Vorakan Burapatana

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.

Effects of acid and alkali promoters on compressed liquid hot water pretreatment of rice straw.

In this study, effects of homogeneous acid and alkali promoters on efficiency and selectivity of LHW pretreatment of rice straw were studied. The presences of acid (0.25%v/v H2SO4, HCl, H3PO4, and oxalic acid) and alkali (0.25 w/v NaOH) efficiently promoted hydrolysis of hemicellulose, improved enzymatic digestibility of the solids, and lower the required LHW temperature. Oxalic acid was a superior promoter under the optimal LHW conditions at 160 °C, leading to the highest glucose yield from enzymatic hydrolysis (84.2%) and the lowest formation of furans. Combined with hydrolyzed glucose in the liquid, this resulted in the maximal 91.6% glucose recovery from the native rice straw. This was related to changes in surface area and crystallinity of pretreated biomass. The results showed efficiency of external promoters on increasing sugar recovery and saving energy in LHW pretreatment.

Enhancing Cellulase Foam Fractionation with Addition of Surfactant

Foam fractionation cannot be used to recover cellulase from an aerated water solution effectively because cellulase by itself can produce only a small amount of foam. The addition of a surfactant can, however, increase the foamate volume and enhance the concentration of cellulase. We studied three detergents individually added to a 200 mg/L cellulase solution to promote foaming. These detergents were anionic, cationic, and nonionic surfactants, respectively. Although contributing to foam production, it was observed that nonionic surfactant (Pluronic F-68) barely concentrated cellulase, leaving the enrichment ratio unchanged, near 1. With anionic surfactant, sodium dedecyl sulfate, and cationic surfactant, cetyltrimethylammonium bromide (CTAB), the enrichment ratio became much larger, but cellulase denaturation occurred, reducing the activity of the enzyme. When CTAB was used to help foam cellulase, beta-cyclodextrin was subsequently added to the foamate to help restore the enzyme activity.

A Proposed Mechanism for Detergent-Assisted Foam Fractionation of Lysozyme and Cellulase Restored With β-Cyclodextrin

Foam fractionation by itself cannot effectively concentrate hydrophilic proteins such as lysozyme and cellulase. However, the addition of a detergent to a protein solution can increase the foam volume, and thus, the performance of the foam fractionation process. In this article, we propose a possible protein concentration mechanism of this detergent-assisted foam fractionation: A detergent binds to an oppositely charged protein, followed by the detergent-protein complex being adsorbed onto a bubble during aeration. The formation of this complex is inferred by a decrease in surface tension of the detergent-protein solution. The surface tension of a solution with the complex is lower than the surface tension of a protein or a detergent solution alone. The detergent can then be stripped from the adsorbed protein, such as cellulase, by an artificial chaperone such as β-cyclodextrin. Stripping the detergent from the protein allows the protein to return to its original conformation and to potentially retain all of its original activity following the foam fractionation process. Low-cost alternatives to β-cyclodextrin such as corn dextrin were tested experimentally to restore the protein activity through detergent stripping, but without success.

Degeneration of β-glucosidase activity in a foam fractionation process

Foam fractionation is a promising technique for concentrating proteins because of its simplicity and low operating cost. One such protein that can be foamed is the enzyme cellulase. The use of inexpensively purified cellulase may be a key step in the economical production of ethanol from biomass. We conducted foam fractionation experiments at total reflux using the cellulase component β-glucosidase to study how continuous shear affects β-glucosidase in a foam such as a fermentation or foam fractionation process. The experiments were conducted at pH 2.4, 5.4, and 11.6 and airflow rates of 3, 6, 15, 20, and 32 cc/min to determine how β-glucosidase activity changes in time at these different conditions. This is apparently a novel and simple way of testing for changes in enzyme activity within a protein foam. The activity did not degenerate during 5 min of reflux at pH 5.4 at an airflow rate of 10 cc/ min. It was established that at 10 min of refluxing, the β-glucosidase denatured more as the flow rate increased. At pH 2.4 and a flow rate of 10 cc/min, the activity remained constant for at least 15 min.

Effect of β-cyclodextrin in artificial chaperones assisted foam fractionation of cellulase

Foam fractionation has the potential to be a low-cost protein separation process; however, it may cause protein denaturation during the foaming process. In previous work with cellulase, artificial chaperones were integrated into the foam fractionation process in order to reduce the loss of enzymatic activity. In this study, other factors were introduced to further reduce the loss of cellulase activity: type of cyclodextrin, cyclodextrin concentration, dilution ratio cyclodextrin to the foamate and holding time. alpha-Cyclodextrin was almost as effective as beta-cyclodextrin in refolding the foamed cellulase-Cetyltrimethylammonium bromide mixture. beta-Cyclodextrin (6.5 mM) was almost as effective as 13 mM beta-cyclodextrin in refolding. The dilution ratio, seven parts foamate and three parts beta-cyclodextrin solution, was found to be most effective among the three ratios tested (7:3, 1:1, and 3:7). The activity after refolding at this dilution ratio is around 0.14 unit/mL. The refolding time study showed that the refolding process was found to be most effective for the short refolding times (within 1 h).

A Comparison of the Activity Reduction Occurring in Two Detergent‐Assisted Protein (Cellulase and Lysozyme) Foam Fractionation Processes

Abstract Foam fractionation has the potential to be an inexpensive alternative to current protein drug concentration or separation methods; however, it has a few drawbacks. One is the fact that not all proteins form a foam layer when aerated at low concentrations. The other is the possible protein denaturation caused during the foaming process. Adding a detergent to the nonfoaming protein solution causes it to foam when aerated. Here, cellulase and lysozyme are studied as model proteins in this process. By themselves, both cellulase and lysozyme solutions hardly form a foam layer when aerated at concentrations below 1000 mg/L (1000 ppm). The addition of 100 mg/L of cetyltrimethylammonium bromide (CTAB) to a 200 mg/L cellulase solution increases the foam volume and makes it possible to almost quadruple (relative to the initial bulk concentration) the concentration of the resulting cellulase foam solution. The foaming, however, reduces the cellulase activity. Diluting the foam with β‐cyclodextrin regains some of the lost activity because β‐cyclodextrin strips CTAB away from the cellulase, which allows the cellulase to refold to its native state. CTAB detergent does not work well with lysozyme, but the addition of SDS detergent leads to a tripling of the concentration of lysozyme solution without any reduction in enzymatic activity.

Effect of Lidocaine on Ovalbumin and Egg Albumin Foam Stability

Foam fractionation is a simple separation process that can remove and concentrate hydrophobic molecules such as proteins, surfactants, and organic wastes from an aqueous solution. Bovine serum albumin and ovalbumin have been widely used as model proteins due to their strong foaming potential and low price. Here, we study the effect of lidocaine on albumin foam, since drugs like lidocaine are known to bind with albumin. We observed that lidocaine not only enhances the amount of foam produced but also the stability of that foam as well. The foam stability was evaluated as the decay rate constant of the foam, determined from a change in height (or volume) of the foam over a given time period.

Breathing Air from Protein Foam

Protein foams can be used to extinguish fires. If foams are to be used to extinguish fires where people are present, such as in high-rise buildings or ships, then a method for allowing people to breathe in a foam-filled environment is needed. It is proposed that the air, used to create the foam be used for breathing. A canister that will break incoming air-filled foam has been designed for attachment to a standard gas mask, in order to provide breathable air to a trapped person. Preliminary results for the modified mask indicate feasibility of breathing air from air-filled protein foam.