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OPTIMASI DAN KARAKTERISASI WHOLE-CELL BIOCATALYST PROTEIN FUSI LPP-OMPA-LC-KUTINASE DAN MUTAN SINONIMNYA PADA ESCHERICHIA COLI EPI300TM PENDEGRADASI POLIETILEN TEREFTALAT (PET)

OPTIMATION AND CHARACTERIZATION OF LPP-OMPA-LC-CUTINASE FUSSION PROTEIN AND ITÂ’S SYNONIMOUS MUTANT IN WHOLE-CELL BIOCATALYST ON ESCHERICHIA COLI EPI300TM FOR POLYETHYLENE TEREPHTHALATE (PET) DEGRADATION

Master Theses from JBPTITBPP / 2017-10-02 10:47:33
Oleh : Yehezkiel Victorio (NIM: 21116027), S2 - Biotechnology-SITH
Dibuat : 2017-10-02, dengan 1 file

Keyword : E. coli, leaf compost cutinase, whole-cell biocatalyst, mutasi sinonim

Polietilen tereftalat (PET) merupakan salah satu jenis polimer plastik yang paling umum digunakan dalam kehidupan sehari-hari. Penggunaan PET saat ini mencapai 7% dari total kebutuhan plastik di dunia atau setara dengan 2,177 juta ton. Lebih dari 50% serat sintetis yang diproduksi di dunia terdiri dari PET dan konsumsi global terhadap PET yang telah dilaporkan mencapai $17 milyar per tahun. Degradasi secara alami yang sulit dan memakan waktu lama serta penggunaannya yang terus meningkat mengakibatkan terjadinya akumulasi limbah yang mencemari lingkungan, akumulasi limbah PET tidak hanya berbahaya bagi lingkungan tetapi juga pada manusia secara langsung, maka solusi untuk mengatasi akumulasi penumpukan PET sangat dibutuhkan segera. Enzim kelompok kutinase diketahui dapat menghidrolisis ikatan ester pada rantai utama polimer pada PET. Berbagai teknologi telah dilakukan untuk melakukan optimasi kerja enzim kutinase, diantaranya adalah penelitian tim iGEM ITB_Indonesia (2014). Tim iGEM ITB_Indonesia (2014) dalam sistemnya mengembangkan rancangan enzim leaf compost kutinase (LC kutinase) sebagai Whole-cell biocatalyst dengan pendekatan surface display. Pendekatan ini dilakukan dengan memfusi enzim LC kutinase dengan outer membrane protein A (OmpA) pada E.coli. Pada penelitian ini dilakukan optimasi dan karakterisasi protein fusi tersebut. Optimasi dilakukan dengan melakukan mutasi sinonim pada urutan nukleotida gen protein fusi dan memindahkan gen ke plasmid pCCI. Hasil protein fusi dan mutan kemudian dikarakterisasi, dimana didapatkan suhu optimum kerja whole-cell biocatalystnya adalah 55oC dan pH optimumnya 8. Efek dari optimasi juga meningkatkan laju pertumbuhan sel dimana pada whole-cell biocatalyst awal memiliki laju pertumbuhan maksimum 626.058 sel detik-1 sedangkan whole-cell biocatalyst mutan 1914,858 sel detik-1. Whole-cell biocatalyst mutan juga memiliki kestabilan terhadap suhu yang lebih tinggi pada pH 8.

Deskripsi Alternatif :

Polyethylene therephthalate (PET) is a synthetic polyester composed of terephthalic acid and ethylene glycol. It is among the most commonly used polymer. The consumption of PET in Europe on 2015 reached 7% of total polymer usage (2,177 million Ton) and increased 10% annually. The majority of PET is used in production of plastic packaging, household and domestic product. PET is mostly used for textile fiber (60%) and plastic bottles (30%). Unavailability of efficient degradation technology and high usage demand with short period of time usage cause PET waste to build up. This build up usually end up on landfills and may cause major impact on environment and towards human, therefore proper degradation technology is urgently required. LC-cutinase ( leaf compost cutinase) that was discovered from metagenomic studies is known for its capability to degrade PET. LC-cutinase is a lipolytic/esterolytic enzyme from cutinase (EC 3.1.1.74) degrade PET by hydrolyzing PET. ITB_Indonesia iGEM team (2014) came up with a proposal of creating whole-cell biocatalyst system to degrade PET. It was done by expressing fusion protein of Lpp-OmpA-LC-cutinase on surface display based system. The aim of this research is to Optimize and characterize the previous system. Optimization of the system was conducted by synonimous mutation using OptimumGeneTm for design and synthesized by GenScript®. Characterization of the enzyme activity is than conducted done by measuring absorbance caused by the degradation of p-Nitrophenyl butyrate (pNPB) in 415 nm wave length. Based on this characterization, the whole-cell biocatalyst highest activity was measured to be 55oC and pH of 8. Synonimous mutation also increased the cell growth rate from 626.058 cells/second to 1914.858 cells/second. Mutant LC-cutinase also had higher thermal stability on pH 8.

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