The occurence of a neutral protease and its inhibitor in rat peritoneal macrophages.

The lysate of the glycogen-induced macrophages in rat peritoneal exudate was fractionated by centrifugation and extraction into a water extract, 1 M KCl extract and residue fractions. Approximately 50% of the neutral protease activity toward casein in the lysate was recovered in the KCl extract fraction, which was practically devoid of acid protease, cathepsin D. The pH optimum of the neutral protease toward casein and urea-denatured hemoglobin was pH 8.5. The activity was inhibited strongly by DFP or chymostatin and only partially by HgCl2 or PCMB. Addition of a salt to the reaction medium caused enhancement of the activity with an optimum concentration of 0.25 M: KCl, KBr, KI, NaCl, NaBr, NaI, and MgCl2 were all almost equally effective. When the enzyme preparation was filtered through a column of Sephadex G-75 gel in the presence of 1 M KCl, a larger molecular weight fraction at the void volume was obtained in addition to a smaller molecular weight fraction showing a caseinolytic activity insensitive to KCl concentration. The former was found to have a specific inhibitory effect on the latter activity.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Potassium Salts Inhibit Growth of the Cyanobacteria Microcystis spp. in Pond Water and Defined Media: Implications for Control of Microcystin-Producing Aquatic Blooms

Ten metals were assayed in 21 Indian ponds which comprised three groups: (i) eutrophic alkaline ponds containing <2.5 mM potassium and thick growths of Microcystis aeruginosa or Microcystis flos-aquae during most of the year, (ii) equally eutrophic alkaline ponds containing >2.8 mM potassium and no detectable Microcystis growth, and (iii) oligo- or mesotrophic ponds with various potassium and hydrogen ion concentrations and no persistent Microcystis blooms. The effects of potassium on Microcystis growth were examined in filter-sterilized pond water and in defined culture media. A 50% reduction in the 10-day yield of cultured M. aeruginosa was observed in DP medium and pond water supplemented with 1 and 3 mM KCl, respectively. In contrast, the addition of 2 to 30 mM NaCl did not suppress the growth of M. aeruginosa in either DP medium or pond water. Both 5 mM KCl and 20 mM KHCO(inf3) in J medium strongly inhibited the growth of M. flos-aquae C3-9, whereas 5 to 30 mM NaCl had no effect and 20 mM NaHCO(inf3) was stimulatory. For pond water cultured with a mixture of M. aeruginosa and the duckweed Wolffia arrhiza, M. aeruginosa dominated in unsupplemented water and W. arrhiza dominated in water supplemented with 4.8 mM KCl. Implications for the ecology and control of Microcystis blooms are discussed.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber,an extremely halophilic bacterium

The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine. We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations. Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low. We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration. NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt. NaCl was stimulatory with optimum activity at 3-3.5 M. However, no activity was found above 2.5 M KCl. Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably. The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.     

Purification and biochemical characterization of a protease secreted by the <Emphasis Type="Italic">Salinivibrio</Emphasis> sp. strain AF-2004 and its behavior in organic solvents

A metalloprotease secreted by the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 when the culture reached the stationary growth phase. This enzyme was purified to homogeneity by acetone precipitation and subsequent Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The apparent molecular mass of the protease was 31 kDa by SDS-PAGE, whereas it was estimated as approximately 29 kDa by Sephacryl S-200 gel filtration. The purified protease had a specific activity of 116.8 mumol of tyrosine/min per mg protein on casein. The optimum temperature and salinity of the enzyme were at 55 degrees C and 0-0.5 M NaCl, although at salinities up to 4 M NaCl activity still remained. The protease was stable and had a broad pH profile (5.0-10.0) with an optimum of 8.5 for casein hydrolysis. The enzyme was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), Pefabloc SC, chymostatin and also EDTA, indicating that it belongs to the class of serine metalloproteases. The protease in solutions containing water-soluble organic solvents or alcohols was more stable than that in the absence of organic solvents. These characteristics make it an ideal choice for applications in industrial processes containing organic solvents and/or salts.     

Halophilic Nuclease from a Moderately Halophilic Micrococcus varians

The moderately halophilic bacterium Micrococcus varians, isolated from soy sauce mash, produced extracellular nuclease when cultivated aerobically in media containing 1 to 4 M NaCl or KCl. The enzyme, purified to an electrophoretically homogeneous state, had both ribonuclease and deoxyribonuclease activities. The nuclease had maximal activity in the presence of 2.9 M NaCl or 2.1 M KCl at 40 C. The enzymatic activity was lost by dialysis against low-salt buffer, whereas when the inactivated enzyme was dialyzed against 3.4 M NaCl buffer as much as 77% of the initial activity could be restored.     

Properties of Intracellular Hatching enzyme in the Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

The intracellular hatching enzyme was confirmed to be particulate-bound in the sea urchin, Hemicentrotus pulcherrimus. The enzyme was solubilized most effectively by sonication in buffer containing 12.5 mM CaCl₂, and 0.5 M KCl. The intracellular hatching enzyme is suggested to be activated by an antipain- or elastatinal-susceptible protease(s) on its solubilization. Since the intracellular hatching enzyme solubilized in the absence of protease inhibitors was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, the active hatching enzyme is concluded to be a chymostatin-sensitive serine protease. The enzyme required CaCl₂, and KCl or NaCl for both stability and activity. The preference of the enzyme of anions as sodium salts was as follows: Cl⁻ > NO₃⁻ > I⁻ > SCN⁻. The apparent molecular weights of the intracellular hatching enzyme (IHE) and the hatching enzyme secreted from the blastula with or without the fertilization envelope (SHE or dSHE) were estimated as 89,000, 135,000, 80,000, respectively. On incubations with isolated fertilization envelopes as an enzyme substrate, the apparent molecular weights of dSHE and IHE increased to 128,000 and 105,000, respectively.     

Protease production by the ontjom fungus,Neurospora sitophila

Summary The production of protease and mycelium byNeurospora sitophila cultured on solid and liquid potato dextrose media was studied. Maximal activity of protease extracted from 4-day-old cultures occurred at pH 6.5 when an unfractionated peanut (groundnut) protein substrate was used. The greatest protease activity and mycelium production occurred during the first day of the 4-day test period. Potato dextrose media containing more than 0.2 M NaCl resulted in decreased protease and mycelium production, while tapioca starch was without affect at concentrations up to 1.4%. Addition of up to 0.3 M sucrose to growth media greatly stimulated protease production and mycelial growth. Maximal proteolytic activity was observed in extracts from mycelium cultured in potato dextrose media adjusted from pH 6.0 to 7.5. Activity was greater when soluble peanut protein was used as a substrate, compared to unfractionated or globulin protein substrates.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp.

A moderately halophilic Acinetobacter sp., capable of producing dextrinogenic amylase, was isolated from sea-sands. Maximum enzyme production was obtained when the bacterium was cultivated aerobically in media containing 1 to 2M NaCl or 1M KCl. Two kinds of amylase, amylases I and II were purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, DEAE-Sephadex A-50 chromatography, and Sephadex G-200 gel filtration. Both enzymes had maximal activity at pH 7.0 in 0.2 to 0.6 M NaCl or KCl at 50 to 55 degrees C. The activities were lost by dialysis against distilled water. Molecular weights for amylases I and II were estimated to be 55 000 and 65 000 respectively by SDS-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose and maltotriose.     

Influence des pretraitements par KCl et des rapports Ca/Na du milieu sur la croissance de vitroplants de tomate en milieu NaCl

The 18-day-old tomato vitroplants were obtained in axenic conditions by culture of expiants (including the terminal bud and the last internode of the stem) on agar-agar nutritive medium with 0 or 75 mM NaCl. The growth and the mineral content of the vitroplants were compared when the expiants were grown on media either with low or high K/Na and Ca/Na ratios, or with low K/Na and Ca/Na ratios after pretreatments of expiants by KC1, NaCl or CaCl₂ (from 0 to – 4.5 bar). The KCl pretreatment (-1.1 bar) during one day brings about an increase in vitroplant growth greater than that produced by a high Ca/Na ratio medium. The Cl accumulation was similar in expiants pretreated by KCl or NaCl. Ion content per gram of fresh matter was similar in 18-day-old vitroplants pretreated by KCl, NaCl or CaCl₂; the Na accumulation by KC1 pretreated vitroplants was not lower than that of 18-day-old vitroplants grown on a high Ca/Na ratio medium. These results show the relation between Na content of expiants and the growth of vitroplants in a NaCl medium.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Effects of neutral salts and alcohols on the activity of Streptomyces caespitosus neutral protease

Streptomyces caespitosus neutral protease (ScNP) is one of the smallest metalloproteinase with a molecular mass of 14 kDa. Effects of solvent composition on ScNP activity were examined using a peptide substrate. The k(cat)/K(m) values of ScNP exhibited bell-shaped pH-dependence with the optimal pH of 6.4-7.0 and the pK(a) values of 5.0 +/- 0.1 and 8.3 +/- 0.1. ScNP activity increased in an exponential fashion with increasing [NaCl]. The relative k(cat)/K(m) value at 3.6 M NaCl to that at 0 M NaCl was 3.7, and the degree of the activation at x M NaCl was expressed as 1.2 (x) (x < 2.0) and 1.4(x) (x > 2.0). On the other hand, ScNP activity decreased with increasing concentrations of LiCl, KCl, NaBr, LiBr, KBr and NaClO(4). Alcohols inhibited ScNP activity with the IC(50) values, the concentration required for decreasing the activity at 50% of the maximum, of 0.77-6.54 M. The order of the inhibitory potency was 1-butanol, 2-methyl-1-propanol, 2-methyl-2-butanol > 2-methyl-2-propanol, 2-butanol, 1-propanol > 2-propanol > ethanol > methanol. The activities recovered completely by the dilution of alcohols, suggesting that the ScNP inhibition by alcohols is reversible. These characteristics of ScNP are compared with those of human matrix metalloproteinase 7 and thermolysin.     

Effects of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa.

The Chenopodiaceae Suaeda salsa L. was grown under different salt concentrations and under osmotic stress. The fresh weight was markedly stimulated by 0.1 M NaCl, 0.4 M NaCl and 0.1 M KCl and reduced by osmotic stress (PEG iso-osmotic to 0.1 M NaCl). Treatment with 0.4 M KCl severely damaged the plants. Membrane vesicle fractions containing tonoplast vesicles were isolated by sucrose gradient from leaves of the S. salsa plants and modulations of V-ATPase and V-PPase depending on the growth conditions were determined. Western blot analysis revealed that V-ATPase of S. salsa consists of at least nine subunits (apparent molecular masses 66, 55, 52, 48, 36, 35, 29, 18, and 16 kDa). This polypeptide pattern did not depend on culture conditions. V-PPase is composed of a single polypeptide (69 kDa). An additional polypeptide (54 kDa) was detected in the fractions of NaCl-, KCl- and PEG-treated plants. It turned out that the main strategy of salt-tolerance of S. salsa seems to be an up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, while V-PPase plays only a minor role. The increase in V-ATPase activity is not obtained by structural changes of the enzyme, but by an increase in V-ATPase protein amount.     

Purification and Some Properties of an Extracellular Amylase from a Moderate Halophile, Micrococcus halobius

A moderate halophile, Micrococcus halobius ATCC 21727, produced an extracellular dextrinogenic amylase when cultivated in media containing 1 to 3 M NaCl. The amylase was purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, diethylaminoethyl-cellulose chromatography, and Bio-Gel P-200 gel filtration. The enzyme had maximal activity at pH 6 to 7 in 0.25 M NaCl or 0.75 M KCl at 50 to 55°C. The activity was lost by dialysis against distilled water. Molecular weight was estimated to be 89,000 by sodium dodecyl sulfate-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose, maltotriose, and maltotetraose, with lesser amount of glucose.     

Effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum (T-strain mycoplasma).

The effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum type VIII was determined by using a well-buffered broth medium containing 10 mM urea. The addition of NH4Cl to the medium at concentrations up to 10 mM did not affect growth; however, addition of larger quantities progressively decreased both the specific growth rate (mu) and the maximum yield of the culture, with concentrations of 80 mM completely inhibiting growth. Addition of either 150 mM KCl or NaCl to the medium did not inhibit growth, indicating that the growth-inhibitory effect was specific to NH4+ and was neither a result of increased Cl- concentration nor increased osmotic pressure. Concentrations of NH4Cl as high as 100 mM did not affect growth of either Acholeplasma laidlawii or Mycoplasma hominis. U. urealyticum was more sensitive to osmotic pressure: osmotic pressures of 710 to 780 mosmol/kg (with KCl, NaCl, or sucrose) resulted in both a substantially lower growth rate and a 5- to 10-fold lower peak yield of organisms. Both A laidlawii and M. hominis were less sensitive to increased osmotic pressure.     

Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular weight was estimated to be 138,000 by Sephadex G-200 gel filtration. The enzyme had marked halophilic properties, showing maximal activities in the presence of 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl. The enzyme hydrolyzed thymidine-5′-monophosphate-p-nitrophenyl ester at a rate that increased with NaCl concentration up to 4.8 M. In the presence of both Mg²⁺ and Ca²⁺, activity was greatly enhanced. The activity was lost by dialysis against water and low-salt buffer, but it was protected when 10 mM Ca²⁺ was added to the dialysis buffer. When the inactivated enzyme was dialyzed against 3.5 M NaCl buffer as much as 68% of the initial activity could be restored. The enzyme exhibited maximal activity at pH 8.5 and at 50°C on DNA and at 60°C on RNA and attacked RNA and DNA exonucleolytically and successively, producing 5′-mononucleotides.     

KCl和NaCl处理对盐生植物碱蓬幼苗生长和水分代谢的影响

研究了用不同浓度KCl和等浓度NaCl处理的盐生植物碱蓬幼苗水分代谢的变化.结果表明,NaCl处理显著促进碱蓬生长,根吸水增加,含水量高于对照.而相同浓度KCl处理却严重抑制碱蓬生长,含水量显著下降,400 mmol/L的 KCl处理后6 d,地上部分发生萎蔫,尔后死亡.KCl处理伤害碱蓬的原因之一是抑制根系吸水,而对蒸腾速率无显著影响,即水分吸收速率与蒸腾速率比值下降,植株缺水引起伤害.