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Title:	Pembangunan mutan prtT dan analisis sekretom untuk meningkatkan kestabilan protein heterolog dalam Aspergillus niger
Authors:	Nurhaida Kamaruddin (P48206)
Supervisor:	Abdul Munir Abdul Murad, Prof. Madya. Dr.
Keywords:	Protein heterolog Aspergillus niger
Issue Date:	9-Sep-2015
Description:	Penghasilan protein heterolog oleh Aspergillus niger sering dihadkan oleh protease yang dihasilkan oleh hos sendiri. Bagi menambah baik penghasilan protein heterolog, gen mengekod protease ataupun faktor transkripsi yang mengawal atur pengekspresan gen protease perlu dinyahaktifkan. Objektif kajian ini adalah untuk menyahaktifkan gen mengekod faktor transkripsi prtT, menentukan penghasilan protease ekstra sel oleh mutan prtT dan menentukan aktiviti serta kestabilan protein heterolog yang dihasilkan oleh mutan prtT. Gen prtT telah dinyahaktifkan dalam A. niger PY11 melalui kaedah penggantian gen dan mutan yang terhasil telah disahkan melalui analisis tindak balas berantai polimerase (PCR) dan pemblotan Southern. Analisis transkripsi berbalik tindak balas berantai polimerase (RT-PCR) menunjukkan bahawa tahap pengekspresan gen protease pepA, pepB dan pepF dalam mutan AnΔprtT didapati berkurangan berbanding A. niger PY11 sama ada dalam keadaan yang teraruh atau tidak teraruh. Pengasaian aktiviti proteolitik ekstra sel secara kualitatif yang dilakukan di atas agar medium minimum yang mengandungi kasein dan gelatin mengesahkan bahawa AnΔprtT mempamerkan fenotip mutan. Ia dicirikan melalui kehilangan pembentukan zon „halo" di sekeliling koloni walaupun AnΔprtT menunjukkan pertumbuhan koloni yang sama dengan A. niger PY11. Pengasaian aktiviti proteolitik ekstra sel turut diukur secara kuantitatif melalui pencernaan bovine serum albumin (BSA) di mana aktiviti protease ekstra sel AnΔprtT didapati kekal rendah berbanding A. niger PY11 yang menunjukkan peningkatan secara linear sepanjang 6 hari pengkulturan. Penyahaktifan prtT ternyata tidak memberi kesan terhadap pertumbuhan AnΔprtT apabila pertumbuhannya didapati setara dengan A. niger PY11. Seterusnya, protease ekstra sel A. niger PY11 dan AnΔprtT dicirikan melalui kaedah kromatografi cecair berprestasi ultra-pengionan elektrosemburan nano aliran-spektrometri jisim selari (UPLC-nanoESI-MS/MS). Keseluruhannya, sebanyak 20 protease ekstra sel dikenal pasti daripada sampel hari ke-5 hingga 9 bagi A. niger PY11 dan AnΔprtT. Daripada jumlah tersebut, 15 merupakan protease yang bergantung kepada prtT manakala lima lagi adalah protease yang tidak bergantung kepada prtT. Bagi membandingkan keefisienan A. niger PY11 dan AnΔprtT sebagai hos untuk penghasilan protein heterolog, gen kutinase daripada Glomerella cingulata di bawah kawalan promoter glukoamilase A telah diintegrasikan ke dalam kedua-dua genom. Aktiviti kutinase daripada transforman A. niger PY11 dan AnΔprtT yang membawa gen kutinase G. cingulata rekombinan masing-masing meningkat kepada 20 dan 36 kali ganda lebih tinggi berbanding strain induk tak tertransformasi. Ini mencadangkan bahawa keupayaan mutan untuk menghasilkan protein heterolog tidak terjejas dengan penyahaktifan prtT. Selain itu, jumlah enzim heterolog juga dihasilkan dengan lebih tinggi dalam AnΔprtT berbanding A. niger PY11. Aktiviti kutinase heterolog daripada kedua-dua strain didapati stabil pada suhu 4°C selama 6 minggu. Manakala semasa penyimpanan pada suhu 25°C, aktiviti kutinase heterolog daripada AnΔprtT didapati kekal sehingga lebih daripada 80% selepas dua minggu penyimpanan berbanding aktiviti kutinase daripada A. niger PY11 yang tinggal kurang daripada 3% dalam tempoh yang sama. Kesimpulannya, pembangunan mutan faktor transkripsi prtT telah mengurangkan aktiviti protease ekstra sel dalam A. niger di samping dapat meningkatkan penghasilan dan kestabilan protein heterolog,Ph.D,Production of heterologous proteins in Aspergillus niger is often limited by the high levels of proteases produced by the fungus. To improve heterologous production, protease genes or a transcription factor controlling expression of extracellular protease-encoding genes has to be deactivated. The objective of this work are to deactivate a gene encoding transcription factor prtT, determine the production of extracellular proteases by the prtT mutant and determine the activity and stability of heterologous protein produced by the prtT mutant. The prtT gene was inactivated in A. niger PY11 via gene replacement and the mutant produced was verified via polymerase chain reaction (PCR) and Southern blotting. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed reduced expression levels of several protease genes, pepA, pepB and pepF in AnΔprtT as compared to A. niger PY11 either in induced or non-induced conditions. A qualitative extracellular proteolytic activity assays conducted on the AnΔprtT mutant confirmed that this strain exhibited the mutant phenotype, characterised by loss of halo production on minimal medium containing casein and gelatine plates even though the mutant has similar colonial growth to the parent strain. Extracellular proteolytic activity was also measured quantitatively via bovine serum albumin degradation whereby the protease activity of A. niger PY11 showed a linear increase throughout six days culturing while AnΔprtT protease activity remained very low. Growth of A. niger PY11 and AnΔprtT was comparable indicating that disruption of prtT did not affect fungal growth. Subsequently, extracellular proteases of A. niger PY11 and AnΔprtT were characterized via Ultra Performance Liquid Chromatography-nano Electrospray Ionisation-Tandem MS/MS (UPLC-nanoESI-MS/MS). Overall, 20 extracellular proteases were identified from day 5 to 9 for samples of both A. niger PY11 and AnΔprtT. Of these, 15 of them were found to be prtT-dependent while the other five were prtT- independent. To compare the efficiency of the A. niger PY11 and AnΔprtT as hosts for heterologous protein production, the Glomerella cingulata cutinase gene under control of the glucoamylase A promoter was integrated into each genome. Cutinase activity of A. niger PY11 and AnΔprtT harbouring the recombinant G. cingulata cutinase gene was increased 20 and 36-fold higher, respectively than the untransformed parental strains, suggesting that the ability of the mutant to produce heterologous protein was not affected by deletion of prtT. Moreover, heterologous total enzyme was produced higher in AnΔprtT compared to A. niger PY11. Heterologous cutinase activity in both strains was stable at 4°C for six weeks. Meanwhile, the heterologous cutinase activity in AnΔprtT was retained with greater than 80% activity after two week incubation at 25°C compared to less than 3% activity remaining in A. niger PY11. In conclusion, development of prtT transcription factor mutant reduced extracellular protease activity in A. niger as well as increased the production and stability of the heterologous proteins.
Pages:	211
Call Number:	QK625.M7N847 2015
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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