Fitness Optimization of Polyethylene Terephtalate (PET)- Degrading Wholecell Biocatalyst through Addition of SsrA Sequence in Lpp-OmpA-LC-Kutinase Fusion Protein

• Main Author: MAWADDA - NIM: 10414022 , NAILI
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29516
• Institution: Institut Teknologi Bandung

<p align="justify">Increasing in accumulation of polyethylene terephtalate (PET) waste causes problem in the environment therefore should be degraded. One of PET degradation method is by using cutinase enzyme. This enzyme has the ability to hydrolize ester-linkage in PET into ethylene glycol and terephtalic acid. iGEM ITB 2014 Team has built PET-degrading whole-cell biocatalyst using Lpp-OmpA-LC-cutinase which is located in the outer membrane of Escherichia coli. Based on this system, previous study reveals that there is fusion protein activities in the cytoplasm. The cytoplasmic protein will only being a cell burden since it could not be used by the PET degradation system. Codon optimization of LC-cutinase by synonimous mutation was succesfully done to increase the efficiency of protein production. Since protein expression is increased, it will cause protein homeostasis disturbance and often lead to inclusion bodies formation. This disturbance will affect the host growth due to increasing in metabolic burden inside the cell. Thus, whole-cell biocatalyst efficiency will be reduced because of decreased in cell fitness. This study perform an approach to increase cell fitness and whole-cell biocatalyst efficiency through addition of degradation tag. Small stable RNA A (ssrA) degradation tag was added in C terminal of Lpp-OmpALC-cutinase protein. The ssrA will be recognized and degraded by cytoplasmic protease, there are ClpXP and ClpAP therefore could reduce metabolic burden inside the cell. Activity assay of ssrA-tag LC-cutinase was done using pNPB (para nitrophenyl butyrate) substrate in various pH and temperatures. The result shows that optimum condition of this protein is pH 8 and 55ºC. The optimum condition then used in latter assay using sample with three different treatments, there were nonsonicated pellet (whole-cell), sonicated pellet (membrane protein), and sonicated supernatant (intraceluller protein). In intraceluller protein fraction, the activity decreased from 2,17 x 10-10 U/Cfu to 1,58 x 10-10 U/Cfu. These results indicated that some intraceluller ssrA-AAV-tagged protein were degraded by protease. Meanwhile, in whole-cell and membrane protein fraction were not detected any inhibition effect in LC-cutinase activity due to ssrA tagging. Effect of ssrA tagging also enhances cell’s growth rate, from 2,04 x 105 cells/second to 7,04 x 105 cells/second. PET degradation test indicates non significant result, that only 0,17%-0,18% weight loss in all PET sample. However, SEM analysis shows there is microscopic holes on the PET sample which indicate degradation activity. SsrA-AAV tagging could increase cell fitness with higher growth rate, but further optimization still needed to enhance wholecell biocatalyst activity in PET degradation. <p align="justify">