The efficiency of processing and secretion of the thermolysin-like neutral protease from Bacillus cereus does not require the whole prosequence, but does depend on the nature of the amino acid sequence in the region of the cleavage site

Using deletion mutants, It is shown that part of the prosequence, the Ω-peptide (-4, -24), of the thermolysin-like neutral protease (TNP) from Bacillus cereus, Cnp, is not required for efficient processing and secretion of fully functional mature protease. It is demonstrated that the rate and selectivity of pro-protein processing is dependent on both the flexibility and primary sequence of the processing site. Processing is found to be particularly sensitive to the nature of the amino acid three residues upstream from the site of cleavage. A consensus sequence for TNP pro-protein processing has been identified, which provides further Insights. Finally, a larger deletion of a portion of the Cnp prosequence upstream from the Ω-peptide that includes amino acids conserved among TNPs reduces the rate of processing and secretion of Cnp and results in the accumulation of export-incompetent pre-proprotein in the cell fraction.     

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

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.     

Role of the pre-pro-region of neutral protease in secretion in Bacillus subtilis

A thermostable neutral protease (NprT) from Bacillus stearothermophilus CU21 is translated as a precursor containing pre-pro-structure. Deletion and insertion mutations were introduced in the pro-region and the effects of the mutations on production of active NprT were studied. The pro-region was important for the production of active NprT and was effective only when it was combined with the signal sequence (pre-region) of NprT. To examine the role of the pre-region, a penicillinase gene from Bacillus licheniformis (penP) was fused with various nprT secretion vectors. It was also found that the pre-region of NprT can potentially function as a signal sequence but is more functional for the production of NprT when it was combined with a normal pro-region. We discuss how the processing between the pro-structure and the mature portion of the protease might be done autoproteolytically.     

The role of calcium ions in the stability and instability of a thermolysin-like protease

Thermolysin and other secreted broad-specificity proteases, such as subtilisin or alpha-lytic protease, are produced as pre-pro-proteins that stay at least partially unfolded while in the cytosol. After secretion, the pro-proteases fold to their active conformations in a process that includes the autolytic removal of the pro-peptide. We review the life cycle of the thermolysin-like protease from Bacillus stearothermophilus in light of the calcium dependent stability and instability of the N-terminal domain. The protease binds calcium ions in the regions that are involved in the autolytic maturation process. It is generally assumed that the calcium ions contribute to the extreme stability of the protease, but experimental evidence for TLP-ste indicates that at least one of the calcium ions plays a regulatory role. We hypothesize that this calcium ion plays an important role as a switch that modulates the protease between stable and unstable states as appropriate to the biological need.     

Laundry detergent compatibility of the alkaline protease from Bacillus cereus

The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C.     

Highly thermostable neutral protease from Bacillus stearothermophilus

Bacillus stearothermophilus MK232, which produced a highly thermostable neutral protease, was isolated from a natural environment. By several steps of mutagenesis, a hyper-producing mutant strain, YG185, was obtained. The enzyme productivity was twice as much as that of the original strain. This extracellular neutral protease was purified and crystallized. The molecular weight of the enzyme was 34,000 by SDS-polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the enzyme activity were 7.5 and 70°C, respectively, and the enzyme was stable at pH 5–10 and below 70°C. The thermostability and specific activity of the new protease are around 10% and 40% higher than those of thermolysin (the neutral protease from Bacillus thermoproteolyticus), respectively. The enzyme was inactivated by EDTA, but not by phenylmethylsulfonyl fluoride. These results indicate that the enzyme is a highly thermostable neutral-(metallo)protease.     

The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase

The complete amino-acid sequence of a neutral proteinase, produced by Bacillus cereus, was determined by protein sequencing. The neutral proteinase consists of 317 amino-acid residues. The primary structure is 70% homologous to thermolysin, a thermostable neutral proteinase and 45% homologous to Bacillus subtilis neutral proteinase. The zinc-binding site and the hydrophobic pocket of the active site are highly similar in all three proteinases. B. cereus neutral proteinase which is 20 degrees C less thermostable (60 degrees C) than thermolysin (80 degrees C) shows only minor differences in calcium binding sites and salt bridges compared to thermolysin (known from its X-ray diffraction analysis), whereas B. subtilis neutral proteinase (50 degrees C) differs considerably. Therefore it was assumed that the difference in thermostability between B. cereus neutral proteinase and thermolysin is not caused by different metal binding properties, or differences in the active site, but by changes within the rest of the molecule. Calculation of secondary structure potentials according to Chou & Fasman, hydrophobicity and bulkiness of the different structural elements and preferred cold----hot amino-acid residue exchanges indicated, that the thermostability of thermolysin compared to B. cereus neutral proteinase is caused by small effects contributed by numerous amino-acid exchanges distributed over the whole molecule, resulting in increased hydrophobicity of beta-pleated sheet and higher bulkiness of alpha-helical regions.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Structural basis of the sphingomyelin phosphodiesterase activity in neutral sphingomyelinase from Bacillus cereus

Sphingomyelinase (SMase) from Bacillus cereus (Bc-SMase) hydrolyzes sphingomyelin to phosphocholine and ceramide in a divalent metal ion-dependent manner. Bc-SMase is a homologue of mammalian neutral SMase (nSMase) and mimics the actions of the endogenous mammalian nSMase in causing differentiation, development, aging, and apoptosis. Thus Bc-SMase may be a good model for the poorly characterized mammalian nSMase. The metal ion activation of sphingomyelinase activity of Bc-SMase was in the order Co2+ > or = Mn2+ > or = Mg2+ > Ca2+ > or = Sr2+. The first crystal structures of Bc-SMase bound to Co2+, Mg2+, or Ca2+ were determined. The water-bridged double divalent metal ions at the center of the cleft in both the Co2+- and Mg2+-bound forms were concluded to be the catalytic architecture required for sphingomyelinase activity. In contrast, the architecture of Ca2+ binding at the site showed only one binding site. A further single metal-binding site exists at one side edge of the cleft. Based on the highly conserved nature of the residues of the binding sites, the crystal structure of Bc-SMase with bound Mg2+ or Co2+ may provide a common structural framework applicable to phosphohydrolases belonging to the DNase I-like folding superfamily. In addition, the structural features and site-directed mutagenesis suggest that the specific beta-hairpin with the aromatic amino acid residues participates in binding to the membrane-bound sphingomyelin substrate.     

Specific detection of the gene for the extracellular neutral protease of Bacillus cereus by PCR and blot hybridization.

A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of the B. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.     

蜡样芽胞杆菌蛋白酶的克隆及在枯草芽胞杆菌系统中的高效表达

基于来源于Bacillus cereus WQ9-2的耐有机溶剂蛋白酶WQ的液相色谱-双质谱(LC-MS-MS)分析结果,设计引物,克隆耐有机溶剂蛋白酶WQ的基因,测序分析表明该蛋白酶的开放阅读框(ORF)大小为1 701 bp,编码566个氨基酸,其中含有信号肽(28个氨基酸)、前肽(220个氨基酸)及成熟肽序列(含954 bp编码318个氨基酸),相对分子质量约为3.7×104。将不带自身信号肽的蛋白酶基因apr WQ插入穿梭质粒p MA5中,构建了表达载体p MA5/apr WQ。该表达载体导入枯草芽胞杆菌WB600中获得阳性重组子WB600-p MA5/apr WQ,通过优化培养基成分以及培养条件,重组子发酵产蛋白酶体积酶活达到17 400 U/m L,约为高产野生菌产酶量的5倍。重组蛋白酶在多种有机溶剂(体积分数为50%)中表现出了良好的耐受性,验证了重组菌表达的蛋白酶与来源于B.cereus WQ9-2的耐有机溶剂蛋白酶性质一致,该耐有机溶剂蛋白酶的高效表达为进一步发挥其高效生物催化作用等实际应用奠定了基础。     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease.

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus

A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28 kDa, pH and temperature optima for this protease were 10 and 60 °C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu²⁺ ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.     

Crystal structure of neutral protease from Bacillus cereus refined at 3.0 A resolution and comparison with the homologous but more thermostable enzyme thermolysin

Neutral protease from Bacillus cereus exhibits a 73% amino acid sequence homology to thermolysin, for which an accurate crystal structure exists. The B. cereus enzyme is, however, markedly less thermostable. The neutral protease was crystallized and diffraction data to 3.0 A resolution were recorded by oscillation photography. The crystal structure was solved by molecular replacement methods using thermolysin as a trial molecule. The solution was improved by rigid-body refinement and model rebuilding into electron density omit-maps. The atomic co-ordinates were refined to R = 21.7% at 3.0 A resolution. Comparison of the resultant model with the thermolysin structure shows that the two enzymes are very similar with a root-mean-square deviation between equivalent C alpha-atoms of 0.88 A. The gamma-turn found in thermolysin is transformed into a beta-turn in the neutral protease by the insertion of a glycine residue. There appear to be no contributions to the enhanced thermostability of thermolysin from additional salt bridges, whereas contributions in the form of extra hydrogen bonding interactions could be important. Other factors that may affect thermostability include the two glycine to alanine exchanges and perturbations in the environment of the double calcium site.     

Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674

Bacillus cereus KCTC 3674 excretes several kinds of extracellular proteases into the growth medium. Two proteases with molecular masses of approximately 36-kDa and 38-kDa, as shown by SDS-PAGE, were purified from the culture broth. The 38-kDa protease was purified from B. cereus cultivated at 37 degrees C, and the 36-kDa protease was obtained from the B. cereus cultivated at 20 degrees C. The 38-kDa protease was identified as an extracellular neutral (metallo-) protease and was further characterized. The 36-kDa protease was shown to be a novel enzyme based on its N-terminal amino acid sequence, its identification as a metallo-enzyme that was strongly inhibited by EDTA and o-phenanthroline, its hemolysis properties, and its optimal pH and temperature for activity of 8.0 and 70 degrees C, respectively.