Physical characterization of liposomes
Structural details were visualized by negative-staining, freeze-fracture and cryo-TEM. Figure 2 shows a representative micrograph of a DPPC MLV containing AMG prepared by the thin film hydration method: a negative-staining image of a sample with an entrapped enzyme-to-lipid ratio of 0.349 mg/mg (Fig. 2A), a freeze-fracture TEM (Fig. 2B), and a cryo-TEM image (Fig. 2C). Cumulative data obtained by these techniques showed that the vesicles are spherical in shape with a wide size distribution ranging from approximately 0.2 to 10 μm. These results may indicate that aggregation of liposomes upon entrapping the enzyme is occurring.
Typically, freeze fracture electron microscopy is an effective method to ascertain vesicle lamellarity. Despite repeated attempts, bisecting cleavage of the vesicles to reveal lamellar structure was not observed in our samples. Therefore, we explored the capacity to use confocal microscopy to demonstrate the lamellar multiplicity. The confocal image in Figure 3 clearly illustrates the presence of multi-lamellar vesicles in samples produced by the thin-film rehydration methods.
Entrapment studies of AMG in liposomal preparations were initiated by incorporation of AMG into the DPPC MLVs. To test the reactivity of entrapped AMG, it was important to ensure that no extravesicular enzyme was present in our preparations. The removal of entrained AMG from the liposome preparations was complete, as less than 0.05% total protein was detected in the final supernatant of the wash preparation.
Table 1 lists AMG entrapment percentage and entrapment efficiency determined after removal of external enzymes by successive centrifugation, washing and re-dispersion steps. Enzyme entrapment is directly proportional to DPPC concentration. However, the entrapment efficiency decreases with increasing DPPC content. The lower entrapment efficiency suggests a disproportionately low increase in capture volume. That is, the addition of more lipid increases the lamellarity of the vesicle population rather than producing more vesicles of the same lamellarity. This is likely an artifact of the thin film rehydration method. As the DPPC concentration increases, the thickness of the lipid film deposited on a round bottom flask wall is likely to increase, resulting in more vesicles of higher lamellarity. With more concentric bilayer shells, the MLVs will have a lower capture volume than MLVs prepared with lesser amounts of DPPC (thinner DPPC film), provided that size distribution, bilayer thickness and interlamellar spaces of these MLVs are constant.
The apparent activity of entrapped AMG was measured as shown in Table 1. It would be expected that the more protein is entrapped, the more activity appears. However, as entrapment percent is increased by 2-fold from 6.83% to 14.45%, the apparent activity decreases from 8.86 units/mg protein to 6.68 units/mg protein. The lower apparent activity may be due to mass transfer limitation.
The entrapment percentage and efficiency as a function of AMG concentration at a constant DPPC concentration content was also investigated. Maximum entrapment percent was reached at 7.8 mg/ml AMG as shown in Figure 4. In addition, an increase in the entrapment efficiency with an increasing AMG concentration was observed.
Thermostability of AMG entrapped in MLV and GUV
To evaluate the thermostability of free and entrapped AMG, the enzyme solution was incubated at the hydrolysis temperature in sealed tubes. Samples were taken after various incubation intervals, the residual enzyme activity was determined, and the relative enzyme activity was estimated by assuming the initial enzyme activity as 100%. Figure 5 shows the thermostability of MLV and GUV samples compared to the free AMG sample on the basis of the estimated relative AMG activities in each sample. AMG entrapped inside MLV and GUV remains preserved for a much longer period of time in comparison to the activity of the free enzyme in aqueous media. At 55°C, the native enzyme retained 55 % activity after 160 h whereas the entrapped enzyme retained 70 % and 100 % activity under identical conditions for GUV and MLV, respectively. Both the GUV and MLV studies show an increase of starch hydrolysis activity within the first few hours of high temperature incubation. This may reflect a change in the state of the entrapped enzyme over time or a change in liposome properties (which would impact starch permeability) at the hydrolysis temperature. Further study is needed to elucidate this phenomenon.
Kinetic parameter estimation
Kinetic constants (Km and Vmax) were determined from a classic enzyme kinetic analysis based on initial velocity measurements of soluble starch hydrolysis by either free AMG or entrapped AMG in MLV or LUV at various starch concentrations (1 – 10 mg mL-1). Results from this analysis are plotted in Figure 6. Ki was determined by fitting experimental product concentration versus time data to the Michaelis-Menten equation. Values for the kinetic parameters are summarized in Table 2. The best-fit kinetic parameters of the Michaelis-Menten model were estimated by non-linear regression as Vmax = 1.28 mg glucose ml-1 min-1 mg-1 protein and Km = 1.55 mg/ml, Vmax = 0.35 mg glucose ml-1 min-1 mg-1 protein and Km = 1.15 mg/ml, Vmax = 0.56 mg glucose ml-1 min-1 mg-1 protein and Km = 1.64 mg/ml, for free AMG, MLV and LUV, respectively. It should be pointed out that kinetic parameters for free AMG are intrinsic while those measured with MLVs and LUVs are apparent values due to mass transfer effects. Consistent with this, values of Vmax were significantly lower in the entrapped samples for both MLVs and LUVs compared to that of the free AMG. This is in agreement with literature data reported for amylase entrapped in soybean phosphatidylcholine liposomes . The decrease in Vmax can be attributed to steric effects resulting from limitation of the accessibility of soluble substrate to the active site. Since MLVs have more lamellarity than LUVs, LUV has a larger value of Vmax (0.56) than that of MLVs (0.35) due to lower mass transfer limitations. The apparent Km values for MLV-entrapped AMG was lower than that for the free enzyme. The apparent Km for GUV-entrapped enzyme was not significantly different from the free enzyme Km. The glucose inhibition constant (Ki) was determined to be 0.10 mg mL-1 for all cases. While it is reasonable to believe that the apparent values differ from the free enzyme Km and Vmax because of the added transport layer(s) in the liposomes, with the data presented, the possibility that the intrinsic enzyme kinetics could be altered in the case of liposome entrapment cannot be completely excluded. The lower apparent Vmax for the multi-layer liposome relative to the single layer is most likely a reflection of the increased mass transfer barrier resulting form the additional transport layers. This provides additional evidence that liposomal kinetics is strongly a function of mass transfer.
Simulation of batch starch hydrolysis
Enzymatic starch hydrolysis by free AMG and entrapped AMG into MLV were investigated for reaction systems containing 1% soluble starch. Glucose concentration was measured versus time. The difference of intrinsic and apparent kinetics was due to the substrate permeability across the bilayer membrane such that the experimental data were used to fit a mass transfer coefficient, by using Equations 2–4. Figure 7 shows the glucose concentration profiles (filled circle and filled square) compared with the simulated results (red and blue lines). The model simulations were in fair agreement with experimental data. In addition, the model and parameter estimation procedure allowed not only the quantification of the substrate permeability in the vesicle system used, but also provided insight into the changes of substrate concentrations inside the vesicles (green line), which would be rather difficult to determine experimentally.
Repeated hydrolysis of starch
Figure 8 shows AMG activity in the repeated enzymatic hydrolysis of starch with vesicle-entrapped enzyme. Enzyme activity is very stable during the first three batch runs. The AMG lost 39.1% of original activity after the fourth batch run (after 96 hrs in processing). Beyond cycle four a great decrease in the degree of hydrolysis was observed. The decrease could be due to either vesicle leakage, loss of enzyme by adsorption to the substrates, or incomplete precipitation. The fact that enzymes are recycled in the process will make an improved process economy possible.
Compared to free AMG, the rate of hydrolysis by entrapped AMG is relatively low. This is either because of the low permeability of substrate across the liposome bilayer or because of the low enzyme activity inside the liposomes. In the current work, while apparent values for Km and Vm are presented, no attempt was made to determine if transport across the lipid layer is rate-limiting, or if a lower reaction rate due to altered enzyme kinetics gives comparable time constants to mass transfer rates. Generally Thiele modulus, φ, is used for this purpose. For example, when φ is sufficiently small (φ .