#TITLE_ALTERNATIVE#

• Main Author: ANNISA UTAMI (NIM: 30712012), RATNA
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/23847
• Institution: Institut Teknologi Bandung

Background: Superoxide dismutase (SOD), a potent antioxidant and is an important constituent of cellular defence mechanism against oxidative stress. One of potential SOD for therapeutic application is from Citrus limon. However, SOD delivery by oral route has limitation for its bioavailability caused by low intestinal permeation due to large size of protein. One approach to overcome this low intestinal permeation is by using permeation enhancers. Purpose : This research was intended to construct two fusion proteins of SOD Citrus limon (SOD_Cl) with gliadin peptides, LGQQQPFPPQQPYPQPQPF (GliSOD_51) and QQPYPQPQPF (GliSOD_61) to enhance their permeation through monolayer CaCO-2 cells, to obtain the three recombinant proteins with high purity, to characterize their specific unit activity determination, thermal and pH stability and stability in the presence of chemicals, their enzyme kinetics, also for their cytotoxicity and permeation capacity through monolayer CaCo-2 cells and to study the mechanism of SOD_Cl, GliSOD_P51 and GliSOD_P61 permeation at molecular level. Methods: The recombinant SOD_Cl and its fusions (GliSOD_P51 and GliSOD_P61) were constructed using genetic engineering approach. The three proteins were overexpressed in Escherichia coli BL21(DE3) and affinity purified using Nickel column. Biochemical characterizations were performed determining residual activity of SOD based on nitro blue tetrazolium-riboflavin oxidation-reduction. Cytotoxicity and permeability assay were conducted using Caco-2 cells. The cytotoxicity of the three SODs in Caco-2 cells in vitro was presented as a percentage of cell viability relative to the negative (without treatment, 100%) as determined using the Alamar Blue and Trypan Blue assay. For permeability testing, Caco-2 cells were grown in Transwell chambers until 6 and 21 days and permeation capacity of the three SODs and paracellular marker Fluorescein Isothiocyanate (FITC)-Dextran 10 kDa (FD 10) and FD 40 was evaluated by incubating proteins and FD together in apical compartment. The presence of proteins in apical and basolateral compartments after permeabiliy assay was analyzed by protein electrophoresis. The permeation of FD 10 and FD 40 was determined by measuring their concentrations in basolateral compartment using spectrofluorometry. Study of molecular mechanism of permeation enhancement was determined by measuring the expression of ZO-1, Claudin-1, Claudin-2 and Occludin genes using quantitative real-time PCR. Results: Research result showed that the level of expression of the three proteins was high about 39.84% - 42.38% of total intracellular proteins of E. coli BL21(DE3). Their electrophoretic purity level was higher than 95%. Results of biochemical characterizations showed that the three proteins shared similar enzymatic properties including activity over a broad pH range from 2.0–8.0, were relatively stable (retained activity of more than 80%) after incubation at temperature below <br /> <br /> <br /> <br /> 50°C and relatively tolerant to chemical agents tested at low concentration. Inhibition by KCN and H2O2 confirmed that SOD_Cl belonged to Cu,Zn-SOD. Of the three proteins, SOD_Cl exhibited the highest specific activity and KM and Vmax values, and the highest thermal stability and stability at alkaline pH. This showed that the introduction of gliadin peptides to SOD_Cl decreased specific activity, thermal stability and stability at alkaline pH (9-11). Cytotoxicity assay results using Alamar Blue and Trypan Blue showed no deleterious effects caused by the three of SODs to the Caco-2 cells. This results suggest that the increase in permeability was not not due to cell deaths lead to high permeability. Based on SDS-PAGE analysis, only GliSOD_P51 and GliSOD_P61 proteins were present in basolateral solutions demonstrating that both proteins but not SOD_Cl was permeated across the Caco-2 cell monolayer. The GliSOD_P51 and GliSOD_P61 increased significantly permeability of FD10 but not FD 40 compared to cell treated with SOD_Cl or negative control indicating the increase of permeability by the two proteins was via paracellular route. Gene expression analysis showed that the GliSOD_P51 increased paracellular permeability without involvement the regulation of claudin-1, claudin-2 and occludin gene expression and the increase expression of ZO-1 gene, while the GliSOD_P61 by decreasing the gene expression of claudin-1, claudin-2 and occludin without involvement of ZO-1 gene expression. Conclusion: In this research, recombinant plasmid carrying DNA encoding for SOD_Cl, GliSOD_P51 and GliSOD_P61 are constructed and the three proteins are expressed in high level in E. coli BL21(DE3). The level of electrophoretic purity of the three recombinant proteins after purification is high. Biochemical characterization of the three recombinant proteins shows high activity, good thermal and pH stability and enzyme kinetics. All of three recombinant protein were not toxic to the Caco-2 cells. In vitro permeability studies showed that gliadin peptides in GliSOD_P51 and GliSOD_P61 increased the permeation capacity of proteins respectively and FD 10. The permeation enhancement mechanism by GliSOD_P51 appears to occur post-translationally or involving other TJ proteins while GliSOD_P61 through decreased expression of occludin gene. Taken all together, based on enhancement of permeation of paracellular marker and SOD proteins, and the results of molecular mechanism study shows that the potency of gliadin peptides in GliSOD_P51 and GliSOD_P61 as non toxic permeation enhancer.