Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

Effects of biomass weight and light intensity on the performance of photosynthetic microbial fuel cells with Spirulina platensis

Microalgae Spirulina platensis were attached to the anode of a membrane-free and mediator-free microbial fuel cell (MFC) to produce electricity through the consumption of biochemical compounds inside the microalgae. An increase in open circuit voltage (OCV) was observed with decreasing light intensity and optimal biomass area density. The highest OCV observation for the MFC was 0.39 V in the dark with a biomass area density on the anode surface of 1.2 g cm⁻². Additionally, it was observed that the MFC with 0.75 g cm⁻² of biomass area density produced 1.64 mW m⁻² of electrical power in the dark, which is superior to the 0.132 mW m⁻² produced in the light. Which also means the MFC can be applied to generate electrical power under both day and night conditions.     

Senescence progression in a single darkened cotyledon depends on the light status of the other cotyledon in Cucurbita pepo (zucchini) seedlings: potential involvement of cytokinins and cytokinin oxidase/dehydrogenase activity

Darkness mediates different senescence-related responses depending on the targeting of dark treatment (whole plants or individual leaves) and on the organs that perceive the signal (leaves or cotyledons). As no data are available on the potential role of darkness to promote senescence when applied to individual cotyledons, we have investigated how darkness affects the progression of senescence in either a single or both individually darkened cotyledons of young 10-day-old Cucurbita pepo (zucchini) seedlings. Strong acceleration of senescence was observed when both cotyledons were darkened as judged by the damage in their anatomical structure, deterioration of chloroplast ultrastructure in parallel with decreased photosynthetic rate and photochemical quantum efficiency of PSII. In addition, the endogenous levels of cytokinins (CKs) and IAA were strongly reduced. In a single individually darkened cotyledon, the structure and function of the photosynthetic apparatus as well as the contents of endogenous CKs and IAA were much less affected by darkness, thus suggesting inhibitory effect of the illuminated cotyledon on the senescence of the darkened one. Apparently, the effect of darkness to accelerate/delay senescence in a single darkened cotyledon depends on the light status of the other cotyledon from the pair. The close positive correlation between CK content and the activity of CK oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) suggested that CKX was essentially involved in the mechanisms of downregulation of endogenous CK levels. Our results indicated that CKX-regulated CK signaling could be a possible regulatory mechanism controlling senescence in individually darkened cotyledons.     

Exposure times in rapid light curves affect photosynthetic parameters in algae

Short-and long-duration light curves were applied to four macroalgae (Ulva sp., Codium fragile, Ecklonia radiata and Lessonia variegata), and two microalgal species (Chlorella emersonii and Chaetoceros muellerii). With increasing light curve duration, the maximal relative electron transport rate increased by a factor of three in E. radiata, and by factors of 1.25 and 1.23 in C. emersonii and L. variegata, respectively, but did not change in C. fragile and Ch. muellerii. The light saturation point E_k increased by 26 μmol photons m⁻² s⁻¹ in C. emersonii and 20 μmol photons m⁻² s⁻¹ in Ch. muellerii and E. radiata with elevated light curve exposure times. Oscillatory patterns of the continuous fluorescence readings reflect accumulation of Q_A. Continuous fluorescence values increased, or decreased, by approximately 10% within light curve increments. However, oscillations of 25% were not uncommon, which shows that cells are changing their photo-physiological response state during steady light conditions. Increasing dark acclimation times prior to light curve application lowered maximal relative electron transport rates in the C. emersonii (from 28 ± 1.7 to 25 ± 1.2 for 15 and 95 min dark acclimation in short-duration light curves respectively). This effect was counterbalanced by longer light curve application. It can therefore be concluded that manipulation of light exposure and dark incubation prior to the experiment affects the photosynthetic response, presumably due to different activation states of photosynthetic and photoprotective mechanisms. The highly species-specific photo-response patterns imply that a common rapid light curve protocol will generate artefacts in some species.     

Chlorophyll fluorescence in the leaves of Tradescantia species of different ecological groups: Induction events at different intensities of actinic light

Chlorophyll fluorescence analysis is one of the most convenient and widespread techniques used to monitor photosynthesis performance in plants. In this work, after a brief overview of the mechanisms of regulation of photosynthetic electron transport and protection of photosynthetic apparatus against photodamage, we describe results of our study of the effects of actinic light intensity on photosynthetic performance in Tradescantia species of different ecological groups. Using the chlorophyll fluorescence as a probe of photosynthetic activity, we have found that the shade-tolerant species Tradescantia fluminensis shows a higher sensitivity to short-term illumination (≤20 min) with low and moderate light (≤200 μE m⁻² s⁻¹) as compared with the light-resistant species Tradescantia sillamontana. In T. fluminensis, non-photochemical quenching of chlorophyll fluorescence (NPQ) and photosystem II operational efficiency (parameter Φ_PSII) saturate as soon as actinic light reaches ≈200 μE m⁻² s⁻¹. Otherwise, T. sillamontana revealed a higher capacity for NPQ at strong light (≥800 μE m⁻² s⁻¹). The post-illumination adaptation of shade-tolerant plants occurs slower than in the light-resistant species. The data obtained are discussed in terms of reactivity of photosynthetic apparatus to short-term variations of the environment light.     

Density dependent effects of an infaunal suspension-feeding bivalve (Austrovenus stutchburyi) on sandflat nutrient fluxes and microphytobenthic productivity

We examined in situ the density dependent effects of an infaunal suspension-feeding bivalve, Austrovenus stutchburyi (hereafter Austrovenus) on sandflat nutrient fluxes and microphytobenthic (MPB) production. Nine experimental plots (0.64 m^− 2) were established at two locations separated by 300 m. Ambient fauna was left intact and Austrovenus added to plots creating a density range from 20 to 2000 ind. m^− 2. Three weeks later, light and dark benthic chambers (area = 0.114 m^− 2) were deployed to measure MPB production and nutrient fluxes. Austrovenus density was positively correlated with organic content and porosity but did not affect other sediment properties (grain size, pigment content) or resident macrofauna. In dark chambers there was a net influx of oxygen (O₂) into the sediments which increased with Austrovenus density (from − 0.45 to − 1.21 mmol m^− 2 h^− 1) whereas in light chambers there was a net efflux from the sediments which decreased with density (from 0.90 to 0.31 mmol m^− 2 h^− 1). Significant (p < 0.01) multiple linear regression models explained respectively 42% and 72% of the variability in the dark and light chamber O₂ fluxes with Austrovenus density as the most important predictor variable. When the effects of significant co-variables (light intensity, grain size) were accounted for, the negative relationship between O₂ flux and Austrovenus density was less steep in light chambers (ANCOVA p < 0.001) suggesting a stimulation of MPB production at higher densities. Estimates of gross MPB primary production indicated a 30% increase in rates of carbon fixation with Austrovenus density (from 36 to 48 mg C m^− 2 h^− 1). Ammonium (NH₄⁺) was released from the sediments in both light and dark chambers and increased with Austrovenus density by a factor of 5.9-6.9×. Multiple linear regression models were significant for light and dark chambers (p < 0.001; r² 86-87%) with Austrovenus again as the most important variable influencing fluxes. ANCOVA results (p < 0.001) indicated that in dark chambers NH₄⁺ efflux increased with Austrovenus density at a rate 1.76× greater than in light chambers. These results indicate that the greater efflux of NH₄⁺ at high densities was being trapped by photosynthesising MPB at the sediment-water interface supporting higher rates of primary production. Our results suggest that a reduction in Austrovenus density will lower nutrient fluxes potentially influencing system productivity by reducing MPB production.     

Light and nitrogen nutrition regulate apical control in Rosa hybrida L.

Apical control is defined as the inhibition of basal axillary bud outgrowth by an upper actively growing axillary axis, whose regulation is poorly understood yet differs markedly from the better-known apical dominance. We studied the regulation of apical control by environmental factors in decapitated Rosa hybrida in order to remove the apical hormonal influence and nutrient sink. In this plant model, all the buds along the main axis have a similar morphology and are able to burst in vitro. We concentrated on the involvement of light intensity and nitrate nutrition on bud break and axillary bud elongation in the primary axis pruned above the fifth leaf of each rose bush. We observed that apical control took place in low light (92 μmol m⁻² s⁻¹), where only the 2-apical buds grew out, both in low (0.25 mM) and high (12.25 mM) nitrate. In contrast, in high light (453 μmol m⁻² s⁻¹), the apical control only operates in low nitrate while all the buds along the stem grew out when the plant was supplied with a high level of nitrate. We found a decreasing photosynthetic activity from the top to the base of the plant concomitant with a light gradient along the stem. The quantity of sucrose, fructose, glucose and starch are higher in high light conditions in leaves and stem. The expression of the sucrose transporter RhSUC2 was higher in internodes and buds in this lighting condition, suggesting an increased capacity for sucrose transport. We propose that light intensity and nitrogen availability both contribute to the establishment of apical control.     

Leaf growth,senescence and decomposition of Juncus maritimus Lam. in a coastal Mediterranean marsh

In this study, the growth, senescence, leaf loss and nutrient dynamics of Juncus maritimus were followed to examine litter decay in a Mediterranean coastal marsh. Decomposition was studied in dead leaves still attached to the plant and in leaves placed in litterbags (detached leaves/litter) on the sediment surface. The dynamics of fungi, meiofauna and epiphytes associated with detached litter were also followed. No significant differences were observed between decay rates in dead leaves attached to plants (0.0017 day⁻¹) and detached leaves (0.0015 day⁻¹) in litter bags. The percentage of ash-free dry weight lost was inversely proportional to the C:N and C:P ratios in plant detritus during decay, indicating N and P limitation for the decomposer community inhabiting decaying J. maritimus litter and uptake of these nutrients from the environment. Water availability and high temperatures on the sediment surface increased the density of meiofauna and epiphyton and decreased fungal biomass during the first 20 days of the experiment. The density of ciliates and nematodes in decomposing litter was inversely related to the C:N ratio and directly related to the percentage of AFDW lost. On the basis of these observations, it was concluded that meiofauna are primary colonizers of J. maritimus leaf litter.     

Functions of the water soluble chlorophyll-binding protein in plants

Functional aspects of water soluble chlorophyll-binding protein (WSCP) in plants were investigated during the courses of leaf senescence, chlorophyll biogenesis, stress response and photoprotection. The cDNA sequence encoding WSCP from cauliflower was cloned into a binary vector to facilitate Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. The resultant transgenic tobacco plants overexpressed the CauWSCP gene under the control of a 35S-promoter. Analyses of protein and pigment contents indicate that WSCP overexpression does not enhance chlorophyll catabolism in vivo, thus rendering a role of WSCP in Chl degradation unlikely. Accumulation of higher levels of protochlorophyllide in WSCP overexpressor plants corroborates a proposed temporary storage and carrier function of WSCP for chlorophyll and late precursors. Although WSCP overexpressor plants did not show significant differences in non-photochemical quenching of chlorophyll fluorescence, they are characterized by significantly lower zeaxanthin accumulation and peroxidase activity at different light intensities, even at high light intensities of 700-900 μmol photons m⁻² s⁻¹. These results suggest a photoprotective function of the functional chlorophyll binding-WSCP tetramer by shielding of chlorophylls from molecular oxygen.     

Effects of light regime and oxygen profile on transformation of oxolinic acid in pond sediment

This study investigated the effects of light (visible light - 5800 lux, 24 h) or dark regime and aerobic or anaerobic condition on the decay of added oxolinic acid (OA) at 5, 10 and 20 mg L⁻¹ in eel pond sediment. An asymptotic decaying exponential model C_t = C_min + C_o × exp (−k × t) was used to facilitate quantitative approach to OA transformation, where C_t is the concentration of OA after t days, C_min the estimated level-off concentration of OA residue, C_o the concentration of added OA and k the decaying coefficient. OA decayed faster under light (C_min = 4.6 mg L⁻¹) than under dark (C_min = 7.8 mg L⁻¹) and also decayed faster under aerobic (C_min = 4.0 mg L⁻¹) than under anaerobic condition (C_min = 8.5 mg L⁻¹). C_min increased with C_o. Sundrying and tilling eel pond bottom should be able to reduce OA residue significantly.     

Night-interrupting light inhibits diapause induction in the Kanzawa spider mite, Tetranychus kanzawai Kishida (Acari: Tetranychidae)

The inhibitory effects of the timing, intensity (I_I) and period (I_T) of night-interrupting light on diapause induction of the Kanzawa spider mite (Tetranychus kanzawai) were investigated in a series of laboratory experiments. During a light and dark period of 8 and 16 h d⁻¹, respectively, a single 1-h night-interrupting light was applied at early (E), middle (M), and late (L) parts of the dark period: i.e., at 3, 7.5, and 12 h after the start of the dark period, respectively. No interrupting light was applied in the control treatment. The incidence of diapause was significantly lower in the M treatment (63%) compared to the control treatment (100%). In the E and L treatments, more than 90% of females entered diapause, which was comparable to the control treatment. Since the longest consecutive dark period during the E and L treatments was longer than the critical dark period (CDP) of 10.5-11 h d⁻¹, during which 50% of females entered diapause, the night-interrupting light probably failed to prevent diapause induction. However, in the M treatment, the longest consecutive dark period was shorter than the CDP; therefore, the night-interrupting light inhibited diapause induction. Moreover, the inhibitory effects of night-interrupting light in the M treatment increased as I_I and I_T increased. The dose of night-interrupting light (I_I × I_T) was significantly negatively related to the incidence of diapause. The median effective dose for 50% disturbance of diapause induction was 2.5 kJ m⁻² at wavelengths between 350 and 1050 nm. Our results suggest that the longest consecutive dark period and the dose of night-interrupting light should both be considered when a lighting-based physical control is applied to inhibit diapause induction and consequent overwintering of T. kanzawai in commercial agricultural fields.     

Lead stress in seedlings of Avicennia marina, a common mangrove species in South China, with and without cotyledons

The effects of lead (Pb; 0-1000 mg L⁻¹) stress on the growth and biochemical responses of seedlings of Avicennia marina were examined, with and without cotyledons. After 50 days exposure to Pb, the growth of A. marina was not affected at low concentrations (0-50 mg L⁻¹ Pb). Roots tolerated to high Pb concentrations, with a significant reduction in biomass only at 1000 mg L⁻¹ Pb. In leaves and stems, 500 mg L⁻¹ Pb already caused a significant decline in biomass (0.6-fold). Accumulation of Pb occurred mainly in roots, with some accumulation in cotyledons but very little in leaves. Pb concentrations in both roots and cotyledons were proportional to the Pb levels in the substrate (y = 25.945x − 4281, r² = 0.67, P = 0.001 for roots, and y = 0.249x + 45.636, r² = 0.879, P < 0.001 for leaves). In treatments with 500 and 1000 mg L⁻¹ Pb, nitrogen concentrations in cotyledons were higher, while the carbon to nitrogen ratios were significantly lower than in the control without Pb. The Pb levels had significant positive effects on sugar content, MDA concentration and POD activity in both roots and leaves, while the removal of cotyledons significantly decreased the POD activity and MDA content in roots A. marina seedlings according to the two-way multivariate analysis of variance test. The sugar content in the cotyledon of Pb-treated seedlings was significantly lower than that in the control (without Pb), suggesting that more carbohydrate reserves (e.g., sugar) stored in cotyledons had been mobilized to leaves and even roots under Pb treatment.     

Photoelectrochemical control of the balance between cyclic- and linear electron transport in photosystem I. Algorithm for P700 induction kinetics

Redox transients of chlorophyll P700, monitored as absorbance changes ΔA₈₁₀, were measured during and after exclusive PSI excitation with far-red (FR) light in pea (Pisum sativum, cv. Premium) leaves under various pre-excitation conditions. Prolonged adaptation in the dark terminated by a short PSII + PSI− exciting light pulse guarantees pre-conditions in which the initial photochemical events in PSI RCs are carried out by cyclic electron transfer (CET). Pre-excitation with one or more 10 s FR pulses creates conditions for induction of linear electron transport (LET). These converse conditions give rise to totally different, but reproducible responses of P700⁻ oxidation. System analyses of these responses were made based on quantitative solutions of the rate equations dictated by the associated reaction scheme for each of the relevant conditions. These provide the mathematical elements of the P700 induction algorithm (PIA) with which the distinguishable components of the P700⁺ response can be resolved and interpreted. It enables amongst others the interpretation and understanding of the characteristic kinetic profile of the P700⁺ response in intact leaves upon 10 s illumination with far-red light under the promotive condition for CET. The system analysis provides evidence that this unique kinetic pattern with a non-responsive delay followed by a steep S-shaped signal increase is caused by a photoelectrochemically controlled suppression of the electron transport from Fd to the PQ-reducing Q_r site of the cytb₆f complex in the cyclic pathway. The photoelectrochemical control is exerted by the PSI-powered proton pump associated with CET. It shows strong similarities with the photoelectrochemical control of LET at the acceptor side of PSII which is reflected by release of photoelectrochemical quenching of chlorophyll a fluorescence.     

Light-induced h secretion and the relation to senescence of oat leaves

When abraded oat (Avena sativa L. cv Victory) leaf segments are floated on KCl solution, white light causes acidification of the solution external to leaf tissue. The presence of mannitol amplifies the light-induced proton secretion. Mature leaves as well as young ones acidify the medium in light, while senescing leaves (after 3 to 4 days incubated in water in the dark) lose the ability to produce this response to light. The decrease in H⁺ secretion is already measureable after as little as 30 minutes in darkness, while the increase in proteolysis rate was detected only after 6 hours in dark. The decrease in capacity to secrete protons is one of the symptoms of leaf senescence. Moreover, fusicoccin mimics light in stimulating H⁺ pumping and delaying the senescence in the dark. On the other hand vanadate, an apparent inhibitor of plasma membrane H⁺ ATPase, blocks the acidification and promotes the chlorophyll and protein degradation in leaf segments during the 2-day period of incubation. These results, which show a parallel between cessation of H⁺ secretion and acceleration of senescence, may suggest a regulatory role for H⁺ secretion in leaf senescence.     

Different effects of dark treatment on pigment composition and photosystem I and II activities in intact cotyledons and primary leaves of Cucurbita pepo (zucchini)

Darkness mediates different senescence-related responses depending on the targeting of dark treatment (whole plants or individual leaves) and the organs that perceive the signal (leaves or cotyledons). In this study, we presented data on the differential effects of 2-day dark treatment on progression of senescence in cotyledons and primary leaves of 14-day-old plants of Cucurbita pepo L. (zucchini). The lack of changes in chlorophyll a, chlorophyll b and carotenoid contents as well as in the PSI activity measured by the far-red induced alterations in the P₇₀₀ oxidation levels and the quantum yield of electron transport from Q _A ⁻ to PSI end acceptors () indicated higher resistance of cotyledons to the applied dark stress compared to the primary leaves. In contrast to cotyledons, PSI activity in the primary leaves was significantly inhibited. Concerning the activity of PSII analyzed by the changes in the JIP-test parameters (the maximal efficiency of PSII photochemistry, ; the performance index, PI_ABS; the efficiency of Q _A ⁻ reoxidation, ψ ₀ and the effective dissipation per reaction center, DI₀/RC), no differences were observed between cotyledons and primary leaves, thus suggesting that PSI activity in the true leaves was more susceptible to the applied dark stress. The transfer of the darkened plants to normal light regime resulted in delayed senescence in cotyledons which was in contrast to results on Arabidopsis, thus implying the existence of specific mechanisms of cotyledon senescence in different monocarpic plants.     

N9-substituted derivatives of kinetin: effective anti-senescence agents

The first isolated cytokinin, 6-furfurylaminopurine (kinetin or Kin), was identified almost 55 years ago. Its biological effects on plant cells and tissues include influences on such processes as gene expression, cell cycle, chloroplast development, chlorophyll biosynthesis, stimulation of vascular development, delay of senescence, and mobilization of nutrients. In the present study we prepared a series of eight N9-substituted Kin derivatives, and characterized them with available physicochemical methods such as CI+ mass spectrometry and ¹H NMR spectroscopy. All compounds were tested in three classical cytokinin bioassays: a tobacco callus assay, an Amaranthus assay, and a senescence assay with excised wheat leaves. The ability of the compounds to interact with Arabidopsis cytokinin receptors CRE1/AHK4 and AHK3 was tested in a bacterial receptor assay. Prepared derivatives with certain substitutions of the N9-atom of the purine moiety enhanced the cytokinin activity of the parent compound in the bioassays to a remarkable degree but negatively affected its perception by CRE1/AHK4 and AHK3. The ability of compounds to delay the senescence of excised wheat leaves in both dark and light conditions, was highly correlated with their ability to influence membrane lipid peroxidation, which is a typical symptom of senescence. Our results were corroborated by gene expression profiling of those genes involved in cytokinin metabolism and perception, plant senescence, and the stress response, and suggest that prepared kinetin derivatives might be used as potent anti-senescence agents.     

Vertical tubular photobioreactor for semicontinuous culture of Cyanobium sp

We evaluated the kinetic culture characteristics of the microalgae Cyanobium sp. grown in vertical tubular photobioreactor in semicontinuous mode. Cultivation was carried out in vertical tubular photobioreactor for 2 L, in 57 d, at 30 °C, 3200 Lux, and 12 h light/dark photoperiod. The maximum specific growth rate was found as 0.127 d⁻¹, when the culture had blend concentration of 1.0 g L⁻¹, renewal rate of 50%, and sodium bicarbonate concentration of 1.0 g L⁻¹. The maximum values of productivity (0.071 g L⁻¹ d⁻¹) and number of cycles (10) were observed in blend concentration of 1.0 g L⁻¹, renewal rate of 30%, and bicarbonate concentration of 1.0 g L⁻¹. The results showed the potential of semicontinuous cultivation of Cyanobium sp. in closed tubular bioreactor, combining factors such as blend concentration, renewal rate, and sodium bicarbonate concentration.     

Properties of ribulose-1,5-bisphosphate carboxylase from dark- and light-exposed soybean leaves

The low activity of ribulose bisphosphate carboxylase from darkened soybean (Glycine max [L.] Merr. cv. Bragg) leaves was not raised to the level of that from leaves in the light by CO₂ and Mg²⁺, even after a 4-h incubation. The extract of darkened leaves, unlike the extract from illuminated leaves, was not fully CO₂/Mg²⁺-activatable after Sephadex gel filtration in the absence of Mg²⁺. (NH₄)₂SO₄ fractionation eliminated the inhibition effect found in the dark extracts resulting in similar rates for the extracts obtained from leaves in the dark and light. Although the V_max values of the gel-filtered extracts from dark and light leaves differed by 3-fold, the K_m(CO₂)-values were the same (12.7 μM), as were the K_m(RuBP)-values (250 μM). These data support the hypothesis that for soybean leaves in the dark a tightly-binding inhibitor renders much of the ribulose bisphosphate carboxylase enzyme catalytically non-functional.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

Substrate recognition and hydrolysis by a fungal xyloglucan-specific family 12 hydrolase

Biochemical studies to elucidate the structural basis for xyloglucan specificity among GH12 xyloglucanases are lacking. Accordingly, the substrate specificity of a GH12 xyloglucanase from Aspergillus niger (AnXEG12A) was investigated using pea xyloglucan and 12 xylogluco-oligosaccharides, and data were compared to a structural model of the enzyme. The specific activity of AnXEG12A with pea xyloglucan was 113 μmol min⁻¹ mg⁻¹, and apparent k_cat and K_m values were 49 s⁻¹ and 0.54 mg mL⁻¹, respectively. These values are similar to previously published results using xyloglucan from tamarind seed, and suggest that substrate fucosylation does not affect the specific activity of this enzyme. AnXEG12A preferred xylogluco-oligosaccharides containing more than six glucose units, and with xylose substitution at the −3 and +1 subsites. The specific activities of AnXEG12A on 100 μM XXXGXXXG and 100 μM XLLGXLLG were 60 ± 4 and 72 ± 9 μmol min⁻¹ mg⁻¹, respectively. AnXEG12A did not hydrolyze XXXXXXXG, consistent with other data that demonstrate the requirement for an unbranched glucose residue for hydrolysis by this enzyme.