Improvement of a useful enzyme (subtilisin BPN′) by an experimental evolution system

In order to improve a natural enzyme so as to fit industrial purposes, we have applied experimental evolution techniques comprised of successive in vitro random mutagenesis and efficient screening systems. Subtilisin BPN, a useful alkaline serine protease, was used as the model enzyme, and the gene was cloned to an Escherichia coli host-vector system. Primary mutants with reduced activities of below 80% of that of the wild type were first derived by hydroxylamine mutagenesis directly applied to subtilisin gene DNA, followed by screening of clear-zone non-forming transformant colonies cultured at room temperature on plates containing skim-milk. Then, secondary mutants were derived from each primary mutant by the same mutagenic procedure, but screened by detecting transformant colonies incubated at 10°C with clear zones that were greater in size than that of the wild type. One such secondary mutant, 12–12, derived from a primary mutant with 80% activity, was found to gain 150% activity (k _cat/K _m value) of the wild-type when the mutant subtilisin gene was subcloned to a Bacillus subtilis host-vector system, expressed to form secretory mutant enzyme in the medium, and the activity measured using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate. When N-succinyl-l-Ala-l-Ala-l-Pro-l-Leu-p-nitroanilide was used, 180% activity was gained. Genetic analysis revealed that the primary and secondary mutations corresponded to D197N and G131D, respectively. The activity variations found in these mutant subtilisins were discussed in terms of Ca²⁺-binding ability. The thermostability was also found to be related to the activity.     

Thermal acclimation and kinetics of a trypsin-like protease in eucarid crustaceans

Summary The properties of a trypsin-like protease in homogenates from midgut glands and gastric fluids of crustaceans were analyzed with special emphasis on thermal acclimation. For comparison, four species from different climatic regions were investigated: Ocypode ryderi (tropical), Cancer pagurus (temperate), Meganyctiphanes norvegica (subarctic-boreal), and Chorismus antarcticus (Antarctic). The pH optimum of the hydrolysis of N-benzoyl-l-arginine-p-nitroanilide is similar in all four species; at 25°C it ranged between pH 8 and 9.5. In the gastric fluids, pH was between 6.4 (Chorismus) and 7.7 (Ocypode); under experimental conditions at 25C, between 25% (Chorismus) and 95% (Ocypode) of maximal activity were observed at these pH values. Temperature optima of protease activity are independent from mean ambient temperature and were found to be around 50°C in Ocypode, 45°C in Cancer, 50–55°C in Meganyctiphanes, and 40°C in Chorismus. At temperatures near 0°C, temperate and tropical species show either a very low or even no activity at all, whereas the Antarctic and subarctic-boreal species display a residual activity of up to 15% of maximum activity. Under natural conditions, approximately 50% of maximal available enzymatic activity are eventually utilized. The kinetic parameters V _max and K _m depend on temperature and show distinct differences between the species. As an immediate response to temperature changes, the affinity for substrate decreases with elevated temperatures. Cold adaptation implies an effective utilization of energy in a low-energy system; the most prominent means of adaptation to low temperatures is the reduction of activation energy. Energies of activation in tropical temperate, and subarctic-boreal species (23.3–31.5 kJ·mol^-1) are significantly higher than in the Antarctic species (11.9–13.6 kJ·mol^-1). The enzymes were inhibited by N-tosyl-l-lysine chloromethyl ketone, copper sulfate, mercury chloride, and silver nitrate. In all enzymes, soybean trypsin inhibitor was the most effective inhibitor. Activation occurred after application of bovine serum albumin or calcium and magnesium chloride. The species-specific reactions after application of different protein or salt solutions support the hypothesis of decisive differences at the molecular level.Abbreviations BSA bovine serum albumin - E_a energy of activation - K _m Michaelis-Menten-constant - l-BAPA N-benzoyl-l-arginine-p-nitroanilide - SB soybean trypsin inhibitor - TLCK N-tosyl-l-lysine chloromethyl ketone - TRIS tris-(hydroxymethyl)-aminomethane - V _max maximal reaction velocity Contribution no. 409 of the Alfred-Wegener-Institute for Polar and Marine Research in Bremerhaven     

Comparative study on the proteases from fish pyloric caeca and the use for production of gelatin hydrolysate with antioxidative activity

Proteases from pyloric caeca extract of three fish species including brownstripe red snapper (Lutjanus vitta), bigeye snapper (Priacanthus tayenus) and threadfin bream (Nemipterus marginatus) were comparatively studied. The extracts from bigeye snapper and threadfin bream exhibited the highest hydrolytic activities toward casein, α-N-benzoyl-dl-arginine-p-nitroanilide and α-N-ρ-tosyl-l-arginine methyl ester at pH 8.0 and 60 °C and pH 8.5 and 55 °C, respectively. The extract of brownstripe red snapper showed the optimal pH and temperature of 8.0 and 60 °C with all substrates used except the optimal temperature was 65 °C when casein was used. All proteases were strongly inhibited by soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-l-lysine chloromethylketone (TLCK) and partially inhibited by N-tosyl-l-phenylalanine chloromethylketone for all substrates tested, suggesting that trypsin-like proteases were the major enzymes. Substrate-gel activity staining of 40–60% ammonium sulfate (AS) fraction revealed that major activity bands were observed with molecular mass of 24, 22 and 20 kDa for brownstripe red snapper, bigeye snapper and threadfin bream, respectively. Those activity bands were partially inhibited by SBTI and TLCK. AS fraction was further used to produce gelatin hydrolysate from the skin of brownstripe red snapper with different degrees of hydrolysis (DH). Hydrolysate with DH of 15% exhibited the highest DPPH and ABTS radical scavenging activities and ferric reducing antioxidant power (p < 0.05). Therefore, the extract from pyloric caeca could be used to produce the gelatin hydrolysates possessing antioxidative activities.     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.     

Chemical composition of Eubacterium nodatum cell wall peptidoglycan

The structure of Eubacterium nodatum cell wall peptidoglycan was investigated. The peptide subunit of E. nodatum peptidoglycan has the following structure: L-Ala-D-Glu (Gly)-L-Orn-D-Ala. The carboxyl group of alanine occupying position 4 is attached to the -amino group of ornithine of an other subunit by the cross-linking bridge L-Ala-L-Ala-L-Orn. All glycine molecules are connected with the -carboxyl group of glutamic acid with the ratio being 0.5–1. The hydrolysis of E. nodatum peptidoglycan by the S. albus G enzyme proceeds primarily due to the activity of alanyl-alanine endopeptidase, ornithyl-ornithine endopeptidase, ornithyl-alanine endopeptidase, N-acetyl-muramyl-alanine amidase, N-acetylmuramidase and N-acetylglucosaminidase.     

Purification and Characterization of Leu-Proteinase, the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves

The leucine specific serine proteinase present in the soluble fraction of leaves from Spinacia oleracea L. (called Leu-proteinase) has been purified by acetone precipitation and a combination of gel-filtration, ion exchange, and adsorption chromatography. This enzyme shows a molecular weight of 60,000 ± 3,000 daltons, an isoelectric point of 4.8 ± 0.1, and a relative electrophoretic mobility of 0.58 ± 0.03. The Leu-proteinase catalyzed hydrolysis of p-nitroanilides of N-α-substituted(-l-)amino acids as well as of chromogenic macromolecular substrates has been investigated between pH 5 and 10 at 23 ± 0.5°C and I = 0.1 molar. The enzyme activity is characterized by a bell-shaped profile with an optimum pH value around 7.5, reflecting the acid-base equilibrium of groups with pK_a values of 6.8 ± 0.1 and 8.2 ± 0.1 (possibly the histidyl residue present at the active site of the enzyme and the N-terminus group). Among the substrates considered, N-α-benzoyl-l-leucine p-nitroanilide shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 1 × 10⁻⁹ molar. In agreement with the enzyme specificity, only N-α-tosyl-l-leucine chloromethyl ketone, di-isopropyl fluorophosphate and phenylmethylsulfonyl fluoride, among compounds considered specific for serine enzymes, strongly inhibit the Leu-proteinase. Accordingly, the enzyme activity is insensitive to cations, chelating agents, sulfydryl group reagents, and activators.     

Proteolytic activity of gut bacteria isolated from the velvet bean caterpillar Anticarsia gemmatalis

The development of proteinase inhibitors as potential insect control agents has been constrained by insect adaptation to these compounds. The velvet bean caterpillar (Anticarsia gemmatalis) is a key soybean pest species that is well-adapted to proteinase inhibitors, particularly serine-proteinase inhibitors, which are abundant in the caterpillar host. The expression of diverse proteolytic enzymes by gut symbionts may allow the velvet bean caterpillar to circumvent proteinase inhibitors produced by the host plant. In this study, we characterized the proteolytic activity of the four nonpathogenic species of gut bacteria isolated from the velvet bean caterpillar—Bacillus cereus, Enterococcus gallinarum, Enterococcus mundtii and Staphylococcus xylosus. Two proteinase substrates, N-α-benzoyl-l-Arg-p-nitroanilide (l-BApNA) and N-α-p-tosyl-l-Arg methyl ester (l-TAME) and five proteinase inhibitors [aprotinin, E-64, ethylenediamine tetraacetic acid (EDTA), pepstatin and N-α-tosyl-l-lysine chloromethyl ketone (TLCK)] as well as CaCl₂, pH and temperature profiles were used to characterize the expressed proteolytic activity of these bacterial strains in vitro. Kinetic parameters for proteolytic activity were also estimated. The results of these experiments indicated that serine- and cysteine-proteinase activities were expressed by all four gut bacteria symbionts of the velvet bean caterpillar. The cysteine- and serine-proteinase activities of these gut symbionts were distinct and different from that of gut proteinases of the caterpillar itself. This finding provides support for the potential involvement of gut symbionts in the mitigation of the negative effects of serine-proteinase inhibitors in the velvet bean caterpillar.     

Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms

Summary Nitric oxide (NO) is generated from l-arginine by NO synthases. Localization of the brain enzyme has been carried out in the rat; however, despite data suggesting that NO is a major regulator of vascular and neural functions in man, there is no information about the localization of NO synthase in human tissues. Rabbit antisera to NO synthase purified from rat brain (antisera A and B) were raised, tested by Western blotting, affinity purification and enzyme immunoprecipitation assay, and used to investigate the distribution of the enzyme in a variety of human tissues by immunohistochemistry. Antisera to two synthetic peptides from cloned neural NO synthase were used to aid specificity testing. Antisera A and B reacted with a 160-kDa protein in Western blots of human brain extracts, gave immunostaining of nerves, and precipitated enzyme activity from rat brain homogenates. Antiserum B to NO synthase also reacted with proteins of M_r between 125 and 140 kDa in extracts of well-vascularised tissues, and immunostained vascular endothelium; the neural and vascular immunoreactivity persisted after affinity purification of antiserum B with the 160 kDa protein. Endothelial staining with antiserum B was seen in respiratory tract, liver, skin and umbilicus; syncytial trophoblasts stained in the placenta. Neural staining with antiserum A and B was seen in the myenteric and submucous plexus, and in nerve fibres in smooth muscle of the gut and in many areas of the central nervous system, particularly cortex, hippocampus, hypothalamus, cerebellum, brain stem and spinal cord. Therefore, antibodies to rat brain enzyme react with the human equivalent and also with other NO synthase isoforms in human endothelium. These findings support the contention that the endothelial enzyme is a different form with partial homology to that in nerves and also provide an anatomical distribution of NO synthase isoforms.Abbreviations NADPH -Nicotinamide adenine dinucleotide phosphate, reduced - FAD flavin adenine dinucleotide - FMN flavin mononucleotide - KLH keyhole limpet haemocyanin - G-MDP N-acetylmuramyl-l-ananyl-d-isoglutamine - TLCK N-p-tosyl-l-lysine chloro-methylketone - DRG dorsal root ganglia     

Highly efficient production ofl-cystathionine fromO-succinyl-l-homoserine andl-cysteine byStreptomyces cystathionine γ-lyase

Summary Utilizing the -replacement reaction ofStreptomyces cystathionine -lyase (EC 4.4.1.1.), an efficient production method forl-cystathionine has been established. Under optimal conditions, 50 mMl-cystathionine was synthesized from 50 mMO-succinyl-l-homoserine and 50 mMl-cysteine, added in four stages to the reaction mixture, with a substrate conversion rate of 100%. This productivity (11 gl^-1 of reaction mixture) is about 3.5 times higher than that withl-homoserine andl-cysteine as substrates.Recipient of a JSPS Fellowship for Japanese Junior Scientists     

Enzymatic production of l-phenylalanine from the racemic mixture of d,l-phenyllactate

Summary The production of l-phenylalanine from the racemate d,l-phenyllactate in an enzyme membrane reactor has been examined. In a first step the racemate is dehydrogenated to the prochiral intermediate phenylpyruvate by the enzymes d-and l-hydroxyisocaproate dehydrogenase. In a second step phenylpyruvate is reductively aminated to l-phenylalanine by l-phenylalanine dehydrogenase. Both steps are dependent on coenzyme, the first one requires NAD, the second one NADH in stoichiometric amounts; in this way the coenzyme is regenerated and only required catalytically. The coenzyme is covalently bound to polyethylene glyco-20 000 and can thus be retained in the reactor analogously to the three enzymes. In order to optimize the continuous production of l-phenylalanine from d,l-phenyllactate, models of the reaction kinetics and of the reactor system have been set up. By means of the reactor model, we can calculate the optimum ratio of the three enzymes, the optimum coenzyme concentration and the optimum phenylpyruvate concentration in the feed.In this process, at a substrate concentration of 50 mM d,l-phenyllactate we reached a spacetime-yield of 28 g l-Phe/(l*d).Abbreviations PEG polyethylene glycol - d-HicDH d-hydroxyisocaproate dehydrogenase - l-HicDH l-hydroxyisocaproate dehydrogenase - PheDH l-phenylalanine dehydrogenase - V _max maximum velocity - K _M Michaelis-Menten constant - K _l inhibition constant - R₁ reaction rate of the d-HicDH forward reaction - R₂ reaction rate of the d-HicDH reverse reaction - R₃ reaction rate of the l-HicDH forward reaction - R₄ reaction rate of the l-HicDH reverse reaction - R₅ reaction rate of the PheDH forward reaction - R₆ reaction rate of the PheDH reverse reaction - d-PLac d-phenyllactate - l-PLac l-phenyllactate - PPy phenylpyruvate - l-Phe l-phenylalanine - NH₄ ammonium - residence time     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

Crystallization and Properties of N-Benzoylglycine Amidohydrolase from Pseudomonas putida

N-Benzoylgiycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170,000 and consisted of four subunits identical in molecular weight (approximately 42,000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-l-alanine and N-benzoyl-l-aminobutyric acid. The Km value for these substrates were 0.72 mm, 0.87 mm, and 0.87mm, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0.     

Trypsin-catalyzed peptide synthesis withm-guanidinophenyl andm-(guanidinomethyl)phenyl esters as acyl donor component

Summary Two series of inverse substrates,m-guanidinophenyl andm-(guanidinomethyl)phenyl esters derived fromN-(tert-butyloxycarbonyl)amino acid, were prepared as an acyl donor component for trypsin-catalyzed peptide synthesis. The kinetic behavior of these esters toward tryptic hydrolysis was analyzed. They were found to couple with an acyl acceptor such asl-alaninep-nitroanilide to produce dipeptide in the presence of trypsin.Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. Within the enzymatic peptide coupling methods, this approach was shown to be advantageous, since the resulting peptides are resistant to the enzymatic hydrolysis.Abbreviations Boc tert-butyloxycarbonyl - Aib -aminoisobutyric acid - DMSO dimethylsulfoxide - Tris tris(hydroxymethyl)aminomethane - MOPS 3-morpholino-l-prop anesulfonate - G guanidinophenyl - GM (guanidinomethyl)phenyl - pNA p-nitroanilide     

New Nematicidal Metabolites from a Fungus,Irpex lacteus

N-Benzoyl-l-alanine amidohydrolase was purified from a cell-free extract of Corynebacterium equi H-7 which was grown in a medium containing hippuric acid as the sole carbon source. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The molecular weight was 230,000 and the enzyme consisted of six subunits, identical in molecular weight (approximately 40,000). The isoelectric point of the enzyme was pH 4.6. The optimum pH of the enzyme reaction was 8.0 and the enzyme was stable from pH 7.0 to 8.0. The enzyme hydrolyzed N-benzoyl-l-alanine, N-benzoylglycine, and N-benzoyl-l-aminobutyric acid. The Km values for these substrates were 4.3 mm, 6.7 mm, and 4.3 mm, respectively. The enzyme was activated by Co²⁺.     

Amino acid metabolism in the thermophilic phototroph,Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases

Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K _i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K _m=20 mM) as well as with l-serine (K _m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K _m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A absorbance - ile isoleucine - PLP pyridoxal-5-phosphate - SDS sodium dodecyl sulfate - TDH threonine dehydratase - U unit     

In vivo inhibition of glutamine synthetase induces sporulation in a protease deficient asporogenous mutant of Bacillus polymyxa

In vivo inhibition of glutamine synthetase (GS) by l-methionine sulfoximine induces sporulation in a protease deficient mutant of Bacillus polymyxa. This induction of sporulation is accompanied by derepression of EDTA insensitive proteases(s) which seems to be specific for differentiation. Some amino acid analogues derepress proteolytic activity without inducing sporulation, but these proteases are sensitive to metal chelators like those in the vegetative cells. When the proteolytic activity is restored, the mutant cells, which are smaller than the parental strain, regain their normal size.Abbreviations GS glutamine synthetase - GYS glucose-yeast extract-salts - MSO l-methionine sulfoximine - Pr protease deficient mutant - DON 6-diazo-5-oxo-l-norleucine - EDTA ethylene-diaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - Tris tris-(hydroxymethyl)-aminomethane     

Effects of metals on elastase fromPseudomonas aeruginosa SES-938-1

Among the numerous virulance factors produced byPseudomonas aeruginosa, elastase is the one most often associated with pathogenesis. In this study, effects of various metal ions on elastase from a new isolate ofP. aeruginosa (Strain SES-938-1) was investigated. Crude elastase was prepared from culture supernatant via salting out by ammonium sulfate, and then desalting and concentrating the sample using a centricon microconcentrator. Activities were measured at 450 nm usingN-succinyl-l-(ala)₃-p-nitroanilide as the substrate. The metal chelating agents EDTA and EGTA inhibited thePseudomonas elastase, which shows that the enzyme is a typical metalloproteinase. At a 10-mM concentration, Mn²⁺, Ni²⁺, and Zn²⁺ strongly inhibited the elastase, whereas Mg²⁺ effect was negligable. There was a gradual decrease in the enzyme activity in accordance with an increase in the concentration of metal ions.