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Title:	Engineering Glomerella cingulata cutinase for enhanced enzymatic performance
Authors:	Chin Iuan Sheau (P34290)
Supervisor:	Farah Diba Abu Bakar, Dr.
Keywords:	Kutinase Glomerella cingulate Enzymatic performance Dissertations, Academic -- Malaysia
Issue Date:	20-Jun-2015
Description:	Cutinase, a natural polyesterase whose unique characteristics are comparable to that of esterases and lipases, is ideally suited to be exploited in a broad spectrum of industrial or biotechnological applications. In this regard, a cutinase gene from a phytopathogenic fungus, Glomerella cingulate, was overexpressed whereby the cutinase enzyme was subsequently purified and characterised. This study describes a number of enzyme engineering techniques employed to improve the catalytic performance of cutinase in ensuring its feasibility for industrial purposes. In the first study, several single-site cutinase variants were generated via megaprimer-based PCR. The A67L variant suggested that the substitution has limited its acceptance towards long chain substrates. The L172K variant showed higher enzymatic performance compared to the recombinant wild type cutinase, especially towards phenyl ester substrates of longer carbon chain lengths. Employing dynamic light scattering measurements, the N177D variant displayed a delayed thermal unfolding compared to the wild type recombinant cutinase. Despite a notable increase in thermal stability, the catalytic activity of the N177D at 25 °C was not compromised. In the second study, the possibility of molecular dynamics (MD) simulations in the rationalisation of the thermal stability enhancement of the N177D was explored. Comparative analyses in thermal unfolding MD simulations carried out on both the wild type and N177D suggested that the enhanced thermal tolerance might be achieved by the formation of an additional salt-bridge between D177 and K154, thereby cooperatively enhancing the strength of the existing dominant salt-bridge network. In the third study, whole gene diversification of the cutinase was carried out by coupling point mutation-based error-prone PCR (epPCR) and recombinative staggered extension process (StEP) in a directed evolution endeavour. Microplate-based enzyme expression and screening was achieved, thus facilitating the screening of a cutinase variant library comprising about 20 000 E. coli clones. Several promising cutinase variants were identified and selected for further characterisation, including the P60H, L169Q and L94H in which enzymatic performances were enhanced. Additionally, the L94H exhibited notable enhancement in thermal tolerance. In the following work, the thermo-stabilising N177D mutation was introduced into cutinase variants, the L172K and L169Q respectively in an attempt to enhance their enzymatic performances. The outcome demonstrated that the L172K/N177D and L169Q/L172K/N177D were capable of enduring elevated temperature, following the introduction of the N177D. This proved advantageous for rational engineering of thermal stability in proteins. As a conclusion, these studies demonstrated that semi-rational design and directed evolution are useful in optimising the catalytic performance of cutinase and ultimately, in generating enzyme variants with superior performance.,Kutinase, iaitu sejenis poliesterase semula jadi yang memperlihatkan ciri-ciri unik setanding dengan esterase dan lipase, adalah sangat sesuai untuk dieksploitasikan dalam spektrum aplikasi industri dan bioteknologi yang luas. Oleh itu, gen kutinase daripada kulat fitopatogen, Glomerella cingulata, telah diekspres secara berlebihan dan seterusnya enzim kutinase telah ditulen serta dicirikan. Kajian ini memperincikan sejumlah teknik kejuruteraan enzim yang telah digunakan untuk meningkatkan prestasi pemangkinan kutinase, dalam memastikan keserasiannya sejajar dengan keperluan industri. Dalam kajian pertama, beberapa varian kutinase tapak tunggal telah dijanakan menerusi PCR bersandarkan pencetus mega. Varian A67L mencadangkan penukaran ini mengekang penerimaan substrat berantai panjang. Varian L172K menunjukkan prestasi pemangkinan yang lebih tinggi berbanding kutinase rekombinan jenis liar, terutamanya terhadap substrat ester fenil berantai karbon panjang. Dengan menggunakan pengukuran hamburan cahaya dinamik (DLS), varian N177D memaparkan penyahlipatan aruhan haba yang tertunda, berbanding kutinase rekombinan jenis liar. Walaupun peningkatan kestabilan haba diperhatikan, aktiviti pemangkinan N177D pada 25 °C tetap tidak berubah. Dalam kajian kedua, penggunaan simulasi dinamik molekul (MD) dalam merasionalkan peningkatan kestabilan haba dengan kehadiran N177D telah dikaji. Analisis perbandingan simulasi MD penyahlipatan haba yang dijalankan terhadap kutinase jenis liar dan N177D mencadangkan bahawa peningkatan toleransi haba dicapai dengan pembentukan titian garam tambahan di antara D177 dan K154, justeru meningkatkan kekuatan secara kerjasama antara rangkaian titian garam dominan yang sedia ada. Dalam kajian ketiga, diversifikasi seluruh gen kutinase dilakukan dengan menggabungkan PCR cenderung kesilapan (epPCR) yang berasaskan mutasi titik dengan proses lanjutan tak serentak (StEP) secara rekombinasi dalam usaha ke arah evolusi terarah. Pengekspresan dan penyaringan aktiviti enzim berasaskan piring mikrotiter berjaya dilakukan, lalu memudahkan penyaringan perpustakaan varian kutinase yang mengandungi kira-kira 20 000 klon E. coli. Beberapa varian kutinase berpotensi berjaya dikenal pasti, lalu dipilih untuk pencirian lanjutan, termasuklah P60H, L169Q dan L94H yang menunjukkan peningkatan prestasi pemangkinan. Tambahan pula, L94H memaparkan peningkatan toleransi haba yang ketara. Dalam kajian seterusnya, mutasi penstabilan haba, N177D telah digabungkan ke dalam varian kutinase L172K dan L169Q untuk meningkatkan prestasi pemangkinan masing-masing. Hasil, menunjukkan bahawa L172K/N177D serta L169Q/L172K/N177D berupaya aktif pada suhu tinggi, lantaran kehadiran mutasi N177D. Dalam kajian seterusnya, mutasi penstabilan haba, N177D telah digabungkan ke dalam varian kutinase L172K dan L169Q untuk meningkatkan prestasi pemangkinan masing-masing. Hasil menunjukkan bahawa L172K/N177D serta L169Q/L172K/N177D berupaya aktif pada suhu tinggi, lantaran kehadiran mutasi N177D. Dengan itu, kejuruteraan secara rasional berfaedah dalam meningkatkan kestabilan haba protein. Secara kesimpulannya, kajian ini menunjukkan reka bentuk semi-rasional dan evolusi terarah bermanfaat dalam mengoptimumkan prestasi pemangkinan kutinase, bagi menjanakan varian enzim dengan prestasi lebih hebat.,Ph.D
Pages:	204
Call Number:	QH324.9.E5 C488 2015
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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