Influence of water activity on the production of extracellular enzymes by Staphylococcus aureus.

Two enterotoxigenic strains of Staphylococcus aureus were examined for their ability to produce a number of extracellular enzymes at various water activity (alphaw) levels. Supernatant, dialyzed culture media were analyzed for total and relative levels of enzyme activity. With the exception of protease, enzyme activity was greatest in spent media obtained from cultures grown at 0.996 alphaw, the highest level tested. Enzyme activity in spent media from an enterotoxin B-producing strain was generally more sensitive to alphaw reduction than activity from an enterotoxin A-producing strain. Unlike the other enzymes assayed, acid and alkaline protease activities were greatest when the organism was grown at 0.94 alphaw.     

Minimal water activity for enterotoxin A production and growth of Staphylococcus aureus.

The minimal water activity (alphaw) for growth was correlated with enterotoxin A formation by two strains of Staphylococcus aureus in a salt mixture broth. Within 7 days at 30 degrees C both strains grew and formed enterotoxin A minimally between alphaw 0.864 and 0.867, but at 25 degrees C, the minimal alphaw for both activities was increased to between 0.870 and 0.887 after a 2-week incubation.     

Effect of Water Activity on Enterotoxin B Production and Growth of Staphylococcus aureus

Staphylococcus aureus C-243, an enterotoxin B-producing strain, was cultured on media adjusted to various water activity (a_w) levels by means of two different solute systems. Total numbers and rate of growth were diminished at low a_w levels, and enterotoxin synthesis was extremely sensitive to reduction in a_w. A reduction of a_w from 0.99 to 0.98 in one medium and from 0.99 to 0.97 in the other medium resulted in extremely low levels of enterotoxin in spent culture media despite the attainment of high numbers of staphylococci.     

Effect of Water Activity on Enterotoxin A Production and Growth of Staphylococcus aureus

Previous studies indicated that enterotoxin B production by staphylococci was strongly inhibited by slight reductions in water activity (a(w)) levels. Similar studies reported herein, employing an enterotoxin A-producing strain, indicated that this organism was capable of producing enterotoxin at a much lower a(w) level than that required for enterotoxin B production. Staphylococcal growth rates were slowed by decreased a(w) levels in all media tested; however, final cell counts did not drop below 10(8)/ml in the media with the lowest a(w) levels.     

Milk-Clotting Enzymes From Microorganisms

A total of 230 cultures of fungi and 43 cultures of bacteria, isolated from such sources as soil, butter, and milk, were screened for their milk-clotting activity. The fungi were cultivated on semisolid media, and the bacteria were grown in milk media in shake culture. Phytic acid, added as calcium phytate, was found to stimulate production of the enzyme in most of the bacterial isolates. Proteolytic activity was invariably found to be associated with the milk-clotting enzyme in bacterial isolates. There was considerable variation in the ratio of the two enzymes from strain to strain.     

Optimization of protease production by endophytic fungus,Alternaria alternata,isolated from an Australian native plant

Endophytes are recognised as potential sources of novel secondary metabolites, including enzymes and drugs, with applications in medicine, agriculture and industry. There is a growing need for new enzymes, including proteases, for use in industry that can function under a variety of conditions. In this study, three fungal endophytes (Alternaria alternata, Phoma herbarum and an unclassified fungus), were isolated from the Australian native plant, Eremophilia longifolia, and assessed for production of proteases. The lyophilised growth media obtained after fungal fermentation were analysed for protease production using enzyme activity assays. Protease production was optimised by assessing the effects of temperature, pH, carbon source and nitrogen source on activity. A. alternata showed the greatest protease activity in a wide range of pH (3–9). The broadest activity between 9 and 50 °C was observed at pH 7, suggesting a neutral protease. Overall, the optimum conditions were 37 °C and pH 7 with a maximum specific activity value of 69.86 BAEE units/mg. The characteristics demonstrated by this fungal endophyte showed that it is a potential source of an enzyme with particular application in the dairy industry. However, further studies of the tolerance to higher temperatures and pH will indicate whether the enzyme is suitable to such applications.     

Extracellular hydrolases of strain Bacillus sp. 739 and their involvement in the lysis of micromycete cell walls

The mycolytic bacterial strain Bacillus sp. 739 produces extracellular enzymes which degrade in vitro the cell walls of a number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process correlated with the levels of the beta-1,3-glucanase and protease activity. The comparative characteristic of Bacillus sp. 739 enzymatic preparations showed that efficient hydrolysis of the fungus cell walls was the result of the action of the complex of enzymes produced by the strain when grown on chitin-containing media. Among the enzymes of this complex, chitinases and beta-1,3-glucanases hydrolyzed most actively the disintegrated cell walls of B. sorokiniana. However, only beta-1,3-glucanases were able to degrade the cell walls of native fungal mycelium in the absence of other hydrolases, which is indicative of their key role in the mycolytic activity of Bacillus sp. 739.     

Superoxide dismutase and catalase levels in halophilic vibrios.

Superoxide dismutase (SOD) and catalase (CAT) levels were determined for several aerobically grown halophilic vibrios and compared with those found in aerobically grown Escherichia coli K-12. The SOD levels ranged from 25 to 103.6 U/mg of protein for the vibrios compared with 44.6 U/mg of protein for E. coli. The CAT levels ranged from 2.1 to 32.1 U/mg of protein. Electrophoretic analysis of cell extracts revealed that the halophilic vibrios tested possessed only one detectable SOD enzyme, except one strain which possessed two distinct enzymes, as compared with the three SOD enzymes in aerobically grown E. coli K-12. A comparison of anaerobically and aerobically grown vibrios revealed a three- to fourfold increase in SOD activity in the aerobic cells, suggesting that oxygen acts as an inducer for SOD in the vibrios as has been reported for E. coli. In one strain, Vibrio parahaemolyticus 27519, both SOD enzymes were observed in low levels in anaerobic and at higher levels in aerobically grown cells as compared with only one SOD enzyme in anaerobically grown E. coli. This suggests that differences in SOD regulation occur between the two genera. Our results indicate that halophilic vibrios possess SOD, which could enhance viruulence by allowing the organisms to survive in oxygenated environments.     

Extracellular Proteases of Mucor pusillus

Mucor pusillus was grown in different media for a period of 92 h, and the media were investigated for both milk-clotting and protease activities. It was observed that the ratio of extracellular milk-clotting activity to protease activity was the highest for 3% corn steep liquor containing 1% glucose as the source of carbon. Variation of both milk-clotting and protease activities was studied during the growth of the organism in the medium stated above. Separation of protease was carried out by ion-exchange chromatography at pH 8.0. Fractions collected were assayed for both activities simultaneously. The findings suggested that, instead of only one major acid protease, as reported by previous workers, two major acid proteases were produced. One of them had significant rennin-like activity, and the other lacked it. The former could be assumed to be the enzyme reported and studied by previous workers. The existence of two proteases was further confirmed by the appearance of two protease activity bands on polyacrylamide gels after electrophoresis. An attempt was made to separate the rennin-like enzyme from nonspecific protease activity by ammonium sulfate fractionation followed by ion-exchange chromatography at pH 6.0. The results indicated that the nonspecific protease activity due to the enzyme that lacked rennin action was substantially removed by the ammonium sulfate fractionation.     

Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K

The physical factors affecting the production of an organic solvent-tolerant protease from Pseudomonas aeruginosa strain K was investigated. Growth and protease production were detected from 37 to 45 degrees C with 37 degrees C being the optimum temperature for P. aeruginosa. Maximum enzyme activity was achieved at static conditions with 4.0% (v/v) inoculum. Shifting the culture from stationary to shaking condition decreased the protease production (6.0-10.0% v/v). Extracellular organic solvent-tolerant protease was detected over a broad pH range from 6.0 to 9.0. However, the highest yield of protease was observed at pH 7.0. Neutral media increased the protease production compared to acidic or alkaline media.     

Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice

Extracellular degradative enzymes released by psychrophilic marine bacteria (growing optimally at or below 15°C and maximally at 20°C) typically express activity optima at temperatures well above the upper growth limit of the producing strain. In the present study, we investigated whether or not near-zero Arctic environments contain extracellular enzymes with activity optimized to temperatures lower than previously reported. By applying fluorescently tagged substrate analogues to measure leucine-aminopeptidase and chitobiase activity, the occurrence of extracellular enzymatic activity (EEA) with remarkably low temperature optima (15°C) was documented in sea-ice samples. An extremely psychrophilic bacterial isolate, strain 34H, yielded an extract of cell-free protease with activity optimized at 20°C, the lowest optimum yet reported for cell-free EEA from a pure culture. The use of zymogram gels revealed the presence of three proteolytic bands (between 37 and 45 kDa) in the extract and the release of the greatest quantities of the proteases when the strain was grown at −1°C, suggesting a bacterial strategy for counteracting the effects of very cold temperatures on the catalytic efficiency of released enzymes. The detection of unusually cold-adapted EEA in environmental samples has ramifications not only to polar ecosystems and carbon cycling but also to protein evolution, biotechnology and bioremediation.     

Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens

While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli. This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions. Hydantoinase and N-carbamoylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in complete medium, enzyme activity can be detected only in early stationary growth phase. In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitrogen source and the presence of inducer, 2-thiouracil, in the growth medium. Growth with (NH4)2SO4 as the nitrogen source repressed the production of both enzymes (nitrogen repression) and also resulted in a rapid, but reversible loss of hydantoinase activity in induced cells (ammonia shock). Mutant strains with inducer-independent production of the enzymes and/or altered response to nitrogen control were isolated. Of greatest importance for industrial application was strain RU-ORPN1F9, in which hydantoinase and NCAAH enzyme activity was inducer-independent and no longer sensitive to nitrogen repression or ammonia shock. Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterologous expression systems.     

A comparison of genetically improved strains of the cellulolytic fungus Trichoderma reesei

Summary Production of cellulases by three different genetically improved strains of Trichoderma reesei: MCG 77, RUT-C30 and CL-847, has been assessed on various fermentation media. The three strains produce high levels of enzymes when grown on purified cellulose as the main carbon energy source; when grown on lactose, decrease in enzyme yield and productivity, differs significantly from strain to strain.     

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.     

Relationship Between Messenger Ribonucleic Acid and Enzyme Levels Specified by the Leucine Operon of Escherichia coli K-12

The levels of leucine-forming enzymes in Escherichia coli K-12 varied over a several thousand-fold range, depending upon conditions of growth. The highest levels were achieved by growing auxotrophs in a chemostat under conditions of leucine limitation. Under such conditions, enzyme levels were increased 45- to 90-fold relative to cells grown in minimal medium containing leucine (the latter values arbitrarily called 1). Leucine operon-specific messenger ribonucleic acid levels were elevated to about the same extent as enzyme levels in cells grown in a chemostat. Growth in media of greater complexity resulted in progressively lower levels of leucine-forming enzymes, reaching a value of less than 0.02 for growth in a medium containing tryptone broth and yeast extract. The levels of leucine operon-specified enzymes and messenger ribonucleic acid were also measured in strains containing about 25 copies of plasmid pCV1(ColE1-leu) per chromosome. For such strains grown in minimal medium, enzyme levels were proportional to the number of plasmids per cell. Furthermore, they followed the same trends as those described above upon derepression in a chemostat or upon repression following growth in rich media. Leucine messenger ribonucleic acid, measured both by pulse-labeling and hybridization-competition experiments, was roughly proportional to enzyme levels over this entire range. For a plasmid-containing strain grown in a chemostat under conditions of leucine limitation (about 100 plasmids per chromosome), about 27% of pulse-labeled ribonucleic acid was coded for by genes in or adjacent to the leucine operon, and 10% of the total protein was β-isopropylmalate dehydrogenase.     

Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. I. α-glucosidase and β-glucosidase

A number of factors which may correlate with the levels of α-glucosidase and β-glucosidase in cultured amniotic fluid cells have been investigated. Fluctuations in enzyme activity occurred as passage numbers increased. Whereas α-glucosidase showed a consistently lower activity in the earlier passages compared to the later ones, the results for β-glucosidase were equivocal. Both enzymes showed an increase in activity correlated with the time taken by the cells to reach confluency in the third passage. When replicate cultures were assayed daily after subculture, neither enzyme showed any change correlated with time. When cultures were grown in parallel in Ham's F10 and Eagle's M.E.M. tissue culture media, the activity of both enzymes was unaffected. Cell strains cultured from serial samples of amniotic fluid from the same woman had differing enzyme levels unrelated to gestational age.     

一株产纤溶酶菌株的分离鉴定及其纤溶组分分析

【目的】筛选性能良好的产纤溶酶菌株,对菌株进行多项分类鉴定,分析其纤溶酶系的组成特征及纤溶能力。【方法】通过酪蛋白培养基初筛,琼脂-纤维蛋白双层平板复筛,从海泥、土壤等环境中筛选纤维蛋白降解菌,以尿激酶为标准测定纤溶酶活性。通过形态学、生理生化特征研究,结合16S rDNA基因序列分析菌株种类及系统分类地位。通过SDS-PAGE和纤维蛋白酶谱法分析胞外纤溶酶系的组成特征。【结果】筛选到一株能降解纤维蛋白的细菌CNY16,鉴定其为沙福芽孢杆菌(Bacillus safensis)。该酶为胞外酶,SDS-PAGE和纤维蛋白酶谱结果表明该纤溶酶系有至少两种分子量大小不同的纤溶酶,分别约33 kD和23 kD。能有效溶解血块中纤维蛋白,并且对红细胞无降解作用。【结论】细菌CNY16是一株新的纤溶酶产生菌,纤溶酶活性及稳定性较好,具有潜在开发价值。为获取新型纤溶酶提供了一种新的菌源。     

Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes

A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60 degrees C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30 degrees C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.     

Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase by Bacteroides gingivalis strain W50

Bacteroides gingivalis strain W50 was grown in batch and continuous culture on complex medium with haemin. In batch culture, cell-bound levels of trypsin-like protease (EC 3.4.21.4), alkaline phosphatase (EC 3.1.3.1) and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) increased during the exponential phase of growth. These enzyme activities were also detected in extracellular vesicles and in extracellular soluble forms in the supernatant fluid, but in lower amounts per unit biomass compared to cell-bound levels. In continuous culture, at high relative growth rates (0.7-0.9 murel), the highest proportions of enzyme activities were cell-bound. In contrast, at low relative growth rates (0.1-0.2 murel), highest enzyme levels were detected in the extracellular vesicle fraction. Levels of extracellular soluble enzymes were always low compared to cell-bound or extracellular vesicle levels, but were highest at low relative growth rates. All three enzymes appeared to be relatively stable in their soluble forms. Vesicle production appeared to be associated with actively growing cells but was influenced by growth rate. The results are consistent with the hypothesis that cell-bound 'periplasmic' enzymes are encapsulated into vesicles which are subsequently released by the cells. Therefore, levels of total extracellular enzyme (extracellular vesicle plus extracellular soluble) may depend on the rate of vesicle formation superimposed on the rates of production of 'periplasmic' enzymes in the cell.     

Identification of a New Marine Bacterial Strain SD8 and Optimization of Its Culture Conditions for Producing Alkaline Protease

While much attention has been given to marine microorganisms for production of enzymes, which in general are relatively more stable and active compared to those from plants and animals, studies on alkaline protease production from marine microorganisms have been very limited. In the present study, the alkaline protease producing marine bacterial strain SD8 isolated from sea muds in the Geziwo Qinhuangdao sea area of China was characterized and its optimal culture conditions were investigated. Strain SD8 was initially classified to belong to genus Pseudomonas by morphological, physiological and biochemical characterizations, and then through 16S rDNA sequence it was identified to be likely Pseudomonas hibiscicola. In addition, the culture mediums, carbon sources and culture conditions of strain SD8 were optimized for maximum production of alkaline protease. Optimum enzyme production (236U/mL when cultured bacteria being at 0.75 mg dry weight/mL fermentation broth) was obtained when the isolate at a 3% inoculum size was grown in LB medium at 20 mL medium/100mL Erlenmeyer flask for 48h culture at 30°C with an initial of pH 7.5. This was the first report of strain Pseudomonas hibiscicola secreting alkaline protease, and the data for its optimal cultural conditions for alkaline protease production has laid a foundation for future exploration for the potential use of SD8 strain for alkaline protease production.