Miniaturization of hydrolase assays in thermocyclers

Lucena, Severino A.; Moraes, Caroline S.; Costa, Samara G.; Souza, Wanderley de; Garcia, Elói S.; Genta, Fernando Ariel

doi:10.1016/j.ab.2012.10.032

Cited by 10 publications

(6 citation statements)

References 14 publications

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“…The assay, therefore, has to be suitable for use in a 96-well plate design. The general advantages of this miniaturization from single reaction tubes to 96-well plates have already been shown for the DNSA and other colorimetric assays [14]. All assays used in this study were suitable for the 96-well plate format.…”

Section: Resultsmentioning

confidence: 89%

A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes

Herlet

Kornberger

Roessler

et al. 2017

Biotechnol Biofuels

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BackgroundGlycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzymes are generally determined by two separate measurements. However, these lead to a two-dimensional assessment of the pH range at one temperature (and vice versa) and do not allow prediction of the relative enzymatic performance at any pH/temperature combination of interest. In this work, we demonstrate a new method that is based on experimental data and visualizes the relationship among pH, temperature, and activity at a glance in a three-dimensional contour plot.ResultsIn this study, we present a method to determine the relative activity of an enzyme at 96 different combinations of pH and temperature in parallel. For this purpose, we used a gradient PCR cycler and a citrate–phosphate-based buffer system in microtiter plates. The approach was successfully tested with various substrates and diverse assays for glycoside hydrolases. Furthermore, its applicability was demonstrated for single enzymes using the endoglucanase Cel8A from Clostridium thermocellum as well as the commercially available complex enzyme mixture Celluclast®. Thereby, we developed a fast and adaptable method to determine simultaneously both pH and temperature ranges of enzymes over a wide range of conditions, an easy transformation of the experimental data into a contour plot for visualization, and the necessary controls. With our method, the suitability of an enzyme or enzyme mixture for any chosen combination of temperature and pH can easily be assessed at a glance.ConclusionsWe propose a method that offers significant advantages over commonly used methods to determine the pH and temperature ranges of enzymes. The overall relationship among pH, temperature, and activity is visualized. Our method could be applied to evaluate exactly what conditions have to be met for optimal utilization of an enzyme or enzyme mixture for both lab-scale and industrial processes. Adaptation to other enzymes, including proteases, should be possible and the method may also lead to a platform for additional applications, such as inactivation kinetics analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0923-9) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 89%

A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes

Herlet

Kornberger

Roessler

et al. 2017

Biotechnol Biofuels

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“…L9634) in a thermocycler with a modified bicinchoninic acid reagent according to ref. [ 28 ]. All assays were performed at 30°C under conditions such that activity was proportional to protein concentration and time.…”

Section: Methodsmentioning

confidence: 99%

Digestion of Yeasts and Beta-1,3-Glucanases in Mosquito Larvae: Physiological and Biochemical Considerations

et al. 2016

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Aedes aegypti larvae ingest several kinds of microorganisms. In spite of studies regarding mosquito digestion, little is known about the nutritional utilization of ingested cells by larvae. We investigated the effects of using yeasts as the sole nutrient source for A. aegypti larvae. We also assessed the role of beta-1,3-glucanases in digestion of live yeast cells. Beta-1,3-glucanases are enzymes which hydrolyze the cell wall beta-1,3-glucan polyssacharide. Larvae were fed with cat food (controls), live or autoclaved Saccharomyces cerevisiae cells and larval weight, time for pupation and adult emergence, larval and pupal mortality were measured. The presence of S. cerevisiae cells inside the larval gut was demonstrated by light microscopy. Beta-1,3-glucanase was measured in dissected larval samples. Viability assays were performed with live yeast cells and larval gut homogenates, with or without addition of competing beta-1,3-glucan. A. aegypti larvae fed with yeast cells were heavier at the 4th instar and showed complete development with normal mortality rates. Yeast cells were efficiently ingested by larvae and quickly killed (10% death in 2h, 100% in 48h). Larvae showed beta-1,3-glucanase in head, gut and rest of body. Gut beta-1,3-glucanase was not derived from ingested yeast cells. Gut and rest of body activity was not affected by the yeast diet, but head homogenates showed a lower activity in animals fed with autoclaved S. cerevisiae cells. The enzymatic lysis of live S. cerevisiae cells was demonstrated using gut homogenates, and this activity was abolished when excess beta-1,3-glucan was added to assays. These results show that live yeast cells are efficiently ingested and hydrolyzed by A. aegypti larvae, which are able to fully-develop on a diet based exclusively on these organisms. Beta-1,3-glucanase seems to be essential for yeast lytic activity of A. aegypti larvae, which possess significant amounts of these enzyme in all parts investigated.

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“…no. L9634) in a thermocycler with a modified bicinchoninic acid reagent (Lucena et al, 2013). The influence of pH in the enzymatic activity was studied using the buffers: sodium citrate (pH 3–7, 200 mM), EPPS (pH 7–9, 200 mM), AMPSO (pH 9–10, 200 mM) and CAPS (pH 10–11, 200 mM) with overlapping pH values to rule out possible inhibition by the buffering species.…”

Section: Methodsmentioning

confidence: 99%

Biochemical and Functional Characterization of Glycoside Hydrolase Family 16 Genes in Aedes aegypti Larvae: Identification of the Major Digestive β-1,3-Glucanase

et al. 2019

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Insect β-1,3-glucanases belong to Glycoside Hydrolase Family 16 (GHF16) and are involved in digestion of detritus and plant hemicellulose. In this work, we investigated the role of GHF16 genes in Aedes aegypti larvae, due to their detritivore diet. Aedes aegypti genome has six genes belonging to GHF16 (Aae GH16.1 – Aae GH16.6), containing two to six exons. Sequence analysis suggests that five of these GHF16 sequences (Aae GH16.1, 2, 3, 5, and 6) contain the conserved catalytic residues of this family and correspond to glucanases. All genomes of Nematocera analyzed showed putative gene duplications corresponding to these sequences. Aae GH16.4 has no conserved catalytic residues and is probably a β-1,3-glucan binding protein involved in the activation of innate immune responses. Additionally, Ae. aegypti larvae contain significant β-1,3-glucanase activities in the head, gut and rest of body. These activities have optimum pH about 5–6 and molecular masses between 41 and 150 kDa. All GHF16 genes above showed different levels of expression in the larval head, gut or rest of the body. Knock-down of AeGH16.5 resulted in survival and pupation rates lower than controls (dsGFP and water treated). However, under stress conditions, severe mortalities were observed in AeGH16.1 and AeGH16.6 knocked-down larvae. Enzymatic assays of β-1,3-glucanase in AeGH16.5 silenced larvae exhibited lower activity in the gut and no change in the rest of the body. Chromatographic activity profiles from gut samples after GH16.5 silencing showed suppression of enzymatic activity, suggesting that this gene codes for the digestive larval β-1,3-glucanase of Ae. aegypti . This gene and enzyme are attractive targets for new control strategies, based on the impairment of normal gut physiology.

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Miniaturization of hydrolase assays in thermocyclers

Cited by 10 publications

References 14 publications

A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes

A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes

Digestion of Yeasts and Beta-1,3-Glucanases in Mosquito Larvae: Physiological and Biochemical Considerations

Biochemical and Functional Characterization of Glycoside Hydrolase Family 16 Genes in Aedes aegypti Larvae: Identification of the Major Digestive β-1,3-Glucanase

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