Effect of heat treatment on lactoperoxidase activity in caprine milk

Using isothermal conditions, inactivation of lactoperoxidase (LPO) in caprine milk was studied in a temperature range of 69–73 °C. In order to evaluate the effect of temperature on the reaction rate, the Arrhenius and thermal death time models were used for data analysis. Thermal inactivation of LPO can be accurately described by a first-order kinetic model, as indicated by the relationships obtained by plotting the retention values as a function of treatment time on a semi-logarithmic scale and confirmed by the high R²-values obtained. D- and k-values decreased and increased, respectively with increasing temperature, indicating a more rapid LPO inactivation at higher temperatures. The corresponding Z- and E_a-values calculated from the slope of the semi-logarithmic plots of D and k as a function of temperature were 9.45 °C and 225.98 kJ/mol, respectively.     

Copper,zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692 bp with an open reading frame of 459 bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with K_m of 0.047 ± 0.008 μM and V_max of 1250 ± 24 units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80 °C with temperature optima at 20 °C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80 °C for 180 min (k_d, 6.54 ± 0.17 × 10⁻³ min⁻¹; t_1/2, 106.07 ± 2.68 min), and had midpoint of thermal transition (T_m) of 70.45 °C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.     

Stability and activity of Dictyoglomus thermophilum GH11 xylanase and its disulphide mutant at high pressure and temperature

The functional properties of extremophilic Dictyoglomus thermophilum xylanase (XYNB) and the N-terminal disulphide-bridge mutant (XYNB-DS) were studied at high pressure and temperature. The enzymes were quite stable even at the pressure of 500 MPa at 80 °C. The half-life of inactivation in these conditions was over 30 h. The inactivation at 80 °C in atmospheric pressure was only 3-times slower. The increase of pressure up to 500 MPa at 80 °C decreased only slightly the enzyme's stability, whereas in 500 MPa the increase of temperature from 22 to 80 °C decreased significantly more the enzyme's stability. While the high temperature (80–100 °C) decreased the enzyme reaction with short xylooligosaccharides (xylotetraose and xylotriose), the high pressure (100–300 MPa) had an opposite effect. The temperature of 100 °C strongly increased the K_m but did not affect the k_cat to the same extent, thus indicating that the interaction of the substrate with the active site suffers before the catalytic reaction begins to decrease as the temperature rises. Circular dichroism spectroscopy showed the high structural stability of XYNB and XYNB-DS at 93 °C.     

Effects of rapid temperature fluctuations prior to breeding on reproductive efficiency in replacement gilts

Rapidly cooling pigs after heat stress (HS) results in a pathophysiological condition, and because rapid temperature fluctuations may be associated with reduced reproductive success in sows, it lends itself to the hypothesis that these conditions may be linked. Objectives were to determine the effects of rapid cooling on thermal response and future reproductive success in pigs. Thirty-six replacement gilts (137.8±0.9 kg BW) were estrus synchronized and then 14.1±0.4 d after estrus confirmation, pigs were exposed to thermoneutral conditions (TN; n=12; 19.7±0.9°C) for 6 h, or HS (36.3±0.5°C) for 3 h, followed by 3 h of rapid cooling (HSRC; n=12; immediate TN exposure and water dousing) or gradual cooling (HSGC; n=12; gradual decrease to TN conditions) repeated over 2 d. Vaginal (T_V) and gastrointestinal tract temperatures (T_GI) were obtained every 15 min, and blood was collected on d 1 and d 2 during the HS and recovery periods at 180 and 60 min, respectively. Pigs were bred 8.3±0.8 d after thermal treatments over 2 d. Reproductive tracts were collected and total fetus number and viability were recorded 28.0±0.8 d after insemination. HS increased T_V and T_GI (P=0.01; 0.98 °C) in HSRC and HSGC compared to TN pigs. During recovery, T_V was reduced from 15 to 105 min (P=0.01; 0.33 °C) in HSRC compared to HSGC pigs, but no overall differences in T_GI were detected (P<0.05; 39.67 °C). Rapid cooling increased (P<0.05) TNFα compared to HSGC and TN pigs during recovery-d 1 (55.2%), HS-d 2 (35.1%), and recovery-d 2 (64.9%). Viable fetuses tended to be reduced (P=0.08; 10.5%) and moribund fetuses tended to be increased (P=0.09; 159.3%) in HSRC compared to HSGC and TN pigs. In summary, rapid cooling prior to breeding may contribute to reduced fetal viability and reproductive success in pigs.     

Spontaneously reduced body temperature and gasping ability as a mechanism of extreme tolerance to asphyxia in neonatal rats

The aim of the investigation was to verify our hypothesis that extreme tolerance of newborn rodents to anoxia is determined by their ability to maintain reduced body temperature and to keep on gasping.Newborn Wistar rats were used. In separate experiments we checked (1) effect of extreme thermal conditions on rectal temperature (T_re) of the newborns in their nests; (2) effect of ambient temperature (T_a) on oxygen consumption; (3) effects of controlled changes in T_re on thermoregulatory and respiratory responses to anoxia and on anoxia tolerance.In their nests rat pups controlled T_re at 32–36 °C while the T_re–T_a difference changed within a range of 1–20 °C. The lowest oxygen consumption of ∼24 ml O₂ kg⁻¹ min⁻¹ was recorded at T_a of 32 °C. Pups, exposed to anoxia at their normal T_re of 33 °C, were able to decrease T_re by another 1.7 °C and they kept on extremely slow and quiescent gasping for scheduled 25 min. In contrast, rats at T_re of 37 °C and 39 °C reached a critical phase of accelerated and shallow gasping after 14.95±0.40 min and 9.25±0.30 min, respectively.In conclusion, reduced T_re and unique gasping ability make newborn rats extremely tolerant to asphyxia.     

Kinetic analysis for suicide-substrate inactivation of microperoxidase-11: A modified model for bisubstrate enzymes in the presence of reversible inhibitors

Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H₂O₂. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism.The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor.Kinetic parameters of inactivation including the initial activity of MP-11, α₀, the apparent inactivation rate constant, k_i and the apparent inhibition rate constant for cysteine, k_I were obtained 0.282 ± 0.006 min^?1, 0.497 ± 0.013 min^?1 and 1.374 ± 0.007 min^?1 at [H₂O₂] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation.     

Characterization of a novel thermostable l-rhamnose isomerase from Thermobacillus composti KWC4 and its application for production of d-allose

d-Allose was considered as a kind of rare sugars with testified potential medicinal and agricultural benefits. l-Rhamnose isomerase (L-RI, EC 5.3.1.14), an aldose-ketose isomerase, played a significant part in producing rare sugar. In this article, a thermostable d-allose-producing L-RI was characterized from a thermotolerant bacterium, Thermobacillus composti KWC4. The recombinant L-RI was activated obviously in the presence of Mn²⁺ with an optimal pH 7.5 and temperature 65 °C. The Michaelis-Menten constant (K_m), turnover number (k_cat) and catalytic efficiency (k_cat/K_m) for l-rhamnose were 33.8 mM, 1189.8 min⁻¹ and 35.2 min⁻¹ mM⁻¹, respectively. At a higher temperature, Mn²⁺ played a pivotal role in strengthening the thermostability of T. composti L-RI. The differential scanning calorimetry (DSC) results showed the denaturing temperature (T_m) of T. composti L-RI was increased by 3 °C in presence of Mn²⁺. Although the T. composti L-RI displayed the optimum substrate as l-rhamnose, it could also effectively catalyze the isomerization between d-allulose and d-allose. When the reaction reached equilibrium, the sole product d-allose was produced from D-alluose by T. composti L-RI.     

Temperature selection by juvenile tuatara (Sphenodon punctatus) is not influenced by temperatures experienced as embryos

Most reptiles thermoregulate to achieve body temperatures needed for biological processes, such as digestion and growth. Temperatures experienced during embryogenesis may also influence post-hatching growth rate, potentially through influencing post-hatching choice of temperatures. We investigated in laboratory settings whether embryonic temperatures (constant 18 °C, 21 °C and 22 °C) influence selected body temperatures (T_sel) of juvenile tuatara (Sphenodon punctatus), providing a possible mechanism for differences in growth rates. We found that incubation temperature does not influence T_sel. Although the average daily mean T_sel was 21.6 ± 0.3 °C, we recorded individual T_sel values up to 33.5 °C in juvenile tuatara, which is higher than expected and above the panting threshold of 31–33 °C reported for adults. We found diel patterns of T_sel of juvenile tuatara, observing a general pattern of two apparent peaks and troughs per day, with T_sel being significantly lower around dawn and at 1500 h than any other time. When comparing our results with other studies on tuatara there is a remarkable consistency in mean T_sel of ~ 21 °C across tuatara of different ages, sizes and acclimatization histories. The ability of juvenile tuatara to withstand a wide range of temperatures supports their former widespread distribution throughout New Zealand and warrants further investigation into their plasticity to withstand climate warming, particularly where they have choices of habitat and the ability to thermoregulate.     

Ways to measure body temperature in the field

Body temperature (T_b) represents one of the key parameters in ecophysiological studies with focus on energy saving strategies. In this study we therefore comparatively evaluated the usefulness of two types of temperature-sensitive passive transponders (LifeChips and IPTT-300) and one data logger (iButton, DS1922L) mounted onto a collar to measure T_b in the field. First we tested the accuracy of all three devices in a water bath with water temperature ranging from 0 to 40 °C. Second, we evaluated the usefulness of the LifeChips and the modified iButtons for measuring T_b of small heterothermic mammals under field conditions. For this work we subcutaneously implanted 14 male edible dormice (Glis glis) with transponders, and equipped another 14 males with data loggers to simultaneously record T_b and oxygen consumption with a portable oxygen analyzer (Oxbox). In one individual we recorded T_b with both devices and analyzed recorded T_b patterns.LifeChips are able to measure temperature within the smallest range from 25 to 40 °C with an accuracy of 0.07±0.12 °C. IPTT-300 transponders measured temperature between 10 and 40 °C, but accuracy decreased considerably at values below 30 °C, with maximal deviations of nearly 7 °C. An individual calibration of each transponder is therefore needed, before using it at low T_bs. The accuracy of the data logger was comparatively good (0.12±0.25 °C) and stable over the whole temperature range tested (0–40 °C). In all three devices, the repeatability of measurements was high.LifeChip transponders as well as modified iButtons measured T_b reliably under field conditions. Simultaneous T_b-recordings in one edible dormouse with an implanted LifeChip and a collar-mounted iButton revealed that values of both measurements were closely correlated. Taken together, we conclude that implanted temperature-sensitive transponders represent an appropriate and largely non-invasive method to measure T_b also under field conditions.     

Tensiomyography of selected lower-limb muscles in professional soccer players

Tensiomyography is a non-invasive method of neuromuscular assessment used to measure muscle action characteristics, muscle tone, and muscle fiber type, and provides information on acute and chronic responses of muscle to different training loads. The aims of the present study were: to analyse differences in muscle response and mechanical characteristics of two major muscles of the lower extremity in a large group of Spanish soccer players according to playing position, and to provide group norms against which clinical findings may be compared. Data were collected from 78 professional soccer players (age 26.6 ± 4.4 years; height: 179.2 ± 5.3 cm; body mass: 75.8 ± 5.3 kg). Tensiomyography was recorded from the rectus femoris (RF) and biceps femoris (BF) muscles after 2 days without take part in any strenuous exercise or training. Five tensiomyographic parameters were analyzed: maximal displacement (D_m), contraction time (T_c), sustain time (T_s), delay time (T_d), and half-relaxation time (T_r). A good to excellent intra-session reliability was found for all contractile parameters (ICC ranged from 0.78 to 0.95). No significant differences between players of any position were observed in absolute values of BF. However, significant differences were observed for T_c, T_r and T_s between the different playing positions on RF (P < 0.05, effect size ranged from 1.3 to 1.6). Professional soccer players showed muscles with ability to rapidly generate force during contractions. The neuromuscular profile provided could help in identifying the normative data that are important for the different positions in order to optimize the training and recovery process of each individual player.     

Investigation on the properties and kinetics of glucose-fed aerobic granular sludge

Aerobic granulation is a process in which suspended biomass aggregate and form discrete well-defined granules in aerobic systems. To investigate the properties and kinetics of aerobic granular sludge, aerobic granules were cultivated with glucose synthetic wastewater in a series of sequencing batch reactors (SBR). The spherical shaped granules were observed on 8th day with the mean diameter of 0.1 mm. With the organic loading rate (OLR) being increased to 4.0 g COD L⁻¹ d⁻¹, aerobic granules grew matured with spherical shape. The size of granules ranged from 1.2 to 1.8 mm, and the corresponding settling velocity of individual granule was 24.2–36.4 m h⁻¹. The oxygen utilization rate (OUR) of mature granules was 41.90 g O₂ kg MLSS⁻¹ h⁻¹, which was two times higher than that of activated sludge (18.32 g O₂ kg MLSS⁻¹ h⁻¹). The experimental data indicated that the substrate utilization and biomass growth kinetics generally followed Monod's kinetics model. The corresponding kinetic coefficients of k (maximum specific substrate utilization rate), K_s (half velocity coefficient), Y (growth yield coefficient) and K_d (decay coefficient) were determined as follows, k_c = 23.65 d⁻¹, K_c = 3367.05 mg L⁻¹, K_N = 0.038 d⁻¹, K_N = 29.65 mg L⁻¹, Y = 0.1927–0.2022 mg MMLS (mg COD)⁻¹ and K_d = 0.00845–0.0135 d⁻¹, respectively. Those properties of aerobic granules made aerobic granules system had a short setup period, high substrate utilization rate and low sludge production.     

Discovery and characterization of a thermostable d-lactate dehydrogenase from Lactobacillus jensenii through genome mining

The demand on thermostable d-lactate dehydrogenases (d-LDH) has been increased for d-lactic acid production but thermostable d-DLHs with industrially applicable activity were not much explored. To identify a thermostable d-LDH, three d-LDHs from different Lactobacillus jensenii strains were screened by genome mining and then expressed in Escherichia coli. One of the three d-LDHs (d-LDH3) exhibited higher optimal reaction temperature (50 °C) than the others. The T₅₀¹⁰ value of this thermostable d-LDH3 was 48.3 °C, much higher than the T₅₀¹⁰ values of the others (42.7 and 42.9 °C) and that of a commercial d-lactate dehydrogenase (41.2 °C). The T_m values were 48.6, 45.7 and 55.7 °C for the three d-LDHs, respectively. In addition, kinetic parameter (k_cat/K_m) of d-LDH3 for pyruvate reduction was estimated to be almost 150 times higher than that for lactate oxidation at pH 8.0 and 25 °C, implying that d-lactate production from pyruvate is highly favored. These superior thermal and kinetic features would make the d-LDH3 characterized in this study a good candidate for the microbial production of d-lactate at high temperature from glucose if it is genetically introduced to lactate producing microbial.     

Estimation of the core temperature control during ambient temperature changes and the influence of circadian rhythm and metabolic conditions in mice

It has been speculated that the control of core temperature is modulated by physiological demands. We could not prove the modulation because we did not have a good method to evaluate the control. In the present study, the control of core temperature in mice was assessed by exposing them to various ambient temperatures (T_a), and the influence of circadian rhythm and feeding condition was evaluated. Male ICR mice (n=20) were placed in a box where T_a was increased or decreased from 27 °C to 40 °C or to −4 °C (0.15 °C/min) at 0800 and 2000 (daytime and nighttime, respectively). Intra-abdominal temperature (T_core) was monitored by telemetry. The relationship between T_core and T_a was assessed. The range of T_a where T_core was relatively stable (range of normothermia, RNT) and T_core corresponding to the RNT median (regulated T_core) were estimated by model analysis. In fed mice, the regression slope within the RNT was smaller in the nighttime than in the daytime (0.02 and 0.06, respectively), and the regulated T_core was higher in the nighttime than in the daytime (37.5 °C and 36.0 °C, respectively). In the fasted mice, the slope remained unchanged, and the regulated T_core decreased in the nighttime (0.05 and 35.9 °C, respectively), while the slopes in the daytime became greater (0.13). Without the estimating individual thermoregulatory response such as metabolic heat production and skin vasodilation, the analysis of the T_a–T_core relationship could describe the character of the core temperature control. The present results show that the character of the system changes depending on time of day and feeding conditions.     

Cellular damage during drying and storage of Trichoderma harzianum spores

Factors that cause cellular damage during the drying and storage of Trichoderma harzianum conidia were independently studied to determine their effects on spore viability. Specifically, thermal stress and dehydration levels (water activity, a_w = 0.1–0.7) were assessed for their effect on spore survival. In addition, environmental conditions, such as water activity and temperature, were evaluated during storage of the spores. T. harzianum spores produced in liquid culture are highly sensitive to thermal stress, but dehydration does not seem to be a factor that influences spore death during desiccation. An inverse correlation between spore survival and the specific concentration of malondialdehyde (MDA) was observed during storage, especially when the conidia moisture levels were lower than the monolayer moisture levels. We prepared spore suspensions without additives and spray-dried the samples. Our data showed that reduced sample viability was mainly caused by the temperature of the drying process, an effect that appears to be independent of water activity.     

Partial permeabilisation and depolarization of Salmonella enterica Typhimurium cells after treatment with pulsed electric fields and high pressure carbon dioxide

The aim of the present study is to investigate the efficiency of the combined pulsed electric fields and high pressure carbon dioxide (PEF + HPCD) treatment on the Gram-negative pathogen Salmonella Typhimurium in a liquid medium, by means of both plate count technique and flow cytometry (FCM). PEF was applied at two conditions: (1) 1 single pulse of 1 ms length at 30 kV/cm and (2) 12 pulses of 4 ms length at 30 kV/cm, while HPCD at 12 MPa, 22 °C and 35 °C for different treating times (0–45 min). At both temperatures, the application of PEF as HPCD pre-treatment was demonstrated to enhance the inactivation kinetics and to decrease the treatment time to inactivate S. Typhimurium if compared to HPCD alone. Further, the approach based on FCM permitted to investigate the functional status of bacterial cells after PEF and HPCD treatments distinguishing among viable bacteria (considered as intact cells), permeabilised cells and depolarised cells simultaneously. It has been demonstrated that the synergistic effect is due to the electroporation effect of PEF which lead to changes in the cell membrane potential but also in a partial structural damage, favoring the subsequent CO₂ penetration into the cells and increasing the inactivation kinetics, thus improving the efficiency of the entire process.     

Daily and annual patterns of thermoregulation in painted turtles (Chrysemys picta marginata) living in a thermally variable marsh in Northern Michigan

The capacity for an ectothermic reptile to thermoregulate has implications for many components of its life history. Over two years, we studied thermoregulation in a population of Midland painted turtles (Chrysemys picta marginata) in a shallow, thermally variable wetland during summer in Northern Michigan. Mean body temperature (T_b) of free-ranging turtles was greater in 2008 (25.8 °C) than in 2010 (19.7 °C). Laboratory determined thermoregulatory set point (T_set) ranged from 25 °C (T_set-min) to 31 °C (T_set-max) and was lower during the fall (17–26 °C). Deviations of T_b distributions from field measured operative temperatures (T_e) and indices of thermoregulation indicated that C. picta marginata were capable of a limited degree of thermoregulation. Operative temperatures and thermal quality (d_e=|T_set-min−T_e| and |T_e−T_set-max|) cycled daily with maximal thermal quality occurring during late morning and late afternoon. The accuracy of thermoregulation (d_b=|T_set-min−T_b| and |T_b−T_set-max|) was maximal (d_b values were minimal) as T_b declined and traversed T_set during the late afternoon–early evening hours and was higher on cloudy days than on sunny days because relatively low T_e values decreased the number of T_b values that were above T_set. Our index of thermal exploitation (E_x=frequency of T_b observations within T_set) was 36%, slightly lower than that reported for an Ontario population of C. picta marginata. Regression of d_b (thermal accuracy) on d_e (thermal quality) indicated that turtles invested more in thermoregulation when thermal quality was low and when water levels were high than when they were low. There were no intersexual differences in mean T_b throughout the year but females had relatively high laboratory determined T_b values in the fall, perhaps reflecting the importance of maintaining ovarian development prior to winter.     

Investigation on the formation and kinetics of glucose-fed aerobic granular sludge

Aerobic granular sludge was cultivated in a glass sequencing batch reactor (SBR) with glucose synthetic wastewater. The spherical shaped granules were observed on 4th day with the mean diameter of 0.1 mm. With the increase of chemical oxygen demand (COD) concentration of the influent, aerobic granules grew matured, the size of which ranged from 1.2 to 1.9 mm. The aerobic granular sludge could sustain high organic loading rate (about 4.0 g COD L⁻¹ d⁻¹), with good settling ability (settling velocity 36 m/h) and high biomass concentration (MLSS 6.7 ±0.2 g/L). Experimental data indicated that the substrate utilization and biomass growth kinetics followed Monod's kinetics model approximately. The corresponding kinetic coefficients of maximum specific substrate utilization rate (k), half velocity coefficient (K_s), growth yield coefficient (Y) and decay coefficient (K_d) were 13.2 d⁻¹, 275.8 mg/L, 0.183–0.250 mg MLSS/mg COD and 0.023–0.075 d⁻¹, respectively, which made aerobic granules have short setup period, high rate of substrate utilization and little surplus sludge.     

Inhibition kinetics of a commercial mixed culture by ammonium sulfate

The inhibitory effect of ammonium sulfate on a commercial mixed culture, used in biological waste-water treatment was studied under aerobic batch conditions. Several mathematical models of enzyme and growth kinetics including a death factor were analyzed through nonlinear regression to find the best fit to corresponding data of inhibition. The best fit model was found to be the generalized Monod type with a death factor having the biokinetic parameters; μ_max 0.681 h⁻¹, K_s 0.224 g dm⁻³, K_i 56240 g dm⁻³, K 0.055 g dm⁻³ and k_d 0.052 h⁻¹ to represent the experimental data accurately. The low saturation coefficient value along with high maximum specific growth rate and inhibition coefficient denotes the competitive characteristics of commercial mixed cultures in the biological treatment of high ammonium polluted waste waters.     

Light thresholds derived from seagrass loss in the coastal zone of the northern Great Barrier Reef,Australia

There is a world-wide trend for deteriorating water quality and light levels in the coastal zone, and this has been linked to declines in seagrass abundance. Localized management of seagrass meadow health requires that water quality guidelines for meeting seagrass growth requirements are available. Tropical seagrass meadows are diverse and can be highly dynamic and we have used this dynamism to identify light thresholds in multi-specific meadows dominated by Halodule uninervis in the northern Great Barrier Reef, Australia. Seagrass cover was measured at ∼3 month intervals from 2008 to 2011 at three sites: Magnetic Island (MI) Dunk Island (DI) and Green Island (GI). Photosynthetically active radiation was continuously measured within the seagrass canopy, and three light metrics were derived. Complete seagrass loss occurred at MI and DI and at these sites changes in seagrass cover were correlated with the three light metrics. Mean daily irradiance (I_d) above 5 and 8.4 mol m⁻² d⁻¹ was associated with gains in seagrass at MI and DI, however a significant correlation (R = 0.649, p < 0.05) only occurred at MI. The second metric, percent of days below 3 mol m⁻² d⁻¹, correlated the most strongly (MI, R = −0.714, p < 0.01 and DI, R = −0.859, p = <0.001) with change in seagrass cover with 16–18% of days below 3 mol m⁻² d⁻¹ being associated with more than 50% seagrass loss. The third metric, the number of hours of light saturated irradiance (H_sat) was calculated using literature-derived data on saturating irradiance (E_k). H_sat correlated well (R = 0.686, p < 0.01; and DI, R = 0.704, p < 0.05) with change in seagrass abundance, and was very consistent between the two sites as 4 H_sat was associated with increases in seagrass abundance at both sites, and less than 4 H_sat with more than 50% loss. At the third site (GI), small seasonal losses of seagrass quickly recovered during the growth season and the light metrics did not correlate (p > 0.05) with change in percent cover, except for I_d which was always high, but correlated with change in seagrass cover. Although distinct light thresholds were observed, the departure from threshold values was also important. For example, light levels that are well below the thresholds resulted in more severe loss of seagrass than those just below the threshold. Environmental managers aiming to achieve optimal seagrass growth conditions can use these threshold light metrics as guidelines; however, other environmental conditions, including seasonally varying temperature and nutrient availability, will influence seagrass responses above and below these thresholds.     

α-Amylase from germinating soybean (Glycine max) seeds – Purification,characterization and sequential similarity of conserved and catalytic amino acid residues

Starch hydrolyzing amylase from germinated soybeans seeds (Glycine max) has been purified 400-fold to electrophoretic homogeneity with a final specific activity of 384 units/mg. SDS–PAGE of the final preparation revealed a single protein band of 100 kDa, whereas molecular mass was determined to be 84 kDa by MALDI–TOF and gel filtration on Superdex-200 (FPLC). The enzyme exhibited maximum activity at pH 5.5 and a pI value of 4.85. The energy of activation was determined to be 6.09 kcal/mol in the temperature range 25–85 °C. Apparent Michaelis constant (K_m(app)) for starch was 0.71 mg/mL and turnover number (k_cat) was 280 s^?1 in 50 mM sodium acetate buffer, pH 5.5. Thermal inactivation studies at 85 °C showed first-order kinetics with rate constant (k) equal to 0.0063 min^?1. Soybean α-amylase showed high specificity for its primary substrate starch. High similarity of soybean α-amylase with known amylases suggests that this α-amylase belongs to glycosyl hydrolase family 13. Cereal α-amylases have gained importance due to their compatibility for biotechnological applications. Wide availability and easy purification protocol make soybean as an attractive alternative for plant α-amylase. Soybean can be used as commercially viable source of α-amylase for various industrial applications.