Purification and characterization of phosphofructokinase of Bacillus licheniformis

The phosphofructokinase (PFK) of Bacillus licheniformis was purified about 50–65-fold and examined for a number of enzymatic and physical characteristics. The enzyme is quite unstable under normal assay conditions, but Mg²⁺, K⁺, adenosine-5′-diphosphate, phosphoenolpyruvate (PEP), and fructose-6-phosphate (fru-6-P) are fairly effective stabilizing agents. Saturation functions for ATP and fru-6-P were hyperbolic. Several attempts to induce positive cooperative binding of fru-6-P were unsuccessful. However, “sigmoidal” saturation kinetics for fru-6-P could be observed under assay conditions that permitted an irreversible inactivation of the PFK during assay. Several divalent cations could support the catalysis of B. licheniformis PFK and the enzyme was activated by both NH₄⁺ and K⁺ ions. B. licheniformis PFK is inhibited by citrate, ATP, PEP, Ca²⁺, and several other metabolic intermediates, but the inhibition caused by citrate and ATP at high fru-6-P concentration and by calcium can be relieved by Mg²⁺ addition while PEP inhibition is specifically relieved by fru-6-P. There are at least three binding sites for PEP on the PFK molecule. The active form of this PFK has a molecular weight of about 134,000 daltons. In the presence of Mg²⁺, adenosine-5′-triphosphate (ATP), and PEP, at 0 °C, the PFK molecule is rapidly dissociated to an inactive form with a molecular weight of about 68,000 daltons. Association of these subunits to yield the active form of PFK occurs spontaneously, and rapidly, when the temperature is raised to 30 °C. Ninety percent of the original activity is recovered after activation. Growth of B. licheniformis on several different substrates resulted in minor variations of PFK activity. In a parallel fashion, sporulation involved no irreversible inactivation of PFK and the level of the activity was about the same throughout the life cycle. Control of this enzyme during sporulation could be affected by any or all of the cell constituents found to regulate PFK activity in vitro, but it is considered likely that the most significant in vivo negative effector is PEP, with this inhibition being reversed by fru-6-P.     

Purification and characterization of the respiratory nitrate reductase of Bacillus licheniformis.

1. Respiratory nitrate reductase of Bacillus licheniformis was extracted from the bacterial membranes by treatment with deoxycholate and purified to a homogeneous state by means of gel chromatography and anion-exchange chromatography. 2. The enzyme (Mr = 193,000, s20, w = 8.6) consists of two subunits, having apparent molecular weight of 150,000 (alpha subunit) and 57,000 (beta subunit), which are present in an equimolar ratio. It does not contain carbohydrate. Ageing of the enzyme appears to result in splitting of the polypeptide chains at specific sites followed by dissociation and reassociation of the digestion products in various combinations. 3. In contrast to Klebsiella aerogenes repiratory nitrate reductase, which is isolated in a tetrameric form that can be reversibly dissociated into a monomeric form by detergents, B. licheniformis nitrate reductase, after isolation, is always present in a monomeric form. This property is related to the difference in membrane localization of the enzyme in the two organisms. 4. B licheniformis nitrate reductase contains 6.9 atoms of non-heme iron, 6.7 atoms of acid-labile sulfide and 0.93 atoms of molybdenum per molecule of enzyme. The molybdenum seems to be part of a low-molecular weight peptide Mo-cofactor) to which it may be bound by interaction with thiol-groups. 5. Antiserum against the native enzyme contains antibodies against both subunits as well as the Mo-cofactor. The Mo-cofactor does not have any antigenic determininants in common with either the alpha or the beta subunit. Also neither subunit cross-reacts with antiserum against the other subunit. Whereas the respiratory nitrate reductases from K. aerogenes and Escherichia coli are immunologically related, the native enzyme from B. licheniformis does not show any cross-reaction with antiserum prepared against either the K. aerogenes or the E. coli enzyme.     

Identification of penicillin-binding protein 5a of Bacillus megaterium KM as a DD-carboxypeptidase.

Measurement of the stabilities of DD-carboxypeptidase activity and the penicillin-binding activity of proteins 5 and 5a in membranes isolated from vegetative cells and stage-V forespores suggests that the unique sporulation-specific protein 5a may be a penicillin-sensitive DD-carboxypeptidase.     

地衣芽孢杆菌胞外蛋白酶的纯化及特性分析

研究不同条件对地衣芽孢杆菌De株产生胞外蛋白酶的量及其酶活性的影响,结果表明在pH为7.4—8.2范围内,温度为30℃时,培养8—12h的菌株所分泌胞外产物中的蛋白酶活性最高。实验先以半透膜法收集芽孢杆菌的胞外产物,然后再经过硫酸铵沉淀过夜S、ephadex G-100凝胶层析和DEAE-Cellulose离子交换层析及聚丙烯酰胺凝胶电泳等四个步骤的分离纯化后,可以得到含有3种主要蛋白质(BLP1、BLP2、BLP3)成分的胞外蛋白酶,其分子量分别为66.2KD、31.0KD及约20.1KD,所得纯化蛋白酶的蛋白浓度为0.773μg/mL,蛋白回收率为11.66%。实验还发现,纯化的胞外蛋白酶在100℃下作用30min,仍可保持其活力,可见具有相当的热稳定性,而其酶活最佳的pH和温度条件分别为7.8和45—65℃。酶活抑制实验显示EDTA、铜、钴、镁离子等均可成为其酶活抑制因子;而丝氨酸蛋白酶抑制剂甲基磺酰氟(PMSF)、铁、锰、钡、钙离子等对酶活性没有明显影响;锌则会令之酶活性其部分丧失。     

Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium ATCC 9790 has an unusually low affinity for penicillin (50% binding occurred at a penicillin level of 8 micrograms/ml after 60 min of incubation, and the protein only became labeled after 20 min of incubation with high concentrations of radioactive penicillin). PBPs with similar properties are carried by strains of Streptococcus durans, Streptococcus faecalis, and Streptococcus lactis but not by strains of groups A, B, C, and G streptococci or Streptococcus pneumoniae. The strains carrying the slow-reacting PBP demonstrated a sensitivity to penicillin that was several hundred times lower than that of strains not carrying it. Spontaneous mutants with minimal inhibitory concentrations of penicillin of 20, 40, and 80 micrograms/ml were isolated from S. faecium ATCC 9790. They all showed a dramatic increase in the amount of slow-reacting PBP produced. Mutants with increased penicillin resistance were also isolated from wild-type strains of S. durans, S. faecalis, and S. faecium. All of them carried a greater amount of the slow-reacting PBP than that carried by the parent. Finally, it was found that resistant S. faecium ATCC 9790 mutants grew normally in the presence of penicillin concentrations that were far above that saturating all PBPs except PBP 5. Cell growth was, on the contrary, inhibited by a penicillin concentration that saturated the slow-reacting PBP by 90%. This penicillin dose was equal to the minimal inhibitory concentration.     

Purification and characterization of PBP4a, a new low-molecular-weight penicillin-binding protein from Bacillus subtilis

Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits DD-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781-788, 1992), which is rapidly inactivated by many beta-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k(2)/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M(-1) s(-1) for the Actinomadura sp. strain R39 peptidase, 1,400 M(-1) s(-1) for B. subtilis PBP4a, and 7,000 M(-1) s(-1) for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k(2)/K = 46,000 M(-1) s(-1)). PBP4a is also much more thermostable than the R39 enzyme.     

Purification and characterization of antimicrobial substances from Bacillus licheniformis BFP011

Bacillus licheniformis BFP011 isolated from papaya (Thailand) could produce extracellular antimicrobial substances which were active against some important phytopathogens, pathogenics and spoilage microorganisms such as Colletotrichum capsici, Escherichia coli O157: H7 and Salmonella typhi ATCC 5784. The antimicrobial substances of this bacterium showed resistance to pronase enzyme and high temperature at 100 and 121°C for 15 min. They were purified by TLC on silica gel plates F254 using the different solvent mixtures. The best solvent mixture was revealed as n-butanol: ethanol: acetic acid: water (30: 60: 5: 30, v/v). The spots F4, F5 and F6 from TLC were able to inhibit growth of S. typhi ATCC 5784 assayed in vitro by the disc diffusion method. The characterization of the active fractions F4, F5 and F6 from TLC and reversedphase HPLC indicated that the antimicrobial substances of B. licheniformis BFP011 contain peptides and unsaturated fatty acids.     

Purification and characterization of two thermostable protease fractions from Bacillus megaterium

Protease enzyme from Bacillus megaterium was successively purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-200. The purification steps of protease resulted in the production of two protease fractions namely protease P1 and P2 with specific activities of 561.27 and 317.23 U mg^?1 of protein, respectively. The molecular weights of B. megaterium P1 and P2 were 28 and 25 KDa, respectively. The purified fractions P1 and P2 were rich in aspartic acid and serine. Relatively higher amounts of alanine, leucine, glycine, valine, thereonine valine and glutamic acid were also present. The maximum protease activities for both enzyme fractions were attained at 50 °C, pH 7.5, 1% of gelatine concentration and 0.5 enzyme concentrations. P1 and P2 fractions were more stable over pH 7.0–8.5 and able to prolong their thermal stability up to 80 °C. The effect of different inhibitors on the protease activity of both enzyme fractions was also studied. The enzyme was found to be serine active as it had been affected by lower concentrations of phenylmethylsulfonyl fluoride (PMSF). Complete dehairing of the enzyme-treated skin was achieved in 12 h, at room temperature.     

Purification and partial characterization of a penicillin-binding protein from Mycobacterium smegmatis.

Penicillin-binding proteins (PBPs), although characterized from several organisms, have so far not been studied in mycobacteria. The present study is the first characterization of a PBP from Mycobacterium smegmatis. The PBP was purified by solubilization of the membranes with Triton X-100 and successive chromatography of the solubilized proteins on ampicillin-linked CH Sepharose 4B and DE-52. The purified PBP (M(r), 49,500) catalyzed a model transpeptidase reaction with the tripeptide acetyl2-L-Lys-D-Ala-D-Ala as the substrate and Gly-Gly as the acceptor. The transpeptidase activity was inhibited by 50% at a benzylpenicillin concentration of 1.8 x 10(-7) M, which was similar to the concentration (1.1 x 10(-7) M) of benzylpenicillin required to saturate to 50% this PBP. Of several antibiotics tested, the concentration of antibiotic required to inhibit [35S]penicillin binding by 90% was found to be the lowest for cefoxitin and Sch 34343.     

Citrate synthase from a Gram-positive bacterium. Purification and characterization of the Bacillus megaterium enzyme.

Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.     

Purification and characterization of a membrane-associated phosphatidylserine synthase from Bacillus licheniformis

A CDP-diacylglycerol-dependent phosphatidylserine synthase was solubilized from Bacillus licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue dextran-Sepharose. The purified preparation showed a single band with an apparent relative molecular mass of 53 000 daltons when subjected to sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Proteolytic digestion of the enzyme yielded a smaller (41 000 daltons) active form. The preparation was free of any phosphatidylglycerophosphate synthase, phosphatidylserine decarboxylase, CDP-diacylglycerol hydrolase, and phosphatidylserine hydrolase activities. The utilization of substrates and the formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential Bi-Bi reaction as opposed to the ping-pong mechanism exhibited by the well-studied phosphatidylserine synthase of Escherichia coli [Larson, T. J., & Dowhan, W. (1976) Biochemistry 15, 5212-5218]. The B. licheniformis enzyme was also found to be markedly dissimilar to the E. coli enzyme with regard to association with detergent micelles, affinity for ribosomes, and antigenicity.     

A frameshift mutation that elongates the penicillinase protein of Bacillus licheniformis

A penicillinase mutant penP102, isolated after ICR (acridine mustard) mutagenesis of Bacillus licheniformis strain 749/C, retains about 50% of the wild-type penicillinase specific activity. The penicillinase produced by this mutant differs from the wild-type protein in its sensitivity to pH and its electrophoretic behaviour. The penP102 mutation appears to have several other phenotypic effects, including an increase in the efficiency of release of the extracellular form of the enzyme.The penP102 penicillinase has been purified and its amino acid sequence compared to that of the wild-type enzyme. The mutation has resulted in the replacement of the last three amino acids of the wild-type enzyme and the addition of 17 residues at the carboxy-terminus. Comparison of the wild-type and mutant amino acid sequences shows that the mutational event is a single nucleotide deletion from the codon for asparagine²⁶⁵. Consideration of the possible nucleotide sequence for the region beyond the carboxy-terminus of the wild-type protein shows that there are no possible termination codons until four and six triplets beyond the codon for the carboxy-terminal lysine, indicating that the carboxy-terminus of the wild-type extracellular penicillinase is generated by proteolytic cleavage of a larger precursor protein.     

The association of penicillin-binding proteins with cell elongation and septum formation in Bacillus megaterium.

The synthesis of a new bifunctional compound in which two aminoacridine chromophores are linked by the bicyclic depsipeptidic backbone of des-N-tetramethylTriostin A is described. The molecule, bis-[(9-acridinyl)-D-seryl-L-alanyl-L-cysteinyl-L-valine] dilactone disulphide, structurally analogous to the antibiotic anti-tumour drug Triostin A, is shown to possess a high affinity to DNA and to act as a bis-intercalator on the basis of spectroscopic, viscosimetric and thermal-denaturation studies. This model constitutes the first attempt of a synergic association between a peptidic moiety that mimics a naturally occurring drug and aminoacridine, the two parts themselves each exhibiting a high affinity for the DNA target.     

Purification and characterization of extracellular beta-amylase of Bacillus megaterium B(6)

The extracellular beta-amylase from starch induced Bacillus megaterium B6 was purified to homogeneity in a very convenient way; through molecular sieving as demonstrated by the presence of a single band of protein in SDS-PAGE and single peak in gel scanning. The molecular mass of the purified enzyme (monomer) was found to be unusually high, around 105,000 Da. The pH and temperature optima of the purified beta-amylase were at 6.9 and 60 degrees C, respectively. Mn2+ and exogenous thiols were found to play a remarkable role in reactivation of thermally and chemically denatured enzyme. The purified enzyme could saccharify both pure and low quality starches, where maltose could be detected as the major end product.     

Purification and characterization of additional low-molecular-weight basic proteins degraded during germination of Bacillus megaterium spores.

Dormant spores Bacillus megaterium contained a group of low-molecular-weight (5,000 to 11,000) basic (pI greater than 9.4) proteins (termed D, E, F, and G proteins) which could be extracted from disrupted spores with strong acids. These proteins were distinct from the previously described A, B, and C proteins which are degraded during spore germination. However, the D, E, F, and G proteins were also rapidly degraded during spore germination, accounting for 10 to 15% of the protein degraded. Proteins similar to the D, E, F, and G species were also present in spores of other bacterial species. In B. megaterium, the D, E, F, and G proteins were low or absent (less than 15% of the spore level) in vegetative and young sporulating cells and appeared only late in sporulation. The D, E, F, and G proteins were purified to homogeneity, and all contained a high percentage of hydrophilic amino acids; one protein (G) contained 31% basic amino acids and also contained tryptophan. All four proteins were rapidly degraded in vitro by dormant spore extracts. Two proteins (D and F) were degraded in vitro by the previously described spore protease which initiates degradation of the A, B, and C proteins in vivo; the spore enzyme (s) degrading proteins E and G have not been identified.     

Saturation of penicillin-binding protein 1 by β-lactam antibiotics in growing cells of Bacillus licheniformis

With the help of a new highly sensitive method allowing the quantification of free penicillin-binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β-lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.