The nucleotide sequence and some properties of the neutral protease gene of Bacillus amyloliquefaciens

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

doi:10.1016/0168-1656(85)90043-4

Cited by 25 publications

(7 citation statements)

References 21 publications

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“…It is likely that Achro- proteases from both gram-positive and gram-negative bacteria have been known to be synthesized as precursor proteins bearing long extensions at the N terminus or the C terminus or both. a-Lytic protease from L. enzymogenes (7,32), proteases A and B from Streptomyces griseus (10), subtilisins (34), and neutral proteases of various Bacillus species (30,36,43) have long polypeptide extensions at their N termini. The immunoglobulin A protease of Neisseria gonorrhoea (26) and a protease from Serratia marcescens (41) have C-terminal extensions.…”

Section: Resultsmentioning

confidence: 99%

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Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus

Norioka

Sakiyama

1990

J Bacteriol

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Two bacteriolytic enzymes secreted by Achromobacter lyticus M497-1 were purified and identified as being very similar (considering their amino acid composition and N-terminal sequence) to a-and I-lytic proteases from Lysobacter enzymogenes. A 1.8-kb EcoRI fragment containing the structural gene for P-lytic protease was cloned from A. lyticus chromosomal DNA. The protein sequence deduced from the nucleotide sequence was identical to the known sequence of I-lytic protease, except for six residues. The nucleotide sequence revealed that the mature enzyme is composed of 179 amino acid residues with an additional 195 amino acids at the amino-terminal end of the enzyme, which includes the signal peptide, thus indicating that the enzyme is synthesized as a precursor protein.Besides lysozyme, which has long been used to lyse bacteria in laboratory and medical procedures, various other bacteriolytic enzymes with different mechanisms of action have been reported from a variety of bacteria (3, 35). These enzymes, termed autolysins (37), are able to hydrolyze particular bonds in cell wall peptidoglycans and are classified into three groups: glycosidases, which hydrolyze the polysaccharide chains; endopeptidases, which cleave the peptide cross-links; and N-acetylmuramoyl-L-alanine amidases, which split the linkage between polysaccharides and peptides. Autolysins are considered to be involved in important biological processes such as cell wall turnover (16), cell separation (8), genetic transformation, formation of flagella, and sporulation (27). These lytic enzymes are interesting not only for their roles in bacterial metabolism (37), but also for their antimicrobial potential (1). In particular, a lytic enzyme preparation, Achromopeptidase, from Achromobacter lyticus is of interest because it can lyse not only gram-positive bacteria, but also some strains of gram-negative bacteria by a different mechanism from that of lysozyme (Y. Yamasaki, personal communication). To investigate the mechanism of action of this type of bacteriolytic enzyme, we undertook the purification of an individual bacteriolytic enzyme from the culture medium of A. lyticus M497-1. Two enzymes were finally purified to homogeneity. From amino acid compositions and amino acid sequence determinations for the 25 N-terminal residues, we demonstrated that these two bacteriolytic enzymes from the Achromobacter strain were identical to a-and P-lytic proteases from Lysobacter enzymogenes (39). In contrast to the extensively investigated a-lytic protease, little is known about the substrate specificity, the active site, and the tertiary structure of 3-lytic protease (40).We then cloned the ,-lytic protease gene from A. lyticus M497-1 and determined its nucleotide sequence. This paper describes the results of our studies on the determination of the primary structure of Achromobacter ,B-lytic protease and its precursor, as deduced from the nucleotide sequence of the cloned gene.

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Section: Resultsmentioning

confidence: 99%

“…To clone the P-lytic protease gene, PCR methodology (28) was adopted. Upstream and downstream primers were deduced from the two known amino acid sequences at positions 46 to 52 and 168 to b Calculated from the amino acid sequences deduced from the cloned a-lytic protease gene (7,30) and from the f-lytic protease gene (this paper).…”

Section: Resultsmentioning

confidence: 99%

Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus

Norioka

Sakiyama

1990

J Bacteriol

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“…When compared to the National Center for Biotechnology Information (NCBI) protein database by BLAST search, the sequences of the purified enzyme did not show significant homology to calf chymosin. However, the sequences of the purified enzyme showed some homology with the sequences of milk-clotting aspartic proteinase (40%) from Rhizomucor pusillus [33], Mucor rennin (33.3%) from Rhizomucor miehei [34] and neutral protease (26.7%) from Bacillus amyloliquefaciens [35]. The differences in those basic amino acids among proteases may result from their differences in their primary structures.…”

Section: N-terminal Amino Acid Sequencementioning

confidence: 99%

Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4

Ren

Guo

et al. 2010

Korean J. Chem. Eng.

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The milk-clotting enzyme from Bacillus amyloliquefaciens D4 was purified to 17.2-fold with 20% recovery by precipitation in ammonium sulfate and ion-exchange chromatography. The molecular mass of the enzyme was 58.2 kDa as determined by SDS-PAGE, and it was proved to be a metalloprotease by EDTA inhibition. The enzyme was active in the pH range 5.5-7.0 and was inactivated completely by heating at 55 o C for 20 min. The highest level of enzyme activity was obtained at 65 o C, pH 5.5, in the presence of 25 mM CaCl 2 . The milk-clotting activity was inhibited only slightly by Na + and K + and significantly by Cu 2+ , Zn 2+ and Sn 2+ . The K m value of this enzyme was 0.471 mg/mL. The high level of milk-clotting activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from B. amyloliquefaciens D4 should be considered as a potential substitute for calf rennet.

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“…The nucleotide sequence contained a large open reading frame 5' of the coding sequence of the mature enzyme, and we proposed that the additional 199 amino acids made up a 33-amino-acid signal sequence (pre) and a 166-amino-acid pro region. Recent studies with secreted proteases from both grampositive and gram-negative bacteria, including several Bacillus species (17,32,34,(36)(37)(38)41), Neisseria gonorrhoeae (28), Streptomyces griseus (13), and Serratia marcescens (40), have shown that all of these bacterial proteases are synthesized as precursors, although the pro region varies in its amino-or carboxyl-terminal location. Recently, the 77-amino-acid amino-terminal pro region of Bacillus subtilis subtilisin E has been shown to be necessary for the production of active protease, suggesting a critical role for the pro region in folding (15).…”

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Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity

et al. 1989

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The a-lytic protease of Lysobacter enzymogenes was successfully expressed in Escherichia cofi by huing the promoter and dgnal sequence of the E. coil phoA gene to the proenzyme portion of the a-lytic preten gene.Following induction, active enzyme was found both within cells and in the extracellular ue , where it slowly accumulated to high levels. Use of a similar gene fusion to express the protease domain alone produced inactive enzyme, indicatin that the large amino-terminal pro region is necessary for activity. The cinations for protein foding are discussed. Furthermore, inactivation of the protse by mutation of the catalytic serine residue resulted in the production of a higher-molecular-weight form of the a-lytic protease, suggesing that the enzyme is self-processing in E. coli. a-Lytic protease is one of a battery of extracellular enzymes secreted by the gram-negative bacterium Lysobacter enzymogenes to lyse and degrade soil microorganisms. By virtue of its well-studied Asp-His-Ser catalytic triad mechanism (1, 2, 5), its degree of structural homology to mammalian serine proteases (6), and its amenity to nuclear magnetic resonance studies (1, 2, 30), a-lytic protease is an ideal candidate for site-specific mutagenesis studies of substrate specificity and structure-function relationships. We recently reported the cloning and sequence analysis of the oa-lytic protease gene from L. enzymogenes (33). The nucleotide sequence contained a large open reading frame 5' of the coding sequence of the mature enzyme, and we proposed that the additional 199 amino acids made up a 33-amino-acid signal sequence (pre) and a 166-amino-acid pro region. Recent studies with secreted proteases from both grampositive and gram-negative bacteria, including several Bacillus species (17,32,34,(36)(37)(38)41), Neisseria gonorrhoeae (28), Streptomyces griseus (13), and Serratia marcescens (40), have shown that all of these bacterial proteases are synthesized as precursors, although the pro region varies in its amino-or carboxyl-terminal location. Recently, the 77-amino-acid amino-terminal pro region of Bacillus subtilis subtilisin E has been shown to be necessary for the production of active protease, suggesting a critical role for the pro region in folding (15). In this report we provide evidence that the pro region of a-lytic protease has a similar function.We subcloned regions of the a-lytic protease gene behind the promoter and signal sequence of the Escherichia coli phoA gene, allowing production of a-lytic protease in E. coli under conditions of phosphate depletion (16). In addition to confirming the requirement for the pro region, this approach has provided evidence that expression of active enzyme is temperature sensitive in E. coli and that a-lytic protease has the ability to proteolytically process itself. The implications of these results for protein folding are discussed. Strains, media, and expression. DG98, an F'-carrying strain used for M13 phage growth, was described previously (33). MH1 (araD139 AlacX74 galU galK hsr rps...

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The nucleotide sequence and some properties of the neutral protease gene of Bacillus amyloliquefaciens

Cited by 25 publications

References 21 publications

Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus

Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus

Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4

Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity

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