Protein content was determinated using the method described by Bradford with bovine serum albumin as standard. Effects of pH and temperature on amylase activity. Amylase activity was measured at different pH and temperature values using starch as substrate.
B.Y. Zaslavsky: Aqueous Two‐Phase Partitioning – Physical Chemistry and Bioanalytical Applications, Marcel Dekker, Inc., New York, Basel, Oxford, ISBN. Aqueous two‐phase partitioning. Physical chemistry and bioanalytical applications. Edited by Boris Y. Zaslavsky, Marcel Dekker, Inc.; New York.
Preparation of the two-phase systems. All the systems, in duplicate, were prepared in graduated centrifuge tubes. The PEG- phosphate salts systems were assumed to be close the original phosphate stock solution pH 6. After separation of the phases by centrifugation for x g, samples of each phase were taken with a pipette for the quantification protein and activity of enzyme. The study of enzyme partitioning at different tie lines was carried out varying the PEG and salts concentrations as show on the Table 1. Determination of the partition coefficient K : The relative distribution of the enzyme in the aqueous two-phase system is characterized by the partition coefficient K , calculated according to the following equation:.
Where C t stands for enzyme activity in the top phase, C b enzyme activity in the bottom phase.
Determination of the purification factor. The purification factor PF is defined as the ratio of the enzymatic specific activity of the crude extract by the enzymatic specific activity after purification. Determination of recovery enzymatic activity. The recovery enzymatic activity is defined as the ratio of the enzymatic activity after purification by enzymatic activity of crude extract multiplied by The development of purification procedure using aqueous two-phase systems involves the variation of several factors until a good result is achieved.
The partition coefficient, recovery of enzyme activity and factor of purification were studied to characterize the distribution of amylase between the two phases of the system.
The tip was immersed in PEG-rich phase containing clay covered glass slide. Vis, M. Mann, S. The application of this experimental approach FIG. Necklace-like shape tubes are observed for all three morphologies of the Lo vesicles. You are using a browser version with limited support for CSS. Use the link below to share a full-text version of this article with your friends and colleagues.
The enzyme partition in the ATPS depends on the size of the biomolecule, superficial properties, molecular load, ionic composition of the phases, polymer concentration and molecular length of the polymer Harries et al. Effect of polymer molecular weight. The partition coefficients showed no regular tendency in all the cases.
This could be attributed to different two effects. One of these effects was the increase in the upper phases hydrophobicty. In fact, as PEG chain length increased there were less hydroxyl groups for the same concentration of the polymer and hence the polymer-richer upper phase increased in hydrophobicy. On the other hand, the chain length increase also caused the increase of the excluded volume, which mean less space available for the protein in the PEG rich phase Marcos et al. Similar behavior was observed by Marcos et al. Effect of the tie line length. In all systems studied in this work at pH 7.
This effect was caused by the increasing PEG concentration witch cause continuous molecular exclusion of amylase from the top to bottom phases, following continuous re-equilibration of the levels in both phases. The same didn't occur in PEG system at pH 8. Study carried by Almeida et al. Ortin et al. Further studies with bovine serum albumin accomplished by Christian et al. Cunha et al. Table 2 , 3 and 4 show the effects of the tie line length in different PEG-phosphate system. Bottom phases, following continuous re-equilibration of the levels in both phases.
As general rule, negatively charged proteins prefer the upper PEG-rich phase and positively charged proteins partition to the lower phase Schmidt et al.
As the pH increases above the isoelectric point pI of a protein, it becomes negatively charged, its interaction with PEG becomes stronger Huddleston et al. However, in systems with the biggest molecular weight, an increase of the coefficient of partition occurred with the increase of pH Figs.
Same results were obtained by Xu et al. Wu et al. Increase of pH resulted a decrease of PF. Similar behavior was obtained in the PEG and at tie line 2 Table 2 and 3. However, except in the PEG at pH 8. In this system was observed recovery maximum of enzymatic activity In present work highest purification factor 5.
Effect of pH and temperature in amylase activity in crude extract and partially purified extract.
The profile of pH and temperature over the enzymatic activity of amylase were evaluated before and after purification. An optimal pH was obtained at pH 5. As shown in Fig. In general, these results were in agreement with studies carried out by Tigue et al. Albertsson, P. New York: Wiley. Nature , , Jo urnal of Chromatography B, , Analytical Biochemistry , 72 , World Journal of Microbiology and Biotechnology , 18 , Bioprocess Engineering , 23 , Journal Biotechnology , , Carbohydrate Polymers , 35 , Folia Microbiology , 37 , Microbiology Letters , , Bioseparation , 7 , Hardcover: Human.
Enzyme Microbiology Technolog y, 13 , Partitioning in aqueous two-phase system: theory, methods, uses, and application to biotechnology. New York: Academic. In: Fiechter, A. Advances in Biochemical Engineering: Reaction Engineering. New York: Spriger. Bioseparation , 3 : 4 , Dx contribution to total refractive index was subtracted and remaining refractive index was used to calculate the concentration of PEG in each phases.
In order to prevent charging during imaging the clay particles, which are nonconductive, we followed the method of Veghte et al. Then, sparse particles over the silicon wafer were imaged.
Samples were vortexed before imaging. Glass coverslips used in microscopy were silanized in gas phase with dichloromethylsilane and used with press-to-seal spacer Therefore we followed the method of Vis et al. Clay particles were deposited on glass slides by vertical deposition method Three-phase macroscopic contact angle between Dx-rich droplets in PEG-rich phase were imaged at least three times per sample, then the contact angle was measured from both left and right sides of the droplets with ImageJ Each measurement repeated at least three times.
Then they were immersed in hydrochloric acid and methanol mixture for an hour. Glass slides were rinsed well and dried with nitrogen. Only surfaces well-covered with clay over their entire surface were used. The tip was immersed in PEG-rich phase containing clay covered glass slide. Reaction progress was followed using an Agilent diode array UV-visible spectrometer with Agilent ChemStation software.
As the reaction progressed, the solution changed color to orange—brown Partitioning of reaction product was determined by measuring the absorbance in each phase. After mixing, phases were separated by centrifugation. The concentration of product in each phase was calculated by measuring absorbance of three samples for each phase using an Agilent diode array UV-visible spectrometer with Agilent ChemStation software. Absorbance values were corrected for each phase.
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