An approach of "monolayer engineering" gives new possibilities for the deposition of molecular organized films of enzymes for biocatalysis [1]. It includes not only classical Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) techniques, but also various combinations of these methods with layer-by-layer adsorption and self-assembly. Specialized instruments can be built to realize these combinations [2]. By the alternation of monolayers with different properties structural stability of the enzyme containing films and their functional activity can be increased [3]. Effective protein encapsulation into the hydrophilic matrix for the purposes of long storage and long-term actitity preservation can be provided as well. In addition, using the modified methods, one can deposit biocatalytic molecular organized media onto the solid supports of quite different materials and shapes and onto the polymeric films of large area and thus this approach is potentially suitable for industrial applications.
In this work, alternate-layer assemblies composed of glutathione S-transferase (GST) layer enclosed between two monolayers of various surfactant compounds as well as monocomponent films of GST deposited by LS technique were deposited and investigated. The top monolayer in alternate-layer assemblies can be completely removed from the surface when the sample is immersed into the aqueous solution for activity measurements if the choice of this monolayer is made in a proper way. Morphology and thickness of the monocomponent films and thickness of the adsorbed GST layers in LB alternate-layer assemblies were controlled using optical microscopy, interference, and ellipsometry techniques while the conditions of their deposition were varied. Namely, the GST films were formed at different values of surface pressure, pH, speed of compression, delay intervals before the deposition and compression, and dose of the solution used for the compound spreading. Time interval of protein adsorption was varied also in the case of deposition of alternate-layer assemblies. At the same time, activity of GST in the prepared structures was measured spectrophotometrically and compared with that of solution and material immobilized in a classical way. Finally, enzymatic activity of LB alternate-layer assemblies was measured in the dependence on the conditions of thermal treatment of the sample.
The results of these studies show that to achieve the preservation of high value of the enzyme activity in thin films prepared by the combination of adsorption and LB deposition techniques is more easy than using the LS technique. On the other hand, the relative activity decrease under the heating is less in the second case. Films of both types are more stable at the increased temperature than the solutions of enzyme. An increase of the exposure time of GST at the air-water interface results in a considerable denaturation of protein molecules. Average thickness of the transferred protein layer when deposited by LS technique is dependent of the surface pressure, but the conditions can be found for the deposition of approximately one monolayer each time. There are also some variations of the adsorbed layer thickness under the increase of adsorption time interval, but the sample activity is independent of this parameter. Some characteristics of the investigated films are shown in Tables 1 and 2.
Table 1. Typical values of GST specific activity (mol min-1 mg-1)* in the alternate-monolayer assemblies. GST layer was adsorbed during 10 minutes.
| Behenic acid / GST / Ba behenate | 11.9 |
| C16H33-BEDT-TTF / GST/ C16H33-BEDT-TTF | 8.6 |
| Behenic acid / GST / Behenic acid | 13.0 |
| C16H33-BEDT-TTF / GST / Behenic acid | 33.0 |
Table 2. Specific activity of GST (mol min-1
mg-1) in the deposited films before and after thermal treatment
at different temperatures in the comparison with the solution activity.
| Structure | initial | 10 min | 40 min | 90 min |
| 60C 80C 100C | 60C 80C | 60C 80C | ||
| C16H33-BEDT-TTF | 32.6 | 23.9 16.6 1.2 | 14.7 2.2 | 6.8 2.2 |
| GST / Behenic acid Monocomponent | 6.9 | 4.6 3.8 3.0 | 3.9 2.4 | 2.9 2.2 |
| GST film (LS) Solution | 64.0 | 1.2 | - | - |
References
1. C. Nicolini. Trends in Biotechnology, 15 (1997) 395.
2. V.I. Troitsky, M. Sartore, T.S. Berzina, D. Nardelli, C. Nicolini. Rev. Sci. Instrum., 67 (1996) 4216.
3. T.S. Berzina, V.I. Troitsky, A. Petrigliano, D. Alliata, A.Yu. Tronin, C. Nicolini. Thin Solid Films, 284-285 (1996) 757.