Determination Of Enzyme Activity Research Articles

不同水氮水平对川西亚高山林地土壤酶活性的影响

PDF HTML阅读 XML下载 导出引用 引用提醒 不同水氮水平对川西亚高山林地土壤酶活性的影响 DOI: 10.5846/stxb201806071284 作者: 作者单位: 中国科学院成都生物研究所,中国科学院成都生物研究所,中国科学院成都生物研究所,中国科学院成都生物研究所,中国科学院成都生物研究所,中国科学院成都生物研究所,中国科学院成都生物研究所 作者简介: 通讯作者: 中图分类号: 基金项目: 四川省重大科技专项（2018SZDZX0030）；国家自然科学基金（31370495）；中国科学院西部之光项目（Y8C2021） Dynamic effects of nitrogen deposition on soil enzyme activities in soils with different moisture content Author: Affiliation: Chengdu Institute of Biology, Chinese Academy of Sciences,Chengdu Institute of Biology, Chinese Academy of Sciences,Chengdu Institute of Biology, Chinese Academy of Sciences,Chengdu Institute of Biology, Chinese Academy of Sciences,Chengdu Institute of Biology, Chinese Academy of Sciences,Chengdu Institute of Biology, Chinese Academy of Sciences,Chengdu Institute of Biology, Chinese Academy of Sciences Fund Project: Supported by Sichuan Science and Technology Program (No. 2018SZDZX0030), the National Natural Science Foundation of China (No. 31370495), West Light Foundation of Chinese Academy of Sciences (Y8C2021) 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为探讨氮沉降在不同土壤水分状况下对林下土壤中参与土壤碳氮磷循环主要酶（β-D-葡萄糖苷酶（β-D-glucosidase，βG）、过氧化物酶（Peroxidase，PER）、多酚氧化酶（Polyphenol oxidase，PPO）、β-N-乙酰葡糖胺糖苷酶（β-N-acetylglucosaminidase，NAG）和酸性磷酸酶（Acid phosphatase，AP））活性的动态影响，于2017年开展了盆栽模拟试验。试验以青杨扦插苗为植物材料，采用两因素（土壤水分和氮沉降）的随机区组设计，土壤水分含量分别为40%（W40）、60%（W60）和80%（W80）最大田间持水量，氮沉降水平分别为：0（N0）、4（N4）和8（N8） g N m-2 a-1。在土壤水分达到预定的水分含量后开始氮沉降处理，于氮沉降后的6 h、24 h和3、7、14、31、62 d采集土壤样品进行土壤酶活性的测定。结果表明：土壤含水量的降低显著降低了βG、NAG和PPO活性，且在W40时达到最低；对AP和PER活性无显著影响。氮沉降抑制了βG、NAG和AP活性，而且施氮浓度越大，抑制效应越强；对PER和PPO活性无显著影响。水氮交互作用对上述5种土壤酶活性均无显著影响。5种土壤酶活性在施氮7 d或14 d内变化较大，之后随处理时间的延长逐步平稳。两个月的实验期间，在不同水氮处理下，5种土壤酶活性基本都呈现升高-降低-升高-降低的双峰模式。该研究可为理解氮沉降对不同水分状况地区森林生态系统中土壤碳氮磷循环的生态学过程提供科学参考。 Abstract:To investigate the effects of nitrogen deposition on activities of soil enzymes (β-D-glucosidase (βG), Peroxidase (PER), Polyphenol oxidase (PPO), β-N-acetylglucosaminidase (NAG), Acid phosphatase (AP)) involved in carbon, nitrogen, and phosphorus cycles in soils with different moisture content, we conducted a pot simulation experiment in 2017. In the experiment, cuttings of Populus cathayana Rehd were used as plant materials and a randomized block design of two factors (soil moisture and nitrogen deposition) was applied. The soil moistures were 40% (W40), 60% (W60), and 80% (W80) of field capacity, respectively; and nitrogen deposition levels were 0 (N0), 4 (N4), and 8 (N8) g N m-2 a-1, respectively. When the soil moisture reached the predetermined moisture content, the simulated nitrogen deposition treatment was started. The soil samples were collected at 6 h, 24 h and 3, 7, 14, 31, and 62 d after nitrogen deposition for the determination of soil enzyme activity. The results showed that the reduction of soil water content decreased significantly the activities of βG, NAG, and PPO, which have the lowest values at W40. Soil moisture had no significant effect on the activities of AP and PER. Nitrogen deposition inhibited the activities of βG, NAG, and AP, and the inhibitory effect was greater with increasing nitrogen concentrations, while it has no significant effect on the activities of PER and PPO. No interaction effect between water moisture and nitrogen were observed on the activities of five soil enzymes. The activities of these five soil enzymes varied greatly at 7 d or 14 d, and then gradually stabilized with the extension of treatment time. During the two-month experimental period, the activities of five soil enzymes in different water and nitrogen treatments showed a "rise-fall-rise-fall" bimodal pattern. This study can provide better understanding for nitrogen deposition effects on the ecological processes of carbon, nitrogen, and phosphorus cycles in forest ecosystem soils under different water conditions, and also supply guidance for forest management. 参考文献 相似文献 引证文献

Sensors for Proteolytic Activity Visualization and Their Application in Animal Models of Human Diseases.

Various sensors designed for optical and photo(opto)acoustic imaging in living systems are becoming essential components of basic and applied biomedical research. Some of them including those developed for determining enzyme activity in vivo are becoming commercially available. These sensors can be used for various fluorescent signal detection methods: from whole body tomography to endoscopy with miniature cameras. Sensor molecules including enzyme-cleavable macromolecules carrying multiple quenched near-infrared fluorophores are able to deliver their payload in vivo and have long circulation time in bloodstream enabling detection of enzyme activity for extended periods of time at low doses of these sensors. In the future, more effective "activated" probes are expected to become available with optimized sensitivity to enzymatic activity, spectral characteristics suitable for intraoperative imaging of surgical field, biocompatibility and lack of immunogenicity and toxicity. New in vivo optical imaging methods such as the fluorescence lifetime and photo(opto)acoustic imaging will contribute to early diagnosis of human diseases. The use of sensors for in vivo optical imaging will include more extensive preclinical applications of experimental therapies. At the same time, the ongoing development and improvement of optical signal detectors as well as the availability of biologically inert and highly specific fluorescent probes will further contribute to the introduction of fluorescence imaging into the clinic.

Differential inhibition of digestive proteases by tannin in two size groups of rohu (Labeo rohita,Hamilton): A biochemical and zymography study

This study was intended to evaluate tannin mediated inhibition of digestive proteases in two different size groups (fingerlings, F, 4.5 ± 0.7 cm; advanced fingerlings, AF, 18.2 ± 1.6 cm) of rohu, Labeo rohita. Graded levels (50, 100, 150 and 200 nM) of tannin (Gallotannin, 99% purity) were added to the enzyme extracts (30°C, 1 hr) prior to determination of enzyme activities. Changes in the activity of trypsin, chymotrypsin and total protease in relation to the control sets were determined through biochemical assay of enzymes and SDS-PAGE zymography. The study revealed that tannin significantly inhibited trypsin, chymotrypsin and total protease activities in a dose dependent manner as evident from the regression equations. The degree of the inhibition appeared to be significantly higher for the F in contrast to the AF (F5,66 = 282.311; p < 0.0001). Trypsin activities were reduced by 8.04 ± 0.19% to 52.68 ± 0.72% and 5.61 ± 0.22% to 39.46 ± 0.19% in F and AF, respectively. The reduction in Chymotrypsin activities ranged between 16.11 ± 0.03% to 38.02 ± 0.27% in F and 6.31 ± 0.07% to 22.80 ± 0.32% in AF. Total protease activities were reduced by 10.9 ± 0.07% to 49.60 ± 0.32% in L. rohitaF, whereas, it ranged between 5.19 ± 0.06% to 32.60 ± 0.13% in AF. On a comparative scale, the difference in tannin induced inhibition in F and AF were more prominent for trypsin and chymotrypsin than total protease. Further, nine protease activity bands (15.9–69 kD) with different electromobility were noticed in both the size groups. Subsequent densitometry analysis revealed that the average densities of the protease activity bands were gradually decreased with increasing level of tannin exposure (50–200 nM). The study might indicate adaptive tolerance to tannin in larger size groups and emphasizes the need for removal of tannin in the plant feedstuffs, especially for feeding the fingerlings of L. rohita.

Environmental drivers and stoichiometric constraints on enzyme activities in soils from rhizosphere to continental scale

Microbial activity and functioning in soils are strongly limited by carbon (C) availability, of which a great proportion is released by living roots. Rhizodeposition and especially root exudates stimulate microbial activity and growth, and may shift the stoichiometric balance between C, N, and P. Thereby, exudates heighten microbial nutrient demand and acquisition of N and P from organic matter, leading to an increase in enzyme production. Aim of this study was to determine environmental controls of extracellular enzyme production, and hence on potential enzyme activities (Vmax) and substrate affinities (Km). To determine the controlling factors, we worked on four spatial scales from the microscale (i.e. rhizosphere) through the mesoscale (i.e. soil depth) and landscape scale (relief positions), and finally to the continental scale (1200 km transect within the Coastal Cordillera of Chile). Kinetics of seven hydrolyzing enzymes of the C, N, and P cycles (cellobiohydrolase, β‑glucosidase, β‑xylosidase, β‑N‑acetylglucosaminidase, leucine‑aminopeptidase, tyrosine‑aminopeptidase, and acid phosphatase) were related to soil texture, C and N contents, pH, and soil moisture via redundancy analysis (RDA). Potential activities of C, N, and P acquiring enzymes increased up to 7-times on the continental scale with rising humidity of sites and C and N contents, while substrate affinities simultaneously declined. On the landscape scale, neither Vmax nor Km of any enzyme differed between north and south slopes. From top- to subsoil (down to 120 cm depth) potential activities decreased (strongest of aminopeptidases under humid temperate conditions with up to 90%). Substrate affinities, however, increased with soil depth only for N and P acquiring enzymes. Affinities of cellobiohydrolase and β‑xylosidase, on the contrary, were 1.5- to 3-times higher in top- than in subsoil. Potential activities of N and P acquiring enzymes and β‑glucosidase increased form bulk to roots. Simultaneously, substrate affinities of N and P acquiring enzymes declined, whereas affinities of β‑glucosidase increased. These trends of activities and affinities in the rhizosphere were significant only for acid phosphatase. The RDA displayed a strong relation of potential activities of C and P acquiring enzymes and β‑N‑acetylglucosaminidase to C and N contents in soil as well as to the silt and clay contents. Aminopeptidase activity was mainly dependent on soil moisture and pH. We conclude that substrate availability for microorganisms mainly determined enzyme activity patterns on the continental scale by the humidity gradient. Patterns on the meso- and microscale are primarily controlled by nutrient limitation, which is induced by a shift of the stoichiometric balance due to input of easily available C by roots in the rhizosphere.

IN VITRO SCREENING OF CELL WALL DEGRADING ENZYME PRODUCTIVITY FROM FUNGAL CULTURE FILTRATES ON DEPROTEINISED PLANT FLUID BY CUP PLATE ASSAY

The present study purpose is to evaluate the potentiality of the Deproteinised juice made from selected plants to induce the enzyme productivity by growing the fungi on it. It is because, in earlier findings, the DPJ was found potential in enhancing fungi and the plant growth when used as the medium. The internal factors are responsible in DPJ which induces plants and fungi growth. During the process of Green Crop Fractionation (GCF), the deproteinised (DPJ) obtained from tissues of cabbage, beet, lucerne (Alfalfa), carrot and Anathum (Dill) forages left after leaf protein extraction employed as a medium for the cultivation of mycelial biomass of Penicillium and Aspergillus fungi. The mycelial growth in vitro was compared with the glucose nitrate medium. The culture filtrates were used to screen different secreted hydrolytic or cell wall degrading enzymes.. The agar ‘cup‐plate’ diffusion technique has been applied to the quantitative determination of enzyme activity, principally to amylase, cellulase and protease. With all enzymes so far examined, the relationship between diameter of zone over a wide range secreted quantitatively was examined. All fungi grew well on DPJ in comparison to their growth on glucose nitrate (GN) medium. Comparatively with GN medium, lucerne DPJ was found having more mycelial cellular proliferation. When the fungi grown on different concentrations of substrates, enriched with carboxymethyl cellulose, casein and starch in deproteinised leaf extracts and GN medium, it was found that there was the enhancement in the mycelial dry weight grown on DPJ as compared with glucose nitrate medium. Penicillium showed more yield of enzyme activities especially of cellulases and amylases as compared to protease by cup plate method. There was enhancement of mycelial biomass when the concentrations of substrates increased from 1% to 2%, while there was no change in the activity of all enzymes by increasing the concentrations of substrates. Activities of the enzymes in vitro can be indicative of the pattern of organogenesis in callus cultures in further studies by fungal culture filtrates cultured on deproteinised fluid medium.

A study on the interaction between cadmium and α-chymotrypsin and the underlying mechanisms.

Because cadmium might interact with proteins and, thus, exert toxicity in organisms, it is vital to understand the molecular mechanism of the interaction between cadmium and biologically relevant proteins as well as the structural and functional changes in these proteins. In this study, the interaction between α-chymotrypsin (α-ChT) and cadmium chloride (CdCl2 ) was investigated by performing enzyme activity determinations, multispectroscopic measurements, isothermal titration calorimetry, and molecular docking studies. It was demonstrated that CdCl 2 binds to α-ChT mainly via electrostatic forces with (21.0 ± 0.982) binding sites, leading to the increase of α-helix and the decrease of β-sheet. The interaction between CdCl 2 and α-ChT loosened the protein skeleton and increased the molecular volume of α-ChT. CdCl 2 first binds to the interface of α-ChT and then interacts with the key residues His 57 or Asp 102 or both in the active sites, leading to the activity inhibition of α-ChT under the exposure of high CdCl 2 concentrations.

Synthesis of N-Peptide-6-Amino-d-Luciferin Conjugates with Optimized Fragment Condensation Strategy

The synthesis of peptide-luciferin conjugates has a pivotal role in the development of bioluminescent detection systems that are based on the determination of protease enzyme activity. This work describes the optimized synthesis of an N-peptide-6-amino-d-luciferin conjugate (Fmoc-Gly-Pro-6-amino-d-luciferin) with a simple fragment condensation method in adequate yields. Fmoc-Gly-Pro-6-amino-d-luciferin was produced from a previously synthesized Fmoc-Gly-Pro-OH and also previously synthesized 6-amino-2-cyanobenzothiazole with an optimized method, to which conjugate cysteine was added in an also improved way. The resulting conjugate was successfully used in a bioluminescent system, in vitro, demonstrating the applicability of the method.

Expression and Enzyme Activity Determination of Apripona Germari Cellulase in Bmnpv/Bac-to-Bac Baculovirus Expression Systems

Expression and Enzyme Activity Determination of Apripona Germari Cellulase in Bmnpv/Bac-to-Bac Baculovirus Expression Systems

Peculiarities of tyrosine metabolism in the rat liver under the condition of protein deficiency

In the present study, a tyrosine content in liver and enzymatic activities of its metabolism: tyrosine aminotransferase, 4-hydroxyphenylpyruvate dioxygenase, aldehyde dehydrogenase (ALDH3A1) were investigated under the conditions of alimentary protein deprivation. The experiments were performed on white rats with a body weight of 100-150 g aging 2.5-3 months. The animals were divided into two experimental groups: I – animals receiving a full-value semi-synthetic ration (C); II – animals receiving a low-protein ration (LPR). In order to simulate the low-protein diet, the animals received a diet containing 4.7% protein, 10% fat and 85.3% carbohydrates for 28 days, calculated according to the American Institute of Nutrition's recommendations. Determination of tyrosine in deproteinized by 6% sulfosalicylic acid extracts of the liver tissue was performed using the automatic analyzer of amino acids T-339 (Microtechnology, Czech Republic). The enzyme activity was determined by spectrophotometric method – tyrosine aminotransferase by the amount of 4-hydroxybenzaldehyde, which has a maximum absorption at 331 nm, 4-hydroxyphenylpyruvate dioxygenase – by the intensity of colored product formation at λ 336 nm, ALDH3A1 activity was measured at 340 nm wavelength. It was established a 5-fold depletion of the tyrosine pool and a 2-fold reduction of the tyrosine aminotransferase activity, which catalyzes the formation of the first transformation of the tyrosine – 4-hydroxyphenylpyruvate transformation in the liver tissue under the condition of protein deficiency. Determination of the key enzymes activity of two possible ways of further 4-hydroxyphenylpyruvate transformation has shown that the activity of 4-hydroxyphenylpyruvate dioxygenase, as an enzyme of the homogentisin pathway of tyrosine metabolism, remains at the control level, while the aldehyde dehydrogenase, as the key enzyme of the synthesis pathway of the benzoyl ring of ubiquinone molecule, is by half reduced. The obtained results allow us to conclude that under the conditions of an alimentary protein deficiency tyrosine is predominantly metabolized in the liver by the homogenetistic pathway, which can be considered as a compensatory reaction directed at the maintaining energy metabolism while simultaneously affecting the use of tyrosine as a precursor in ubiquinone synthesis. Key words: alimentary protein deficiency, liver, mitochondria, tyrosine, ubiquinone, tyrosine aminotransferase, 4-hydroxyphenylpyruvate dioxygenase, aldehyde dehydrogenase

Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6

Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2 + substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments. How to cite: Li S, Qin K, Li H, et al. Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6. Electron J Biotechnol 2018;34.

Constraint-Based Modeling Highlights Cell Energy, Redox Status and α-Ketoglutarate Availability as Metabolic Drivers for Anthocyanin Accumulation in Grape Cells Under Nitrogen Limitation.

Anthocyanin biosynthesis is regulated by environmental factors (such as light, temperature, and water availability) and nutrient status (such as carbon, nitrogen, and phosphate nutrition). Previous reports show that low nitrogen availability strongly enhances anthocyanin accumulation in non carbon-limited plant organs or cell suspensions. It has been hypothesized that high carbon-to-nitrogen ratio would lead to an energy excess in plant cells, and that an increase in flavonoid pathway metabolic fluxes would act as an “energy escape valve,” helping plant cells to cope with energy and carbon excess. However, this hypothesis has never been tested directly. To this end, we used the grapevine Vitis vinifera L. cultivar Gamay Teinturier (syn. Gamay Freaux or Freaux Tintorier, VIVC #4382) cell suspension line as a model system to study the regulation of anthocyanin accumulation in response to nitrogen supply. The cells were sub-cultured in the presence of either control (25 mM) or low (5 mM) nitrate concentration. Targeted metabolomics and enzyme activity determinations were used to parametrize a constraint-based model describing both the central carbon and nitrogen metabolisms and the flavonoid (phenylpropanoid) pathway connected by the energy (ATP) and reducing power equivalents (NADPH and NADH) cofactors. The flux analysis (2 flux maps generated, for control and low nitrogen in culture medium) clearly showed that in low nitrogen-fed cells all the metabolic fluxes of central metabolism were decreased, whereas fluxes that consume energy and reducing power, were either increased (upper part of glycolysis, shikimate, and flavonoid pathway) or maintained (pentose phosphate pathway). Also, fluxes of flavanone 3β-hydroxylase, flavonol synthase, and anthocyanidin synthase were strongly increased, advocating for a regulation of the flavonoid pathway by alpha-ketoglutarate levels. These results strongly support the hypothesis of anthocyanin biosynthesis acting as an energy escape valve in plant cells, and they open new possibilities to manipulate flavonoid production in plant cells. They do not, however, support a role of anthocyanins as an effective mechanism for coping with carbon excess in high carbon to nitrogen ratio situations in grape cells. Instead, constraint-based modeling output and biomass analysis indicate that carbon excess is dealt with by vacuolar storage of soluble sugars.

Facile and Selective Determination of Dipeptides Using 3-Methylcatechol as a Novel Fluorogenic Reagent

Selective determination of peptides in mixtures or biological samples requires specific techniques for analysis. Herein, we report 3-methylcatechol (3-MC) as a novel fluorogenic reagent for the selective determination of dipeptides by a simple fluorescence (FL) derivatization reaction. After extensive screening of 31 different catechol analogues, 3-MC was found to generate FL with peptides. The reaction was performed at 100 °C for 10 min in the presence of borate buffer (pH 7) and sodium periodate. The resulting FL intensities were measured by spectrofluorometer at excitation wavelengths of 380 nm and emission wavelengths of 500 nm. Different reaction conditions such as concentration of sodium periodate, reaction time and pH of the borate buffer were studied to determine the optimum reaction conditions. Linearity was obtained between FL intensity and peptide concentrations from 10 to 160 µM with a lower detection limit of 10 µM (S/N = 3). Dipeptides containing Ala, Phe, Leu and Val at the N-termini generated significant FL in comparison to the reagent blank (**p < 0.005, ***p < 0.0005). The reaction is simple, rapid, selective and sensitive which can be applied for the determination of the dipeptides as biomarkers or to determine enzyme activity.

Inorganic Vanadium Supplementation in Crossbred Calves: Effects on Antioxidant Status, Immune Response and Haemato-Biochemical Attributes.

The aim of the study was to assess the effect of inorganic vanadium (V) supplementation on antioxidant enzymes, immune status, and haemato-biochemical attributes of growing crossbred calves. Twenty-four male Karan Fries calves (Tharparkar × Holstein Friesian) (initial body mass 72.83 ± 2.5kg; age 3-9month) were randomly allocated to four groups: the control (received basal diet devoid of supplemental V), the 3ppm (received basal diet with 3mg/kg V), the 6ppm (received basal diet with 6mg/kg V) and the 9ppm group (received basal diet with 9mg/kg V). All the calves were fed for 150days as per ICAR (2013) feeding standards to meet their nutrient requirements for 500g growth rate/day. Peripheral blood samples were collected at the start of experiment and subsequently at 30, 60, 90, 120 and 150days post-V supplementation for determination of antioxidant enzyme activity, immunological parameters and haemato-biochemical attributes. Results indicated that dietary supplementation of V did not affect daily gain, feed intake and haematological parameters. Crossbred calves fed with 9mg V/kg diet showed reduced (P <0.05) plasma total cholesterol concentration; however, plasma total protein and glucose concentration remained unaltered. Glutathione peroxidase (GPx) activity as well as immunoglobulin G (IgG) concentration was significantly (P <0.05) higher in group supplemented with 9mg V/kg DM; however, superoxide dismutase (SOD), catalase activity and total plasma immunoglobulin (Ig) concentration were similar in all experimental group. Dietary V supplementation showed a negative relation with plasma thiobarbituric acid reactive substances (TBARS) concentration, whereas non-esterified fatty acid (NEFA) concentration remained unaltered among all groups. Plasma V level increased (P <0.05) with increasing dietary V levels without affecting levels of Ca, Mg, Fe, Cu and Zn. In conclusion, a dietary addition of 9mg V/kg DM reduced cholesterol content and improved antioxidant and immune response in growing crossbred calves.

Electrochemistry of surface-confined enzymes: Inspiration, insight and opportunity for sustainable biotechnology

Redox enzymes can generate electricity from sunlight and produce valuable chemicals, including fuels, from low-value materials. When an electrode takes the role of an enzyme's natural redox partner, these properties inspire creative approaches to generate renewable resources. Enzymatic fuel cells produce electricity, enzyme electrosynthesis drives chemical transformations and biophotovoltaics harness solar energy. Underpinning rational development of these applications, time-dependent currents resolved by dynamic electrochemistry provide quantitative insight into the determinants of enzyme activity. This article reviews popular and emerging routes to sequester, study and exploit redox enzymes on two- and three-dimensional electrode materials. Studies are highlighted that draw on synergies of these different aspects of enzyme electrochemistry.

Induction of defense enzymes and control of anthracnose in cucumber by Corymbia citriodora aqueous extract

ABSTRACT Secondary compounds of medicinal plants can activate defense mechanisms in plants against pathogens. The aqueous extract (AE) of Corymbia citriodora has shown that activity, but there is scarce information about the involved mechanisms of action. Therefore, this study aimed to evaluate the effect of AE on the induction of defense enzymes and protection of cucumber from Colletotrichum lagenarium. Thus, the AE, autoclaved or not, was evaluated for its capability of protecting and inducing peroxidases, polyphenoloxidases, chitinases and β-1.3-glucanases, and phenylalanine ammonia-lyase. Distilled water and acibenzolar-S-methyl (50 mg a.i. L-1) were used as controls. The effect of AE at the concentrations of 1, 5, 10, 15 and 20% was also evaluated, as well as its local and systemic effect, and the effect of one or two applications. Pathogen inoculation or sample collection for determination of enzyme activity was performed at 72 hours after treatments. The AE reduced the severity of C. lagenarium and induced peroxidases and β-1.3-glucanases by 37.6, 67.2 and 122.7%, respectively. There was a reduction in the disease severity and an increase in peroxidases from the concentration of 5% AE. The latter showed only local effect and greater reduction in severity when two applications were performed. These results suggest the effect of AE in inducing resistance in cucumber and inducing peroxidases and β-1.3-glucanases.

Factors determining enzyme activities in soils under Pinus halepensis and Pinus sylvestris plantations in Spain: a basis for establishing sustainable forest management strategies

Water availability and soil pH seem to be major constraints for enzyme activities in calcareous soils under Pinus halepensis and acidic soils under Pinus sylvestris plantations respectively. Proposals for improving enzyme activities may include the promotion of broadleaf species to increase soil pH and the modulation of stand density or the implementation of soil preparation techniques to facilitate water infiltration. Soil enzymes play a key role in nutrient turnover in forest ecosystems, as they are responsible for the transformation of organic matter into available nutrients for plants. Enzyme activities are commonly influenced by temperature, humidity, nutrient availability, pH, and organic matter content. To assess the differences between enzyme activities in calcareous soils below Pinus halepensis and acidic soils below Pinus sylvestris plantations in Spain and to trace those differences back to edapho-climatic parameters to answer the questions: Which environmental factors drive enzyme activities in these soils? How can forest management improve them? The differences in climatic, soil physical, chemical, and biochemical parameters and the correlations between these parameters and enzyme activities in soils were assessed. Low pH and high level of phenols in acidic soils under Pinus sylvestris and water deficit in calcareous soils under Pinus halepensis plantations appeared to be the most limiting factors for enzyme activities. Options such as the promotion of native broadleaf species in the Pinus sylvestris stands and the modulation of Pinus halepensis stand density or the implementation of soil preparation techniques may improve enzyme activities and, therefore, nutrient availability.

Simultaneous determination of multiple soil enzyme activities for soil health-biogeochemical indices

Enzyme activities (EAs) are soil health indicators of changes in soil biogeochemical cycling potential. β-glucosidase, β-glucosaminidase, acid phosphomonoesterase, and arylsulfatase are commonly assayed as indices of C, C and N, P and S cycling, respectively. These EAs can be measured in air-dried soil with appropriate substrates following similar assay conditions. Although the assays are similar, the current protocol is to measure each single EA independently. Subsequently, different approaches have been used to obtain a single index for all EAs (e.g., geometric mean of the sum of all EAs divided by the number of enzymes) to represent biogeochemical cycling and soil organic matter (SOM) dynamics. However, the burgeoning interest in soil health has created a need for high-throughput (and simpler) assays that are more cost effective compared to measuring multiple enzymes independently. Therefore, we evaluated simultaneous determination of two to three EAs in the same soil sample in the following combined assays: 1) β-glucosidase and acid phosphomonoesterase (C and P cycling), 2) β-glucosaminidase and arylsulfatase (C, N and S cycling), 3) β-glucosidase and β-glucosaminidase (C and N cycling), and 4) β-glucosidase, acid phosphomonoesterase and β-glucosaminidase (biogeochemical potential index). The results from combined EAs showed significant correlations with the sum of EAs calculated from individual assays (r > 0.931, p < 0.001). The combined EAs also showed positive significant correlations with soil organic C (r = 0.765–0.96, p < 0.001). We provide four options to assay multiple enzymes simultaneously, which reduces the time, resources, and chemical wastes generated from assaying the four enzyme activities individually.

Optimized angiotensin-converting enzyme activity assay for the accurate diagnosis of sarcoidosis.

Serum angiotensin-converting enzyme (ACE) activity determination can aid the early diagnosis of sarcoidosis. We aimed to optimize a fluorescent kinetic assay for ACE activity by screening the confounding effects of endogenous ACE inhibitors and interfering factors. Genotype-dependent and genotype-independent reference values of ACE activity were established, and their diagnostic accuracies were validated in a clinical study. Internally quenched fluorescent substrate, Abz-FRK(Dnp)P-OH was used for ACE-activity measurements. A total of 201 healthy individuals and 59 presumably sarcoidotic patients were enrolled into this study. ACE activity and insertion/deletion (I/D) genotype of the ACE gene were determined. Here we report that serum samples should be diluted at least 35-fold to eliminate the endogenous inhibitor effect of albumin. No significant interferences were detected: up to a triglyceride concentration of 16 mM, a hemoglobin concentration of 0.71 g/L and a bilirubin concentration of 150 μM. Genotype-dependent reference intervals were considered as 3.76-11.25 U/L, 5.22-11.59 U/L, 7.19-14.84 U/L for II, ID and DD genotypes, respectively. I/D genotype-independent reference interval was established as 4.85-13.79 U/L. An ACE activity value was considered positive for sarcoidosis when it exceeded the upper limit of the reference interval. The optimized assay with genotype-dependent reference ranges resulted in 42.5% sensitivity, 100% specificity, 100% positive predictive value and 32.4% negative predictive value in the clinical study, whereas the genotype-independent reference range proved to have inferior diagnostic efficiency. An optimized fluorescent kinetic assay of serum ACE activity combined with ACE I/D genotype determination is an alternative to invasive biopsy for confirming the diagnosis of sarcoidosis in a significant percentage of patients.

Mass spectrometry based methods for the enzyme substrate screening and kinetics analysis

Revealing the relationship between the enzyme and substrate could provide insights into signaling networks. Mass spectrometry has been widely applied for the large scale interrogation of biological systems in proteomics, and also has taken an important role in determination of enzyme activities and screening of enzyme inhibitors. In this review, we introduced the recent advance in the mass spectrometry based methods for enzyme substrate screening and enzyme kinetics analysis, including in vitro substrate screening using protein-derived library and peptide-derived library, in vivo substrate screening and high-throughput kinetics analysis, and then gave our opinions on the outlook of mass spectrometry in the study of enzyme kinetics in complex system.

Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER.

Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding of activities of enzymes involved in the central carbon metabolism might lead to increased product yields. Currently, cell-free lysates are widely used for the determination of intracellular enzyme activities. However, due to thick cell walls in cyanobacteria, lysis of cyanobacterial cells is inefficient and often laborious. The present protocol describes an easy and efficient method to permeabilize cyanobacterial cells, without lysing them, and direct usage of the permeabilized cells for the determination of metabolic enzyme activities in vivo.