Thermophilic protease-producing Geobacillus from Buranga hot springs in Western Uganda

Two proteolytic thermophilic aerobic bacterial strains, PA-9 and PA-5, were isolated from Buranga hot springs in western Uganda. The cells were rods, approximately 10–12 μm in length and 3 μm in width. Isolate PA-9 grew at between 38°C and 68°C (optimum, 62°C), and PA-5 grew at between 37°C and 72°C (optimum, 60°C). Both isolates grew optimally at pH 7.5–8.5. Their 16S rRNA gene sequences indicated that they belong to the newly described genus Geobacillus. Zymogram analysis of the crude enzyme extracts revealed the presence of two extracellular proteases for isolate PA-5, and at least eight for isolate PA-9. The optimum temperature and pH for casein-degrading activity were 70°C, pH 6.5 for isolate PA-9, but caseinolytic activity could also be observed at 2°C. In the case of isolate PA-5, optimal activity was observed over a temperature and pH range of 50–70°C and pH 5–10, respectively. Received: 26 November 2001 / Accepted: 12 December 2001     

Effects of pH,NH<Subscript>4</Subscript>-N concentration,temperature, and storage period on basidiospore germination in an ectomycorrhizal ammonia fungus <Emphasis Type="Italic">Hebeloma vinosophyllum</Emphasis>

Basidiospore germination in an ectomycorrhizal ammonia fungus Hebeloma vinosophyllum was stimulated by 10–500 mM NH₄Cl aqueous solution at pH 4.5–9.0, but not by pure water. The basidiospores germinated at 10°–35°C with an optimum at 25°–30°C. The highest germination percentage (83.0%) was observed in 100 mM NH₄Cl aqueous solution adjusted to pH 8.0 by KOH, when the basidiospores were incubated at a density of 10⁶ spores/ml at 30°C for 14 days. The percent germination value decreased with the increased duration of storage under both dry and wet conditions. Humidity and temperature affected the longevity of H. vinosophyllum basidiospores. The basidiospores maintained their germination ability longer under a dry condition than under a wet condition. The greatest longevity was accomplished by storage at 15°C under a dry condition.     

Conversion and degradation of shellfish wastes by <Emphasis Type="Italic">Serratia</Emphasis> sp. TKU016 fermentation for the production of enzymes and bioactive materials

A chitosanase and a protease were purified from the culture supernatant of Serratia sp. TKU016 with shrimp shell as the sole carbon/nitrogen source. The molecular masses of the chitosanase and protease determined by SDS–PAGE were approximately 65 and 53 kDa, respectively. The chitosanase was inhibited completely by Mn²⁺, but the protease was enhanced by all of tested divalent metals. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase and protease were (pH 7, 50°C, pH 6–7, <50°C) and (pH 8–10, 40°C, pH 5–10, <50°C), respectively. SDS (2 mM) had stimulatory effect on TKU016 protease activity. The result demonstrates that TKU016 protease is SDS-resistant protease and probably has a rigid structure. Besides, TKU016 culture supernatant (2% SPP) incubated for 2 days has the highest antioxidant activity, the DPPH scavenging ability was about 76%. With this method, we have shown that shrimp shell wastes can be utilized and it’s effective in the production of enzymes, antioxidants, peptide and reducing sugar, facilitating its potential use in biological applications and functional foods.     

Proteolytic Activity from an Alkali-Thermotolerant <Emphasis Type="Italic">Streptomyces gulbargensis</Emphasis> sp. nov.

Multiple proteases were produced and partially purified from an alkali-thermotolerant novel species of Streptomyces (i.e., Streptomyces gulbargensis DAS 131) after 48 h of growth at 45°C. The enzyme preparation exhibited activity over a broad range of pH (4–12) and temperature (27–55°C). Optimum activity was observed at a pH of 9.0 and a temperature of 45°C. Starch and protease peptone was found to be a good source of carbon and nitrogen to enhance the enzyme activity. Two active zones in the range of 19 to 35 kDa were detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

Degradation of DNA during the autolysis of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The autolysis of yeast cells has practical implications in the production of fermented foods and beverages and flavourants for food processing. Protein and RNA degradation during yeast autolysis are well described but the fate of DNA is unclear. Yeast cells (Saccharomyces cerevisiae) were autolysed by incubating suspensions at 30–60°C (pH 7.0), and at pH 4.0–7.0 (40°C) for 10–14 days. Up to 55% of total DNA was degraded, with consequent leakage into the extracellular environment of mainly 3′- and 5′-deoxyribonucleotides, and lesser amounts of polynucleotides. The rate and extent of DNA degradation, composition of the DNA degradation products and DNase activity were affected by temperature and pH. The highest amount of DNA degradation occurred at 40°C and pH 7.0, where the highest DNase activity was recorded. DNase activity was lowest at 60°C and pH 4.0, where the proportion of polynucleotides in the degradation products was higher. Electronic Publication     

Biodegradation of shellfish wastes and production of chitosanases by a squid pen-assimilating bacterium, <Emphasis Type="Italic">Acinetobacter</Emphasis><Emphasis Type="Italic">calcoaceticus</Emphasis> TKU024

Two chitosanases (CHSA1 and CHSA2) were purified from the culture supernatant of Acinetobacter calcoaceticus TKU024 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHSA1 and CHSA2 determined by SDS-PAGE were approximately 27 and 66 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHSA1 and CHSA2 were (pH 6, 50°C, pH 4–10, <90°C) and (pH 7, 60°C, pH 6–11, <70°C), respectively. CHSA1 and CHSA2 had broad pH and thermal stability. CHSA1 and CHSA2 were both inhibited by EDTA and were inhibited completely by 5 mM Mn²⁺. CHSA1 and CHSA2 degraded chitosan with DD ranging from 60 to 98%, and also degraded some chitin. The most susceptible substrate was 60% deacetylated chitosan. Furthermore, TKU024 culture supernatant (1.5% SPP) incubated for 5 days has the most reducing sugars (0.63 mg/ml). With this method, we have shown that shellfish wastes may have a great potential for the production of bioactive materials.     

Cloning,expression, and characterization of an insoluble glucan-producing glucansucrase from <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> NRRL B-1118

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K _M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching.     

Effects of extreme changes of sea water temperature and salinity on the development of the sand dollar <Emphasis Type="Italic">Scaphechinus mirabilis</Emphasis>

The combined effects of temperatures of 14, 17, 20, 22, and 25°C and salinities of 36–12‰ on embryos and larvae of the sand dollar Scaphechinus mirabilis was studied. Embryonic development is the most sensitive stage in the early ontogenesis of S. mirabilis. It is completed at a temperature of 14–20°C in a salinity range of 36–24‰ and at temperature of 22°C to 26‰. The fertilization proceeds in wider ranges of temperature and salinity. Among the swimming larvae, blastulae showed the greatest resistance to variations of these environmental factors. All the larvae survived at a temperature of 14–22°C and a salinity of 36–20‰, and more than 70% of them at 18‰. The pluteus I is the most vulnerable stage; probably this is related to the formation of the larval skeleton and transition to phytoplankton feeding. The survival of larvae at the age of 20 days was 100% at 14–22° C and a salinity of 36–24‰, most of them survived at 14–20°C and a salinity 18‰. The temperature 25 ° C is the most damaging for early development of S. mirabilis. The duration of development of that species lasts 28.5–29 days at 20°C and a salinity of 32.2–32.6‰. At 20 and 22°C, the larvae settled and completed metamorphosis more quickly if sand from the parental habitat was present. The larvae did not settle during the experiment (14 days) at 14 ° C and in the absence of sand.     

<Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> isolated from black tiger prawn larvae (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) in Vietnam

 A marine fungus was isolated from the black tiger prawn Penaeus monodon at Nha Trang, Vietnam, on March 20, 2001 and named isolate NJM 0131. The fungus was identified as Haliphthoros milfordensis from the characteristics of asexual reproduction, and its physiological characteristics were investigated. Although the optimum temperature for growth of the isolate was 25°–30°C, the fungus grew at a wide range of temperatures (15°–40°C). H. milfordensis grew well in 50%–100% seawater, but poorly in PYG agar containing 1.0%–5.0% NaCl and KCl. The fungus grew at a wide range of pH (4.0–11.0) with the optimum pH value of 7.0–9.0. The isolate also showed pathogenicity to swimming crab larvae (Portunus trituberculatus) by artificial infection, but mortality was not high. This is the first report of disease in the black tiger prawn P. monodon in Vietnam caused by H. milfordensis. Received: July 22, 2002 / Accepted: January 21, 2003 Correspondence to:K. Hatai     

Degradation of RNA during the autolysis of Saccharomyces cerevisiae produces predominantly ribonucleotides

Autolytic degradation of yeast RNA occurs in many foods and beverages and can impact on the sensory quality of the product, but the resulting complex mixture of nucleotides, nucleosides and nucleobases has not been properly characterised. In this study, yeast autolysis was induced by incubating cell suspensions of Saccharomyces cerevisiae at 30–60 °C (pH 7.0), and at pH 4.0–7.0 (40 °C) for 10–14 days, and the RNA degradation products formed during the process were determined by reversed-phase HPLC. Up to 95% of cell RNA was degraded, with consequent leakage into the extracellular environment of mainly 3′-, 5′- and 2′-ribonucleotides, and lesser amounts of polynucleotides, ribonucleosides and nucleobases. The rate of RNA degradation and the composition of the breakdown products varied with temperature and pH. RNA degradation was fastest at 50 °C (pH 7.0). Autolysis at lower temperatures (30 °C and 40 °C) and at pH 5.0 and 6.0 favoured the formation of 3′-nucleotides, whereas autolysis at 40 °C and 50 °C (pH 7.0) favoured 5′- and 2′-nucleotides. The best conditions for the formation of the two flavour-enhancing nucleotides, 5′-AMP and 5′-GMP, were 50 °C (pH 7.0) and pH 4.0 (40 °C), respectively.     

Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated <Emphasis Type="Italic">Bacillus</Emphasis> species Y

Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about 1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn²⁺, Co²⁺ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating the potential role of this enzyme for detergent application.     

Thermal preference and avoidance in cladoceran <Emphasis Type="Italic">Daphnia magna</Emphasis> strauss (crustacea,cladocera) acclimated to constant temperature

The final preferable temperature (FPT) and avoidance temperature (AT) were determined in parthenogenetic females of the crustacean Daphnia magna Strauss. The animals were preliminary acclimated to constant temperature of 23.4°C followed by keeping them in a thermo-gradient device for 24 days. It was revealed that daphnia select FPT with overshoot. In the first four days, daphnia selected temperatures 0.6–1.6°C higher than the acclimation temperature and 4–7.4°C higher than FPT. Two zones of FPT are revealed: the first zone by the time of 5–13 days (17.6 ± 1.2°C); the second, by 16–24 days (20 ± 1.5°C). The dynamics of AT diapason followed the dynamics of FPT. Two zones of the AT plateau were observed: over five to 17 days (temperatures <14°C and >25.8°C were avoided) and for 21–24 days (<8.5°C and >26°C).     

Characterization of plant growth-promoting rhizobacterium <Emphasis Type="Italic">Exiguobacterium</Emphasis> NII-0906 for its growth promotion of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>)

A phosphate solubilizing and antagonistic bacterial strain, isolated from a Western Ghat forest soil in Kerala province, India (designated as NII-0906), showed cold tolerance and grew from 10 to 37°C (optimum temperature 30°C). It was a Gram-positive, rod shaped, 0.8–1.6 μm in size, and exhibited tolerance to a wide pH range (5–12; optimum 7.0) and salt concentration up to 7% (w/v). The isolate showed maximum similarity with Exiguobacterium marinum TF-80^T based on 16S rRNA analysis. It solubilized tricalcium phosphate under in vitro conditions. The phosphate solubilization was estimated along a temperature range (5–40°C), and maximum activity (84.7 μg mL⁻¹ day⁻¹) was recorded at 30°C after 10 days of incubation. The phosphate solubilizing activity coincided with a concomitant decrease in pH of the medium. The isolate also exhibited antifungal activity against phytopathogenic fungi in Petri dish assays and produced siderophore and hydrogen cyanide. The strain’s plant growth promotion properties were demonstrated through a cowpea-based bioassay under greenhouse conditions. The bacterial inoculation resulted in significant increment in plant root, stem and as well as in plant biomass. Further, scanning electron microscopic study revealed the root colonization in cowpea. These results could offer potential perspective for the strain to be used as plant growth-promoting rhizobacteria, which could be used as an inoculant for regional crops.     

Characterization of a xylanase from a thermophilic strain of <Emphasis Type="Italic">Anoxybacillus pushchinoensis</Emphasis> A8

A facultatively anaerobic, thermophilic, xylanolytic bacterium was isolated from a sample collected from the Diyadin Hot Springs, Turkey. According to morphological, biochemical and molecular identification, this new strain was suggested to be representative of the Anoxybacillus pushchinoensis and it was designated as Anoxybacillus pushchinoensis strain A8. It exhibited 97% similarity to 16S rRNA gene sequence of A. pushchinoensis and 77% DNA homology by DNA-DNA hybridization studies. Q-sepharose and CM-sepharose chromatography was used to purify an extracellular xylanase to >90% purity from this species. The enzyme had a molecular mass of approximately 83 kDa. The enzyme showed optimum activity at pH 6.5 and it was 96% stable over a broad pH range of 6.5–11 for 24 hours. The enzyme had optimum activity at 55°C and it was 100% stable at temperature between 50–60°C up to 24 hours. Kinetic characterization of the enzyme was performed at temperature optima (55°C) and V_max and K _m were found to be 59.88 U/mg protein and 0.909 mg/mL, respectively. Oat spelt xylan but not xylooligosaccharides was degraded by the enzyme and xylose was the only product detected from oat xylan degradation. This suggested that the enzyme was an exo-acting xylanase.     

Production of a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> QM9414 xylanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its application in biobleaching of wheat straw pulp

The purpose of this study was to produce a Trichoderma reesei xylanase (XYN2) in Pichia pastoris and to test its potential application for pulp bleaching. The recombinant xylanase was purified by a two-step process of ultrafiltration and gel filtration chromatography. The molecular mass of the recombinant enzyme was 21 and 25 kDa by SDS–PAGE analysis, due to different glycosylation of the native protein. The optimum pH and temperature of the recombinant XYN2 was 5.0 and 50 °C. Enzyme activity was stable at 50 °C and at pH 5.0–7.0. The bleaching ability of the recombinant xylanase was also studied at 50 °C and pH 6.0, using wheat straw pulp. Biobleaching of the xylanase produced chlorine dioxide savings of up to 60%, while retaining brightness at the control level and led to a lower kappa number and small enhancements in tensile, burst and tear strength of pulp fibers.     

The response of <Emphasis Type="Italic">Corylus avellana</Emphasis> L. phenology to rising temperature in north-eastern Slovenia

Knowledge of plant–weather relationships can improve crop management, resulting in higher quality and more stable crop yields. The annual timing of spring phenophases in mid-latitudes is largely a response to temperature, and reflects the thermal conditions of previous months. The effect of air temperature on the variability of hazelnut (Corylus avellana L.) phenophases (leafing, flowering) was investigated. Meteorological and phenological data for five cultivars were analysed over the periods 1969–1979 (P1) and 1994–2007 (P2) in Maribor, Slovenia. Phenological data series were correlated strongly to the temperature of the preceding months (R ²: 0.64–0.98) and better correlated to daily maximum and mean temperatures than to daily minimum temperatures. About 75% of phenophases displayed a tendency towards earlier appearance and a shorter flowering duration during P2, which could be explained by the significant temperature changes (+0.3°C/decade) from December to April between 1969 and 2007. An increase in air temperature of 1°C caused an acceleration in leafing by 2.5–3.9 days, with flowering showing higher sensitivity since a 1°C increase promoted male flowering by 7.0–8.8 days and female flowering by 6.3–8.9 days. The average rate of phenological change per degree of warming (days earlier per +1°C) did not differ significantly between P1 and P2. An estimation of chilling accumulation under field conditions during 1993–2009, between 1 November and 28 February, showed that all four of these months contributed approximately similar amounts of accumulated chilling units. The growing degree days (GDD) to flowering were calculated by an estimated base temperature of 2°C and 1 January as a starting date, given the most accurate calculations. In general, thermal requirements were greater in P2 than in P1, although this difference was not significant. Longer-time series data extended to other agricultural and wild plants would be helpful in tracking possible future changes in phenological responses to local climate.     

Direct fermentation of potato starch in wastewater to lactic acid by<Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

The fungal species ofRhizopus oryzae 2062 has the capacity to carry out a single stage fermentation process for lactic acid production from potato starch wastewater. Starch hydrolysis, reducing sugar accumulation, biomass formation, and lactic acid production were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/L at pH 6.0 and 30°C was favourable for starch fermentation, resulting in a lactic acid yield of 78.3%–85.5% associated with 1.5–2.0 g/L fungal biomass produced in 36 h of fermentation.     

Production and partial purification of invertase using <Emphasis Type="Italic">Cympopogan caecius</Emphasis> leaf powder as substrate

The present study investigates the efficiency of Aspergillus niger to produce invertase, an industrially important enzyme by using powdered stem of Cympopogan caecius (Lemon grass) as sole substrate and sole carbon source for the microorganism. The molecular weight of invertase was estimated to be 66–70 kDa by sodium do decyl sulphate poly acrylamide gel electrophoresis (SDS PAGE). The production of the enzyme was studied at different pH scales ranging from pH 4.0 to 7.0 at a constant temperature of 30°C and 2% substrate concentration. The maximum production of invertase (specific activity −0.0516 μk/mg protein) was obtained at pH 5.5 at 30°C temperature, and incubation for 48 h. The activity was found to be stable at pH 5.5 for 30 min. The enzyme was found to be stable in the temperature range of 20–55°C. The effect of divalent metal ions Cu²⁺, Fe²⁺, Co²⁺ on the activity of the enzyme invertase showed that these ions affected the activity by a certain factor. The study can be further industrially exploited in a country-like India where lemon grass is found in plenty and can be used as substrate for enzyme production. Moreover, the preparation of the substrate is also a simple process.     

Conformational Flexibility of Lipase Lip1 from <Emphasis Type="Italic">Candida Rugosa</Emphasis> Studied by Electronic Spectroscopies and Thermodynamic Approaches

We have used second-order orthogonal designs to obtain empirical models that describe the combined effect of pH and temperature on the secondary structure of a lipase (Lip1) from Candida rusosa. The equations that describe lipase conformational flexibility were derivated from the enzyme alpha helix fraction obtained from the experimental matrix. The thermal unfolding of lipase at different pH values was followed by measuring the circular dichroism signal as a function of temperature over a temperature range of 20–80 °C. The results showed a melting temperature of 58.9 °C at pH 5.5, while at pHs 7.0 and 8.6, the temperature values were 50.2 °C and 36.1 °C respectively. The optimum experimental conditions of conformations with high content of alpha helix were found at high temperature and pH, both at zero time and at one-hour incubation time of enzyme. Important variations in the enzyme secondary structure were induced for the pH and temperature. In contrast, minor changes were observed during the incubation time. This behaviour suggests that the medium pH induces a modification in the enzyme secondary structure and not due to a result of a progressive denaturation process. From the values of thermodynamic functions at different pHs, the system at initial state of unfolding process is previously disordered by the pH effect.