Toxocara canis: proteolytic enzymes secreted by the infective larvae in vitro

Second-stage larvae of the dog nematode Toxocara canis are infective to man and cause the syndromes of visceral larva migrans and ocular toxocariasis. Larvae cultured in vitro secrete proteases which degrade components of a model of extracellular matrix and basement membranes. These enzymes have been characterized using a variety of techniques. Multiple enzyme activities were demonstrated by substrate gel electrophoresis, associated with proteins of molecular weights of 120 and 32 kDa. The enzyme activity was inhibited both in substrate gels and in a radiogelatin microplate assay by phenylmethylsulfonyl fluoride. Optimal activity occurred at pH 9, with minor activities apparent at pH 5 and 7; the relationship between these proteolytic activities is currently under investigation.     

Two time-resolved fluorometric high-throughput assays for quantitation of GDP-L-fucose

Two rapid and simple procedures for the quantitative analysis of GDP-l-fucose (GDP-Fuc) are described. The methods are based on time-resolved fluorescence and microplate assay technology. The first assay relies on measuring the enzyme activity of alpha1, 3-fucosyltransferase. In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasaccharide, which is detected and quantified by relevant antibodies on a microplate. The formation of the reaction product is directly dependent on the presence of GDP-Fuc in the concentration range of 10-10,000 nM. In the second method GDP-Fuc inhibits the binding of fucose-specific Aleuria aurantia lectin to fucosylated glycan on a microwell. The lectin-based assay is less sensitive than the enzyme assay, but it is cheaper and faster. We used these assays in monitoring the amount of GDP-Fuc in crude lysates of transgenic yeast, which expresses the enzymes producing GDP-Fuc. The newly developed assays are versatile and applicable to measure also other nucleotide sugars or glycosyltransferase activities in a high-throughput manner.     

Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. In order to examine extracellular enzyme production in this chronically low-temperature environment, fungi were isolated from ornithogenic, pristine and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica during the austral summer in February 2007. Twenty-eight isolates of psychrophilic and psychrotolerant fungi were obtained and screened at a culture temperature of 4°C for activity of extracellular hydrolase enzymes (amylase, cellulase, protease), using R2A agar plates supplemented with (a) starch for amylase activity, (b) carboxymethyl cellulose and trypan blue for cellulase activity or (c) skim milk for protease activity. Sixteen isolates showed activity for amylase, 23 for cellulase and 21 for protease. One isolate showed significant activity across all three enzyme types, and a further 10 isolates showed significant activity for at least two of the enzymes. No clear associations were apparent between the fungal taxa isolated and the type of source soil, or in the balance of production of different extracellular enzymes between the different soil habitats sampled. Investment in extracellular enzyme production is clearly an important element of the survival strategy of these fungi in maritime Antarctic soils.     

Radiolabeled heparan sulfate immobilized on microplate as substrate for the detection of heparanase activity

We developed a quantitative assay to monitor the enzymatic activity of heparanase, a protein responsible for the degradation of heparan sulfate (HS) present on cell surface and extracellular matrix. Our assay is based on a new procedure to immobilize radiolabeled HS to a solid support by a single end which is adaptable to a microplate format, thus allowing the rapid analysis of numerous samples. First, HS was radiolabeled by partial de-N-acetylation and re-N-acetylation with [3H] acetic anhydride, second, after reductive amination at the reducing terminus, it was covalently linked to an amino-reactive biotin analog, and third it was immobilized on a streptavidin-coated plate. The degradation of our solid-phase tritiated HS by heparanase was monitored by measuring the soluble radioactivity released in the well. The heparanase-induced release of radioactivity was linear with respect either to time or to the amount of enzyme and was inhibited by heparin or high ionic strength. The linearity of this assay for time and enzyme concentrations could be useful to determine the potency of heparanase inhibitors. Moreover, this assay was shown to be suitable for monitoring HS-degrading activity of either heparanase endogenously expressed by the HCT 116 tumor cell line or recombinant forms of this protein.     

A new method to screen polysaccharide cleavage enzymes

The activity of polysaccharide cleavage enzymes has usually been evaluated by qualitative plate screening methods and quantitative colorimetric or chromatographic assays. The recent development of protein engineering has shown the limits of these techniques when applied to high throughput screening. Here we propose a microplate method to measure the activity of polysaccharide cleavage enzymes through small variations in viscosity. Polysaccharide solutions are co-incubated with magnetic particles in enzyme buffers. The cleavage action of polymer-degrading enzymes increases the mobility of the particles in a magnetic field, even at low levels of enzyme activities. This reproducible, sensitive technique was used to evaluate enzymatic specificity towards substrates. BioFilm indices (BFI) determined by associated software were used to follow enzyme kinetics and measure the usual variables.     

A comparison of methods for total community DNA preservation and extraction from various thermal environments

The widespread use of molecular techniques in studying microbial communities has greatly enhanced our understanding of microbial diversity and function in the natural environment and contributed to an explosion of novel commercially viable enzymes. One of the most promising environments for detecting novel processes, enzymes, and microbial diversity is hot springs. We examined potential biases introduced by DNA preservation and extraction methods by comparing the quality, quantity, and diversity of environmental DNA samples preserved and extracted by commonly used methods. We included samples from sites representing the spectrum of environmental conditions that are found in Yellowstone National Park thermal features. Samples preserved in a non-toxic sucrose lysis buffer (SLB), along with a variation of a standard DNA extraction method using CTAB resulted in higher quality and quantity DNA than the other preservation and extraction methods tested here. Richness determined using DGGE revealed that there was some variation within replicates of a sample, but no statistical difference among the methods. However, the sucrose lysis buffer preserved samples extracted by the CTAB method were 15-43% more diverse than the other treatments.     

Effect of the electrical currents generated by the intestinal smooth muscle layers on pancreatic enzyme activity: an in vitro study

Gut enzymes in the small intestine are exposed to extremely low electrical currents (ELEC) generated by the smooth muscle. In the present study, the in vitro tests were undertaken to evaluate the effect of these electric currents on the activity of the proteolytic pancreatic digestive enzymes. A simulator generating the typical electrical activity of pig gut was used for these studies. The electric current emitted by the simulator was transmitted to the samples, containing enzyme and its substrate, using platinum plate electrodes. All samples were incubated at 37 degrees C for 1 h. The changes in optical density, corresponding to enzyme activity, in samples stimulated for 1 h with ELEC was compared with that not exposed to ELEC. The obtained results show that the electrical current with the characteristics of the myoelectrical migrating complex (MMC) has an influence on pancreatic enzyme activity. Increased endopeptidase and reduced exopeptidase activity was noticed in samples treated with ELEC. This observation can be of important as analyzed factors which can alter enzymatic activity of the gut, can thus also affect feed/food digestibility.     

No temperature acclimation of soil extracellular enzymes to experimental warming in an alpine grassland ecosystem on the Tibetan Plateau

Alpine grassland soils store large amounts of soil organic carbon (SOC) and are susceptible to rising air temperature. Soil extracellular enzymes catalyze the rate-limiting step in SOC decomposition and their catalysis, production and degradation rates are regulated by temperature. Therefore, the responses of these enzymes to warming could have a profound impact on carbon cycling in the alpine grassland ecosystems. This study was conducted to measure the responses of soil extracellular enzyme activity and temperature sensitivity (Q₁₀) to experimental warming in samples from an alpine grassland ecosystem on the Tibetan Plateau. A free air-temperature enhancement system was set up in May 2006. We measured soil microbial biomass, nutrient availability and the activity of five extracellular enzymes in 2009 and 2010. The Q₁₀ of each enzyme was calculated using a simple first-order exponential equation. We found that warming had no significant effects on soil microbial biomass C, the labile C or N content, or nutrient availability. Significant differences in the activity of most extracellular enzymes among sampling dates were found, with typically higher enzyme activity during the warm period of the year. The effects of warming on the activity of the five extracellular enzymes at 20 °C were not significant. Enzyme activity in vitro strongly increased with temperature up to 27 °C or over 30 °C (optimum temperature; T_opt). Seasonal variations in the Q₁₀ were found, but the effects of warming on Q₁₀ were not significant. We conclude that soil extracellular enzymes adapted to seasonal temperature variations, but did not acclimate to the field experimental warming.     

Kytococcus sedentarius,the organism associated with pitted keratolysis,produces two keratin-degrading enzymes

AIMS: To determine characteristics of the extracellular enzyme activity of Kytococcus sedentarius on human callus. METHODS AND RESULTS: A concentrate of a continuous culture supernatant fluid of K. sedentarius, which had callus-degrading activity, was subjected to a series of chromatographic purification procedures. The enzyme activity was found to be attributable to two proteases. These were capable of degrading both native callus and extracted keratin polypeptides and were purified to homogeneity, as shown by SDS-PAGE with silver staining. The enzymes P1 and P2 were 30 kDa and 50 kDa in size with isoelectric points of 4.6 and 2.7, respectively. The optimum conditions for callus-degrading activity were 40 degrees C, pH 7.1 for P1 and 50 degrees C, pH 7.5 for P2. P2 displayed increased activity in the presence of 800 mmol l(-1) NaCl and both enzymes were inhibited by PMSF (1 mmol(-1) Phenylmethylsulphoryl fluoride) and 1 mmol l(-1) EDTA. The main enzyme cleavage sites were Lys-Trp, Val-Lys, Gly-Asp and Asp-Arg, as determined after incubation of P1 and P2 with the beta-chain of insulin. CONCLUSIONS: K. sedentarius produces two extracellular enzymes that independently degrade natural, insoluble human callus. Both enzymes are serine proteases and have cleavage preference sites that are present in a range of human keratins. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification, in K. sedentarius cultures, of two enzymes which can degrade human callus strengthens the hypothesis that this organism is responsible for the pitting in human epidermis observed in pitted keratolysis. These enzymes may be of commercial use in the biodegradation of a range of keratin polymers, biological washing powders and in the treatment of unwanted callus on human skin.     

Persistence of microbial extracellular enzymes in soils under different temperatures and water availabilities

Microbial extracellular enzyme activity (EEA) is critical for the decomposition of organic matter in soils. Generally, EEA represents the limiting step governing soil organic matter mineralization. The high complexity of soil microbial communities and the heterogeneity of soils suggest potentially complex interactions between microorganisms (and their extracellular enzymes), organic matter, and physicochemical factors. Previous studies have reported the existence of maximum soil EEA at high temperatures although microorganisms thriving at high temperature represent a minority of soil microbial communities. To solve this paradox, we attempt to evaluate if soil extracellular enzymes from thermophiles could accumulate in soils. Methodology at this respect is scarce and an adapted protocol is proposed. Herein, the approach is to analyze the persistence of soil microbial extracellular enzymes at different temperatures and under a broad range of water availability. Results suggest that soil high‐temperature EEA presented longer persistence than enzymes with optimum activity at moderate temperature. Water availability influenced enzyme persistence, generally preserving for longer time the extracellular enzymes. These results suggest that high‐temperature extracellular enzymes could be naturally accumulated in soils. Thus, soils could contain a reservoir of enzymes allowing a quick response by soil microorganisms to changing conditions. This study suggests the existence of novel mechanisms of interaction among microorganisms, their enzymes and the soil environment with relevance at local and global levels.     

Microplate enzyme assay for screening lipoxygenases to degrade wood extractives

Lipoxygenases are non-heme iron-containing dioxygenases, capable of catalyzing the oxidation of unsaturated fatty acids. The enzyme has the potential to degrade problematic wood extractives in the paper-making process. However, commercially available lipoxygenase is currently too expensive for this application. A 96-well UV microplate assay was developed to screen enzymes from fungal sources for a more cost-effective alternative lipoxygenase. The substrate used for this assay was linoleic acid, a predominant fatty acid in wood. The enzyme activity and reaction kinetics determined by this microplate assay were compared to those obtained from a conventional bench scale assay. A number of hydrolytic enzymes and other oxidases were also tested using this protocol, to examine the specificity of the assay. The results show that the microplate assay developed can provide an inexpensive method for accelerated screening of a large number of enzymes to identify potential oxidative enzymes with specific action in degrading wood extractives.     

Microplate enzyme assay for screening lipoxygenases to degrade wood extractives

Lipoxygenases are non-heme iron-containing dioxygenases, capable of catalyzing the oxidation of unsaturated fatty acids. The enzyme has the potential to degrade problematic wood extractives in the paper-making process. However, commercially available lipoxygenase is currently too expensive for this application. A 96-well UV microplate assay was developed to screen enzymes from fungal sources for a more cost-effective alternative lipoxygenase. The substrate used for this assay was linoleic acid, a predominant fatty acid in wood. The enzyme activity and reaction kinetics determined by this microplate assay were compared to those obtained from a conventional bench scale assay. A number of hydrolytic enzymes and other oxidases were also tested using this protocol, to examine the specificity of the assay. The results show that the microplate assay developed can provide an inexpensive method for accelerated screening of a large number of enzymes to identify potential oxidative enzymes with specific action in degrading wood extractives.     

Complexation,Stabilization, and UV Photolysis of Extracellular and Surface-Bound Glucosidase and Alkaline Phosphatase: Implications for Biofilm Microbiota

Biofilm-produced and commercially-purified a- and b-glucosidase and alkaline phosphatase were subjected to different spectral portions of natural and artificial light and exposed to various humic substances to elucidate their impact on enzyme activities. Photochemical degradation of all enzymes occurred under different portions of the light spectrum. UVB irradiance produced the greatest overall photochemical degradation of enzymes, with significant rates occurring with UVA and PAR irradiance. The complexation of enzymes with humic substances resulted in inhibition, stabilization, and photochemical protection of the enzyme. Inhibition of enzyme activity occurred via reductions in overall enzyme activity in the presence of humic substances. However, humic-enzyme complexation also resulted in stabilization by restricting enzyme degradation while retaining high activities. Enzymes exposed to natural and artificial light sources had significantly lower reductions in enzyme activities in the presence of humic substances, which indicates that humic-enzyme complexes may protect enzymes from light-induced photochemical degradation. Bacterial surface-bound a- and b-glucosidase activities were significantly reduced in the presence of humic substances. Photosynthetically induced pH changes within biofilm communities can cause large reductions in a- and b-glucosidase activities while enhancing the hydrolytic activity of alkaline phosphatase.     

Soil minerals and humic acids alter enzyme stability: implications for ecosystem processes

In most ecosystems, the degradation of complex organic material depends on extracellular enzymes produced by microbes. These enzymes can exist in bound or free form within the soil, but the dynamics of these different enzyme pools remain uncertain. To address this uncertainty, I determined rates of enzyme turnover in a volcanic soil with and without added enzymes. I also tested whether or not soil minerals and humic acids would alter enzyme activity. In soils that were gamma-irradiated to stop enzyme production, 35–70% of the enzyme activity was stable throughout the 21-day incubation. The remaining enzyme fraction decayed at rates ranging from − 0.032 to − 0.628 day⁻¹. In both the irradiated soils and in soils with added enzymes, addition of the mineral allophane had a strong positive effect on most enzyme activities. Another added mineral, ferrihydrite, had a weak positive effect on some enzymes. Added humic acids strongly inhibited enzyme activity. These findings suggest that minerals, especially allophane, enhance potential enzyme activities in young volcanic soils. However, the actual activity and function of these enzymes may be low under field conditions if stabilization results in less efficient enzyme-substrate interactions. If this is the case, then much of the measured enzyme activity in bulk soil may be stabilized but unlikely to contribute greatly to ecosystem processes.     

Crude and purified proteasome activity assays are affected by type of microplate

Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate.     

An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid,and its application in finding inhibitors which interact directly with the enzyme

Abstract

Myeloperoxidase (MPO) is a pro-oxidant enzyme involved in inflammation, and the measurement of its activity in biological samples has emerged essential for laboratory and clinical investigations. We will describe a new method which combines the SIEFED (specific immunological extraction followed by enzymatic detection) and ELISA (ELISAcb) techniques to measure the active and total amounts of MPO on the same human sample and with the same calibration curve, as well as to define an accurate ratio between both the active and total forms of the enzyme.

The SIEFED/ELISAcb method consists of the MPO extraction from aqueous or biological samples by immobilized anti-MPO antibodies coated onto microplate wells. After a washing step to eliminate unbound material, the activity of MPO is measured in situ by adding a reaction solution (SIEFED). Following aspiration of the reaction solution, a secondary anti-MPO antibody is added into the wells and the ELISAcb test is carried out in order to measure the total MPO content.

To validate the combined method, a comparison was made with SIEFED and ELISA experiments performed separately on plasma samples isolated from human whole blood, after a neutrophil stimulation.

The SIEFED/ELISAcb provides a suitable tool for the measurement of specific MPO activity in biological fluids and for the estimation of the inhibitory potential of a fluid. The method can also be used as a pharmacological tool to make the distinction between a catalytic inhibitor, which binds to MPO and inhibits its activity, and a steric inhibitor, which hinders the enzyme and prevents its immunodetection.     

我国主要蜜环菌生物种胞外酶活性对温度的种特异性响应

蜜环菌生物种与天麻的共生关系受到温度的调节。为了揭示我国蜜环菌生物种的温度适应性,本研究以我国8个蜜环菌生物种为实验材料,采用2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐（ABTS）法和3,5-二硝基水杨酸（DNS）法研究了胞外漆酶、果胶酶和纤维素酶的活性对温度的响应特征。结果表明,各生物种的胞外酶活性存在显著性差异,各胞外酶活性具有随时间先升后降的变化趋势;各胞外酶的分泌时间和活性大小对温度的响应存在着很大的差异;且在培养过程中,首先分泌的是果胶酶和纤维素酶,然后才是漆酶。温度对蜜环菌胞外漆酶和果胶酶活性大小有影响,对产酶时间没有影响;对纤维素酶活性达到峰值的时间和活性大小都有很大影响。除此之外,蜜环菌胞外酶活性对温度的响应和我国蜜环菌的地域分布特点具有相关性,分布于秦岭-淮河线以南和以北的蜜环菌生物种胞外酶活性对温度的响应具有差异。本研究对开展蜜环菌生物学和我国天麻的野外种植区划的研究具有指导意义。     

Inhibitor fingerprinting of metalloproteases using microplate and microarray platforms: an enabling technology in Catalomics

One of the most fundamental properties of an enzyme is its selectivity, a property that has proved highly challenging to understand. Recent developments offer methodologies to rapidly establish activity-dependent profiles of enzymes. In this protocol, we describe methods to elucidate inhibitor fingerprints of enzymes. By taking advantage of well-defined small-molecule inhibitor libraries and the screening throughput offered from microplate and microarray platforms, we provide step-by-step application of the methodology toward the global characterization of metalloproteases, an important class of enzymes involved in numerous diseases and cellular processes. The same strategy is nonetheless applicable to virtually any given enzyme class, provided suitable experimental design and chemical inhibitor libraries are carefully implemented. We are able to routinely fingerprint as many as 2,000 independent enzyme interactions on the microplate platform within a span of approximately 7 h; however, the same throughput is attained within 5 h on the microarray platform.     

嗜盐古菌几种常见胞外酶研究进展

嗜盐古菌是一类生活于极端高盐环境的化能异养型原核微生物,其所分泌的胞外酶(外泌酶)具有在高盐条件下仍能保持活性的特点,在制革工业、高盐有机废水处理和泡菜加工等腌制食品方面发挥重要用途。本文对嗜盐古菌的胞外蛋白酶、淀粉酶、酯酶等几种常见胞外酶的来源和基本酶学性质的最新研究进展进行综述,为更好地开发利用嗜盐古菌胞外酶资源提供参考。     

Microplate diffusion assay for screening of beta-glucanase-producing microorganisms

A method is described to screen fungal strains rapidly for overexpression of extracellular beta-1,4-endoglucanase in the presence of high levels of sugar compounds. The semi-quantitative assay utilizes microplates in a 96-well format and an azurine dye covalently cross-linked (AZCL) chromogenic substrate. The digestion of AZCL-hydroxyethyl-beta-1,4-endoglucanase results in the release of a blue dye directly proportional to the amount of enzyme activity present in the sample. Sample absorbance was read at 590 nm. and the enzyme activity was determined by reference to a standard curve. The results from the microplate diffusion assay were similar to the results derived from the Ostazin Brilliant Red-hydroxyethyl cellulose assay. The technique described allowed the rapid comparison and screening beta-1,4-glucanase activity directly in spent fungal supernatant, from cultures grown in potato dextrose broth. The method could also be easily adapted for the screening of the presence of other activities such as beta-1,3-glucanase activity by using either AZCL-beta-glucan or AZCL-pachyman in place of the AZCL-hydroxyethyl-cellulose. This assay could be used to measure supernatant within an activity range of 0.1-2 U/mL