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

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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.

Introduction

Cellulases are a group of enzymes that hydrolyze cellulose to soluble sugars, including glucose. At least two steps in cellulose degradation by microorganisms begin with the preparatory prehydrolytic first step involving an enzyme (C1) which swells and/or hydrates anhydroglucose chains. The second step uses hydrolytic enzymes (Cx) and beta glucosidase (cellobiase).

Microorganisms including fungi, bacteria and actinomycetes produce three main types of cellulase components: endo-1,4-β-d-glucanase, exo-1,4-β-d-glucanase and β-glucosidase, either separately or in the form of a complex (Bhat and Bhat, 1997). The relative proportion of each component in the complex depends on the source of the cellulases (Bhat and Bhat, 1997). Each component may have different isoforms, which are different in their three dimensional structures. For example, Trichoderma reesei cellulases consist of at least six genetically different cellulases: two cellobiohydrolases (exo-1,4-β-d-glucanases) (CBH I and II) and four endo-glucanases (endo-1,4-β-d-glucanases) (EG I, II, III and V) (Medve et al., 1998). The isoforms may have different isoelectric points (pI), and their catalytic and adsorption properties may be similar (Medve et al., 1998). Thus the cellulase system is very complex in both composition and function. Cellulase has a wide variety of applications and is widely used in the food, fuel, paper, pharmaceutical and chemical industries. The breakdown process of cellulose into glucose using cellulase has significant potential for low cost energy usage when glucose is converted to ethanol via the traditional yeast fermentation process. Presently, cellulase, itself, is expensive because its purification and concentration is both costly and time-consuming (Johansson and Reczey, 1998). Foam fractionation is a potentially low cost and effective purification method for concentrating cellulases from dilute solutions (Loha, 1999a, Loha, 1999b). It is currently unknown, however, whether all the components in the cellulase complex have the same foaming properties. Depending on foamability, the non-foaming components may be retained in the residue, permitting separation of the complex. β-glucosidase, a relatively hydrophilic enzyme, is often the limiting catalytic component in the cellulase complex from Trichoderma reesei. In this study we will compare the surface tension–pH profiles of cellobiase (from Aspergillus niger) to the cellulase complex and relate how those profiles affect the ability to foam fractionate cellobiase and cellulase.

Loha, 1999a, Loha, 1999b found that the effectiveness of the batch foam fractionation of cellulases was strongly dependent on the pH of the initial solution. With an increase in pH from 2 to 7, the separation ratio (defined as the ratio of the foamate and residue concentrations) decreased to a minimum. That ratio increased from pH 7–11. The enzymatic activity of cellulase decreased to some degree following foam fractionation, depending on the pH. At extreme pH values (lower than 3 or higher than 10), the activity of the cellulase dropped dramatically, presumably due to denaturation (Loha, 1999a, Loha, 1999b). The role of each of the proteins in the cellulase complex of several proteins in the foam fractionation process is not readily apparent. We will, thus, study the interactive effect of one of these, cellobiase, and the complex itself to gain a better understanding of the foam fractionation of such proteins.

Section snippets

Methods

β-glucosidase (from Aspergillus niger) was purchased from the Fluka Chemical Corp. USA (Catalog Number: 49291). β-glucosidase solution was prepared by dissolving β-glucosidase powder in deionized water. The pH of the solution was adjusted by adding either 1.0 M NaOH or 1.0 M HCl. The surface tension of the β-glucosidase solution was determined at 50, 100, 200 and 300 mg/l over a pH range from 2 to 12 using a computer-controlled Wilhelmy plate tensiometer (KSV Sigma 70, KSV Instruments Ltd.,

Surface tension

The effect of pH on the surface tension of β-glucosidase solutions at 50, 100, 200 and 300 mg/l is shown in Fig. 2. At 50 mg/l, the surface tension showed only small decreases relative to that of pure water (≈72.6 mN/m) at all pH levels, indicating negligible surface excess, Γ, mg/cm2, at the air–water interface. At 100 mg/l, the surface tension decreased significantly below pH 4. This decrease may be due to the increase in hydrophobicity of β-glucosidase molecules at this acidic condition.

Conclusions

The surface tension–pH profile of the Aspergillus niger β-glucosidase solution shows that a local minimum is present around pH 3.6. This corresponds to the isoelectric point of Aspergillus niger β-glucosidase of 4.05, reported by Glass and Romanowska (1997) from chromatography measurements. For foaming to occur in significant amounts, high concentrations of β-glucosidase were needed (200 mg/l or more). Two local minima were observed for the cellobiase surface tension versus pH trajectory at the

Acknowledgments

The authors wish to thank Professor George Tsao for raising the issue of whether there are two minima in the surface tension–pH profile for β-glucosidase as there are for the cellulase complex. The authors also gratefully acknowledge the support of the National Science Foundation, (NSF Grant No. CTS-9712486). In addition, the authors thank the Vanderbilt University Dean of Engineering and the Department of Agriculture (USDA Grant No. 2001-52104-11476) for the Summer Fellowship and the

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