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Title:	Penulenan enzim kutinase menggunakan kromatografiturus terpadat dan kromatografi membran
Authors:	Suhaila Johar (P59402)
Supervisor:	Abdul Wahab Mohammad, YBhg. Prof. Ir. Dato’ Dr.
Keywords:	enzim kutinase Chromatographic analysis
Issue Date:	1-Oct-2013
Description:	Kromatografi merupakan salah satu kaedah pemisahan yang berkesan dalam menghasilkan peleraian yang tinggi antara komponen-komponen dalam sampel protein. Walau bagaimanapun, daya pemprosesan yang rendah akibat penggunaan media jenis gel partikel mengekang kecekapan kaedah ini. Pembangunan membran yang mempunyai daya pemprosesan yang tinggi sebagai media alternatif dapat mengatasi masalah tersebut. Kajian ini memfokuskan kepada penulenan enzim kutinase secara kromatografi menggunakan media konvensional dan membran dengan interaksi hidrofobik sebagai mekanisma pemisahan pilihan. Turus yang digunakan bagi kromatografi konvensional (HIC) dipilih secara empirikal terlebih dahulu. Melalui pemilihan secara empirikal, turus Butyl sepharose dipilih untuk selanjutnya dioptimumkan keadaan operasi prosesnya. Sementara itu, turus Sartobind Phenyl nano pula digunakan bagi penulenan kutinase menggunakan kromatografi membran (HIMC). Sampel protein campuran kutinase dan albumin serum bovin (BSA) digunakan bagi mensimulasikan proses penulenan. Pengoptimuman operasi kromatografi pada kedua-dua jenis medium ditentukan dengan menggunakan kaedah statistik reka bentuk rencam berpusat (CCD) untuk mendapatkan keadaan optimum parameter fasa bergerak (pH dan kepekatan garam ammonium sulfat) untuk menghasilkan ketulenan dan peratus pemulihan kutinase yang maksimum. Kaedah statistik ini berjaya mendapatkan keadaan optimum yang dikehendaki iaitu pada pH 4.0; 1.1 M ammonium sulfat bagi HIC dan pH 6.0; 1.3 M ammonium sulfat untuk HIMC. Model statistik bagi setiap sambutan (ketulenan dan pemulihan) mempunyai nilai pekali korelasi, R2 yang memuaskan iaitu masing-masing 0.742 dan 0.828 pada proses HIC, manakala 0.934 dan 0.916 pada proses HIMC. Pengoptimuman RSM meramalkan hasil ketulenan dan pemulihan sebanyak 3.8-kali dan 62% bagi HIC manakala 27-kali dan 85% bagi HIMC. Ketulenan dan pemulihan sebenar pada keadaan optimum HIC ialah 4.8-kali dan 66% manakala HIMC pula 24-kali dan 72%. Ini menunjukkan bahawa proses HIMC berjaya menghasilkan keluaran yang tinggi dari segi peleraian dan juga output proses. Ujikaji pengesahan membandingkan nilai ujikaji dengan ramalan dan terdapat sebanyak 21% dan 6% ralat diperoleh pada proses HIC, manakala ralat proses HIMC pula ialah sebanyak 13% dan 18%. Bentuk puncak yang kecil dan lebar diperoleh pada proses HIC, berlainan dengan puncak tajam yang terbentuk semasa proses HIMC. Ini menunjukkan bahawa HIMC berjaya menambah baik resolusi kromatogram yang terhasil. Kualiti sampel dianalisis menggunakan kaedah elektroforesis gel poliakrilamida-natrium dodesil sulfat (SDS-PAGE) dan dapat dilihat bahawa kandungan sampel tertulen proses HIC mempunyai sedikit protein kontaminan manakala sampel tertulen HIMC hanya mengandungi protein sasaran. Faktor-faktor yang mempengaruhi pemisahan dan interaksi hidrofobik dalam proses dikaji dengan menggunakan analisis interaksi pilihan. Interaksi ini melihat trend plot ln k’ melawan kepekatan garam selain mendapatkan anggaran nilai bilangan molekul air (-(v1)) dan ion garam (-(Δv++Δv_)) untuk melihat pemisahan dan keterpilihan kutinase semasa proses kromatografi. Hasil keseluruhan analisis ini mendapati pengaruh pH terhadap keterpilihan protein pada turus HIC adalah sangat signifikan berbanding kepekatan garam. Oleh itu, proses tidak memerlukan kepekatan garam yang tinggi bagi menghasilkan ketulenan dan pemulihan kutinase yang maksimum. Manakala, keterpilihan kutinase dalam media HIMC pula adalah lebih kompleks di mana pengaruh kedua-dua fasa bergerak (pH dan kepekatan garam) serta fasa pegun (ligan dan medium) saling berkait antara satu sama lain.,Chromatography is a highly efficient method for purification process due to high resolution of protein separation. However, some limitations are observed in process throughput, caused by the application of gel particle as its support matrix. The development of membrane as an alternative chromatographic media could overcome such problem. This study is focused the purification of cutinase using both conventional and membrane chromatography with hydrophobic interaction as the chosen separation mechanism. For conventional hydrophobic interaction chromatography (HIC), the column of choice was selected empirically before optimisation of the process condition could be proceed. Through some experiments, Butyl sepharose column was chosen to be optimized for its operation condition. As for hydrophobic interaction membrane chromatography (HIMC), the column used was Sartobind Phenyl nano. To simulate protein purification, a mixture of cutinase and bovine serum albumin (BSA) was used as crude feed sample. Optimisation of chromatographic operation parameters (pH and ammonium sulfate concentration) for each type of media was determined using a statistical approach of central composite design (CCD) which target to maximise both cutinase purification factor and percent recovery. The statistical method conducted has successfully determined the optimum condition within the experimental design space. The resulting optimum conditions are pH 4.0; 1.1 M ammonium sulfate for HIC and pH 6.0; 1.3 M ammonium sulfate for HIMC. The correlation coefficient, R2 of the statistical model for the purity and recovery responses showed adequacy of model to describe the experimental data. For purity and recovery of HIC, R2 values of 0.742 and 0.828 were obtained while R2 of 0.934 and 0.916 were recorded for the respective responses in the HIMC system. The predicted maximum purity and recovery for HIC are 3.8-fold and 62% whilst 27-fold and 85% for HIMC. The obtained purity and recovery at optimum values were 4.8-fold and 66% for HIC while 24-fold and 72% recovery were recorded in HIMC process. This shows that HIMC has managed to produce both high resolution and throughput of purified cutinase. The percent error of real and predicted responses for purity and recovery of HIC and HIMC are 21%; 6% and 13%; 18% respectively. Broad and small peaks were obtained in the HIC chromatogram while HIMC process produced much sharper peaks which shows that HIMC could produce chromatogram with improved resolution. The quality of the purified sample was analysed by sodium dedocyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was seen that the HIC-purified cutinase contained a little amount of contaminant protein, while only cutinase was present in the purified sample of HIMC. Factors that influence separation and hydrophobic interaction of the processes was studied using preferential interaction analysis to observe the trend of ln k’ versus salt concentration plots and to estimate the number of released water (-(Δv1)) and salt ions ((Δv++Δv_)) during the process. Throughout the analysis, it was observed that pH is the most significant factor for cutinase separation in HIC as compared to salt concentration, thus process does not require high concentration of ammonium sulphate to produce a maximum cutinase recovery and purification fold. Meanwhile, cutinase selectivity in HIMC was seen as an influence of multiple factors such as mobile phase composition (pH and salt concentration) and stationary phase (ligand and medium).,Master/Sarjana
Pages:	138
Call Number:	QD272.C4S839 2013 3 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Engineering and Built Environment / Fakulti Kejuruteraan dan Alam Bina

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