In the Laboratory
Immobilized Lactase in the Biochemistry Laboratory* Matthew J. Allison and C. Larry Bering** Department of Chemistry, Clarion University, Clarion, PA 16214
The use of catalysts that allow reactions to proceed at lower temperatures and ambient pressures (and thus lower costs) is widespread in industry. Many of these catalysts are quite expensive and must be recovered after use. If the catalyst is present in the solid phase, recovery is greatly facilitated. Enzymes are biological catalysts that offer high substrate specificity and stereospecificity, with limited side reactions. In the laboratory, we often study enzymes on a small scale in solution, and are not concerned with recovery after use. However, an expensive genetically engineered enzyme or one involving a complex purification procedure may be too valuable for an industrial-scale setting. Thus enzymes that have been immobilized onto a solid-phase particle are of great interest. If an enzyme can be immobilized onto a chromatography bead, it may be packed into a column and used to generate a continuous flow-through reactor, or it may be added as a heterogeneous-phase catalyst and recovered after use by filtration or other means. An excellent introduction to this subject for students is found in the monograph by Woodward (1). Immobilized enzyme technology is not new. Many enzymes, or whole bacterial cells representing “bags” of enzyme, have been immobilized and used in several industrial processes such as antibiotic transformations, steroid modification using ketosteroid dehydrogenase, D-amino acid production using aminoacylase, and production of high-fructose corn syrup using glucose isomerase or invertase (1–9). Unique biosensors have been developed by immobilizing enzymes onto films that cover electrodes (6 ). For instance, an enzyme catalyzing a reaction that produces oxygen or protons may be immobilized onto an oxygen or glass electrode, respectively. Mifflin et al. (10) described a laboratory experiment that utilizes penicillinase immobilized onto a glass electrode as a sensor for penicillin. Other biosensors that can detect a number of analytes such as lactate and uric acid have been developed in this manner. Immobilized enzymes have also found their way into the marketplace. The tapes that monitor urine glucose levels in diabetics (Clini-Stix, TesTape) contain immobilized glucose oxidase coupled with a dye that gives a color whose intensity is directly proportional to glucose concentration. There are numerous ways in which an enzyme may be immobilized (1, 3). It may be adsorbed onto or covalently attached to a gel particle (Silica gel, Sephadex, etc.), crosslinked with reagents such as glutaraldehyde to produce an aggregated particle, or encapsulated into liposomes. Perhaps the most widely used procedure is entrapment in a matrix such as the seaweed products κ-karageenen or calcium alginate, or in polyacrylamide, which is readily available in most labs. Using immobilized enzymes in a biochemistry laboratory can be an excellent tool for studying catalysis, as well as a
way of introducing an important technique in modern biotechnology. Immobilized enzyme experiments are not found in most biochemistry laboratory textbooks, although Boyer (11) does include an experiment using immobilized peroxidase. Several papers have appeared in this Journal that describe the use of immobilized enzymes in undergraduate laboratories (10, 12–15). Lactase (β-galactosidase) is a popular enzyme for use in the undergraduate or secondary-school laboratory (16 ). Russo and Moothart (17 ) described an enzyme kinetics experiment using an over-the-counter preparation of lactase as the source of the enzyme. A previous report in the literature describes an experiment utilizing β-D-galactosidase immobilized on silica gel, that involves a fairly complicated grafting of the enzyme onto the matrix (18). In this paper, we describe a simple and inexpensive experiment in which lactase from an over-the-counter preparation is entrapped in polyacrylamide, then packed into a column to generate a continuous flowthrough reactor. When the disaccharide lactose is added to this column, it is hydrolyzed into glucose and galactose as it passes through the column: lactose + H2O lactase → glucose + galactose The product glucose in the eluant can be detected using a clinical test kit that utilizes the following reactions: glucose + O2 → glucuronic acid + H2O2 glucose oxidase
H2O2 + 4-aminophenazone + phenol → quinone complex peroxidase The quinone complex has an absorbance at 500 nm which is proportional to the concentration of glucose in the sample. Experimental Procedure Acrylamide, bis-acrylamide, and ammonium persulfate were purchased from Fisher Biotech (Pittsburgh, PA). N,N,N′, N′-tetramethylethylenediamine (TEMED) and o-nitrophenyl β-D-galactopyranoside (ONPG) were purchased from Sigma (St. Louis, MO). The Stanbio Glucose Liquicolor enzymatic–colorimetric test kit was purchased from Stanbio Laboratories (San Antonio, TX). SAFETY NOTE: Students should be cautioned that acrylamide and bis-acrylamide are highly neurotoxic, and that they should not allow the solid or the solution to come into contact with the skin. They should use gloves whenever handling acrylamide, and although the polymer is nontoxic, it should also be handled with gloves because residual unpolymerized acrylamide may be present. TEMED is flammable and can cause irritation.
Special Equipment *Presented at the 14th Biennial Conference on Chemical Education, Clemson, SC, 1996. **Corresponding author: 814/226-2565; email: bering@mail. clarion.edu
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Microcentrifuge High speed centrifuge Waring blender
Journal of Chemical Education • Vol. 75 No. 10 October 1998 • JChemEd.chem.wisc.edu
In the Laboratory
15 cm chromatography column (1 cm diameter) Spec 20 or other spectrophotometer
Preparation of Immobilized Lactase One Dairy-Ease tablet was ground in 0.02 M phosphate buffer, pH 6.5, using a mortar and pestle. The extract was transferred to one or more microcentrifuge tubes and spun at top speed (12,400 rpm; 13,600 × g) for 30 seconds. The tubes were removed and the clarified supernatant was transferred to a test tube using a Pasteur pipet. To a 125-mL vacuum flask, 10 mL of 30% acrylamide solution (prepared by adding 58.4 g of acrylamide and 1.6 g of bis-acrylamide to 200 mL of distilled water), 9.5 mL of 0.06 M phosphate buffer, pH 6.5, 1.5 mL of the lactase, and 9.0 mL of distilled water were added. The flask was stoppered and degassed for 15 minutes. During the degassing step, a fresh solution of 100 mg/mL of ammonium persulfate (APS) was prepared. After degassing, the flask was removed from the vacuum line and 150 µL of APS solution and 15 µL of TEMED were added. The flask was swirled and the acrylamide was allowed to polymerize (about 15 minutes). The flask was stoppered during the polymerization because oxygen in the air can inhibit the polymerization. After the gel had polymerized, any unpolymerized liquid was poured into an appropriate organic waste container, and the solid gel was scraped from the flask and transferred to a Waring blender. Two hundred milliliters of 0.02 M phosphate buffer was added to the gel, which was then ground in the blender at high speed for 15–20 seconds. Longer grinding times will produce very fine beads, which when packed into a column will lead to very slow flow rates. We have found that the beads produced by the shorter grinding times give a flow rate that will allow students to wash the column and obtain satisfactory hydrolysis of the lactose substrate within the time constraints of the teaching lab. After grinding, the slurry was spun in a high-speed centrifuge for 5 minutes at 5000 × g. The supernatant was decanted and the gel was washed by resuspending it in about 100 mL of 0.02 M phosphate buffer, pH 6.5. The suspension was spun again in the centrifuge using the conditions described above. These washings remove most of the lactase that is not entrapped in the acrylamide matrix. The gel was resuspended in a minimal amount of 0.02 M phosphate buffer and packed into a chromatography column (15 cm × 1 cm). Students were reminded not to let the column run dry. The column was packed about 1/2 full, resulting in a gel volume of approximately 4 mL (approximately l mL void volume.) The column was washed with approximately 25 mL of 0.02 M phosphate buffer. To test for any unbound lactase still present, a 1-mL fraction of the eluant was collected in a test tube, and 0.1 mL of the ONPG solution added to the tube. The contents were mixed and allowed to stand for 2 minutes. A blank tube was also prepared alongside the eluant using phosphate buffer and ONPG. If a yellow color appeared in the eluant sample, washing of the column with phosphate buffer was continued. The eluant was occasionally tested in this manner until no yellow color appeared. This indicated that no free lactase was present in the void volume (mobile phase) of the column. Any subsequent lactase activity would be due to immobilized lactase. Once the column was sufficiently washed, 0.03 M lactose was added to the column. The lactose may be added as a 1-mL
aliquot and chased through the column with phosphate buffer, or may be added continuously to mimic a continuous flow-through reactor. Students were split into two groups. Half of the students operated the column as flow-through reactor while the other half added 1 mL of the lactose solution. In both cases, 2-mL fractions were collected immediately upon addition of lactose. Collection was continued until 10 fractions were collected. If the enzyme is immobilized in the acrylamide in an active form, the collected fractions should contain glucose and galactose due to lactose hydrolysis. To measure the amount of the glucose product in eluant, the clinical glucose test kit was utilized according to the directions in the kit. Using a glucose standard, and the instructions with the clinical kit, the concentration of glucose was determined spectrophotometrically for each sample. Results During the first year in which we used this experiment, students working in pairs conducted the lab at various time slots during a one-week period. This allowed us to detect problems early in the week that could be corrected later in the week. The only real problem that we encountered was differences in the flow rate through the column. This was shown to be due to the time that the acrylamide was ground in the blender. A one-minute grinding time produced very fine particles, which packed tightly (even if the column was filled to less than 1/2 capacity with the gel). With subsequent groups the grinding time was cut to 20 seconds, with excellent results. Regardless of the flow rate, every student group obtained lactose hydrolysis, and a set of controls described below indicated that the hydrolysis was indeed due to immobilized lactase. Owing to the many variables such as diffusion and band broadening in the column, our glucose concentration was below typical concentrations found in biological fluids, and we adjusted the assay to reflect this. Each student prepared the following tubes in the assay:
Tube
Glucose Reagent/ mL
Phosphate Buffer/mL
Sample/ mL
Glucose Standard/ mL
1
1.0
2.0
—
—
2
1.0
2.0
—
0.01
3–12
1.0
1.0
1.0
—
In the Stanbio kit that we utilized, the concentration of glucose can be determined by use of the formula (A u/A s ) × 0.01 = glucose concentration in mg/mL where A u = absorbance of unknown and A s = absorbance of standard. When lactose was added continuously to the column, it was found that the concentration of glucose in the 2-mL fractions reached a constant value after the fourth or fifth fraction and then remained at a constant value. When a 1-mL aliquot was added, the absorbance rose to a maximum value in the fourth fraction and then slowly decreased, although some glucose was still detectable in the 10th fraction. Typical absorbance values are shown below.
JChemEd.chem.wisc.edu • Vol. 75 No. 10 October 1998 • Journal of Chemical Education
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In the Laboratory Continuous Mode: Fraction
1-mL Aliquot Added: A500
Fraction Glucose standard
A500
Glucose standard
0.061
0.055
1
0.068
1
0.049
5
0.810
2
0.684
3
0.655
4
0.741
5
0.620
6
0.435
7
0.348
8
0.259
9
0.210
10
0.151
Although students were instructed to collect ten 2-mL fractions, in many cases time constraints prevented them from collecting all the fractions, especially if the flow rate was slow. In such cases, where ten fractions were collected from the 1-mL aliquot experiment, the students stopped the experiment after the tenth fraction and proceeded with their assays. Thus they were not able to determine when the glucose level in the eluant dropped to a nondetectable level. Calculation of glucose concentrations shows that in the continuous mode, fractions with a glucose concentration of approximately 0.13 mg/mL are obtained from the column. This represents a yield of 2.45%. If all the lactose added to the column is hydrolyzed, one would expect 5.4 mg/mL of glucose in the eluant. This of course does not take into consideration factors such as band broadening, the flow rate of substrate through the column, and access of the substrate to the bound enzyme, all of which are problems encountered with immobilized enzymes. If one adds a 1-mL aliquot of lactose, the expected yield of glucose would be 5.4 mg. The total glucose obtained in the ten fractions is 0.753 mg, corresponding to a 13.9% yield. The over-the-counter preparation of lactase is remarkably stable. A packed column with the gel stored in 0.02 M phosphate buffer was left on the lab bench at room temperature and tested for activity from time to time. At four weeks, virtually the same lactase activity was still observed. We also prepared a column with immobilized β-galactosidase (purchased from Sigma), but this pure enzyme lost activity on the column after about two days. Several control experiments were carried out during the development of this experiment. No glucose oxidase activity was seen in any of the controls, and the colorimetric assay was positive only for glucose as the product of hydrolysis by the immobilized enzyme. No color was seen with the phosphate buffer, either before or after passage through the column. Lactose gave no color. If lactose was passed through a column containing polyacrylamide beads without any immobilized lactase, no color was seen. Finally, if the enzyme was heated in a boiling water bath for one minute, then immobilized in the polyacrylamide matrix, no color was seen when lactose was passed through the column. Discussion With students working in pairs, this experiment can easily fit into a three- or four-hour laboratory. The costs are minimal, 1280
approximately $5.00 per student. The enzyme source not only makes this a very cost-effective teaching lab, but students gain an appreciation for the real-world applications of enzymes that can be found at a local pharmacy. We have encouraged students to examine labels of certain products for their active ingredients—for example, subtilisin in enzymatic contact lens cleaning tablets, or glucose oxidase in Clini-Stix. A typical biochemistry laboratory would have a supply of acrylamide and other chemicals needed for preparing polyacrylamide, chromatography columns, centrifuges, and spectrophotometers. The only chemicals that might need to be ordered would be ONPG (about $25) and the glucose colorimetric assay kit (about $50). There are no major safety concerns other than handling acrylamide as indicated above. Although immobilized enzymes are a specialized application of biotechnology, the students found this experiment to be one of their favorite during the semester. We usually run this experiment fairly late in the term, after students are exposed to most of the principal techniques in biochemistry. By the time they got to this experiment, they needed minimal instruction, as they had already made acrylamide (for electrophoresis), done column chromatography, and were familiar with a centrifuge and spectrophotometer. Anonymous written comments about the immobilized enzyme experiment were solicited from the students. Their comments supported the importance of reinforcement of techniques and skills. Some of the comments are listed below. We could utilize techniques we had learned in previous laboratory experiments. I also enjoyed the fact that the lab made use of well-known substances such as Dairy-Ease and lactose. This made the lab less abstract, and therefore, more interesting. I liked the fact that it was a combination of several laboratory techniques and principles (centrifugation, column chromatography, spectrophotometry…). Because of its combination of laboratory principles and techniques, it would serve as a good final exam.
Literature Cited 1. Immobilized Cells and Enzymes: A Practical Approach; Woodward, J., Ed.; IRL: Oxford, 1985. 2. Mosbach, K. Sci. Am. 1971, 224, 26. 3. Klibanov, A. M. Science 1983, 219, 722. 4. Fukui, S.; Tanaka, A. Annu. Rev. Microbiol. 1982, 36, 145. 5. Weetall, H. H.; Pitcher, W. J. Science 1986, 232, 1396. 6. Enzyme Engineering: Immobilized Biosystems; Gemeiner, P., Ed.; Ellis Horwood: New York, 1992. 7. Immobilized Macromolecules: Application Potentials; Sleytr, U. B.; Messner, P.; Pum, D.; Sara, M., Eds.; Springer: London, 1993. 8. Uses of Immobilized Biological Compounds; Guilbault, G. G.; Mascini, M., Eds.; Kluwer: Boston, 1993. 9. Immobilization of Enzymes and Cells; Bickerstaff, G. F., Ed.; Humana: Totowa, NJ, 1997. 10. Mifflin, T. E.; Andriano, K. M.; Robbins, W. B. J. Chem. Educ. 1984, 61, 638. 11. Boyer, R. F. Modern Experimental Biochemistry, 2nd ed.; Benjamin/ Cummings: New York, 1992. 12. DeJong, P. J.; Kumler, P. L. J. Chem. Educ. 1974, 51, 200. 13. Conlon, H. D.; Walt, D. R. J. Chem. Educ. 1986, 63, 368. 14. Grunwald, P. J. Chem. Educ. 1986, 63, 775. 15. Manoharan, A.; Dreisbach, J. H. J. Chem. Educ. 1988, 65, 98. 16. Bullerwell, L.; Raunig, V.; Hagar, W. Sci. Teacher 1994, 61, 27. 17. Russo, S.F.; Moothart, L. J. Chem. Educ. 1986, 63, 242. 18. Lartillot, S. Biochem. Educ. 1993, 21, 157.
Journal of Chemical Education • Vol. 75 No. 10 October 1998 • JChemEd.chem.wisc.edu
