be inhibited by EDTA, 1,10-phenanthroline (a zinc-specific chelator), and dithiothreitol. It retained 65% of its activity after treatment at 65C for 20 min. Sequence comparison indicates that the mature form of this protease has 66% homology with the two thermostable neutral proteases from B. thermoproteolyticus and B. stearothermophilus. It also shares 65, 61, and 56% homology with the thermolabile neutral proteases from B. cereus, B. amyloliquefaciens, and B. subtilis, respectively. The zinc-binding site and the catalytic residues are all conserved among these proteases. Sequence homology extends into the "propeptide" region. The nprB gene was mapped between metC and glyB and was not required for growth or sporulation.The recent efforts to clone and characterize protease genes from Bacillus subtilis have clearly established that B. subtilis secretes at least five extracellular proteases into the culture medium at the end of the exponential phase. They are the neutral protease A (48), subtilisin (alkaline protease) (34), extracellular protease (45), metalloprotease (31), and bacillopeptidase F (32, 47). The structural genes encoding these proteases have been cloned and characterized. All of these proteases were synthesized in forms of "prepro" enzyme. Both subtilisin and neutral protease A are considered major proteases since their activity accounts for 20 and 70%, respectively, of total protease activity in the culture medium (14,34,48). While these proteases are thermolabile, a thermostable neutral protease (thermolysin) from B. thermoproteolyticus has been purified (4) and characterized (21,22,26 Bacterial strains and media. B. subtilis DB102 (his nprR2 nprEI8) and DB104 (his nprR2 nprEJ8 aprAA3) (7) and E.coli DH5a [4+80dlacZ M15 endAl recAl hsdRJ7 (r-m-) supE44 thi-1, A-gyrA relAl F-A(lacZYA-argF)U169] were used for routine transformation. WB30 (his nprR2 nprE18 aprAA3 sacU1200) (16) was used for expression of the cloned nprB. B. subtilis strains were cultivated on tryptose blood agar base (Difco) or Schaeffer sporulation (SG) (17) agar plates. For detection of proteolytic activity, SG plates containing 1% skim milk were used. E. coli carrying plasmids was cultured on YT agar plates (19) or in LB broth containing 75 ,ug of ampicillin per ml.Cloning of nprB. Chromosomal DNA was...

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Bacillus cereus neutral protease

Cited by 43 publications

References 16 publications

Substrate Specificity in the Highly Heterogeneous M4 Peptidase Family Is Determined by a Small Subset of Amino Acids

Substrate Specificity in the Highly Heterogeneous M4 Peptidase Family Is Determined by a Small Subset of Amino Acids

The structure of neutral protease from Bacillus cereus at 0.2‐nm resolution

Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis

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