In the Laboratory
Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase Suhasini Kulkarni, Vu Tran, Maggie K.-M. Ho, Chieu Phan, Elizabeth Chin, Zeke Wemmer, and Monika Sommerhalter* Department of Chemistry and Biochemistry, California State University, East Bay, Hayward California 94542 *
[email protected] Enzymes can catalyze a remarkable variety of challenging chemical reactions with high substrate-, regio-, and enantioselectivity under mild, environmentally benign conditions. It is therefore desirable to introduce enzymes as biocatalysts in industrial chemical processes. Already established enzyme applications include the use of proteases to tan leather (1), amyloglucosidase to produce low-calorie beer (2), and rennin to manufacture cheese (3). The immobilization of enzymes to a solid support renders industrial enzyme applications even more attractive because of the ease of recovery and reusability of the enzyme. Using sol-gels as immobilization matrices has several advantages. Sol-gels are low in cost, easy to prepare, chemically inert, optically transparent, and can be cast into various shapes (4). Biosensors using sol-gel encapsulated enzymes have been developed to detect many chemical species, including glucose, hydrogen peroxide, and cholesterol (5). Sol-gel encapsulated enzymes can also be used as biocatalysts. Lipases, for example, were entrapped in hydrophobic sol-gel materials to catalyze hydrolysis reactions (6). In this laboratory experiment, students encapsulate wheat germ acid phosphatase (APase) in sol-gel beads. They compare the catalytic activity of the immobilized enzyme to free APase in solution and investigate other parameters that are critical to assess the performance of the immobilized enzyme, such as enzyme retention and reusability. APase catalyzes the hydrolysis of monoesters and anhydrides of phosphoric acid to produce inorganic phosphate (7). APase acts on a large variety of substrates and can be used to improve the quality of feedstock or to recycle phosphate from sewage sludge (8).
published that target material science, instrumental analysis, and physical chemistry courses (9-14). This experiment, however, targets biochemistry students and interlinks sol-gel technology with enzyme catalysis. Surprisingly, few articles provide experiments on enzyme immobilization for undergraduate laboratories. In this Journal, experiments that use chitosan (15), collagen (16), and acrylamide (17-19) as solid supports have been published. Other experiments employ chemical linkers to immobilize enzymes on glass electrodes (20). A major advantage of the enzyme sol-gel encapsulation process is that virtually any enzyme can be incorporated into the sol-gel mesh. Also, it is not necessary to modify the enzyme with chemical linkers before the encapsulation process. Some enzymes, however, are more susceptible than others with respect to the decline in enzymatic activity upon sol-gel encapsulation. APase was previously shown to be compatible with the sol-gel encapsulation process (21, 22). Wheat germ APase was chosen, because students extracted and purified this enzyme from wheat germ in a previous experiment. Wheat germ APase is also commercially available at a reasonable price (Sigma-Aldrich). Because of the universal nature of the sol-gel encapsulation process, the course instructor can easily adjust the experiment to another enzyme deemed more relevant for a particular course. The research literature contains many examples for the successful sol-gel encapsulation of enzymes, for example, lipase (6) or horseradish peroxidase (23).
Rationale
To prepare the APase sol-gel beads, a sonicated acidic solution of an alkoxysilane precursor molecule (here tetramethoxysilane, TMOS) is mixed with a buffered APase enzyme solution of pH 7.2. A rise in pH triggers the polymerization process (Scheme 1). As the sol-gel network forms, the enzyme is directly incorporated into the pores of the transparent silica mesh. Sol-gels can be cast into various forms, for example, beads (as chosen in our experiment), thin sheets, monoliths, or ground into fine powder. Once the APase sol-gel beads are solidified, they are transferred into a buffered solution. First, the students have to investigate how well the APase sol-gel beads retain the encapsulated enzyme. They remove the buffered solution on top of the beads at different time intervals (after several hours, after several days) and test for enzymatic activity. The activity measurement is based on a colorimetric assay that monitors the formation of yellow p-nitrophenolate after APase cleaves the phosphate group from the colorless substrate p-nitrophenylphosphate (Scheme 2). This assay can be performed as a kinetic
This experiment was successfully performed in an upperlevel undergraduate biochemistry laboratory course. Typically 10-26 students are enrolled in the course. The experiment requires two 2-3 h laboratory sessions. It combines multiple learning goals in a straightforward, low-cost, and fun activity. Solid-phase chemistry concepts (the polymerization and formation of the sol-gel) and biochemistry essentials (the measurement of enzyme activity) are placed into a biotechnological context. Sol-gel technology in combination with bioactive components is a rapidly evolving research area with an impressive range of diverse applications including biocatalysis, biosensing, and medical crafting (4). It is therefore timely to include this laboratory experiment, which exposes students to this lively and exciting field, in the curricula. In this Journal, several experiments on sol-gels, or solgels doped with small molecules as chromophores, have been 958
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In the Laboratory
Scheme 1. The Two Stage Sol-Gel Process: (A) The Partial Hydrolysis of an Alkoxysilane Precursor and (B) The Polycondensation into a Branched Silica Network
Scheme 2. APase Catalyzes the Hydrolysis of p-Nitrophenylphosphate into p-Nitrophenolate
(as chosen in our experiment) or a fixed-time assay (24). Students determine the molar absorptivity of p-nitrophenolate at 405 nm. They plot the absorbance increase at 405 nm versus the time and calculate the enzymatic activity in International Units (IUs) using the slope of this plot and their previously determined molar absorptivity of p-nitrophenolate. One IU corresponds to the generation of 1 μmol p-nitrophenolate per 1 min of time. Next, students evaluate the catalytic performance of the sol-gel encapsulated enzyme by comparing its activity to the activity of a sample containing the same quantity of free APase in solution. The activity assay with sol-gel encapsulated APase is repeated at least three times to explore the reusability of the immobilized biocatalyst. Hazards The sol-gel precursor TMOS is flammable, toxic by inhalation, irritating to the respiratory system and the skin, and has the potential to cause serious eye damage. Acid phosphatase is an irritant. Diluted hydrochloric acid is corrosive. TRIS buffer is an irritant and the product of the assay, p-nitrophenolate, is considered harmful. Results and Discussion A typical result from this experiment is shown in Figure 1. Notably, the sol-gel beads retain the enzyme very well. Even after several days, enzyme leakage from the APase sol-gel beads is almost negligible. However, the activity of sol-gel encapsulated APase is diminished by almost 70% in comparison to the activity of free APase in solution. There are two possible reasons. First, the enzyme may be damaged during the encapsulation process due to the released alcohol (methanol, if TMOS is used). Second, the sol-gel material exhibits limited material transport properties in comparison to the solution sample. Depending on the pore size of the silica mesh, the substrate molecules may be hindered in reaching the encapsulated enzyme, and the release of the product may be delayed as well. The loss of enzyme activity is a common drawback of the enzyme sol-gel encapsulation process, but this disadvantage is counterbalanced by the reusability of the immobilized biocatalyst. In the laboratory, the
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Figure 1. Results of the APase sol-gel encapsulation activity on the hydrolysis of p-nitrophenylphosphate. Combined data from five student teams are shown.
colorimetric assay was performed three times with the APase sol-gel beads without loss in the catalytic performance of the APase sol-gel beads.1 Furthermore, sol-gel encapsulation often confers a higher stability to the enzyme against thermal denaturation or denaturation by organic solvents or chaotropic agents. If time permits, students can investigate the influence of temperature, pH, or chemical additives (for example inhibitors) on the catalytic performance of free and sol-gel encapsulated APase.2 The experiment can be easily expanded to include a heat shock test: heat exposure at 60 °C for 1 h and then retest the enzyme activity. This new aspect can be used to stimulate a discussion on protein stability and folding-unfolding processes of proteins. A further variation of the experiment is to crush the APase sol-gel beads after running an activity assay and to determine the APase activity of the crushed beads in a fresh reagent mixture. Students are thereby able to address the influence of the beading process and the limited material transport properties of the sol-gel material on the catalytic performance. An important goal of this experiment is to stimulate students' reflection on the advantages and disadvantages of enzyme sol-gel encapsulation. The examination of students' laboratory reports demonstrated that 8 out of 10 students wrote engaged, well-balanced, and thoughtful discussions on this topic. All students evaluated their enzyme activity data properly. During the experiment, several students commented positively on the arts-and-crafts appeal of casting the sol-gel beads. In survey questions solicited at the end of the course, the experiment on sol-gel encapsulation of APase was found to be the most popular experiment out of the eight experiments conducted in the course. In particular, students valued the practical relevance of the experiment and the exposure to a new research field. Acknowledgment This work was supported by a Faculty Research Grant and an Interdisciplinary Sieber-Tombari Research award from California State University, East Bay. Suhasini Kulkarni received a graduate student research grant from California State University, East Bay. Elizabeth Chin gratefully acknowledges financial
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support from the CSU-LSAMP program, which is supported by the National Science Foundation under Grant No. HRD0331537. Notes 1. In our research laboratory, nine catalytic cycles were conducted without significant loss in the catalytic performance of the APase sol-gel beads. 2. In our research laboratory, the effect of heat exposure on free and sol-gel encapsulated APase was tested. Heat shock exposure at 60 °C for 1 h resulted in a 9-fold activity loss for free APase. In contrast, only a 3-fold activity loss was observed for sol-gel encapsulated APase. The main reason for the improved thermal stability of encapsulated enzymes is that their confinement in the pores of the silica mesh hinders larger conformational changes that are necessary for protein unfolding (25).
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Supporting Information Available Student handout; notes for the instructor. This material is available via the Internet at http://pubs.acs.org.
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