10 Sequential action of multiple encapsulated enzymes
10.2 Glucoamylase and Glucose Oxidase
Separate molecular layers of glucoamylase (GA) and GOD (pI of GA and GOD are 4.2 and 4.3, respectively) were assembled on an ultrafilter (Molcut II LC, cut‐off molecular weight 5 kDa, Millipore) by LbL adsorption using PEI as a polycation [85]. The Molcut II system is composed of an upper cup, an ultrafilter and a lower cup (Figure 20). Protein alternate films were assembled on the filter, which was fixed on the bottom of the upper cup. Aqueous PEI solution was placed in the upper cup for 15 min at room temperature, and then the inside of the upper cup was washed carefully with water for 2 min. Aqueous PSS solution was then placed in the upper cup for 15 min at room temperature and the cup was washed again. These procedures were repeated three times to produce precursor layers of (PEI/PSS)4, then continued to build the following film structures using aqueous GA and GOD solutions:
(1) Film 1: filter + (PEI/PSS)4+ (PEI/GOD)2+ (PEI/PSS)2 + (PEI/GA)2+ PEI (2) Film 2: filter + (PEI/PSS)4+ (PEI/GOD)2+ (PEI/PSS)10 + (PEI/GA)2+ PEI (3) Film 2: filter + (PEI/PSS)4 + (PEI/GA)2 + (PEI/PSS)2 + (PEI/GOD)2 + PEI (4) Film 2: filter + (PEI/PSS)4 + (PEI/GA)2 + (PEI/PSS)10 + (PEI/GOD)2 + PEI
Film formation on the filter was confirmed by X‐ray Photoelectron Spectroscopy (XPS) elemental analysis. Only carbon and oxygen were observed from a bare filter membrane (cellulose). After polyions and proteins had been assembled, nitrogen and sulfur were also detected.
Figure 20: Sequential enzymatic process based on glucoamylase (GA) and glucose oxidase (GOD) and its experimental setup [85]. “Reprinted from reference 85, Copyright 1996, with permission from Elsevier.”
The enzymatic activities of these films were examined using the experimental setup depicted in Figure 20. An aqueous solution of “water‐soluble starch” was placed on the enzyme‐immobilized ultrafilter in the upper cup. Filtration was started by applying pressure to the upper cup with a syringe to achieve a constant flow rate. Then, 1 ml of PIPES buffer was added to the upper cup and the same pressure was applied. The latter procedure was repeated several times in order to wash out the filterable components. As per the reaction scheme shown in Figure 20, the glycosidic bonds in starch are hydrolysed by GA producing glucose, which is converted to gluconolactone by GOD with H2O2 as a co‐product. Then unreacted starch, H2O2 and glucose were quantified.
The highest reaction yield was obtained with film 2, and the second highest yield was with film 1. In these two films, the arrangement of protein layers against the direction of flow agrees with the order of the sequential enzymatic reactions. In contrast, when the order of the two protein layers was reversed, as in films 3 and 4, lower yields were obtained. These results indicated that appropriate arrangement of the two enzymes is crucial for obtaining high yields in the sequential reactions, since starch is hydrolyzed to glucose in the outer GA layers and the glucose produced is subsequently converted to gluconolactone and H2O2 in the inner GOD layers. In contrast, in the case of films 3 and 4, starch had to pass through GOD layers and separator layers, (PEI/PSS)n, before it reached GA layers, and the glucose produced had to diffuse back to GOD layers against the
flow of the solution. This, of course, slows down the whole process. In particular, diffusion of high molecular‐weight starch through multilayers would be rate limiting, and facile contact of starch with GA layers is important.
The role of the spacer layer that separates the GA layers from the GOD layers becomes evident from a comparison of the activities of films 1 and 2. It is curious that higher yields of glucose and H2O2 were obtained using film 2 rather than using film 1, regardless of the initial starch concentration. This cannot be explained by the ease of diffusion. Reports have shown that for multi‐component assemblies, film growth is somewhat smaller in the first few steps when the assembled pair is changed. The GA‐PEI layers might not be ideally prepared in film 1 because film 1 has thinner spacer layers of PEI‐PSS than film 2, so a smaller amount of GA will be immobilized on film 1, reducing the total activity. Another plausible mechanism is related to the inhibition of GA activity by gluconolactone. Small molecules such as gluconolactone and H2O2 should be able to diffuse backward to the GA layers relatively easily. They are inhibitors of GA and reduce its enzymatic activity. A mixed type inhibition of the GA by gluconolactone was reported [87]. The extent of this inhibition must be larger in film 1 than in film 2, because the spacer layer of the latter is thicker. This novel approach of combining ultrafiltration with the use of a layered protein film allowed successfully the separation of substrate and products without any further procedure [85].
The concept of sequential action of GA and GOD was implemented by Sun et al., to develop a maltose sensor [88]. In principal, GA breaks maltose into 2 molecules of D‐glucose, which are subsequently oxidized by GOD to gluconic acid with the production of hydrogen peroxide:
Maltose + H2O 2 D‐glucose
Β‐D‐glucose + O2
gluconic acid + H2O2
H2O2→O2 + 2 e‐ + 2 H+
The oxidation current of H2O2 produced in the enzymatic reaction has a linear relationship with the concentration of maltose within a certain range. The electrode structure is illustrated in Figure 21a, the enzyme multilayer films are fabricated on a gold electrode, so that GOD and GA are alternatingly sandwiched between the bipolar quaternary ammonium salt NC6BPC6N (Figure 21b).
In detail, a gold electrode was polished with aluminum powder, sonicated in fresh water and
allowed to air‐dry. The clean gold electrode was immersed for 24 h at room temperature in an ethanolic solution of 3‐mercaptopropionic acid to give one monolayer of self‐assembled film.
Afterwards it was transferred into the NC6BPC6N solution (pH = 8), thus a positively charged surface was obtained. The modified cationic electrode was deposited with two layers of GOD followed by two layers of GA alternating with NC6BPC6N. The resulting modified electrode was applied as a maltose sensor. So when it was immersed into the maltose solution, maltose first reacted with outside GA layers to produce glucose. The resulting b‐D‐glucose then diffused into the assembly and reacted with interior GOD layers to produce H2O2, which oxidized on the surface of the gold electrode.
Figure 21: (a) Schematic illustration of the structure of the bienzyme multilayer film. El refers to glucose oxidase and E2 refers to glucoamylase, (b) The bipolar quaternary ammonium salt NC6BPC6N [88]. “Source:
Macromol Chem Phys, 197, 1996, 147‐153. Sun, Y.; Zhang, X.; Sun, C.; Wang, B.; Shen, J. Copyright [1996].
This material is reproduced with permission of John Wiley & Sons, Inc.”.
It was shown that the time required to reach 95% of the steady‐state current is less than 60 s after the addition of the maltose sample. The calibration curve for the determination of maltose was measured. A linear relationship up to 6 mMol L‐1 was obtained between the current response and maltose concentration. In the experiment, the authors also found that no current is observed when the bare gold electrode, the electrode with 3‐mercaptopropionic acid and NC6BPC6N layers, and finally the electrode with a protein bilayer of GA alone were immersed in the experimental solutions that contained maltose. The above experimental results showed that only when GA and
(a)
(b)
GOD were fabricated rationally on the surface of the gold electrode, the sensor can be used to determine maltose [88].