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

Abstract

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.

Crosstalk of Cellulose and Mannan Perception Pathways Leads to Inhibition of Cellulase Production in Several Filamentous Fungi

“…The optimal β-mannopyranosidase activity of GH2-1 at different combinations of temperatures and pHs, in parallel, was assayed according to the setup used before (74) with modifications. The reactions consisted of 50 mM KP buffer at different pHs (pH 5, 5.5, 6, 6.5, 7.5, and 8), 80 μg 4-nitrophenyl-β- d -mannopyranoside (O-PNPBM; Megazyme, Ireland), and 0.0025 μg of purified enzyme.…”

pH profiles of cellulases depend on the substrate and architecture of the binding region

“…To enable activity and stability measurements at different pH (but otherwise similar conditions) we prepared mixed buffers of citrate and phosphate (so‐called McIlvaine buffer). This system is expedient due to its wide buffer range and low‐temperature dependence (Herlet et al, ). Eleven solutions with pH 2–7 were made with 0.5 pH units interval by mixing 0.1 M citric acid (VWR chemicals, product no.…”

“…Clarification of pH effects on saccharification will require both extensive empirical work under process relevant conditions and a better fundamental understanding of the enzymatic process. Progress in the former area was recently achieved as Herlet et al () introduced an effective methodology for screening pH and temperature effects on saccharification. In the current work, we address the latter and report systematic measurements of activity and stability of selected (monocomponent) cellulases, as a function of pH, temperature, as well as, the type and load of the substrate.…”

Addition of β-galactosidase boosts the xyloglucan degradation capability of endoglucanase Cel9D from Clostridium thermocellum

“…The total reaction volume of 1.2 ml comprised 1 ml 1% (w/v) tamarind XG substrate obtained from Megazyme (Bray, Ireland) solubilized in ddH 2 O, 100 µl citrate–phosphate buffer pH 6.4, prepared as described previously [28], and 100 µl of the respective diluted enzymes (Cel9D, 0.12–24 U; Bga2B, 0–155 mU). All components were mixed on ice before starting the assay reaction at 60 °C.…”

“…All components were mixed on ice before starting the assay reaction at 60 °C. Suitable reaction conditions for both enzymes were determined using pH vs. temperature plots as described previously [28]. Incubation was performed by shaking at 800 rpm at 60 °C for 6 h using a HLC MHR 23 thermomixer (Ditabis, Pforzheim, Germany).…”