BG Activity Research Articles

Vector analysis of ecoenzyme activities reveal constraints on coupled C, N and P dynamics

The primary goal of this study was to devise a quantitative method of interpreting the simultaneous microbial C, N, and P acquisition through the activities of four, key extracellular enzymes, 1,4-β-glucosidase (BG), leucine aminopeptidase (LAP), 1,4-ß-N-acetylglucosaminidase (NAG) and acid/alkaline phosphatases (AP). To this end, the proportional activity of C vs. N acquiring enzymes (BG/[BG + NAG + LAP]) was plotted against C vs. P acquiring enzymes (BG/[BG + AP]). We then calculated the length and angle of the vector created by connecting a line between the plot origin and point represented by these proportions; the length quantifies relative C vs. nutrient limitation and the angle quantifies the relative P vs. N limitation. Analyses of large data sets obtained from soil, freshwater periphyton and aquatic sediments revealed that logarithmic, arcsine, arcsine-square-root and logit transformations did little to improve the statistical distribution of data over raw proportions. More importantly, the vector characteristics of enzyme activity loci are much easier to interpret for raw proportions than when data have been previously transformed. Analyses also revealed the importance of using consistent methods, i.e., omitting NAG assays for an acidic soil led to overestimates of P limitations, and the importance of understanding the nature of the system, i.e., soils of the Antarctic Dry Valleys had low BG activities, likely because there is little to no local production of cellulose. Further, analyses of four sites in Luquillo Forest, Puerto Rico, showed no differences among sites in relative C demand despite differences in BG activities, and higher P demand in the cloud than lower montane forest, despite higher AP activity at the latter site. Finally, analyses of three decomposing litter types over time revealed contrasting patterns of change in relative C, N and P demand over time, reflecting both stoichiometric and C quality effects on decomposer communities. Thus enzyme activity vectors reflect the simultaneous, relative resource demands of the microbial community independent of variations in total enzyme activity, such as may result from variations in total microbial biomass.

Control Effect and Possible Mechanism of the Natural Compound Phenazine-1-Carboxamide against Botrytis cinerea.

To develop new agents against strawberry grey mould and to aid in the development of biological pesticides, we investigated the inhibitory effect of a natural compound, phenazine-1-carboxamide (PCN), against Botrytis cinerea using a growth rate assay. Additionally, indoor toxicity and the in vitro control effect of PCN were further studied to determine its potential mechanisms of action on B. cinerea. PCN was inhibitory against B. cinerea with a 50% effective concentration (EC50) of 108.12 μg/mL; the toxicity of PCN was equivalent to that of carbendazim (CBM). The best in vitro control effect of PCN against grey mould in strawberry (fruit) reached 75.32%, which was slightly higher than that of CBM. The field control effect of PCN against grey mould reached a maximum of 72.31% at a PCN concentration of 700 μg/mL, which was 1.02 times higher than that of CBM. Fungistatic activity was observed at low concentrations of PCN, while high concentrations of PCN resulted in fungicidal activity against B. cinerea. This natural compound strongly inhibited both spore and sclerotium germination of B. cinerea, with the best relative inhibition rates of 77.03% and 82.11%, respectively. The inhibitory effect of PCN on mycelial growth of B. cinerea was significant and reached levels of 87.32%. Scanning electron microscopy observations revealed that after 48 h of PCN treatment, the mycelia appeared loose, locally twisted, and folded, with exudation of contents; the mycelia was withered and twisted, with edge burrs, deformations, ruptures and a sheet-like structure. Transmission electron microscopy observations revealed that after 48 h of PCN treatment, the structure of the cell nucleus was unclear and the vacuoles had ruptured; additionally, various organelles exhibited disordered structures, there were substantial non-membrane transparent inclusions, the cells were plasmolysed, the cell walls were collapsed in some cases, and the hyphal tissue was essentially necrotic. A PCN dosage of 35–140 μg/mL had no effect on the cell membrane permeability of the mycelia, while a PCN dosage of 700 μg/mL resulted in significant permeability. PCN inhibited B. cinerea toxin; the mycotoxin level was approximately 0.41 of the value recorded for the control at a PCN dosage of 700 μg/mL. PCN affected the activity of pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β-glucosidase (BG); the lowest activities of PMG, PG, BG and Cx reached 0.3 U/mg, 0.62 U/mg, 0.64 U/mg, and 0.79 U/mg, respectively, after treatment with 700 μg/mL PCN.

Changes in soil microbial community, enzyme activities and organic matter fractions under long-term straw return in north-central China

Field nutrients management practices influence soil biological properties and organic matter fractions. A long-term experiment (30-year) was conducted to investigate changes in soil microbial community, enzyme activities and organic carbon fractions under straw return in north-central China. Treatments included no-fertilizer control (CK) and maize straw return at rates of 0 (S0), 2250 (S1), 4500 (S2), and 9000kgha−1 (S3) under combined nitrogen and phosphorus fertilization. All fertilization treatments increased total phospholipid fatty acid (PLFA) and the abundances of Gram-negative (Gm–) bacteria and fungi over the CK treatment. The S3 treatment increased total PLFA compared with the S0 treatment. The S2 and S3 treatments increased Gm− bacterial abundance by 11.6 and 9.3%, respectively, and increased fungal abundance by 68.2 and 113.6%, respectively, compared with the S0. Fertilization increased the activities of β-glucosidase (BG), β-xylosidase (XYL), and N-acetyl-glucosaminidase (NAG) over the CK. The S2–S3 increased the activities of BG, XYL, and NAG by 10.5–20.7, 19.0–32.5, and 21.6–32.8% compared with the S0, respectively. Although the S1 and S3 had lower activities of phenol oxidase than the CK, the activities did not differ among the S0–S3 treatments. The S0–S3 treatments increased the concentration of total organic C (TOC) than the CK, and the S2–S3 increased TOC than the S0. There were no differences in soil light fraction (LF) and the light fraction organic C (LFOC) among the CK, S0, and S1. The LF and LFOC in the S2 increased by 14.7 and 33.9%, respectively, and these values in the S3 increased by 48.0 and 81.3%, respectively, relative to the S0. The S0–S3 treatments increased the heavy fraction organic C (HFOC) over the CK and the HFOC in the S2–S3 increased by 39.2–43.1% compared with the S0. The LFOC/TOC ratio was lower than the HFOC/TOC ratio for each treatment. Overall, low rates of straw return did not affect, while high rates of straw changed microbial community structure and increased the activities of most hydrolytic enzymes and the concentration of LFOC and HFOC under chemical fertilizer application.

Facile large scale synthesis of Bi2S3 nano rods–graphene composite for photocatalytic photoelectrochemical and supercapacitor application

Bi2S3 nano rods–graphene (BG) composite material was synthesized by a simple one step precipitation method. The crystallanity, structural and morphological properties were studied by the X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), high resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy techniques. The photocatalytic activity of BG was evaluated by the photocatalytic degradation of malachite green dye (MG) aqueous solution under the visible light irradiation. The effect of graphene content on the photoelectrochemical property of Bi2S3 nano rods was also studied. The enhancement of photocurrent and photocatalytic properties of BG composite attributed to the synergistic effect between the Bi2S3 nano rods and graphene sheets which improves the charge separation efficiency in Bi2S3 nano rods. The supercapacitor behavior was studied using cyclic voltametry and galvanostatic charge discharge studies. The BG composite exhibits a maximum specific capacitance of 290Fg−1 at a current density of 1Ag−1. The present study may provide as a new approach in improving the performance of BG composite in supercapacitor, solar cells and photocatalytic applications.

Responses of enzymatic activities within soil aggregates to 9-year nitrogen and water addition in a semi-arid grassland

Soil microorganisms secrete enzymes used to metabolize carbon (C), nitrogen (N), and phosphorus (P) from the organic materials typically found in soil. Because of the connection with the active microbial biomass, soil enzyme activities can be used to investigate microbial nutrient cycling including the microbial response to environmental changes, transformation rates and to address the location of the most active biomass. In a 9-year field study on global change scenarios related to increasing N inputs (ambient to 15 g N m−2 yr−1) and precipitation (ambient to 180 mm yr−1), we tested the activities of soil β-glucosidase (BG), N-acetyl-glucosaminidase (NAG) and acid phosphomonoesterase (PME) for three soil aggregate classes: large macroaggregates (>2000 μm), small macroaggregates (250–2000 μm) and microaggregates (<250 μm). Results showed higher BG and PME activities in micro-vs. small macroaggregates whereas the highest NAG activity was found in the large macroaggregates. This distribution of enzyme activity suggests a higher contribution of fast-growing microorganisms in the micro-compared with the macroaggregates size fractions. The responses of BG and PME were different from NAG activity under N addition, as BG and PME decreased as much as 47.1% and 36.3%, respectively, while the NAG increased by as much as 80.8%, which could imply better adaption of fungi than bacteria to lower soil pH conditions developed under increased N. Significant increases in BG and PME activities by as much as 103.4 and 75.4%, respectively, were found under water addition. Lower ratio of BG:NAG and higher NAG:PME underlined enhanced microbial N limitation relative to both C and P, suggesting the repression of microbial activity and the accompanied decline in their ability to compete for N with plants and/or the accelerated proliferation of soil fungi under elevated N inputs. We conclude that changes in microbial activities under increased N input and greater water availability in arid- and semi-arid grassland ecosystems where NPP is co-limited by N and water may result in substantial redistribution of microbial activity in different-sized soil particles. This shift will influence the stability of SOM in the soil aggregates and the nutrient limitation of soil biota.

Enzymatic cellulose hydrolysis: enzyme reusability and visualization of β-glucosidase immobilized in calcium alginate.

The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde cross-linked BG was entrapped in calcium alginate gel particles. More than 60% of the enzyme activity could be recovered under optimized conditions, and glutaraldehyde cross-linking decreased leakage of BG from the calcium alginate particles. The immobilized BG aggregates were visualized by confocal laser scanning microscopy (CLSM). The CLSM images, which we believe are the first to be published, corroborate that more BG aggregates were entrapped in the matrix when the enzymes were cross-linked by glutaraldehyde as opposed to when they are not cross-linked. The particles with the immobilized BG were recycled for cellulase catalyzed hydrolysis of Avicel. No significant loss in BG activity was observed for up to 20 rounds of reaction recycle steps of the BG particles of 48 h each, verifying a significant stabilization of the BG by immobilization. Similar high glucose yields were obtained by one round of enzymatic hydrolysis of hydrothermally pretreated barley straw during a 72 h reaction with immobilized BG and free BG.

Comparison of Salivary Beta Glucuronidase Activity in Chronic Periodontitis Patients with and without Diabetes Mellitus

The aim of the study was to estimate the salivary beta glucuronidase (β) activity in patients with chronic periodontitis with and without diabetes mellitus and to evaluate the relationship between Beta Glucuronidase activity and Periodontal clinical parameters. The study consisted of 80 patients of both sexes with age ranging from 20-60 years and they were divided into four groups. Clinical parameters such as Gingival index, Probing depth and Clinical attachment loss were measured. Salivary Beta Glucuronidase activity was measured using spectrophotometer with reagents like phenolphthalein glucuronic acid, phosphate and glycine buffer. The mean BG activity of Group IV (1.17 ± 0.27) was significantly higher than mean BGA levels of Group I, II, III. The p-value was < 0.05. The mean BGA levels of Group III (0.78 ± 0.17) was significantly higher than mean BGA levels of Group I, Group II at 5 % level. There was a significant positive linear relationship between salivary β Glucuronidase level and Probing Depth, clinical attachment level in the experimental Groups. The salivary β Glucuronidase level was higher in Diabetic patients with periodontitis than nondiabetic periodontitis patients.

Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A mesocosm simulation study

Bioaugmentation (amendment with selected bacterial strains) and/or biostimulation (nutrients addition and/or air supply) are relatively new fields in environmental microbiology for preventing pollution and cleanup contamination. In this study, the efficiency of application of bioaugmentation/biostimulation treatments, for recovery of crude oil-polluted seawater, was evaluated. Three different series of experiments were performed in a “Mesocosm Facility” (10.000 L). Natural seawater was artificially polluted with crude oil (1000 ppm) and was amended with inorganic nutrients (Mesocosm 1, M1), inorganic nutrient and an inoculum of Alcanivorax borkumensis SK2T (Mesocosm 2, M2) and inorganic nutrient and an inoculum of A. borkumensis SK2T and Thalassolituus oleivorans MIL-1T (Mesocosm 3, M3), respectively. During the experimental period (20 days) bacterial abundance (DAPI count), culturable heterotrophic bacteria (CFU count), MPN, microbial metabolic activity [Biochemical Oxygen Demand and enzymatic activity (leucine aminopeptidase LAP, β-glucosidase BG, alkaline phosphatase AP)] and quali-, quantitative analysis of the composition of total extracted and resolved hydrocarbons and their derivates (TERHCs) were carried out. The microbiological and physiological analysis of marine microbial community found during the three different biostimulation and bioaugmentation assays performed in mesocosms show that the load of crude oil increases total microbial abundance, inhibits the activity of some enzymes such as LAP while stimulates both AP and BG activities. The biodegradation results show that bioaugmentation with A. borkumensis SK2T alone is able to produce the highest percentage of degradation (95%) in comparison with the biostimulation treatment (80%) and bioaugmentation using an Alcanivorax-Thalassolituus bacterial consortium (70%). This result highlights the reduced biodegradation capability of the consortium used in this study, suggesting an unfavourable interaction between the two bacterial genera.

Bioactive compounds, in vitro antioxidant capacity and Maillard reaction products of raw, boiled and fried garlic (Allium sativum L.)

SummaryGarlic is used in many dishes but some of its bioactive compounds are lost when exposed to heat. This study evaluated bioactive compounds, antioxidant activity and Maillard reaction products of raw (RG), boiled (BG) and fried garlic (FG). Total phenolic compounds, flavonoids, allicin and phytosterol were quantified as well as dietary fibres and mineral content. Antioxidant activities of RG, BG and FG were evaluated by oxygen radical absorbance capacity (ORAC), oxidation of β‐carotene/linoleic acid and Rancimat methods. The main results were the following: thermal processing of RG decreased both bioactive compounds and antioxidant capacity, boiling was found to be less aggressive than frying, and strong correlations were obtained between total phenolic compounds and the antioxidant assays. However, phytosterol content was higher in FG than in RG and BG and higher temperatures increased the formation of Maillard reaction products. It was concluded that RG and BG should be preferred for human consumption as they present higher antioxidant capacity.

Effects of conventional tillage and no tillage permutations on extracellular soil enzyme activities and microbial biomass under rice cultivation

Extracellular soil enzymes hold a cardinal position in nutrient dynamics by regulating bioavailability of elements, and hence are linked with soil health. The present study aimed to analyze the responses of extracellular enzymes involved in mineralization of carbon (β-d-glucosidase (BG), cellobiohydrolase (CBH), polyphenol oxidase (PPO)), nitrogen (urease (UR), glycine-amino peptidase (GAP)) and phosphorous (alkaline phosphatase (ALP)) under four permutations of conventional tillage and no tillage under rice–wheat system in eastern Indo-gangetic plains during rice cultivation period. The permutations were: tillage before sowing/transplantation of each crop (RCT–WCT), tillage before transplantation of rice and no tillage before sowing of wheat (RCT–WNT), tillage before sowing wheat and no tillage before sowing of rice (RNT–WCT) and no tillage before sowing of each crop (RNT–WNT). Microbial biomass carbon and nitrogen and activities of BG, CBH, ALP and UR increased with reduction in tillage frequency, becoming the highest under RNT–WNT and the lowest under RCT–WCT. Principal component analyses (PCA) condensed the variables in to two components, apparently described by soil temperature and moisture content under all the tillage permutations. Most of the enzymes and soil properties identified to be associated under PCA followed linear relationships. Under RCT–WCT, CBH, UR and ALP were related with BG. Different orders of residue incorporation and tillage under RCT–WNT, RNT–WCT and RNT–WNT masked these relations. Results indicated that reduction in tillage frequency made the soil healthier. Relationship of BG with other enzymes appeared as a probable indicator to reflect deviations from the conventional cultivation practice in the study region.

Immobilization of β-glucosidase on mercaptopropyl-functionalized mesoporous titanium dioxide

Abstract Mesoporous titanium dioxide (M-TiO 2 ) and mercaptopropyl-functionalized M-TiO 2 (SH-M-TiO 2 ) were prepared as carriers for immobilization of β-glucosidase (BG). Analyses suggested that in SH-M-TiO 2 the grafted mercaptopropyl groups attributed to about 3.5% of the total weight of the matrices. For the immobilization of BG, SH-M-TiO 2 or M-TiO 2 powder was added with free BG solution and stirred at 4 °C for 8 h, followed by centrifugation and washing. BG was effectively immobilized on SH-M-TiO 2 with an enzyme activity of 10.9 U/g, which was 92.8% of the total enzyme activity in the starting solution of free BG. BG immobilized on SH-M-TiO 2 (BG-SH-M-TiO 2 ) was more stable in pH, thermal, and storage tests than BG on M-TiO 2 or free BG in solution. The K m and v max of BG-SH-M-TiO 2 were similar to values for free BG in solution. Batch hydrolysis of cellobiose-containing substrate by BG-SH-M-TiO 2 was conducted. Conversion rates of cellobiose in 10 batches were consistently around 90%. Although the residual enzyme activity of BG-SH-M-TiO 2 decreased gradually, only a small amount of BG activity was detected in supernatants. This indicated effective adsorption of enzyme molecules to SH-M-TiO 2 matrices.

The effects of simulated nitrogen deposition on extracellular enzyme activities of litter and soil among different-aged stands of larch

Aims Nitrogen (N) addition could affect the rate of forest litter and soil organic matter decomposition by regulating extracellular enzyme activity (EEA). The impact of N addition on EEA may differ across different age stands with different organic matter quality. We were interested in whether the impact of N addition on EEA in litter and mineral soil during the growing season was dependent on stand age of a larch plantation in North China. Methods We added three levels of N (0, 20 and 50 kg N ha −1 year −1 ) in three age stands (11, 20 and 45 years old) of Larix principis-rupprech- tii plantation in North China. We measured potential activities of β-1,4-glucosidase (BG), cellobiohydrolase (CB), β-1,4-N-acetyl- glucosaminidase (NAG) and phenol oxidase (PO) in litter (organic horizon) and mineral soil (0-10 cm) during the second growing sea- son after N amendment. We also measured C and N concentrations, microbial biomass C and N, and KCl-extractable ammonium and nitrate in both litter and mineral soil. temperature. Stand age had a strong effect on EEA in both litter and mineral soil, and this effect differed between litter and mineral soil as well as between different enzymes. N addition did not signifi- cantly affect the activities of BG or CB but significantly suppressed the activity of NAG in litter. We also found stand age-specific responses of PO activity to N addition in both litter and mineral soil. N addition suppressed PO activity of the high C:N ratio litters in 20- and 45-year-old stands but had no significant effect on PO activity of the low C:N ratio litter in 11-year-old stand. Moreover, N addition inhibited PO activity of the high C:N ratio soil in 20-year- old stand but had no significant impact on PO activity of the low C:N ratio soils in 11- and 45-year-old stands. Overall, stand age had a greater effect on EEA in litter and mineral soil compared to 2 years of N addition. Moreover, the effect of N addition on PO activity is stand age dependent, which may affect the long-term soil carbon storage in this forest.

Microbial enzyme activity at the watershed scale: response to chronic nitrogen deposition and acute phosphorus enrichment

Microbial enzymes play a critical role in organic matter decomposition and enzyme activity can dynamically respond to shifts in inorganic nutrient and substrate availability, reflecting the nutrient and energy limitation of the microbial community. We character- ized microbial enzyme response to shifting nitrogen (N) and phosphorus (P) availability across terrestrial and aquatic environments at the Bear Brook Watershed in Maine, the site of a whole-watershed N enrichment experiment. We compared activity of b-1,4-glucosi- dase (BG); b-1,4-N-acetylglucosaminidase (NAG); acid phosphatase (AP) in soil, leaf litter in terrestrial and stream habitats and stream biofilms in a reference and N enriched watershed, representing whole-eco- system response to chronic N enrichment. In addition, we used shorter, experimental P enrichments to address potential P limitation under ambient and elevated N availability. We found that BG and NAG activity were not affected by the long-term N enrichment in either habitat. Enhanced P limitation due to N enrichment was evident only in the aquatic habitats with 5- and 8-fold higher treated watershed AP activity in stream biofilms and stream litter, respectively. Acute P additions reduced AP activity and increased BG activity and these effects were also most pronounced in the streams. The stoichiometry of enzyme activity was constrained across ecosystem compartments with regression slopes for lnBG:lnNAG, lnBG:lnAP, and lnNAG:lnAP close to 1, ranging 1.142-1.241. We found that microbial enzyme response to shifting N and P availability varied among watershed compartments, typically with stronger effects in aquatic habitats. This suggests that understanding the response of ecosystem function to disturbance at the watershed scale requires simultaneous consideration of all compartments.

Identification of a Genomic Region Containing a Novel Promoter Resistant to Glucose Repression and Over-Expression of β-Glucosidase Gene in Hypocrea orientalis EU7-22

A high concentration of glucose in the medium could greatly inhibit the expression of cellulase in filamentous fungi. The aspartic protease from fungus Hypocrea orientalis EU7-22 could efficiently express under both induction condition and glucose repression condition. Based on the sequence of structure gene of aspartic protease, the upstream sequence harboring the putative promoter proA for driving the expression of aspartic protease was obtained by genome walking. The upstream sequence contained the typical promoter motifs “TATA” and “CAAT”. The β-glucosidase gene (Bgl1) from H. orientalis was cloned and recombined with promoter proA and terminator trpC. The expression cassette was ligated to the binary vector to form pUR5750-Bgl1, and then transferred into the host strain EU7-22 via Agrobacterium tumefaciens mediated transformation (ATMT), using hygromycin B resistance gene as the screening marker. Four transformants Bgl-1, Bgl-2, Bgl-3 and Bgl-4 were screened. Compared with the host strain EU7-22, the enzyme activities of filter paper (FPA) and β-glucosidase (BG) of transformant Bgl-2 increased by 10.6% and 19.1% under induction condition, respectively. The FPA and BG activities were enhanced by 22.2% and 700% under 2% glucose repression condition, respectively, compared with the host strain. The results showed that the putative promoter proA has successfully driven the over-expression of Bgl1 gene in H. orientalis under glucose repression condition.

Interacting Cortical and Basal Ganglia Networks Underlying Finding and Tapping to the Musical Beat

Humans are able to find and tap to the beat of musical rhythms varying in complexity from children's songs to modern jazz. Musical beat has no one-to-one relationship with auditory features-it is an abstract perceptual representation that emerges from the interaction between sensory cues and higher-level cognitive organization. Previous investigations have examined the neural basis of beat processing but have not tested the core phenomenon of finding and tapping to the musical beat. To test this, we used fMRI and had musicians find and tap to the beat of rhythms that varied from metrically simple to metrically complex-thus from a strong to a weak beat. Unlike most previous studies, we measured beat tapping performance during scanning and controlled for possible effects of scanner noise on beat perception. Results showed that beat finding and tapping recruited largely overlapping brain regions, including the superior temporal gyrus (STG), premotor cortex, and ventrolateral PFC (VLPFC). Beat tapping activity in STG and VLPFC was correlated with both perception and performance, suggesting that they are important for retrieving, selecting, and maintaining the musical beat. In contrast BG activity was similar in all conditions and was not correlated with either perception or production, suggesting that it may be involved in detecting auditory temporal regularity or in associating auditory stimuli with a motor response. Importantly, functional connectivity analyses showed that these systems interact, indicating that more basic sensorimotor mechanisms instantiated in the BG work in tandem with higher-order cognitive mechanisms in PFC.

Role of GLP-1 in the Hypoglycemic Effects of Wild Bitter Gourd

This study aimed to examine the role of GLP-1 in the hypoglycemic activity of wild bitter gourd (Momordica charantia L., BG). In vitro, the GLP-1 secretion in STC-1, a murine enteroendocrine cell line, was dose dependently stimulated by water extract (WE), its fractions (WEL, >3 kD and WES, <3 kD), and a bitter compounds-rich fraction of BG. These stimulations were partially inhibited by probenecid, a bitter taste receptor inhibitor, and by U-73122, a phospholipase Cβ2 inhibitor. These results suggested that the stimulation might involve, at least in part, certain bitter taste receptors and/or PLCβ2-signaling pathway. Two cucurbitane triterpenoids isolated from BG, 19-nor-cucurbita-5(10),6,8,22-(E),24-pentaen-3β-ol, and 5β,19-epoxycucurbita-6,24-diene-3β,23ξ-diol (karavilagenine E,) showed relative high efficacy in the stimulation. In vivo, mice fed BG diet showed higher insulinogenic index in an oral glucose tolerance test. A single oral dose of WE or WES pretreatment significantly improved intraperitoneal glucose tolerance. A single oral dose of WES significantly decreased glucose and increased insulin and GLP-1 in serum after 30 min. This acute hypoglycemic effect of WES was abolished by pretreatment with exendin-9, a GLP-1 receptor antagonist. Our data provide evidence that BG stimulates GLP-1 secretion which contributes, at least in part, to the antidiabetic activity of BG through an incretin effect.

The susceptibility of soil enzymes to inhibition by leaf litter tannins is dependent on the tannin chemistry, enzyme class and vegetation history

By inhibiting soil enzymes, tannins play an important role in soil carbon (C) and nitrogen (N) mineralization. The role of tannin chemistry in this inhibitory process, in conjunction with enzyme classes and isoforms, is less well understood. Here, we compared the inhibition efficiencies of mixed tannins (MTs, mostly limited to angiosperms) and condensed tannins (CTs, produced mostly by gymnosperms) against the potential activity of β-glucosidase (BG), N-acetyl-glucosaminidase (NAG), and peroxidase in two soils that differed in their vegetation histories. Compared with CTs, MTs exhibited 50% more inhibition of almond (Prunus dulcis) BG activity and greater inhibition of the potential NAG activity in the gymnosperm-acclimatized soils. CTs exhibited lower BG inhibition in the angiosperm-acclimated soils, whereas both types of tannins exhibited higher peroxidase inhibition in the angiosperm soils than in gymnosperm soils. At all of the tested tannin concentrations, irrespective of the tannin type and site history, the potential peroxidase activity was inhibited two-fold more than the hydrolase activity and was positively associated with the redox-buffering efficiency of tannins. Our finding that the inhibitory activities and mechanisms of MTs and CTs are dependent on the vegetative history and enzyme class is novel and furthers our understanding of the role of tannins and soil isoenzymes in decomposition.

A comparative study of hydrolysis and transglycosylation activities of fungal β-glucosidases

β-glucosidases (BGs) from Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and investigated for their (simultaneous) hydrolytic and transglycosylation activity in samples with high concentrations of either cellobiose or glucose. The rate of the hydrolytic process (which converts one cellobiose to two glucose molecules) shows a maximum around 10-15mM cellobiose and decreases with further increase in the concentration of substrate. At the highest investigated concentration (100mM cellobiose), the hydrolytic activity for the different enzymes ranged from 10% to 55% of the maximum value. This decline in hydrolysis was essentially compensated by increased transglycosylation (which converts two cellobiose to one glucose and one trisaccharide). Hence, it was concluded that the hydrolytic slowdown at high substrate concentrations solely relies on an increased flow through the transglycosylation pathway and not an inhibition that delays the catalytic cycle. Transglycosylation was also detected at high product (glucose) concentrations, but in this case, it was not a major cause for the slowdown in hydrolysis. The experimental data was modeled to obtain kinetic parameters for both hydrolysis and transglycosylation. These parameters were subsequently used in calculations that quantified the negative effects on BG activity of respectively transglycosylation and product inhibition. The kinetic parameters and the mathematical method presented here allow estimation of these effects, and we suggest that this may be useful for the evaluation of BGs for industrial use.

Do enzyme activities during decomposition follow predicted patterns? A test of the conceptual model of litter decay.

AbstractSurprisingly, there remains a paucity of research examining specific interactions between the relationship between microbial community behavior and plant litter chemistry during decomposition. A more mechanistic understanding of the relationship between these drivers will ultimately help determine the trajectory of litter decomposition and the conditions in which soils serve as either a source or sink for atmospheric C. In order to examine these relationships, a laboratory incubation was established using Acer saccharum litter and a sandy soil (&lt; 1.5% organic matter). Extracellular enzyme activities ([BETA]-glucosidase, N-acetyl glucosaminidase, leucine-amino peptidase, acid phosphatase, phenol oxidase, and peroxidase) were monitored on a consistent basis along with instantaneous rates of carbon dioxide production, microbial biomass (carbon and nitrogen) and phospholipid fatty acid biomarkers (PLFA), and nitrogen and phosphorus availability. Microbial biomass and microbial respiration peaked within the first week of the experiment. This was likely due to the high availability of water soluble substrates early in decay that can be obtained without the production of extracellular enzymes. [BETA]-glucosidase (BG), N-acetyl glucosaminadase (NAG), and acid phosphatase activities increased quickly following the first week and peaked within the first month (at approximately 15% mass loss). Leucine amino peptidase was not detected during the incubation, which may be due to its strong positive correlation with soil pH, while other hydrolytic enzymes tend to track concentrations of soil organic matter. Phenol oxidase and peroxidase activities were not measurable until the second month of the experiment (&gt; 25% mass loss), likely following the depletion of more labile substrates. A second increase in BG activity was observed between Days 83-111, which may be due to an increase in the availability of cellulose that was previously shielded by lignin, since oxidative enzyme activity was first detected on Day 68. We also observed some shifts in microbial PLFAs along with enzyme activities during decomposition. Prior to the increases in enzyme activity we observed a high proportion of PLFA 18:1[omega]7c, which is a bacterial biomarker. As enzyme activities increased, we observed a decrease in this biomarker and an increase in 18:2[omega]6,9c, a fungal biomarker that was correlated with BG and NAG activity. We did not observe any clear relationships between PLFAs and lignolytic enzyme activity, however. Overall, we observed a distinct functional shift in microbial substrate use that may be associated with either changes in composition of the microbial community or community shifts in enzyme production.

A Microscale Platform for Integrated Cell-Free Expression and Activity Screening of Cellulases

Recent advances in production of cellulases by genetic engineering and isolation from natural microbial communities have necessitated the development of high-throughput analytical technologies for cellulase expression and screening. We have developed a novel cost-effective microscale approach based on in vitro protein synthesis, which seamlessly integrates cellulase expression with activity screening without the need for any protein purification procedures. Our platform achieves the entire process of transcription, translation, and activity screening within 2-3 hours in microwell arrays compared with days needed for conventional cell-based cellulase expression, purification, and activity screening. Highly sensitive fluorescence-based detection permits activity screening in volumes as low as 2-3 μL with minimal evaporation (even at temperatures as high as 95 °C) leading to two orders of magnitude reduction in reagent usage and cost. The platform was used for rapid expression and screening of β-glucosidases (BGs) and cellobiohydrolases (CBHs) isolated from thermophilic microorganisms. Furthermore, it was also used to determine optimum temperatures for BG and CBH activities and to study product inhibition of CBHs. The approach described here is well suited for first-pass screening of large libraries to identify cellulases with desired properties that can subsequently be produced on a large scale for detailed structural and functional characterization.