Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Industrial media and fermentation processes for improved growth and protease production by Tetrahymena thermophila BIII

Tetrahymena thermophila was cultivated on industrial by-product media. The composition of the best medium (with milk proteins) was optimised by a central composite design for growth and protease secretion. The optimal combination [1.07% (w/v) of yeast extract and 0.99% (w/v) of skimmed milk] improved biomass production by 46%. In a fermentation strategy, the pH must be regulated to produce no cell damage, lengthening the stationary phase and resulting in a more abundant protease production. To increase cell concentration and protease secretion, a continuous culture with cell recycling by microfiltration was successfully tested on ciliated protozoa. Journal of Industrial Microbiology & Biotechnology (2000) 24, 285–290. Received 28 July 1999/ Accepted in revised form 20 January 2000     

Regulation of exoprotease production by temperature and oxygen in Vibrio alginolyticus

The production of an extracellular collagenase and an alkaline protease by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by a lack of oxygen. The stability of the exoproteases was unaffected by incubation at 37°C and aeration. The optimum growth temperature for the V. alginolyticus strain was 33.5°C Aeration enhanced the rate of growth of exponential phase cells. Temperature and oxygen did not affect the growth of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. There was no rapid release of the exoproteases after temperature shift down and chloramphenicol inhibited the production of the enzymes when added at time of temperature shift down from 37 to 30°C. The regulation of exoprotease production by temperature and oxygen was specific and has implications regarding the ecology of V. alginolyticus. Cerulenin, quinacrine and O-phenanthroline inhibited the production of the exoproteases.     

Production of Extracellular Acid Proteases by Aspergillus Clavatus

The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 °C. The highest acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1%(w/v) at 30 °C at the third day of incubation. Cultures developed in Vogel medium with glucose at 2%(w/v) showed at about 45% of proteolytic activity when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 30, 10 and 5 min, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence for bacterial urea production in marine sediments

Abstract The quantitative importance of bacteria in urea production and turnover in a defaunated sediment from a Danish estuary was studied. After collecting the sediment, benthic infauna was removed by sieving, followed by anoxic pre-incubation for a week. Yeast extract was added to half of the samples. The urea concentration, urea production/turnover rates and the net ammonium production rate, were followed during a 338 h incubation. The urea concentration was highest in the enriched sediment, although urea production and turnover rates were the same in the enriched and control sediment, indicating that the urea concentration was controlled by the urea turnover rate constant, k _urea. Net ammonium production, urea concetrations and teh urea turnover declined towards the end of the experiment. Comparison of the net ammonium production rate and the urea turnover rate in the enriched and control treatments, showed that urea hydrolysis accounted for up to 100% of the ammonium produced, from all sources. More than 98% of the urea which was hydrolyzed, originated from production during incubation. This suggests that urea was a major nitrogen excretion product from bacteria in this sediment, and that bacteria could also be an important source of urea in other marine sediments.     

Effect of nutritional conditions on extracellular protease production by the haloalkaliphilic archaeon Natrialba magadii

AIMS: The effect of various nitrogen sources and nutritional starvation was examined on the production of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii. METHODS AND RESULTS: Cell growth and proteolytic activity were measured in cells grown with different nitrogen sources. Proteolytic activity was produced in complex and easily metabolized nitrogen sources such as yeast extract, casein and casamino acids; meanwhile, ammonium repressed enzyme production. The time course and amount of protease accumulated showed an inverse correlation with growth rate and nutrient concentration. Starvation did not induce extracellular protease production. CONCLUSION: The accumulation of Nab. magadii extracellular protease is stimulated by nutrient limitation and slow growth rate indicating that it is probably induced in response to a deficit in the energetic status of the cells. Nutritional starvation did not induce protease accumulation suggesting that de novo synthesis of this protease and/or factor/s necessary for its activation are required. This enzyme may be regulated by nitrogen catabolite repression and it does not require protein substrates for induction. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the basic knowledge on protease regulation in haloalkaliphilic archaea and will help to optimize the production of this extremozyme for biotechnological applications such as protease-catalysed peptide synthesis.     

Nutrient-induced competition between two species of marine diatoms

Competition experiments betweenPhaeodactylum tricornutum andSkeletonema costatum showed that even at temperatures higher than 10°C (i.c. 14°C), the development ofSkeletonema can be favoured by adjusting nutrient levels and nutrient ratios. Low NSi ratios were found to favourSkeletonema. Additionally, high NP ratios further enhanced the ability ofSkeletonema to dominate the cuftures. Contrary to some statements in literature, it seems that high concentrations of silicates are more important for the dominance ofSkeletonema costatum in large-scale cultures than just low temperatures. This finding is important with regard to stimulating the blooming ofSkeletonema costatum in natural phytoplankton populations as food for bivalve molluscs.     

A suite of citrate-derived siderophores from a marine Vibrio species isolated following the Deepwater Horizon oil spill

Nearly all microbes require iron for growth. The low concentration of iron found in the ocean makes iron acquisition a particularly difficult task. In response to these low iron conditions, many bacteria produce low-molecular-weight iron-binding molecules called siderophores to aid in iron uptake. We report herein the isolation and structural characterization of a suite of amphiphilic siderophores called the ochrobactins-OH, which are produced by a Vibrio species isolated from the Gulf of Mexico after the 2010 Deepwater Horizon oil spill. The citrate-based ochrobactins-OH are derivatives of aerobactin, replacing the acetyl groups with fatty acid appendages ranging in size from C8 to C12, and are distinctly different from the ochrobactins in that the fatty acid appendages are hydroxylated rather than unsaturated. The discovery of the marine amphiphilic ochrobactin-OH suite of siderophores increases the geographic and phylogenetic diversity of siderophore-producing bacteria.     

Production and optimization of process parameters for alkaline protease production by a newly isolated Bacillus sp. under solid state fermentation

Production of alkaline protease employing the laboratory isolate, Bacillus sp. under solid state fermentation (SSF) was optimized. The effect of wheat bran and lentil husk was examined. Wheat bran showed highest enzyme production. The appropriate incubation time, inoculum size, moisture level and buffer solution level were determined. Maximum yields of 429.041 and 168.640 U g⁻¹ were achieved by employing wheat bran and lentil husk as substrates in 0.1 M carbonate/bicarbonate buffer at pH 10 with 30 and 40% initial moisture level at 24 h. Inoculum size and buffer solution level were found to be 20 and 25% and 0.5:1 for wheat bran and lentil husk, respectively.     

Sterigmatocystin production by nine newly described Aspergillus species in section Versicolores grown on two different media

Nine recently described Aspergillus species and four known species in section Versicolores were tested for their ability to produce sterigmatocystin on two liquid media, Czapek w/20 % Sucrose Broth and Yeast Extract Broth grown in the dark for 1 week at 25 °C. Detection and quantification of ST were performed by reversed-phase liquid chromatography coupled with electrospray ionization ion trap mass spectrometry. Limit of detection was 3 ng/mL and limit of quantification was 10 ng/mL. Nine newly described Aspergillus species from various substrates, A. amoenus, A. creber, A. cvjetkovicii, A. fructus, A. jensenii, A. puulaauensis, A. subversicolor, A. tennesseensis and A. venenatus in section Versicolores were found to produce sterigmatocystin. Production was confirmed in recently collected isolates of A. protuberus and A. versicolor. A. austroafricanus and A. tabacinus did not produce sterigmatocystin.     

Characterization of two forms of hemagglutinin/protease produced by Vibrio cholerae non-O1

Two forms (34 kDa and 32 kDa) of hemagglutinin/protease produced by Vibrio cholerae non-O1 were characterized. The hemagglutinin/protease purified by immunoaffinity column chromatography using a monoclonal antibody was essentially a 34-kDa form. By incubation of the purified 34-kDa form at 37 degrees C, it was processed (autodigested) to the 32-kDa form. The N-terminal 20 amino acid sequences of both the 34- and 32-kDa forms were identical, suggesting that proteolytic processing at the C-terminal region of the 34-kDa hemagglutinin/protease resulted in the 32-kDa form. With this shift, protease activity increased, but hemagglutinating activity decreased, suggesting that the C-terminal region of the hemagglutinin/protease is related to hemagglutinating activity.     

一株海洋细菌产碱性蛋白酶的研究

对1株产碱性蛋白酶的海洋细菌Pseudomonas sp.7-11发酵产生碱性蛋白酶的条件和酶的调控机制进行了初步研究。结果显示,7－11为好氧菌,具有嗜低温特性,且适宜在偏碱性的条件下生长并产酶,该菌产生碱性蛋白酶表现出初级动力学持征;酷蛋白培养基适合菌株产酶;K^ 对于菌株产酶有关键作用;复合氨基酸对产酶有抑制作用;初步显示,7－11产生蛋白酶受诱导及阻遏两种方向的调控。     

Effect of culture media on protease and oxytetracycline production with mycelium and protoplasts of Streptomyces rimosus

For the simultaneous production of protease and oxytetracycline, mycelium and protoplasts of Streptomyces rimosus TM-55 were cultivated in basal medium containing soluble starch, corn steep liquid, ammonium sulphate, calcium carbonate, sodium chloride and soybean oil. Protease and oxytetracycline production increased with decreasing in ratio of culture broth to vessel volume from 1:2 to 1:5. Each ml of broth with 0.286 mg fresh mycelia yielded 168–204 units of protease and 785–972 g of oxytetracycline after replacement of corn steep liquor, sodium chloride and soybean oil with beef extract and sunflower oil, while each ml of broth with 7.5 × 10⁷ protoplasts produced 141–153 units of protease and 504–615 g of oxytetracycline. Protease and oxytetracycline production were low when the pH was 5.1 or 9.0. Soluble starch and ammonium sulphate were the best carbon and nitrogen sources, respectively. Supplementation with calcium carbonate enhanced protease and oxytetracycline production. The productivity of protoplasts decreased sharply when the incubation temperature increased from 28 to 34 °C, while the productivity of mycelium was almost unchanged.     

Isolation,partial purification,biochemical characterization and detergent compatibility of alkaline protease produced by Bacillus subtilis,Alcaligenes faecalis and Pseudomonas aeruginosa obtained from sea water samples

In the current study, bacteria isolated from sea water samples of Murdeshwar, Karnataka, were screened for the production of alkaline protease by culturing them onto skim milk agar media. Of the isolated bacteria, Bacillus subtilis, Pseudomonas aeruginosa and Alcaligenes faecalis showed distinct zones of hydrolysis due to enzyme production. They were each inoculated into enzyme production media under submerged fermentation conditions at 37?°C for 48?h with a constant agitation of 120?rpm. Partial purification of alkaline protease was carried out by isoelectric precipitation. Enzyme activity was determined under varying conditions of pH, incubation temperature, different substrates, carbon and nitrogen sources and salt concentrations using sigma’s universal protease activity assay. Enzyme immobilization was carried out using 2% Sodium alginate and 0.1?M ice cold CaCl₂ and its activity under varying pH, temperature conditions and detergent compatibility was assayed. Efficacy of enzyme in stain removal was tested and haemolysis was observed within of 60?s which resulted in removal of the stain. Among the three organisms, enzyme from Bacillus subtilis showed highest activity in all cases indicating that it was the most ideal organism for enzyme production.     

Arrowroot (Marantha arundinacea) starch as a new low-cost substrate for alkaline protease production

The feasibility of arrowroot (Marantha arundinacea) starch for alkaline protease production using an alkalophilic Bacillus lentus isolate was evaluated in batch fermentations in shake flasks and in a bioreactor under a range of conditions. A new arrowroot starch-casein medium (pH 10.2) was formulated having a composition (%, w/v): arrowroot starch 1, casein 1, sodium succinate 0.25, NH₄Cl 0.05, NaCl 0.05, KH₂PO₄ 0.04, K₂HPO₄ 0.03, MgCl₂ 0.01, yeast extract 0.01 and Na₂CO₃ 1.05. The isolate produced a maximum protease yield (6754.7 U ml^–1) in this medium when grown for 72 h at 250 rev/min and 37 °C. Scaling-up studies in a bioreactor showed a 5-fold increase in alkaline protease yields (31899 U ml^–1) at a lower production time of 45 h, aeration of 1 v/v/m and agitation of 400 rev/min at 37 °C.     

Extracellular enzyme production by Rhizopus and Mucor species on solid media

Solid media were employed to determine the presence and absence of extracellular enzyme production by two genera of fruit-rot fungi, Rhizopus and Mucor. The results of this investigation revealed that phosphatase was released into the cultural medium by all the fungi examined; however, only R. oryzae, R. tritici, M. mucedo, and M. piriformis showed the possibility of being high producers of the enzyme. Protease, urease, ribonuclease, pectate lyase, and polygalacturonase, at varying levels of activity, were detected, in the majority of the fungi, in the cultural medium.     

Production of extracellular alkaline proteases by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH₄NO₃ showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.     

Seawater requirement for the production of lipoxazolidinones by marine actinomycete strain NPS8920

A novel marine actinomycete strain NPS8920 produces a new class of 4-oxazolidinone antibiotics lipoxazolidinone A, B and C. Lipoxazolidinone A possesses good potency (1-2 microg/mL) against drug-resistant pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Strain NPS8920 exhibits different morphologies in both agar and submerged cultures. The ability of strain NPS8920 to sporulate on saline-based agar media but not on deionized water-based agar medium supported that strain NPS8920 is a marine actinomycete. While strain NPS8920 does not require seawater for growth, the production of lipoxazolidinones by strain NPS8920 can only be detected in the seawater-based media. The optimal production of lipoxazolidinones was observed in the natural seawater-based medium. Strain NPS8920 produced 10-20% of lipoxazolidinones in the synthetic sea salt Instant Ocean-based medium and no production in the sodium chloride-based and deionized water-based media.     

Study on the relationship of protease production and luminescence in Vibrio harveyi

AIMS: To demonstrate that Vibrio harveyi produces various types of toxins and how the production of those toxins is related with luminescence. METHODS AND RESULTS: Luminescence and toxicity of eight V. harveyi were evaluated. We demonstrated that all V. harveyi emitting luminescence were isolated from marine organisms and also showed that they were highly pathogenic when compared with culture collection V. harveyi based on cytotoxic assay test. On the contrary, V. harveyi isolated from shrimp farm showed no luminescence but showed high pathogenicity based on toxicity test. The effect of protease inhibitors on pathogenicity and luminescence was also investigated. We demonstrated that light emission of pathogenic V. harveyi remarkably decreased after addition of protease inhibitor. Furthermore, extracellular proteins from cell-free culture supernatant of luminescent and nonluminescent V. harveyi were compared using SDS-PAGE analysis. Results showed that there were differences in molecular weight and amount of proteins. CONCLUSIONS: Vibrio harveyi parasiting marine organisms have both luminescence and pathogenicity. Based on this study, luminescence and protease toxin activity in V. harveyi are related. Moreover, this paper clarified that V. harveyi produces various types of toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study demonstrated that V. harveyi produces two kinds of toxins, haemolysin and protease toxin. It may be clear roots of V. harveyi toxin.