Procedures

1. Preparation of immobilized enzyme beads: Follow the immobilization protocols provided to prepare 100 ml of immobilized invertase beads. See Note 1. The process of bead formation may be easily automated with a pump, as shown in Figure 1.
2. Construction of immobilized enzyme reactor: The construction of the reactor to be used in this experiment is schematically shown in Figure 2. First, plug the bottom end of the glass tubing and fill with immobilized enzyme beads. Place the top plug with the outlet tubing, whose end is screened to prevent the loss of beads. The optional water jacket for temperature studies can also be added.
3. Construction of automated sugar analysis: The automated continuous reducing sugar analysis is based on the same DNS wet chemical principle used in the previous experiments. The solution to be sampled and the DNS reagent are pumped with peristaltic pumps and mixed at a ratio of 1:1. The precise value of this mixing ratio is not critical as long as the user calibrates the automated analysis system with the same ratio as he uses during the run; otherwise the mixing ratio of the sample and color developing reagent must be determined by disconnecting the tubing before the point of mixing and measuring the flow rates individually with graduated cylinders. After sample and DNS reagent are mixed, air is added to the stream to segment the liquid, thus, ensuring a plug flow. The mixture is then passed through a water bath at 90ºC. The water bath can be constructed from a 1000~ml beaker heated on a magnetic stirrer. The length of tubing to be immersed in this hot water batch is such that the sample stays in the heated bath for 10 minutes. It is critical that the color reaction is allowed to proceed to completion. (The best way to determine the length is simply to turn on the pumps for 10 minutes and note the front of the sample at the end of this period.) The tubing is cooled in the next water bath to room temperature. After passing through a bubble eliminator, the colored sample solution is pumped into a flow-through cell in a spectrophotometer. The absorbance is measured continuously with a computer. If the absorbance is not within the desired linear range of the spectrophotometer, either a flow-through cell with a smaller light path should be used or water may be mixed with the colored sample stream prior to absorbance measurement. For an optically dense sample, both strategies can be combined.
4. Calibration of automated sugar analysis: Dip the end of the sample tubing into a solution of equimolar mixture of glucose and fructose. The movement of the sample and the gradual development of the characteristic deep red-brown color in the heated bath can be followed visually if translucent silicone tubing or transparent Tygon tubing are used. The reacted sample can be seen reaching the spectrophotometer. While the transient response in the absorbance shows the inherent dynamics of the measurement system, the steady absorbance reading is used to generate a calibration curve to relate the measured absorbance to the concentration of the equivalent hydrolyzed sugar solution. After the stable absorbance reading, the end of the tubing is dipped into water for about 10 seconds. This cleans the tubing to avoid cross contamination. The end of the tubing is then transferred into the next standard sugar solution. Note that the small segment of water suctioned between two different standard solutions of sugar can be used as an indicator of the boundary between these solutions that may otherwise be difficult to follow visually. This calibration process is repeated until the concentration of the reducing sugar solution covers a range similar to that used in the main part of the experiment. See Table 1.
5. On-line data acquisition: The on-line data-acquisition program with graphic display capabilities is provided. You are encouraged to study its source code and perhaps make suggestions for improvements for future experiments. The program continues to take data until the end of the sampling interval is reached. At that time the voltage or absorbance values are averaged, and the program displays this averaged value on the screen, both numerically and graphically, and simultaneously sends it to a sequential file to be stored for later retrieval. The program then updates the count by one and goes on to the next sampling interval, and so on, until a pre-defined set of signals (i.e., the simultaneous pressing of left and right shift keys) is sent to the computer to indicate the end of the run.
6. Effect of flow rate: Change the residence time to up to 10 minutes. The substrate volumetric flow rate needed to achieve the specified residence time depends on volume of the packed bed. Because the measurement of the extent of reaction is already automated, the entire experiment can be automated if the speed of the substrate feed pump can be programmed to shift in a stepwise manner. Table 2 suggests some flow rates to be used based on a packed bed volume of 100~ml.
7. Due to time constraints, only the effect of flow rate is studied above. Other interesting experiments that can be conducted to reveal the performance of an immobilized enzyme reactor include varying the substrate concentration in the feed, pH, temperature, superficial liquid velocity (which depends on the reactor geometry: diameter, bed height), % polymer in the gel bead, matrix pore size, bead size, and bed fluidization. In addition, the enzyme reactor can be operated over many days/weeks to measure the rate of enzyme deactivation and leakage.

Notes

1. Note that the standard protocol produces only approximately 10 ml of beads. The amount of reagent can be multiplied accordingly to obtain the desired amount of immobilized enzymes. Enzyme immobilization should be completed before the class so that the students may concentrate on the measurement of enzyme kinetic parameters during the laboratory period. The beads should be prepared with enough time to allow the alginate beads to harden sufficiently.

Discussions

Numerous methods exist for enzyme immobilization, sometimes referred to as enzyme insolubilization. The overwhelming majority of the methods can be classified into four main categories: matrix entrapment, microencapsulation, adsorption, and covalent binding. Of these methods, matrix entrapment is the focus of this experiment.

Many entrapment methods are used today, and all are based on the physical occlusion of enzyme molecules within a "caged" gel structure such that the diffusion of enzyme molecules to the surrounding medium is severely limited, if not rendered totally impossible. What creates the "wires" of the cage is the cross-linking of polymers. A highly cross-linked gel has a fine "wire mesh" structure and can more effectively hold smaller enzymes in its cages. The degree of cross-linking depends on the condition at which polymerization is carried out. Because there is a statistical variation in the mesh size, some of the enzyme molecules gradually diffuse toward the outer shell of the gel and eventually leak into the surrounding medium. Thus, even in the absence of loss in the intrinsic enzyme activity, there is a need to replenish continually the lost enzymes to compensate for the loss of apparent activity. In addition, because an immobilized enzyme preparation is used for a prolonged period of operation, there is also a gradual, but noticeable, decline in the intrinsic enzyme activity even for the best method. Eventually, the entire immobilized enzyme packing must be replaced.

Besides the leakage of enzymes, another problem associated with the entrapment method of immobilization is the mass transfer resistance to substrates, products, and inhibitors. Because the average diameter of a typical bead of enzyme impregnated gel is much larger compared to the average diffusion length, substrate cannot diffuse deep into the gel matrix, as in any other conventional non-biological immobilized catalysts. At the same time, the diffusional resistance encountered by the product molecules can sometimes cause the product to accumulate near the center of the gel to an undesirable high level, leading to product inhibition for some enzymes. Thus, ideally the network of cross-linking should be coarse enough so that the passage of substrate and product molecules in and out of a gel bead is as unhindered as possible. For this reason, entrapment is not suitable for special cases where the substrate has a large molecular weight such that it cannot easily move freely in the gel matrix.

Unlike the adsorption and covalent bonding methods, most polymerization reactions that cause cross-linking and gel formation in entrapment methods do not directly involve the formation of bonds between the support material and the enzyme molecules. There are reports that these bonds change the conformation of the enzyme protein and modify the enzyme properties. Since the enzyme molecules do not themselves participate in the polymerization reaction in the entrapment methods, the same entrapment techniques can be successfully applied to a wide range of enzymes with only minor modifications between different enzymes.

Questions

1. Plot the transient response of sucrose hydrolyzed as a function of time and estimate the time constant of the immobilized enzyme reactor. How does this compare with the residence time of the substrate?
2. Plot the degree of conversion (in %) and productivity as a function of flow rate.
3. From a plot of reaction rate versus residual substrate concentration, report the value for the apparent Michaelis-Menten constant.
4. Comment on ways to improve the experiment.

Data Forms

   Table 1. AUTOMATED SUGAR ANALYSIS
       -------------------
        Invert
        Sugar   Absorbance
        Conc.
        (g/l)    (A.U.)
       -------------------
         0.0
        10.0
        15.0
        20.0
        25.0
        30.0
        35.0
        40.0
        50.0
       -------------------

      Table 2. FLOW RATE
      ---------------------------------------------------------------------
      VolumetricResidence               Invert
        Flow      Time      Absorbance   Sugar  Conversion   Productivity
        Rate                             Conc.
      (ml/min)    (min)     (A.U.)       (g/l)      (%)         (g/l-hr)
      ---------------------------------------------------------------------
      100.0      1.0
       75.0      1.3
       66.7      1.5
       50.0      2.0
       40.0      2.5
       33.3      3.0
       25.0      4.0
       20.0      5.0
       16.7      6.0
       14.3      7.0
       12.5      8.0
       11.1      9.0
       10.0     10.0
      ---------------------------------------------------------------------