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

Abstract

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...

A 10.3 kbp Segment from nprB to argJ at the 102 Region of the Bacillus Subtilis Chromosome

“…This, therefore, strongly suggests that a DNA rearrangement occurred downstream of the dap operon, giving rise to the difference between strain 168 and strain GS11. Finally, it is important to note that nprB was, in fact, previously mapped at 95" (Tran et al, 1991), whilst the location of ipi on the chromosome was not reported earlier.…”

Hetero- and Autoprocessing of the Extracellular Metalloprotease (Mpr) in Bacillus subtilis

“…Two major extracellular proteases are produced during the onset of sporulation, the alkaline serine protease subtilisin (AprE) (18,38) and a neutral protease (NprE) (40). The minor extracellular proteases include Epr (2,27), bacillopeptidase F (Bpr) (30,31,39), metalloprotease (Mpr) (20,23,28), neutral protease B (NprB) (35), wall-associated extracellular protease (WprA) (17), and Vpr (29). Expression of these proteolytic enzymes seems to be tightly regulated (33), and their major function is thought to be supplying amino acids for growth via degradation of extracellular proteins.…”

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

“…Furthermore, we show that existing differences in specificity and activity between two individual members can be canceled by a single amino acid substitution. (25), the nprT gene encoding the TLP of Bacillus stearothermophilus CU21 (26) (TLP-ste), the nprC gene encoding the TLP of Bacillus cereus (3)(TLP-cer), and the nprB gene encoding the TLP-sub of Bacillus subtilis (27) were cloned, subcloned, and expressed as described previously (28). The purified TLP-sau of Staphylococcus aureus (29) (aureolysin, EC 3.4.24.29) was kindly provided by Dr. J. Potempa.…”