Veara Loha

The effect of pH on the foam fractionation of β-glucosidase and cellulase

Abstract The surface tension–pH profile of β-glucosidase was established to determine its relationship to the corresponding profile of cellulase and to the foam fractionation of that cellulase. The goal of this work was to determine the optimal foaming points for both cellulase and cellobiase. This data may prove useful in the separation of certain components of cellulase, since the non-foaming hydrophilic β-glucosidase does not foam as well as the hydrophobic components of cellulase at low concentrations. A key finding from these experiments was that there are two local minima in the surface tension–pH trajectory for Trichoderma reesei cellulase, as contrasted to the usual single minimum. The lower of these minimum points corresponds to the cellulase isoelectric point. The double minimum surface tension–pH profile was also observed for cellobiase alone. The optimal foaming pH for cellobiase alone was determined to be around 10.5, while for cellulase it was between 6 and 9.

Modeling a Protein Foam Fractionation Process

A simple staged model for the protein foam fractionation process is proposed in this article. This simplified model does not detail the complex foam structure and gas-liquid hydrodynamics in the foam phase but, rather, is built on the conventional theoretical stage concept considering upward bubbles with entrained liquid and downward liquid (drainage) as counter-current flows. To simulate the protein concentration distribution in the liquid along the column by the model, the bubble size and liquid hold-up with respect to the position must be known, as well as the adsorption isotherm of the protein being considered. The model is evaluated for one stage by data from the semibatch foam fractionation of egg albumin and data from the continuous foam fractionation of bovine serum albumin. The effect of two significant variables (superficial gas velocity and feed protein concentration) on enrichment is well predicted by the model, especially for continuous operation and semibatch operation when initial concentration is high.

Preserving the activity of cellulase in a batch foam fractionation process.

Foam fractionation isone of the low operating-cost techniques for removing proteins from a dilute solution. The initial bulk solution pH and air superficial velocity play an importantrole in the foam-fractionation process. Denaturation of proteins (enzymes) can occur, however, during the foamfractionation process from the shear forces resulting from bursting air bubbles. At the extreme bulk solution pHs (lower than 3.0 and higher than 10.0), the en zymatic activity of cellulase in the foamate phase drops significantly. Within these two pH boundsan increase in the air superficial velocity, Vo, and a decrease in the bulk solution pH leads to a decrease in the separation ratio (SR), defined as theratio of the protein concentration in the foamate to the protein concentration in the residue. On the other hand, an increase in Vo provides a higher foamate-protein recovery. The process efficiency is defined as the product of foamate-protein recovery times the SR times the cellulase activity. The optimal operating condition of the cellulase foamfractionation process is taken into account at the maximum value of the processefficiency. In this study, that optimal condition is atan air superficial velocity of 32 cm/min and a bulk-solution pH of 10.0. At this condition, the recovered foamate is about 80% of the original protein mass, the SR is about 12, and the en zymatic activity is about 60% of the original cellulase activity.

The effect of pectinase on the bubble fractionation of invertase from α-amylase

Fermentation broth normally contains many extracellular enzymes of industrial interest. To separate such enzymes on-line could be useful in reducing the cost of recovery as well as in keeping their yield at a maximum level by minimizing enzyme degradation from broth proteases (either the desired enzymes or the proteases could be removed selectively or both removed together and then separated). Several large-scale separation methods are candidates for such on-line recovery such as ultrafiltration, precipitation, and two-phase partitioning. Another promising technique for on-line recovery is adsorptive bubble fractionation, the subject of this study. Bubble fractionation, like ultrafiltration, does not require contaminating additives and can complement ultrafiltration by preconcentrating the enzymes using the gases normally present in a fermentation process. A mixture of enzymes in an aqueous bubble solution can, in principle, be separated by adjusting the pH of that solution to the isoelectric point (pI) of each enzyme as long as the enzymes have different pIs. The model system investigated here is comprised of three enzyme separations and the problem is posed as the effect of pectinase (a charged enzyme) on the bubble fractionation of invertase (a relatively hydrophilic enzyme) from α-amylase (a relatively hydrophobic enzyme).The primary environmental variable studied, therefore, is the pH in the batch bubble fractionation column. Air was used as the carrier gas. This prototype mixture exemplifies an aerobic fungal fermentation process for producing enzymes. The enzyme concentration here is measured as total protein concentration by the Coomassie Blue (Bradford) solution method (1), both as a function of time and column position for each batch run. Since, from a previous study (2), it was found that invertase and α-amylase in a two-enzyme system can be partially separated in favor of one vs the other at two different pHs (pH 5.0 and 9.0) with significant separation ratios, emphasis is placed on the effect of pectinase at these pHs. In this study, the addition of pectinase reduced the total separation ratio of the α-amylase-invertase mixture at both pHs.

Batch foam fractionation of kudzu (Pueraria lobata) vine retting solution

The aqueous protein solution from kudzu (Pueraria lobata) vine retting broth, without the addition of other surfactants, was foam-fractionated in a vertical tubular column with multiple sampling ports. Time-varying trajectories of the total protein levels were determined to describe the protein behavior at six positions along the 1-m column. The lowest two trajectories of this batch process represented a loss of proteins from the bulk liquid and tended to merge and decay together in time; the other trajectories displayed a gain in proteins in the foam phase. These upper column port protein concentration trajectories generally increased in time up to 45 mm, followed by a decrease, reflecting the removal of proteins from the column ports. The foam became dryer as it passed up the column to the top port. The protein concentration was about 5-8x higher in the top port foam than in the initial bulk solution, mainly as a result of liquid drainage from the foam along the column axis. This concentration increase in the collected foam was dependent on the initial pH of the bulk solution. The mol-wt profile of the proteins in the concentrated foam effluent was determined by one-dimensional gel electrophoresis. An analysis of the gel electropherograms indicated that the most abundant proteins could be cellulases and pectinases.

Batch Foam Recovery of Sporamin from Sweet Potato

The major sweet potato root protein, sporamin (which comprises about 80-90% of the total protein mass in the sweet potato) easily foams in a bubble/foam-fractionation column using air as the carrier gas. Control of that foam fractionation process is readily achieved by adjusting two variables: bulk solution pH and gas superficial velocity. Varying these parameters has an important role in the recovery of sporamin in the foam. Changes in the pH of the bulk solution can control the partitioning of sporamin in the foam phase from that in the bulk phase. A change in pH will also affect the amount of foam generated. The pH varied between 2.0 and 10.0 and the air superficial velocities (V0) ranged between 1.5 and 4.3 cm/s. It was observed in these ranges that, as the pH increased, the total foamate volume decreased, but the foamate protein (mainly sporamin) concentration increased. On the other hand, the total foamate volume increased significantly as the air superficial velocity increased, but the foamate concentration decreased slightly. The minimum residual protein concentration occurred at pH 3.0 and V0 = 1.5 cm/s. On the other hand, the maximum protein mass recovery occurred at pH 3.0 and at V0 = 4.3 cm/s.

Partitioning invertase between a dilute water solution and generated droplets

Water droplets or mist occur naturally in the air at seashores. These water droplets carry inorganic and organic substances from the sea to the land via the air, creating fertile land in sandy coastal areas (1). The same phenomenon occurs in an air-fluidized bed bioreactor (2). In an air-fluidized bed reactor, proteins can be transferred from the bioreactor semisolid bulk phase to an enriched droplet phase. This protein transfer process (droplet fractionation) can be experimentally simulated by shaking a separatory funnel containing a dilute solution of a given protein, which can be an enzyme like invertase. The created droplets become richer in invertase (protein) than that of the original dilute solution. The droplets can then be coalesced by trapping them and recovering the concentrated protein in the new liquid phase. Typically, in such a droplet fractionation process a collected enzyme can be degraded in its ability to catalyze a chemical reaction. In this article, we explore whether the initial solution pH control variable can be adjusted to minimize the decrease of enzyme activity in this process. The protein droplet recovery problem is one in which the recovered amount of desired protein (enzyme) in the droplet is maximized, subject to the minimization of the enzyme activity loss. The partition coefficient, which is the ratio between the protein concentration in the droplets and the residual solution, is maximized at approx 4.8 and occurs at pH 3.0. Here, the partition coefficient for invertase decreases as the initial solution pH increases, between pH 3.0 and 8.0. Interestingly, the initial solution surface tension seems to be inversely proportional to the partition coefficient. The partition coefficient reaches a maximum value at a surface tension value of approx 63 mN/m at pH 3.0. The enzymatic activity of the initial, the residual, and the droplet solutions all decrease as the bulk solution pH increases. A decrease of enzymatic activity was observed in the residual bulk solution when compared with that in the initial bulk solution at all pH levels. Also, up to 90% of the invertase activity was lost in the droplets when compared to the initial bulk solution.

The effect of fermentation (retting) time and harvest time on kudzu (Pueraria lobata) fiber strength

The noncommercial kudzu plant has been growing wild in the southern United States since the 1930s. In this article, the kudzu fibrous vine is investigated for possible economic applications. The feasibility of removing the fibers by microbial retting is investigated as a low-cost method for recovery of these fibers. The harvest season, vine diameter, and the natural retting time are the three main variables investigated to determine the optimal fiber tensile strength. To extract these light yellow, unidirectional, and multicellular kudzu fibers, retting using a culture screened from a naturally occurring mixed culture was investigated. Late fall, winter, and spring retted fibers were subjected to tensile strength tests. The kudzu fibers were harvested from the wild in both Nashville, TN and Muscle Shoals, AL.

Sonic Wave Separation of Invertase from a Dilute Solution to Generated Droplets

It has previously been shown that a droplet fractionation process, simulated by shaking a separatory funnel containing a dilute protein solution, can generate droplets richer in protein than present in the original dilute solution. In this article, we describe an alternative method that can increase the amount of protein transferred to the droplets. The new method uses ultrasonic waves, enhanced by a bubble gas stream to create the droplets. The amount of protein in these droplets increases by about 50%. In this method, the top layer of the dilute protein solution (of the solution-air interface) becomes enriched in protein when air is bubbled into the solution. This concentrating procedure is called bubble fractionation. Once the protein has passed through the initial buildup, this enriched protein layer is transferred into droplets with the aid of a vacuum above the solution at the same time that ultrasonic waves are introduced. The droplets are then carried over to a condenser and coalesced. We found that this new method provides an easier way to remove the protein-enriched top layer of the dilute solution and generates more droplets within a shorter period than the separatory funnel droplet generation method. The added air creates the bubbles and carries the droplets, and the vacuum helps remove the effluent airstream from the condenser. The maximum partition coefficient, the ratio of the protein concentration in the droplets to that in the residual solution (approx 8.5), occurred at pH 5.0.