4.2 Methods
4.2.4 Characterization of Enzymes
4.2.3.7 SDS PAGE
SDS-PAGE, more precisely SDS polyacrylamide gel electrophoresis, is a discontinuous electrophoresis method for analysis of proteins. Dividing the gel into a resolving gel with smaller pores and a stacking gel with larger pores is the reason for discontinuity. Proteins form stacks in the stacking gel in order of their mobility, but later they destack and separate according to electrophoresis principles in the resolving gel. Addition of SDS, sodium dodecyl sulphate, to the polyacrylamide gel renders all proteins negatively charged, therefore prevents the migration of positively charged proteins towards the cathode and allows the proteins separation to be based exclusively on molecular weight. (Westermeier, 2005) In this project SDS-PAGE was performed to evaluate the purity of the produced enzyme solutions before and after chromatographic purification. This was accomplished by exposing ready-to-use Mini-PROTEAN® TGX™ Precast Gels from BIO-RAD to 200 V and 0.3 A. The standard used to provide a reference for the molecular weight of proteins was BIO-RAD Precision Plus Protein™ Unstained Protein Standard (Figure 15).
Figure 15 Precision Plus Protein Unstained Protein Standard, BIO-RAD
4.2.4 Characterization of Enzymes
sample was diluted with RO-H2O in a way that the final protein concentration was between 0.2 and 0.8 mg/mL. The absorption of each sample after incubation was measured at a wavelength of 595 nm.
4.2.4.1.2 Activity assays
Enzyme activity assays require precisely standardized conditions, as stated below, to provide reproducible and comparable data.
4.2.4.1.2.1 Laccase activity assay using ABTS
This assay was used to determine the activity in crude extracts as well as purified preparations of the enzymes BliLac, BaLac and Cgl1. The reaction is detected spectrophotometrically by the oxygen dependent oxidation of the electron donor ABTS to its green cation radical. The diammonium salt of ABTS was purchased from amresco® (Solon, USA). The enzyme sample had to be added at an appropriate dilution (approximately 0.1-2 U/mL). One unit of laccase activity was defined as the amount of laccase required to obtain 1 µmol of product (ABTS+) per minute.
Table 32 Pipetting scheme for Laccase activity assay
Reagent Volume [µL] Reaction conditions
ABTS (10 mM) 100
The ABTS solution can be stored on ice for only 12 hours.
ABTS and buffer were incubated for 15 minutes at 30°C in a water bath. Adding the sample started the reaction and the increase in absorption was recorded for 150 seconds at 420 nm and by using an enzyme activity factor of 83.333298.
50 mM sodium citrate buffer, pH 5.5 880
Laccase solution (diluted) 20
4.2.4.1.2.2 Carbohydrate oxidase activity assay using ABTS
This assay was used to determine the activity of the enzymes PcPOx and PnGOx in crude extracts as well as purified preparations. The reaction is detected by the oxygen dependent oxidation of D-glucose with concomitant production of H2O2 from O2. H2O2 subsequently oxidizes the chromogene ABTS in a peroxidase-catalyzed reaction, resulting in the formation of its green radical, which is detected at a wavelength of 420 nm using a spectrophotometer.
Figure 16 Carbohydrate oxidase assay principle (Danneel et al., 1992)
The diammonium salt of ABTS was purchased from amresco® (Solon, USA). The enzyme sample had to be added at an appropriate dilution (approximately 0.005-0.02 U/mL). One unit of either PcPOx or PnGOx activity was defined as the amount of enzyme necessary for oxidation of 2 µmol of ABTS, which is equivalent to the oxidation of 1 µmol of D-glucose per minute at the conditions described above (Danneel et al., 1992). pH 5.5 was chosen for the standard enzyme characterization as the average of the initial and final pH during sourdough fermentation (approximately pH 5 at the beginning and pH 6 in the end).
Table 33 Pipetting scheme for carbohydrate oxidase activity assay
Reagent Volume [µL] Reaction conditions
1 mM ABTS solution, pH 5.5
(with 5.7 U/mL of peroxidase) 970 ABTS and D-Glucose solution were incubated for 15 minutes at 30°C in a water bath. The reaction was then started by adding the sample and the rise in absorption was recorded for 180 seconds at 420 nm and by using an enzyme activity factor of 69.444443.
1 M D-Glucose, pH 5.5 20
P2O or GOx solution (diluted) 10
4.2.4.1.3 Temperature optimum and stability
The temperature optimum and stability of PcPOx were determined, as Pisanelli et al. (2009) did not conduct those experiments for the heterologous expression of the PcPOx gene in E.coli.
Artolozaga et al. (1997) found a temperature optimum of 55°C for the native enzyme at pH 8.
For the temperature optimum experiment the activity of PcPOx was examined using the carbohydrate oxidase activity assay at pH 5.5 and incubation at various temperatures between 30°C and 80°C in equidistant steps and with additional measuring points close to the supposed optimum. An additional cuvette with water was incubated alongside the cuvettes filled with assay reagents to enable manually checking the actual temperature after incubation with a thermometer. However, for the thermostability experiments the PcPOx solution (7.0 U/mg) was incubated constantly at either 28°C, 37°C, 60°C, 70°C or 80°C and aliquots of the differently incubated enzyme solutions were taken at appropriate time intervals. Those aliquots were
analyzed using a P2O activity assay at 30°C and pH 5.5, as described above. The incubation at 37°C was chosen as it is the physiological temperature of higher organisms and thus the working temperature of many enzymes; this temperature is interesting as it is frequently used in other publications, so obtained results within this work should be more easily comparable with results from other working groups. The incubation was at 28°C as the 30°C incubator was fixed to this temperature. Nevertheless, one can deduce the behavior of enzymes at 30°C sourdough fermentation temperature from the present measurements at 28°C.
4.2.4.2 Rheological Characterization
The measurements were carried out with a rye arabinoxylan standard solution of 2% (w/v), to which various amounts of different oxidases and D-Glucose, if required as electron donor, were added. If the amount of added enzyme solution would have caused a significant dilution effect (BliLac, CDH Oxyplus and PnGOx) and therefore would have changed the concentration of the arabinoxylan standard, a higher concentrated WEAX standard solution, e.g. 4%, was prepared to yield a 2% WEAX solution after enzyme addition. Following Carvajal-Millan et al. (2005), AX solution supplemented with enzyme was at first put on ice to prevent cross-linking prior to recording of the reaction. The solution was then poured onto the measuring area of a Kinexus Rotational Rheometer, which was also maintained at 4°C. A cone/plate measuring geometry (CP1/60) was used. WEAX gelation was started by a sudden increase of temperature from 4°C to 25°C and was monitored at 25°C for 5000 seconds recording the storage (G’) and loss (G’’) moduli. Small amplitude oscillatory shear was used to follow the gelation process of the rye arabinoxylan standard solution. Although the temperature in the fermentation chamber will be 30°C, a measuring temperature of 25°C was chosen as the core of the dough usually has 25 to 26°C and as dough can only be heated slowly. A constant strain of 10% and constant frequency of 1 Hz were applied throughout the measurement. The height of the measuring gap was set at 0.2 mm.