Ilmi, Afifah Nurul (2025) Karakterisasi Crude Enzim Wild Type Untuk Produksi Maltooligosakarida dan Kloning Gen Penyandi Glukoamilase dari Aspergillus awamori Berbasis Whole Genome Sequencing. Masters thesis, Institut Teknologi Sepuluh Nopember.
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Abstract
Permintaan global terhadap enzim terus meningkat, dengan glukoamilase menyumbang sebagian besar pasar enzim karbohidrat. Aspergillus awamori KT 11, koleksi BRIN, berpotensi memproduksi enzim amilase, termasuk glukoamilase yang dapat menghidrolisis pati menjadi glukosa dan fruktosa untuk sirup gula cair. Namun, sebagai fungi wild type, aktivitas enzimnya masih rendah, sehingga diperlukan pendekatan rekayasa genetika seperti kloning dan ekspresi rekombinan. Penelitian ini bertujuan mengkaji data Whole Genome Sequencing A. awamori KT 11, mengkloning gen penyandi glukoamilase dengan primer spesifik, serta mengkarakterisasi enzim wild type. Metode yang digunakan meliputi produksi
enzim, karakterisasi enzim (uji aktivitas, TLC, HPLC, SEM, FTIR), analisis data WGS, kloning, transformasi, dan sequencing. Hasil penelitian menunjukkan, crude enzim dari A. awamori KT 11 memiliki pH optimum 6 (aktivitas 16,38 U/mL) dan suhu optimum 70°C (aktivitas 170,17 U/mL). Hasil TLC menunjukkan produk hidrolisis berupa glukosa, maltosa, dan maltotriosa, sedangkan HPLC menunjukkan glukosa dan maltosa sebagai produk dominan, analisis FTIR menunjukkan pemecahan ikatan O–H dan C–O, serta SEM mengonfirmasi perubahan struktur pati setelah hidrolisis. WGS menggunakan platform Illumina menghasilkan genom sepanjang 34,9 Mb dengan GC content 49,18%, terdiri dari 331 contig. Selain itu, insert gen glukoamilase berhasil dikloning ke dalam vektor pCRTM Blunt II-TOPO dan ditransformasi ke E. coli DH5α.
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Global demand for enzymes continues to increase, with glucoamylase accounting for a large portion of the carbohydrate enzyme market. Aspergillus awamori KT 11, a BRIN collection, has the potential to produce amylase enzymes, including glucoamylase, which can hydrolyze starch into glucose and fructose for liquid sugar syrup. However, as a wildtype fungus, its enzyme activity is still low, requiring genetic engineering approaches such as cloning and recombinant expression. This study aims to examine the Whole Genome Sequencing data of A. awamori KT 11, clone the glucoamylase-encoding gene with specific primers, and characterize the wild-type enzyme. The methods used include enzyme production, enzyme characterization (activity test, TLC, HPLC, SEM, FTIR), WGS data analysis, cloning, transformation, and sequencing. The results showed that the crude enzyme from A. awamori KT 11 has an optimum pH of 6 (activity of 16.38 U/mL) and an optimum temperature of 70°C (activity of 170.17 U/mL). TLC results showed that hydrolysis products were glucose, maltose, and maltotriose, while HPLC showed glucose and maltose as the dominant products, FTIR analysis showed the cleavage of O–H and C–O bonds, and SEM confirmed changes in starch structure after hydrolysis. WGS using the Illumina platform produced a 34.9 Mb genome with a GC content of 49.18%, consisting of 331 contigs. In addition, the glucoamylase gene insert was successfully cloned into the pCRTM-Blunt II TOPO vector and transformed into E. coli DH5α.
| Item Type: | Thesis (Masters) |
|---|---|
| Uncontrolled Keywords: | Aspergillus awamori, Enzim, Glukoamilase, Maltooligisakarida, Whole Genome Sequencing, Aspergillus awamori, Enzyme, Glucoamylase, Maltooligosaccharide, Whole Genome Sequencing |
| Subjects: | Q Science > QH Biology Q Science > QH Biology > QH301 Biology |
| Divisions: | Faculty of Science and Data Analytics (SCIENTICS) > Biology > 46101-(S2) Master Thesis |
| Depositing User: | Afifah Nurul Ilmi |
| Date Deposited: | 06 Aug 2025 04:15 |
| Last Modified: | 06 Aug 2025 04:15 |
| URI: | http://repository.its.ac.id/id/eprint/127738 |
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