Pembangunan mutan prt T dan analisis sekretom untuk meningkatkan Kestabilan Protein Heterolog dalam Aspergillus Niger (IR)
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 ob...
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| Format: | thesis |
| Language: | zsm |
| Published: |
2015
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| Subjects: | |
| Online Access: | https://ir.upsi.edu.my/detailsg.php?det=1244 |
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| Summary: | 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 prt T mutant. The prt T gene was inactivated in A. niger PYll via gene replacement and the mutant produced was verified via polymerase chain reaction (PCR) and Southern blotting. Reverse transcriptasepolymerase chain reaction (RT-PCR) analysis showed reduced expression levels of several protease genes, pepA, pepB and pepF in An/sprt'T as compared to A. niger PYll either in induced or non-induced conditions. A qualitative extracellular proteolytic activity assays conducted on the An/sprt'I' 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 PYll showed a linear increase throughout six days culturing while Arusprt'I' protease activity remained very low. Growth of A. niger PYll and An/sprt'I' was comparable indicating that disruption of prtT did not affect fungal growth. Subsequently, extracellular proteases of A. niger PYll and An/sprt'T 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 PYll and An/sprt'I'. 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 PYll and An/xprt'T 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 PYll and Arusprt'I' 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 An/sprt'T compared to A. niger PYll. Heterologous cutinase activity in both strains was stable at 4C for six weeks. Meanwhile, the heterologous cutinase activity in An/sprt'I' was retained with greater than 80% activity after two week incubation at 25C compared to less than 3% activity remaining in A. niger PY 11. In conclusion, development ofprtT transcription factor mutant reduced extracellular protease activity in A. niger as well as increased the production and stability of the heterologous proteins. |
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