Peptides from several italian cheeses inhibitory to proteolytic enzymes of lactic acid bacteria, pseudomonas fluorescens ATCC 948 and to the angiotensin I-converting enzyme

Water-soluble peptides from Mozzarella, Italico, Crescenza, and Gorgonzola cheeses were fractionated by reverse-phase fast protein liquid chromatography. Peptide fractions with inhibitory activity to amino- and endo-peptidases from Lactobacillus delbrueckii ssp. bulgaricus B397, Streptococcus thermophilus 305, and Lactococcus lactis ssp. cremoris Wg2 were found. Enzymes from Lactobacillus casei ssp. casei 2752 were less sensitive. Endopeptidase from Lactobacillus casei ssp. casei 2752 also had a different response to the effect of some inhibitors. It probably showed limited differences in catalysis and substrate positioning. Most of these inhibitory peptides were also effective in reducing the activity of the Pseudomonas fluorescens ATCC 948 endopeptidase and the angiotensin I-converting enzyme. Inhibitory peptide fractions from Mozzarella, Italico, and Crescenza cheeses had a certain degree of hydrophobicity while the peptide fraction from Gorgonzola cheese eluted in the initial part of the acetonitrile gradient. One of the inhibitory peptides contained in the water-soluble extract of Crescenza cheese was further purified and sequenced. It corresponded to the β-casein fragment 58-72.     

Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at −5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme.     

Characterization of two extracellular proteinases from Aspergillus terreus and their role in the formation of low molecular weight endoglucanases

Two extracellular proteolytic activities from the wood degrading fungus Aspergillus terreus have been characterized. Proteinase I (serine thiol-dependent enzyme) was active over a broad pH range (7·0–10·0) and at 55°C. The second proteinase (metalloproteinase) showed optimal activity at pH 6·0–7·0 and at 65–70°C. Both proteins had isoelectric points at acid pH and contained carbohydrate moieties. The metalloproteinase possessed a uniquely high content of serine and threonine and an extremely low percentage of glutamate and aspartate. The metalloproteinase was involved in the formation of the low molecular mass endoglucanases of A. terreus.     

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown “petite-negative” yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40°C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles.

2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerolgrown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae.

3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the “petite-positive” yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126.

4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 μM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 μM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor.

5. We conclude that “petite-positive” and “petite-negative” yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Heat-shock protein expression in a perennial grass commonly associated with active geothermal areas in western North America

Heat-shock protein (hsp) expression in response to short- (hours) versus long-term (weeks) heat was examined in Dichanthelium lanuginosum. Expression of cytoplasmic class I small hsps (shsps) and hsp 101 was elicited by 2 h above 40°C, along with an increase over basal levels of hsp 70. Elevated levels of heat-induced shsp and hsp 101 persisted for 5–7 days after plants were returned to control temperatures. Protein extracts from roots exposed to 42°C for several weeks displayed increased levels of shsp but decreased hsp 101 levels. This decrease in hsp 101 did not occur in D. lanuginosum from normothermic environments.     

Amino Acid esters prevent thermal inactivation and aggregation of lysozyme

Small potent inhibitors of aggregation are eagerly demanded for preventing the inactivation of proteins. This paper shows that amino acid esters (AAEs) prevent heat-induced aggregation and inactivation of hen egg lysozyme. Lysozyme was completely inactivated (<1% original activity) during heat treatment at 98 degrees C for 30 min in a solution containing 0.2 mg/mL lysozyme in 50 mM Na-phosphate buffer (pH 6.5). The residual activities only slightly increased (<5%) in the presence of 100 mM commonly used additives such as arginine, guanidine, urea, and sugars. However, in the presence of 100 mM AAEs, the residual activities were >60% and no aggregates were observed during the heat treatment at 98 degrees C for 30 min. This fact provides new information on the scaffold for designing additives to prevent heat-induced aggregation.     

Meta-pathway degradation of phenolics by thermophilic Bacilli

Thermophilic bacteria capable of degrading phenol as the sole carbon source were isolated from sewage effluent. The isolates were aerobic, sporulating, motile rod-shaped bacteria characterized as Bacillus species with growth temperature optima of 50–60°C. The enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase, was detected in all isolates grown in the presence of phenol. One strain, designated Bacillus strain Cro3.2, was capable of degrading phenol, o-, m-, and p-cresol via the meta-pathway and tolerated phenol at concentrations up to 0.1% (w/v) without apparent inhibition of growth. Phenol degradation activities in strain Cro3.2 were induced 3–5 h after supplementation by phenol, orcinol, and the cresols but not by halo- or nitro-substituted phenols. Maximal rates of phenol degradation in stirred bioreactors (10 μmol/min⁻¹/g⁻¹ cells) were achieved at an O₂ delivery rate of 1.0 vvm and temperatures of 45–60°C; however, catechol-2,3-dioxygenase (but not 2-hydroxymuconic semialdehyde dehydrogenase) was rapidly inactivated at high oxygen concentrations. Whole cells of Bacillus strain Cro3.2 entrapped in calcium alginate, polyacrylamide, and agarose gels showed widely different rates of phenol degradation. In calcium alginate gels, rapid loss of phenol-degrading activity was attributed to calcium-induced inactivation of catechol-2,3-dioxygenase. No stabilization with respect to oxygen-induced inactivation was observed under any of the immobilization conditions. It is concluded that the counteractive effects of oxygen limitation at low dO₂ and inactivation of catechol-2,3-dioxygenase at high dO₂ levels pose a significant impediment to the use of resting thermophile cells in the treatment of phenolic waste streams.     

Comparative thermodynamic elucidation of the structural stability of thermophilic proteins

Differential scanning calorimetry, circular dichroism, and visible absorption spectrophotometry were employed to elucidate the structural stability of thermophilic phycocyanin derived from Cyanidium caldarium, a eucaryotic organism which contains a nucleus, grown in acidic conditions (pH 3.4) at 54°C. The obtained results were compared with those previously reported for thermophilic phycocyanin derived from Synechococcus lividus, a procaryote containing no organized nucleus, grown in alkaline conditions (pH 8.5) at 52°C. The temperature of thermal unfolding (t_d) was found to be comparable between C. caldarium (73°C) and S. lividus (74°C) phycocyanins. The apparent free energy of unfolding (ΔG_[urea]=0) at zero denaturant (urea) concentration was also comparable: 9.1 and 8.7 kcal/mole for unfolding the chromophore part of the protein, and 5.0 and 4.3 kcal/mole for unfolding the apoprotein part of the protein, respectively. These values of t_d and ΔG_[urea]=0 were significantly higher than those previously reported for mesophilic Phormidium luridum phycocyanin (grown at 25°C). These findings revealed that relatively higher values of t_d and ΔG_[urea]=0 were characteristics of thermophilic proteins. In contrast, the enthalpies of completed unfolding (ΔH_d) and the half-completed unfolding (ΔH_d)1/2 for C. caldarium phycocyanin were much lower than those for S. lividus protein (89 versus 180 kcal/mole and 62 versus 115 kcal/mole, respectively). Factors contributing to a lower ΔH_d in C. caldarium protein and the role of charged groups in enhancing the stability of thermophilic proteins were discusse.     

Temperature and carbon source affect the production and secretion of a thermostable β-xylosidase by Aspergillus fumigatus

The effect of the temperature of growth and carbon source on the production and secretion of β-xylosidase (EC 3.2.1.37) by the thermotolerant fungi Aspergillus fumigatus was studied in submerged cultures. In cultures developed at optimal temperature (30 °C), the enzyme was predominantly cell-bound, while in cultures developed at higher temperature (42 °C), the β-xylosidase activity was predominantly found in the cell-free filtrates. The use of corn cob powder instead of xylan as substrate increased considerably the secretion of enzyme. The highest level of extracellular β-xylosidase (45 U/ml or 360 U/mg protein) was obtained in 3% corn cob cultures grown at 42 °C for 72 h. The partially purified enzyme was active and stable at high temperatures. The presence of high titres of β-xylosidase activity in association with xylanase in the culture filtrates enhanced the efficiency of the pulp hydrolysis process.     

Some effects of thyroidectomy on growth, heat production and the thermoregulatory ability of the immature fowl (Gallus domesticus)

1. 1.|The effect of thyroidectomy at 12 days of age on weight gain, and on heat production and thermoregulatory ability of 4- to 5-week-old chickens at temperatures within and below the thermo-neutral zone was investigated.

2. 2.|Despit the absence of thyroid tissue, as demonstrated with radioiodine, a small amount of thyroxine was found in the plasma of some thyroidectomized (TX) birds.

3. 3.|Thyroidectomy depressed weight gain; pair-fed controls grew significantly faster than TX birds.

4. 4.|Resting heat production of TX birds at thermoneutrality (30°C) was depressed by 18% (P < 0.001) and body temperature by 0.4°C (P < 0.001).

5. 5.|At 12°C heat production of TX birds was similar to that of controls but the body temperature of TX birds was 0.7°C lower (P < 0.001).

6. 6.|Thyroidectomized birds were unable to regulate body temperature at 5°C even if thyroxine was provided on the day before and at the time of cold-exposure. This inability to thermoregulate was probably due to inadequate insulation and poor nutritional status.

Exercise in the heat: Effects of dinitrophenol administration

1. 1.|Dinitrophenol (DNP) was administered to rats in two equal dosages (20 mg/kg, 30 min interval); the second injection was followed immediately by exercise (9.14 m/min) in the heat (30°C) or at room temperature (21°C).

2. 2.|At 21°C control (saline-treated) rats manifested a mean endurance of 94 min which was reduced to 32 min among DNP-treated animals.

3. 3.|At 30°C, control rats ran for 65 min (δT_re/min = 0.05°C) while DNP-treated animals had a mean endurance of only 12 min (δT_re/min = 0.22°C).

4. 4.|DNP-treated rats (30°C) manifested no decrements in tail-skin heat loss (δT_sk/min = 0.17°C vs 0.10°C) or saliva secretion (0.78 g/min, DNP vs. 0.19 g/min, control) for their brief treadmill duration.

5. 5.|The increased metabolic heat production of DNP severely reduced performance.

Determination of the thermodynamics of carbonic anhydrase acid-unfolding by titration calorimetry

The enthalpy of unfolding (Δ_uH) of carbonic anhydrase II was determined by titrating the protein with acid and measuring the heat using isothermal titration calorimetry (ITC) in the temperature range of 5 to 59 °C. By combining the ITC results with our previous findings by differential scanning calorimetry (DSC) in the temperature range of 39 to 72 °C, the Δ_uH dependence over a wide temperature range was obtained. The temperature dependence of the enthalpy displays significant curvature indicating that the heat capacity of unfolding (Δ_uC_p) is dependent on temperature. The T-derivative of Δ_uC_p was equal to 100 ± 30 J/(mol × K²), with the result that the Δ_uC_p is equal to 15.8 kJ/(mol × K) at 5 °C, 19.0 kJ/(mol × K) at 37 °C and 21.8 kJ/(mol × K) at 64 °C. The enthalpy of unfolding is zero at 17 °C. At lower temperatures, the Δ_uH becomes exothermic.

This method of determining protein unfolding thermodynamics using acid-ITC, significantly widens the accessible T-range, provides direct estimate of the thermodynamic parameters at physiological temperature, and gives further insight into the third T-derivative of the Gibbs free energy of unfolding.     

Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay.

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.     

Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.     

Efficiency of asbestos shading for growth of Barki rams during hot summer

Twenty-four growing Barki rams, 5–6 months old with an average body weight of 22 kg, were divided into asbestos shaded and unshaded groups during summer. Ad libitum feeding on roughage with 0.5 kg of concentrates per head per day was practiced. Drinking water was available twice daily in the early morning and afternoon. Biweekly observations included rectal temperature (RT; °C), and respiration rate (RR; breaths per minute) at 06:00 h and 14:00 h were recorded. Meteorological data, plasma glucose, calcium, inorganic phosphorus and packed cell volume were also measured. Results indicated that experimental animals developed hyperthermia during June to September, as evidenced by higher (P < 0.01) RT and RR than normal (40 ± 0.05 and 103.9 ± 3.87 vs. 38.9 ± 0.10 and 40 ± 6.56, respectively). Diurnal variations in these physiological phenomena were closely associated (P < 0.05) with ambient temperature changes. As compared with the unshaded group, providing an asbestos shed reduced (P < 0.05) RT and RR in the hyperthermic animals during the day (39.9 ± 0.07 and 94.7 ± 3.75 vs. 40.1 ± 0.08 and 113.1 ± 4.74, respectively), but it increased (P < 0.05) these parameters during summer nights (39.5 ± 0.05 and 82.4 ± 0.95 vs. 39.2 ± 0.07 and 71.6 ± 2.41, respectively). It also increased (P < 0.05) packed cell volume 1 h after morning drinking (35 ± 0.97 vs. 33.2 ± 0.60); reduced (P < 0.05) plasma glucose (43.3 ± 5.88 vs. 53.2 ± 6.31); caused hypocalcemia (10.9 ± 0.35 vs. 11.5 +- 0.43; P < 0.05) and hypophosphatemia (3.9 ±0.35 vs. 4.6 ± 0.34; P < 0.05) as a result of hyperthermia. Monthly variations in all parameters studied were higher (P < 0.01) during early summer. It is concluded that providing an asbestos shed for growing Barki rams under Egyptian summer conditions will not protect the animals from hyperthermia by day and night, as it interferes with extra body heat dissipation to the surroundings during summer nights. Although the unshaded animals were more hyperthermic during summer days, they tended to be normal during the night.     

Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris

The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644 U mL^-1 and the optimal secretion time was 4 days at 25°C. SDS-PAGE showed two bands, of 32.2 kDa and 29.6 kDa, both larger than the predicted mass of 22.4 kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6-9 and 50°C, respectively.     

不同地下水矿化度对柽柳光合特征及树干液流的影响

为揭示柽柳(Tamarix chinensis)光合能力及耗水特征对地下水矿化度的响应规律, 以黄河三角洲建群种——柽柳3年生植株为研究对象, 在1.2 m的潜水水位下, 模拟设置淡水、微咸水、咸水和盐水4种不同的地下水矿化度, 测定柽柳叶片光合-光响应、蒸腾速率和树干液流的日变化。结果表明: 地下水矿化度通过影响土壤盐分可显著影响柽柳光合特性及耗水性能。随地下水矿化度升高, 柽柳叶片净光合速率(P_n)、最大P_n、蒸腾速率、气孔导度、表观量子效率和暗呼吸速率均先升高后降低, 而水分利用效率(WUE)持续降低。淡水、微咸水和盐水处理下, 柽柳P_n光响应平均值分别比咸水处理降低44.1%、15.1%和62.6%; 微咸水、咸水和盐水处理下, 柽柳WUE光响应平均值分别比淡水处理降低25.0%、29.2%和41.7%。随地下水矿化度升高, 柽柳叶片光饱和点先升高后降低, 而光补偿点持续升高, 光照生态幅变窄, 光能利用率变低。淡水和盐水处理下, 柽柳P_n下降分别是非气孔限制和气孔限制引起的。柽柳树干液流速率随地下水矿化度升高而先升高后降低, 咸水处理下树干液流速率日变幅最大, 日液流量最高。淡水、微咸水和盐水处理下日液流速率平均值分别比咸水处理降低61.8%、13.1%和41.9%。咸水矿化度下柽柳有较高的光合特性, 在蒸腾耗水较严重的情况下可实现高效生理用水, 适宜柽柳较好地生长。     

Effects of groundwater salinity on the characteristics of leaf photosynthesis and stem sap flow in Tamarix chinensis

AimsThe objective of this study was to investigate the change pattern of leaves photosynthesis and stem sap flow of Tamarix chinensisin under different groundwater salinity, which can be served as a theoretical basis and technical reference for cultivation and management of T. chinensis in shallow groundwater table around Yellow River Delta.MethodsThree-year-old T. chinensis, one of the dominated species in Yellow River Delta, was selected. Plants were treated by four different salinity concentrations of groundwater—fresh water (0 g∙L^-1), brackish water (3.0 g∙L^-1), saline water (8.0 g∙L^-1), and salt water (20.0 g∙L^-1) under 1.2 m groundwater level. Light response of photosynthesis and the diurnal courses of leaf transpiration rate, stem sap flux velocity and environment factors under different groundwater salinity were determined via LI-6400XT portable photosynthesis system and a Dynamax packaged stem sap flow gauge based on stem-heat balance method, respectively.Important findings The result showed that groundwater salinity had a significant impact on photosynthesis efficiency and water consumption capacity of T. chinensis by influencing the soil salt. The net photosynthetic rate (P_n), maximum P_n, transpiration rate, stomatal conductance, apparent quantum yield and dark respiration rate increased first and then decreased with increasing groundwater salinity, while the water use efficiency (WUE) continuously decreased. The mean P_n under fresh water, brackish water and salt water decreased by 44.1%, 15.1% and 62.6%, respectively, compared with that under saline water (25.90 µmol∙m^-2∙s^-1). The mean WUE under brackish water, saline water and salt water decreased by 25.0%, 29.2% and 41.7%, respectively, compared with that under fresh water (2.40 µmol∙mmol^-1). With the increase of groundwater salinity from brackish water to salt water, light saturation point of T. chinensisdecreased while the light compensation point increased, which lead to the decrease of light ecological amplitude and light use efficiency. Fresh water and brackish water treatment helped T. chinensis to use low or high level light, which could significantly improve the utilization rate of light energy. The decrease in P_n of T. chinensis was mainly due to non-stomatal limitation under treatment from saline water to fresh water, while the decrease in P_n of T. chinensis was due to stomatal limitation from saline water to salt water. With increasing groundwater salinity, stem sap flux velocity of T. chinensis increased firstly and then decreased, reached the maximum value under saline water. The mean stem sap flux velocity under fresh water, brackish water and salt water decreased by 61.8%, 13.1% and 41.9%, respectively, compared with that under saline water (16.96 g·h^-1). Tamarix chinensis had higher photosynthetic productivity under saline water treatment, and could attained high WUE under severe water deprivation by transpiration, which was suitable for the growth of T. chinensis.     

The temperature and pH properties of the extracellular hemicellulose-Degrading enzymes of aureobasidium pullulans NRRL Y 2311-1

Aureobasidium pullulans grown on arabinoxylan accumulates β-xylanase, p-nitrophenyl xylosidase, - -arabinofuranosidase and acetyl esterase activity in the culture fluid. The pH and temperature optima of these arabinoxylan-degrading enzymes were determined. The temperature optima of β-xylanase and p-nitrophenyl xylosidase were between 45 and 50°C whereas the optima for acetyl esterase and - -arabinofuranosidase were 55 and 60°C, respectively. β-xylanase, p-nitrophenyl xylosidase and - -arabinofuranosidase were stable over 3 h at 35°C, 35°C and 60°C, respectively, whereas acetyl esterase remained stable at 55°C for h. The enzymes were inactivated at higher temperatures. The pH optima for β-xylanase, p-nitrophenyl xylosidase and - -arabinofuranosidase were pH 4·0, between 4·0 and 7·0 and 5·0, respectively. β-xylanase, p-nitrophenyl xylosidase, - -arabinofuranosidase and acetyl esterase were most stable at pH 5·0 4·0–5·0, 6·0 and 5·0–6·0, respectively. The most suitable conditions for the use of the enzymes together to hydrolyze arabinoxylan would be 35 °C and pH 5.     

Oestradiol and the preovulatory surges of luteinising hormone and follicle stimulating hormone in ewes during the breeding season and transition into anoestrus

This study was designed to see if giving exogenous oestradiol, during the follicular phase of the oestrous cycle of intact ewes, during the breeding season or transition into anoestrus, would alter the occurrence, timing or magnitude of the preovulatory surge of secretion of luteinising hormone (LH) or follicle stimulating hormone (FSH). During the breeding season and the time of transition, separate groups of ewes were infused (intravenously) with either saline (30 ml h⁻¹; n = 6) or oestradiol in saline (n = 6) for 30 h. Infusion started 12 h after removal of progestin-containing intravaginal sponges that had been in place for 12 days. The initial dose of oestradiol was 0.02 μg h⁻¹; this was doubled every 4 h for 20 h, followed by every 5 h up to 30 h, to reach a maximum of 1.5 μg h⁻¹. Following progestin removal during the breeding season, peak serum concentrations of oestradiol in control ewes were 10.31 ± 1.04 pg ml⁻¹, at 49.60 ± 3.40 h after progestin removal. There was no obvious peak during transition, but at a time after progestin removal equivalent to the time of the oestradiol peak in ewes at mid breeding season, oestradiol concentrations were 6.70 ± 1.14 pg ml⁻¹ in ewes in transition (P < 0.05). In oestradiol treated ewes, peak serum oestradiol concentrations (24.8 ± 2.1 pg ml⁻¹) and time to peak (41.00 ± 0.05 h) did not differ between seasons (P > 0.05). During the breeding season, all six control ewes and four of six ewes given oestradiol showed oestrus with LH and FSH surges. The two ewes not showing oestrus did not respond to oestrus synchronisation and had persistently high serum concentrations of progesterone. During transition, three of six control ewes showed oestrus but only two had LH and FSH surges; all oestradiol treated ewes showed oestrus and gonadotrophin surges (P < 0.05). The timing and magnitude of LH and FSH surges did not vary with treatment or season. In blood samples collected every 12 min for 6 h, from 12 h after the start of oestradiol infusion, mean serum concentrations of LH and LH pulse frequency were lower in control ewes during transition than during mid breeding season (P < 0.05). Oestradiol treatment resulted in lower mean serum concentrations of LH in season and lower LH pulse frequency in transition (P < 0.05). We concluded that enhancing the height of the preovulatory peak in serum concentrations of oestradiol during the breeding season did not alter the timing or the magnitude of the preovulatory surge of LH and FSH secretion and that at transition into anoestrus, oestradiol can induce oestrus and the surge release of LH and FSH as effectively as during the breeding season.