AMG from Aspergillus niger (A-3042) and dipalmitoylphosphatidylcholine (DPPC) were purchased from Sigma (St. Louis, MO). Soluble starch was obtained from Aldrich (Milwaukee, WI). The fluorescent probe, 1,1'-dioctadecyl-3, 3,3', 3'-tetramethylindocarbocyanine perchlorate (DiIC18 (3)) was obtained from Molecular Probes (Eugene, OR). All other chemicals were of reagent grade and obtained from Fisher Scientific (Hanover Park, IL). All water used was purified using a Barnstead commercial deionization system (Boston, MA).
Preparation of enzyme-containing liposomes
Multilamellar vesicles (MLVs)
MLVs were produced using the thin-film hydration method . Various amounts of lipids (usually 4–22 mg DPPC) were dissolved into 10 ml of chloroform in a 100 ml round bottom flask. The solvent was removed to form a thin lipid film on the wall of the flask under reduced pressure using a rotary evaporator. Any residual solvent was removed either under vacuum overnight or under a stream of N2. AMG solution was prepared by diluting stock solution (Sigma A-3042) 10-fold with distilled deionized water. This solution (4 mL) was slowly added to the flask, followed by a small quantity of glass beads to provide mechanical agitation. The flask was returned to the rotary evaporator, immersed in a warm water bath and vigorously rotated to reconstitute the lipid film above the lipid's main transition temperature (Tc = 41.3°C), approximately 5–10 minutes. A milky suspension of multilamellar vesicles (MLVs) was produced.
Large unilamellar vesicles (LUVs)
LUVs containing entrapped enzyme were produced via extrusion. In this process, AMG-containing MLV samples were centrifuged and suspended in AMG solution (0.1 X) to preserve the intravesicular enzyme concentration during extrusion. The suspension was passed 21 times through a stacked pair of polycarbonate filters (Avestin Inc., Ottawa, Canada) mounted in an extrustion device (Avestin, Lipo-fast), first with 1000 nm filters, then again using 200 nm filters . A significant decrease in size and lamellarity can be achieved, resulting in a relatively monodisperse vesicles.
Giant unilamellar liposomes (GUVs)
In some experiments, giant unilamellar liposomes (GUVs) were prepared according to . In brief, a desired quantity of DPPC was dissolved in 1 ml of chloroform and 200 μl of methanol. The aqueous phase containing 4 mL AMG was then carefully added along the flask walls. The organic solvent was removed in a rotary evaporator. After evaporation for 2 min, an opalescent fluid was obtained. The resulting aqueous solution contained GUVs.
Separation of the free and entrapped enzymes
Liposomal suspensions were centrifuged at 16 000 g for 30 min at 4°C to separate unencapsulated AMG from liposomes. The supernatant was removed and 4 mL water was added to the pellet to resuspend the liposomes before repeating the centrifugation. This wash process was repeated three times to ensure complete removal of the free enzyme from the liposome preparations. Free enzyme removal was verified by total protein assay and starch hydrolysis activity, the details of which are described in the later section, for each supernatant. After the final centrifugation, the pellet was resuspended in 2 mL water prior to determination of entrapped protein and starch hydrolysis activity.
Determination of entrapment percent and entrapment efficiency
Entrapment percent and entrapment efficiency were determined after removal of external enzyme by repeated centrifugation and washing. The entrapment percent (EP) and entrapment efficiency (EF) can be calculated by subtracting the amount of free enzyme in the supernatant from the total amount of the added enzyme as:
Characterization of enzyme-containing liposomes
Negative staining electron microscopy
A drop of freshly prepared liposome was applied to a Formvar-coated copper grid. After 20 seconds, the excess liquid was absorbed at the periphery of the grid by filter paper. The remaining sample was air-dried at room temperature and the liposome was negatively stained with 0.5% uranyl acetate or ammonium molybdate for 20 s, after which most of the staining solution was absorbed at the periphery of the grid by means of filter paper. Samples were examined with a Hitachi H-600 transmission electron microscope (TEM).
Freeze-fracture and electron microscopy
A small aliquot of a liposome sample was placed between two copper strips, a double wing replica holder. The holder was plunged into liquid propane (-190°C) for approximately 10 seconds. Frozen samples were inserted into a hinged double replica device and transferred into a Balzer 301 freeze-fracture apparatus. Fracturing was performed at -120°C and about 10-7 torr by releasing a spring that opened the two sides of the replica holder. Samples were immediately replicated with platinum and coated with carbon. Replicas were cleaned in an acidic mixture of nitric acid, sulfuric acid and acetic acid and washed with distilled water. The clean replicas were collected onto uncoated 400 mesh electron microscope copper grids and were examined with a Hitachi H-600 TEM.
Cryo-transmission electron microscopy (cryo-TEM)
A drop of liposome sample was applied to a standard electron microscopy grid coated with a perforated carbon film. Excess liquid was removed by blotting with filter paper, leaving a thin layer of aqueous sample covering the holes of the carbon film. The grid was rapidly frozen in liquid ethane, resulting in vesicles embedded in a thin film of vitreous ice. Images of the vesicles in ice were obtained under cryogenic conditions using a Gatan cryo-holder in a Hitachi H-600 TEM and a defocus of -1.5 μm.
Fluorescent probe (DiIC18(3)) was added to the lipid at a concentration of 0.1 mol %. Confocal images were obtained with a MRC 1024 confocal microscope (Bio-Rad) with a 585 LB emmision filter at 488 nm excitation. For three-dimensional image projection of a vesicle, z-scans in 0.5 μm steps were taken through the upper half of a liposome and projected by using the Confocal Assistant 4.02 software.
Hydrolytic activity and protein assays
AMG activity was determined by the starch-iodine method as described in . The assay solution consisted of 0.1 mL enzyme-containing sample and 1.0 mL starch solution (1%, w/v) in distilled water at pH 4.5. Assay tubes were incubated for 4 min at 55°C in a dry bath incubator. Appropriate negative controls, samples prepared without enzyme were made in all cases. One unit of enzyme activity was defined as the amount of enzyme, which hydrolyzed 1 mg of starch per minute under specified conditions.
Protein content of enzyme solution was determined using the Bio-Rad (Richmond, CA) DC Protein Assay kit with bovine serum albumin as a standard.
Sugars produced by the enzymatic hydrolysis of starch were identified and quantified by a Shimadzu HPLC system (Liquid Chromatograph LC-10AT, Diode Array SPD-M10A, and RID 6A) equipped with an Aminex HPX-87H cation-exchange column (300 mm × 7.8 mm, Bio-Rad Laboratories, Richmond, CA). The column was maintained at 50°C using a Bio-Rad column heater. Samples were eluted isocratically with 5 mM H2SO4 at a flow rate of 0.4 ml min-1. Maltooligosaccharides were purchased from Sigma Chemical Co. (St. Louis, MO) and used as standards as described previously .
Intrinsic enzyme kinetics
Experiments for kinetic parameter estimation were performed by hydrolyzing starch solutions of varying concentrations (1 – 10 mg mL-1) at 55°C in a dry bath incubator. The reaction medium consisted of 0.1 mL free or entrapped AMG, and 1.0 mL starch solution. Starch solutions were prepared in distilled, deionized H2O (pH 4.5). For each specified concentration, a series of identical reaction tubes containing starch solution was prepared. Incubation of the tubes was initiated at the same time. Samples, consisting of one reaction tube per time point, were taken at 0.5 min intervals for 10 min. The reaction in a tube was stopped by adding 1 mL HCl (1.0 M). The amount of unreacted starch was estimated by the starch-iodine assay, and glucose concentrations by HPLC as described above. Each experiment was conducted in triplicate. Initial rates were calculated using the linear portion of substrate vs. time plot.
Starch hydrolysis by entrapped AMG
Starch hydrolysis was carried out in liposomal suspensions. Soluble starch was used as substrate, and the process conditions were 55°C and pH 4.5 prior to the addition of entrapped AMG. Samples (1 mL) were taken at 10–60 min intervals, and the reaction was stopped by quenching in ice and adding 1.0 mL HCl (1.0 M). Samples were further analyzed by HPLC as described above.
Repeated hydrolysis of starch
The capacity for recovery and recycle of MLV-entrapped AMG activity from a process stream was also determined. The rate of starch hydrolysis by entrapped enzyme was determined at 55°C in 1.0 % (w/v) starch. After each batch of hydrolysis, the entrapped enzyme preparations were recovered from the reaction mixture by centrifugation at 16000 g for 10 min. The pellet was then resuspended into fresh starch solution for the subsequent run of starch hydrolysis. The whole process was repeated for six runs.
Kinetic parameters for starch hydrolysis were estimated using the nonlinear parameter estimation software of TableCurve 2D (Jandel Scientific, San Rafael, CA) based on initial rate data. Polymath 5.0  (Special Education Version, Prentice Hall, New Jersey) was used to solve the ordinary differential equations. The programs gave concentration profiles of substrate (starch) and product (glucose) for each set of conditions evaluated.