Using hydrothermal time concept to describe sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) seed germination response to temperature and water potential

To quantify both temperature (T) and water potential (ψ) effects on sesame (Sesamum indicum L.) seed germination (SG) and also to determine the cardinal T _s for this plant, a laboratory experiment was carried out using hydrothermal time model (HTT). For this purpose, four sesame cultivars (‘Asbomahalleh’, ‘Darab’, ‘Dashtestan’ and ‘Yellowhite’) were germinated at seven constant T _s (20, 25, 30, 35, 37, 39 and 43 °C) at each of the following ψ _s (0, ? 0.12, ? 0.24 and ? 0.36 MPa; provided by PEG 8000). Germination rate (GR) and germination percentage (GP) significantly influenced by ψ, T and their interactions in all cultivars (P ≤ 0.01). There was no significant difference, based on the confidence intervals of the model coefficients, between cultivars, so an average of cardinal T _s was 14.7, 35.4 and 47.2 °C for the minimum (T _b), optimum (T _o) and maximum (T _c) T _s, respectively, in the control condition (0 MPa). Hydrotime values in all cultivars decreased when T was increased to T _o and then remained constant at T _s > T _o (15 MPa h^?1). An average value of ψ _b(50) was estimated to be ? 1.23 MPa at T _s ≤ T _o and then increased linearly (0.1041 MPa°Ch^?1, the slope of the relationship between ψ _b(50) and supra-optimal T _s) with T when T _s increased above T _o and finally reached to zero at T _c. The T _b and T _o values were not influenced by ψ, but T _c value decreased (from 47.2 for zero to 43.5 °C for ? 0.36 MPa) at supra-optimal T _s as a result of the effect of ψ on GR. Based on our findings, this model (as a predictive tool) and or the estimated parameter values in this study can easily be used in sesame SG simulation models to quantitatively characterize the physiological status of sesame seed populations at different T _s and ψ _s.     

A chimeric α-amylase engineered from <Emphasis Type="Italic">Bacillus acidicola</Emphasis> and G<Emphasis Type="Italic">eobacillus thermoleovorans</Emphasis> with improved thermostability and catalytic efficiency

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K _cat: 4 × 10⁴ s^?1 and K _cat/K _m: 5 × 10⁴ mL^?1 mg^?1 s^?1] and higher thermostability than Ba-amy. The melting temperature (T _m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir–Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca²⁺ independence, and better than the other known bacterial acidic α-amylases.     

Gas exchange,carbon balance and stomatal traits in wild and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Carbon balancing within the plant species is an important feature for climatic adaptability. Photosynthesis and respiration traits are directly linked with carbon balance. These features were studied in 20 wild rice accessions Oryza spp., and cultivars. Wide variation was observed within the wild rice accessions for photosynthetic oxygen evolution or photosynthetic rate (A), dark (R _d), and light induced respiration (LIR) rates, as well as stomatal density and number. The mean rate of A varied from 10.49 μmol O₂ m^?2 s^?1 in cultivated species and 13.09 μmol O₂ m^?2 s^?1 in wild spp., The mean R _d is 2.09 μmol O₂ m^?2 s^?1 and 2.31 μmol O₂ m^?2 s^?1 in cultivated and wild spp., respectively. Light induced Respiration (LIR) was found to be almost twice in wild rice spp., (16.75 μmol O₂ m^?2 s^?1) compared to cultivated Oryza spp., Among the various parameters, this study reveals LIR and A as the key factors for positive carbon balance. Stomatal contribution towards carbon balance appears to be more dependent on abaxial surface where several number of stomata are situated. Correlation analysis indicates that R _d and LIR increase with the increase in A. In this study, O. nivara (CR 100100, CR 100097), O. rufipogon (IR 103404) and O. glumaepatula (IR104387) were identified as potential donors which could be used in rice breeding program. Co-ordination between gas exchange and patchiness in stomatal behaviour appears to be important for carbon balance and environmental adaptation of wild rice accessions, therefore, survival under harsh environment.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

Synthesis of disaccharides using β-glucosidases from <Emphasis Type="Italic">Aspergillus niger</Emphasis>, <Emphasis Type="Italic">A. awamori</Emphasis> and <Emphasis Type="Italic">Prunus dulcis</Emphasis>

Objective

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l^?1.

Results

The reactions’ time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l^?1, the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l^?1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l^?1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l^?1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides.

Conclusion

β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

Production,characteristics and applications of phytase from a rhizosphere isolated <Emphasis Type="Italic">Enterobacter</Emphasis> sp. ACSS

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40–80 °C) and pH (2.0–6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m of the pure phytase were 0.21 mM, 131.58 nmol mg^?1 s^?1, 1.64 × 10³ s^?1, and 7.81 × 10⁶ M^?1 s^?1, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca⁺², Mg⁺² and Mn⁺², but inhibited by Zn⁺², Cu⁺², Fe⁺², Pb⁺², Ba⁺² and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.     

Genetic engineering of polyamine metabolism changes <Emphasis Type="Italic">Medicago truncatula</Emphasis> responses to water deficit

In the current scenario of climate change and increasing water scarcity there is an increased need to combine research efforts for the development of abiotic stress resistant crops, specifically plants able to support water deficit (WD). Polyamines (PAs) have been described as being involved in the regulation of many physiological processes and a variety of stress responses in plants. Arginine decarboxylase (ADC) is considered a key enzyme of the polyamine (PA) biosynthetic pathway. In this study, a T₂ transgenic homozygous line of Medicago truncatula expressing the oat Adc under the control of CaMV 35S was obtained and was shown to have higher leaf accumulation of putrescine, spermidine and norspermidine compared to wild type plants. The photosynthetic parameters, leaf internal CO₂ concentration (Ci), net CO₂ assimilation rate (A), transpiration (E) and stomatal conductance (gs) of transformed and untransformed lines during WD and water deficit recovery experiments were measured by IRGA (infrared gas analyzer) and compared over time. Two light intensities were used, growth light intensity (391 μmol m^?2 s^?1) and saturating light intensity (1044 μmol m^?2 s^?1). Independently of the light intensity, and under WD, the transgenic line stood out with increased Ci, A, E and gs; suggesting a possible benefit of the augmented PAs under such disturbing environmental conditions. We showed that the constitutive expression of the oat Adc gene improve the physiological responses to WD and that WD recovered transgenic plants had higher seed yield, suggesting a possible benefit of PA metabolism manipulation in legumes.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

Impact of elevated CO<Subscript>2</Subscript> in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> rooted stem cuttings inoculated with <Emphasis Type="Italic">Frankia</Emphasis>

Impact of different levels of elevated CO ₂ on the activity of Frankia (Nitrogen-fixing actinomycete) in Casuarina equisetifolia rooted stem cuttings has been studied to understand the relationship between C. equisetifolia, Frankia and CO₂. The stem cuttings of C. equietifolia were collected and treated with 2000 ppm of Indole Butyric Acid (IBA) for rooting. Thus vegetative propagated rooted stem cuttings of C. equisetifolia were inoculated with Frankia and placed in the Open top chambers (OTC) with elevated CO₂ facilities. These planting stocks were maintained in the OTC for 12 months under different levels of elevated CO₂ (ambient control, 600 ppm, 900 ppm). After 12 months, the nodule numbers, bio mass, growth, and photosynthesis of C. equisetifolia rooted stem cuttings inoculated with Frankia were improved under 600 ppm of CO₂. The rooted stem cuttings of C. equisetifolia inoculated with Frankia showed a higher number of nodules under 900 ppm of CO₂ and cuttings without Frankia inoculation exhibited poor growth. Tissue Nitrogen (N) content was also higher under 900 ppm of CO₂ than ambient control and 600 ppm levels. The photosynthetic rate was higher (17.8 μ mol CO₂ m^?2 s^?1) in 900 ppm of CO₂ than in 600 ppm (13.2 μ mol CO₂ m^?2 s^?1) and ambient control (8.3 μ mol CO₂ m^?2 s^?1). This study showed that Frankia can improve growth, N fixation and photosynthesis of C. equietifolia rooted stem cuttings under extreme elevated CO₂ level conditions (900 ppm).     

Physiological and Biochemical Mechanisms Preventing Cd Toxicity in the New Hyperaccumulator <Emphasis Type="Italic">Abelmoschus manihot</Emphasis>

Abelmoschus manihot, an ornamental plant, was examined for phytoremediation purposes in accordance with the ability to accumulate cadmium and physiological mechanisms of cadmium tolerance. A net photosynthetic rate (A _N) glasshouse experiment for 60 days was conducted to investigate the influence of different cadmium amounts (0–100 mg kg^?1) on the growth, biomass, photosynthetic performance, reactive oxygen species (ROS) production, antioxidative enzyme activities, Cd uptake and accumulation of A. manihot. Exposure to cadmium enhanced plant growth even at 100 mg kg^?1, without showing symptoms of visible damage. The cadmium concentration of shoots (stems or leaves) and roots was more than the critical value of 100 mg kg^?1 and reached 126.17, 185.26 and 210.24 mg kg^?1, respectively. BCF values of A. manihot plants exceeded the reference value 1.0 for all the Cd treatments, and TF values were greater than 1 at 15–60 mg kg^?1 Cd treatment. The results also showed that cadmium concentrations of 60 mg kg^?1 or less induced a significant enhancement in plant net photosynthetic rate (A _N), stomatal conductance (G _s), transpiration rate (T _r), photosynthetic pigments and F _v/F _m. These parameters were slightly decreased at the higher concentration (100 mg kg^?1). The ROS production (O₂ ^?, H₂O₂) and antioxidative response including SOD, CAT and POD were significantly enhanced by increasing cadmium. These results suggest that A. manihot can be considered as a Cd-hyperaccumulator and the hormetic effects may be taken into consideration in remediation of Cd contamination soil.     

Effects of low nocturnal temperature on photosynthetic characteristics and chloroplast ultrastructure of winter rapeseed

We investigated the effects of low nocturnal temperature on photosynthetic apparatus of winter rapeseed (Brassica campestris L.). An artificial climate chamber was used to simulate the effects of low nocturnal temperature on seedling and stomatal morphology, chloroplast ultrastructure, photosynthetic parameters, and dry matter distribution and accumulation in two winter rapeseed cultivars, Longyou-7 (ultra coldresistant) and Tianyou-2 (weak cold resistance). Compared with those at diurnal/nocturnal temperatures of 20°/10°C (control), rapeseed seedlings at 20°/5°C had increased leaf chlorophyll content, deepened green leaf color, decreased stomatal conductance (G_s), intercellular CO₂ concentration (C_i), and photosynthetic rate (P_n), and improved root/shoot ratio; the majority of stomata remained open in Longyou-7 while those in Tianyou-2 were mostly closed or semi-closed. At diurnal/nocturnal temperatures of 20°/–5°C, rapeseed seedlings had decreased leaf chlorophyll content with increased C_i but decreased G_s and P_n; Tianyou-2 exhibited ruptured chloroplast membrane, dissolved grana, broken stroma lamella, and decreased root/shoot ratio, whereas Longyou-7 had chloroplasts retaining partial structure of grana with a small amount of starch granules in guard cells. Low nocturnal temperature damaged the photosynthetic membrane of chloroplasts and reduced P_n in the leaves of winter rapeseed influencing photosynthetic processes in this crop. The reduction of P_n was mainly related to stomatal limitation at diurnal/nocturnal temperatures of 20°/5°C and non-stomatal limitation at diurnal/nocturnal temperatures of 20°/–5°C.     

Response of effective quantum yield of photosystem 2 to <Emphasis Type="Italic">in situ</Emphasis> temperature in three alpine plants

The response of effective quantum yield of photosystem 2 (ΔF/F_m’) to temperature was investigated under field conditions (1 950 m a.s.l.) in three alpine plant species with contrasting leaf temperature climates. The in situ temperature response did not follow an optimum curve but under saturating irradiances [PPFD >800 µìmol(photon) m^?2s^?1] highest ΔF/F_m’ occurred at leaf temperatures below 10°C. This was comparable to the temperature response of antarctic vascular plants. Leaf temperatures between 0 and 15°C were the most frequently (41 to 56%) experienced by the investigated species. At these temperatures, ΔF/F_m’ was highest in all species (data from all irradiation classes included) but the species differed in the temperature at which ΔF/F_m’ dropped below 50% (Soldanella pusilla >20°C, Loiseleuria procumbens >25°C, and Saxifraga paniculata >40°C). The in situ response of ΔF/F_m’ showed significantly higher ΔF/F_m’ values at saturating PPFD for the species growing in full sunlight (S. paniculata and L. procumbens) than for S. pusilla growing under more moderate PPFD. The effect of increasing PPFD on ΔF/F_m’, for a given leaf temperature, was most pronounced in S. pusilla. Despite the broad diurnal leaf temperature amplitude of alpine environments, only in S. paniculata did saturating PPFD occur over a broad range of leaf temperatures (43 K). In the other two species it was half of that (around 20 K). This indicates that the setting of environmental scenarios (leaf temperature×PPFD) in laboratory experiments often likely exceeds the actual environmental demand in the field.     

Carbon Dioxide and Methane Fluxes From Tree Stems,Coarse Woody Debris,and Soils in an Upland Temperate Forest

Forest soils and canopies are major components of ecosystem CO₂ and CH₄ fluxes. In contrast, less is known about coarse woody debris and living tree stems, both of which function as active surfaces for CO₂ and CH₄ fluxes. We measured CO₂ and CH₄ fluxes from soils, coarse woody debris, and tree stems over the growing season in an upland temperate forest. Soils were CO₂ sources (4.58 ± 2.46 µmol m^?2 s^?1, mean ± 1 SD) and net sinks of CH₄ (?2.17 ± 1.60 nmol m^?2 s^?1). Coarse woody debris was a CO₂ source (4.23 ± 3.42 µmol m^?2 s^?1) and net CH₄ sink, but with large uncertainty (?0.27 ± 1.04 nmol m^?2 s^?1) and with substantial differences depending on wood decay status. Stems were CO₂ sources (1.93 ± 1.63 µmol m^?2 s^?1), but also net CH₄ sources (up to 0.98 nmol m^?2 s^?1), with a mean of 0.11 ± 0.21 nmol m^?2 s^?1 and significant differences depending on tree species. Stems of N. sylvatica, F. grandifolia, and L. tulipifera consistently emitted CH₄, whereas stems of A. rubrum, B. lenta, and Q. spp. were intermittent sources. Coarse woody debris and stems accounted for 35% of total measured CO₂ fluxes, whereas CH₄ emissions from living stems offset net soil and CWD CH₄ uptake by 3.5%. Our results demonstrate the importance of CH₄ emissions from living stems in upland forests and the need to consider multiple forest components to understand and interpret ecosystem CO₂ and CH₄ dynamics.     

Screening and identification of an <Emphasis Type="Italic">Enterobacter ludwigii</Emphasis> strain expressing an active <Emphasis Type="Italic">β</Emphasis>-xylosidase

Researchers have expressed increasing interest in the xylanolytic enzymes used in hemicellulose hydrolysis that convert wood and agricultural residues to second-generation biofuels. In our study, 32 isolates showed clear hydrolysis zones on agar plates containing xylan after Congo red staining. Among these isolates, strain LY-62 exhibited the highest β-xylosidase activity (1.29?±?0.05 U/mL). According to the phylogenetic analysis of the 16S rDNA, strain LY-62 belongs to the Enterobacter genus. Using a combination of electron microscopy, Gram-staining, and conventional physiological and biochemical examinations, the strain LY-62 was identified as Enterobacter ludwigii. The β-xylosidase gene from Enterobacter ludwigii LY-62 was cloned, and the full-length protein was expressed in Escherichia coli as an N-terminal or C-terminal His-tagged fusions protein. Optimal β-xylosidase activity was achieved at pH 7.0 and 40 °C. The Michaelis constant K_M values for His-Xyl62 and Xyl62-His were 1.55 and 2.8 mmol/L, respectively. The k_cat values for His-Xyl62 and Xyl62-His were 8.51 and 6.94 s^?1, respectively. The catalytic efficiencies of His-Xyl62 and Xyl62-His were 5.49 and 2.48 s^?1?×?mM^?1, respectively. Thus, Xyl62 is a functional β-xylosidase, and our study represents the first report of a β-xylosidase from Enterobacter ludwigii.     

Gas exchanges of an endangered species <Emphasis Type="Italic">Syringa pinnatifolia</Emphasis> and a widespread congener <Emphasis Type="Italic">S. oblata</Emphasis>

Net photosynthetic rate (P_N), transpiration rate (E), water use efficiency (WUE), stomatal conductance (g_s), and stomatal limitation (L_s) were investigated in two Syringa species. The saturation irradiance (SI) was 400 µmol m^-2s^-1 for S. pinnatifolia and 1 700 µmol m^-2s^-1 for S. oblata. Compared with S. oblata, S. pinnatifolia had extremely low g _s . Unlike S. oblata, the maximal photosynthetic rate (P_max) in S. pinnatifoliaoccurred around 08:00 and then fell down, indicating this species was sensitive to higher temperature and high photosynthetic photon flux density. However, such phenomenon was interrupted by the leaf development rhythms before summer. A relatively lower P_N together with a lower leaf area and shoot growth showed the capacity for carbon assimilation was poorer in S. pinnatifolia.     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Purification and characterization of a β-1,4-glucosidase from a newly isolated strain of <Emphasis Type="Italic">Fomitopsis pinicola</Emphasis>

An efficient ß-1,4-glucosidase (BGL) producing strain, Fomitopsis pinicola KMJ812, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular BGL was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The relative molecular weight of F. pinicola BGL was determined to be 105 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, or 110 kDa by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 4.5 and a temperature optimum of 50°C. The enzyme showed high substrate specificity and high catalytic efficiency (k _cat?=?2,990 s^?1, K _m?=?1.76 mM, k _cat/K _m?=?1,700 mM^?1 s^?1) for p-nitrophenyl-β-d-glucopyranoside. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 3, indicating that the F. pinicola BGL is a member of glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, F. pinicola BGL is distinguished from other BGLs by its high catalytic efficiency and strict substrate specificity.     

Potential Outdoor Cultivation of Green Microalgae Based on Response to Changing Temperatures and by Combining with Air Temperature Occurrence

In this study, our working hypothesis was to examine whether temperature alters biomass and metabolite production by microalgae according to strain. We also addressed whether it is possible to choose a strain suitable for growing in each season of a given region. A factorial experiment revealed a significant interaction between chlorophylls a and b (Chl a and Chl b), carotenoid/Chl (a?+?b) ratio, biomass and total lipid productivity of six green microalgae (four Chlorella spp., Chlorella sorokiniana and Neochloris oleoabundans) after 15 days at four temperatures. At 39/35 °C, two Chlorella sp. strains (IPR7115 and IPR7117) showed higher total carotenoids/Chl (a?+?b) (0.578 and 0.830), respectively. N. oleoabundans had the highest Chl a (8210 μg L^?1) and Chl b (1909 μg L^?1) at 19/15 °C and highest maximum dry biomass (2900 mg L^?1), specific growth rate (0.538 day^?1) and total lipids (1003 mg L^?1) at 15/8 °C. We applied a method to infer the growth of these six green microalgae in outdoor ponds, as based on their response to changing temperatures and by combining with historical data on day/night air temperature occurrence for a given region. We conclude that the use of regionalized maps based on air temperature is a good strategy for predicting microalgal cultivation in outdoor ponds based on their features and tolerance to changing temperature.