FUNCTIONALIZED MEMBRANE WITH LIPID BILAYER FOR IMMOBILIZED ENZYMATIC SYSTEMS

Abstract

Enzymes, immobilized within membrane pores, offer improved kinetics due to pressure driven convective flow. On the other hand, supported lipid bilayer (SLB), owing to its fluidity and native “cell-membrane” environment, facilitates the activity and stability of enzymes. In this study, the advantages of biomimetic characteristics of SLB and convective flow through microporous membrane were integrated within the same configuration. We report development of a novel biomimetic membrane by incorporating SLB within membrane pores using two different approaches – (i) direct deposition of lipid bilayer within bare membrane pores, and (ii) immobilization of a polymer cushion prior to lipid bilayer within membrane pores. As a model enzyme, glucose oxidase (GOx) was immobilized into the phospholipid bilayer network within the functionalized membrane pores. In-depth activity study was conducted with different GOx-functionalized membranes while monitoring the production of H2O2 by oxidation of glucose. In-depth activity study revealed that the biomimetic membrane containing polymer cushion supported SLB and operating under convective mode of flow exhibited benefits in terms of activity, stability and operating range of pH. Therefore, we further extended our investigation and developed a novel bienzymatic system containing two enzymes, electrostatically co-immobilized, within phospholipid bilayer functionalized membrane pores. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were used as model enzymes, and the two enzymes were immobilized using negative and positive charges, respectively. Again, two different approaches of direct deposition and polymer cushion supported lipid bilayer attachment within membrane pores were considered. Two different membrane configurations were also examined – (i) stack configuration, and (ii) co-immobilized configuration. Bienzymatic system containing polymer cushion supported lipid bilayer and present in co-immobilized configuration was superior to the other alternatives. The electrostatically immobilized bienzymatic system also exhibited improved stability and enabled a broader range of operating pH than the free enzymes. The major observation from GOx-HRP bienzymatic system was that two enzymes containing opposite charges can be electrostatically co-immobilized with zwitterionic DMPC molecules of SLB to give better activity and satisfying storage stability along with a broader range of operating pH. This finding was further extended to investigate the possibility of development of multienzymatic system. ii A multienzymatic system containing glucoamylase (GA), GOx and HRP enzymes was developed by electrostatically incorporating all three enzymes within biomimetic membrane pores. CL membrane was selected to develop GA-GOx-HRP multienzymatic system. GOx-HRP enzymes were coimmobilized within a membrane, whereas GA was immobilized within a separate membrane. Both of these membranes were used in stack configuration, with the GA-membrane on top of the GOx-HRP membrane, for activity analysis of multienzymatic system. Activity analysis and storage stability were observed to be better for immobilized GA-GOx-HRP multienzymatic system than free enzymes due to the presence of the lipid bilayer functionalized membrane pores. Operating range of pH was also observed to be broader as observed earlier for immobilized GOx and GOx-HRP bienzymatic system. The study was further extended for exploring the reusability of the membrane as well as the other functional components. The functionalized architecture was constructed based on electrostatic interactions, which facilitate reversible attachment-detachment sequence of the functional moieties. To demonstrate potential application, an enzyme, glucose oxidase (GOx), was electrostatically immobilized within the phospholipid bilayer functionalized membrane and enzymatic catalysis was conducted. Then, the enzyme was detached keeping the functional SLB backbone intact. Detachment of the enzyme without affecting the functional activity of SLB backbone permits attachment of fresh enzyme. In addition, reusability of the phospholipids is also of great importance as they have wide range of applications, but their usage is limited by higher cost. We have demonstrated detachment of the SLB from the membrane using a simple technique. Characterization of the detached phospholipid confirmed retention of the original structural and functional properties as exhibited before attachment. Comparison of the results obtained in this study with other literature reported results establishes the benefits of the phospholipid bilayer functionalized biomimetic membrane operated under convective flow. To the best of our knowledge, this is the first study that investigates the advantages of incorporation of phospholipid bilayer within membrane pores for immobilized enzymatic catalysis and discusses the potential of reusability of the biomimetic membrane as well as the phospholipid. With further improvement in the strategy of functionalization and process optimization, this technique promises to deliver an efficient bioprocess for green synthesis of chemicals at larger scale.