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Title:	Pencirian endoglukanase dan β-glukosidase rekombinan aspergillus niger dan aplikasi kedua-duanya dalam pencuraian tandan kosong kelapa sawit
Authors:	Shazilah Kamaruddin (P43129)
Supervisor:	Abdul Munir Abdul Murad Prof. Madya Dr.
Keywords:	Endoglukanase β-glukosidase Aspergillus niger
Issue Date:	13-Aug-2014
Description:	Keberkesanaan pencuraian secara enzimatik biojisim pertanian kepada gula ringkas memerlukan kefahaman sifat biokimia setiap enzim yang digunakan dalam tindak balas. Objektif kajian ini ialah untuk mencirikan endoglukanase (EglB) dan β-glukosidase (BglA) Aspergillus niger rekombinan dan menganalisis potensi kedua-duanya untuk mencurai biojisim pertanian iaitu tandan kosong kelapa sawit (TKKS). cDNA endoglukanase (eglB) dan β-glukosidase (bglA) telah disintesis daripada mRNA Aspergillus niger strain ATCC 10574. Penghasilan enzim selulase rekombinan telah dilakukan menggunakan dua hos pengekspresan iaitu Escherichia coli dan Pichia pastoris mengikut kesesuaian gen yang hendak diekspreskan. eglB telah diekspres dalam sistem pengekspresan E. coli dan protein rekombinan yang terhasil menunjukkan aktiviti enzim dipengaruhi oleh penanda tioredoksin yang terlakur pada protein rekombinan tersebut. Pemotongan penanda tioredoksin daripada protein EglB rekombinan menunjukkan peningkatan aktiviti sehingga ~20 kali ganda. Oleh itu, enzim ini adalah tidak sesuai untuk aplikasi pencuraian biojisim pertanian kerana memerlukan proses pemotongan penanda tioredoksin bagi memperoleh aktiviti optimum. Justeru, gen eglB dan bglA telah diekspres dalam sistem pengekspresan P. pastoris dan protein rekombinan masing-masing bersaiz ~46 dan ~116 kDa telah dihasilkan. Pencirian biokimia seperti penentuan suhu dan pH optimum, kestabilan pada suhu dan pH berbeza, kesan ion logam dan reagen kimia serta kespesifikan substrat telah dilakukan. EglB dan BglA mempunyai aktiviti optimum pada pH 4.0 tetapi pada suhu yang berbeza iaitu masing-masing pada 50°C dan 60°C. Kedua-dua enzim didapati stabil pada suhu sekitar 50°C hingga 60°C dan tiada aktiviti dapat dikesan apabila dieram pada suhu 70°C ke atas. Kedua-dua enzim menunjukkan profil kestabilan pH yang berbeza di mana EglB lebih stabil pada pH 5 hingga 8 manakala BglA pula lebih stabil pada pH 3 hingga 6. Tiada peningkatan aktiviti yang signifikan dapat dilihat apabila kedua-dua enzim ini ditambah dengan ion logam ataupun reagen kimia. EglB menunjukkan kespesifikan substrat yang tinggi terhadap arabinogalaktan dikuti dengan karboksimetil selulosa dengan nilai pemalar kespesifikan masing-masing ialah 136.66 mLmg-1s-1 dan 55.65 mLmg-1s-1. BglA pula menunjukkan kespesifikan substrat yang tinggi terhadap substrat sintetik, p-nitrofenil-β-D-glukopiranosida diikuti dengan selobiosa dengan nilai pemalar kespesifikan masing-masing 466.19 mLmg-1s-1 dan 6.01 mLmg-1s-1. Kedua-dua enzim rekombinan seterusnya digunakan bersama enzim selobiohidrolase yang diperoleh daripada kajian lepas untuk menghidrolisis TKKS. Selobiohidrolase ditambah kerana ia perlu bagi melengkapkan tindak balas sinergi tiga komponen utama selulase untuk hidrolisis TKKS. Pengoptimuman nisbah enzim untuk tindak balas ditentukan menggunakan Kaedah Gerak Balas Permukaan (Response Surface Methodology, RSM). Hasil hidrolisis menunjukkan hidrolisis TKKS pada suhu 50°C dan pH 4.0 menggunakan enzim pada nisbah 641.4 unit CMCase : 10.14 unit Avicelase : 93.8 unit β-glukosidase, menghasilkan gula terturun dan glukosa tertinggi, masing-masing sebanyak 63 mg dan 40 mg per gram substrat TKKS. Kesimpulannya, sifat biokimia endoglukanase dan β-glukosidase rekombinan daripada A. niger telah berjaya dicirikan dan kedua-dua enzim ini berpotensi untuk digunakan bersama selobiohidrolase untuk pencuraian TKKS secara enzimatik.,The effectiveness of agricultural biomass enzymatic degradation into simple sugars requires understanding of the biochemical properties of every enzyme used in the reaction. The objectives of this study were to characterise the recombinant Aspergillus niger endoglucanase (EglB) and β-glucosidase (BglA) and to analyse their potential in degrading agricultural biomass as an oil palm empty fruit bunch (OPEFB). The cDNA of endoglucanase (eglB) and β-glucosidase (bglA) were synthesised from the mRNA of Aspergillus niger strain ATCC 10574. The production of recombinant cellulases was carried out using two expression hosts, Escherichia coli and Pichia pastoris, depending on the genes to be expressed. eglB was expressed in E. coli expression system and the recombinant protein showed the activity of the enzyme was influenced by the thioredoxin tag that was fused to the recombinant protein. Removal of the thioredoxin tag from the recombinant EglB showed an enhanced activity up to ~20 fold. Hence, the enzyme is not suitable for the degradation of agricultural biomass as it requires the removal of thioredoxin tag for an optimal activity. Thus, eglB and bglA genes were expressed in P. pastoris expression system and recombinant proteins with the size of ~46 and ~116 kDa respectively, were produced. Biochemical characterizations such as determination of optimum temperature and pH, stability at different temperature and pH, effect of metal ions and chemical reagents and substrate specificity had been carried out. EglB and BglA showed an optimum activity at pH 4.0 but at different temperatures, 50°C and 60°C, respectively. Both enzymes are stable at the temperatures around 50°C to 60°C and no activity was detected when incubated at 70°C and above. Both enzymes showed different pH stability profiles where EglB was more stable at pH 5 to 8 while BglA was more stable at pH 3 to 6. No increase in activity was observed when the two enzymes were treated with metal ions or chemical reagents. EglB showed a higher specificity towards arabinogalactan followed by carboxymethyl cellulose with the specificity constant of 136.66 and 55.65 mLmg-1s-1, respectively. BglA, in turn, showed higher specificity towards synthetic substrate, p-nitrophenyl-β-D-glucopyranoside followed by cellobiose with the specificity constant of 466.19 and 6.01 mLmg-1s-1, respectively. Both enzymes were subsequently used in combination with a recombinant cellobiohydrolase that was obtained from a previous study for the hydrolysis of OPEFB. Cellobiohydrolase was added to complete the synergistic reaction of three cellulase component to degrade OPEFB. Response Surface Methodology (RSM) was used to determine the best enzyme ratio for OPEFB hydrolysis. Hydrolysis results showed OPEFB hydrolysed at 50°C and pH 4.0 using enzyme ratio of 641.4 CMCase unit: 10.14 Avicelase unit: 93.8 β- glucosidase units, resulted in the highest production of reducing sugars and glucose at 63 mg and 40 mg per gram OPEFB, respectively. In conclusion, the biochemical properties of the recombinant endoglucanase and β-glucosidase from A. niger has been successfully characterised and both enzymes have the potential to be used together with cellobiohydrolase for enzymatic degradation of OPEFB.,Bachelor
Pages:	212
Call Number:	TP248.65.C44S534 2014 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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