Pengaruh Variasi Konsentrasi Substrat Pati terhadap Aktivitas Enzim Pullulanase Asal Mikroba Indigenous Ketan Terfermentasi dan Pemurnian Parsialnya

Dilan, Dina Revon (2023) Pengaruh Variasi Konsentrasi Substrat Pati terhadap Aktivitas Enzim Pullulanase Asal Mikroba Indigenous Ketan Terfermentasi dan Pemurnian Parsialnya. Other thesis, Institut Teknologi Sepuluh Nopember.

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Abstract

Organisme membutuhkan enzim untuk dapat memanfaatkan gula dari pemotongan molekul polisakarida menjadi glukosa sederhana. Enzim pullulanase dapat mengkatalisis hidrolisis α-1,6-penghubung pullulan dan polisakarida lain untuk menghasilkan maltotriosa (produk akhir). Dalam penelitian ini, pullulanase yang efisien telah dihasilkan dari mikroba indigenous ketan fermentasi dan dimurnikan untuk potensi penggunaan dalam sintesis pati. Mikroba indigenous ketan fermentasi merupakan isolat koleksi Laboratorium Genomik, Pusat Riset Mikrobiologi Terapan, Badan Riset dan Inovasi Nasional (BRIN). Tujuan dari penelitian ini adalah mengetahui pengaruh konsentrasi substrat pati terhadap produksi pullulanase menggunakan bakteri indigenous ketan fermentasi dengan parameter aktivitas enzim spesifik dan karakterisasinya seperti kadar protein, berat molekul protein dan titik isoelektriknya. Variasi konsentrasi substrat pati yang dilakukan terhadap produksi pullulanase terdiri dari 3 taraf yaitu penambahan pati dengan konsentrasi 1%, 2%, dan 3%. Kemudian crude pullulanase yang menunjukkan hasil aktivitas tertinggi (spektrofotometer Uv-Vis λ = 600 nm) dilakukan pemurnian dengan presipitasi amonium sulfat pada fraksi 40%, 60%, dan 80% dan dilanjutkan pemurnian dengan dialisis. Pullulanase yang sudah dimurnikan, dikarakterisasi kembali berdasarkan kadar protein menggunakan reagen BCA (Bicinchoninic acid), dan pengukuran berat molekul protein dengan SDS PAGE serta penetuan titik isoelektriknya yang mengiindikasikan pH asam amino berada pada bentuk amfoter (zwitter ion). Data yang didapatkan dianalisis secara deskriptif. Hasil penelitian menunjukkan variasi konsentrasi substrat pati yang dilakukan menggunakan bakteri indigenous ketan fermentasi (P. pentosaceus) berpengaruh terhadap produksi pullulanase. Konsentrasi terbaik dalam memproduksi pullulanase yaitu pada konsentrasi pati 2% dengan nilai aktivitas spesifik pullulanase sebesar 23,56 U/mg dan kadar protein 2,1 mg/ml. Aktivitas spesifik crude pullulanase menunjukkan peningkatan setelah dilakukan pemurnian dengan presipitasi dan juga dialisis sebesar 1478,84 U/mg dan kadar protein sebesar 0,38 mg/ml. Berat molekul subunit pullulanase (Pull) yang dimurnikan didapatkan sebesar 42,88 kDa dengan titik isoelektrik menunjukkan pengendapan pada pH 5.
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Organisms need enzymes to be able to utilize sugar from cutting polysaccharide molecules into simple glucose. The pullulanase enzyme can catalyze the hydrolysis of α-1,6-linkages of pullulan and other polysaccharides to produce maltotriose (the final product). In this study, an efficient pullulanase was produced from fermented and purified indigenous glutinous microbes for potential use in starch synthesis. Indigenous glutinous fermented microbes are isolates from the collection of the Genomics Laboratory, Center for Applied Microbiology Research, National Research and Innovation Agency (BRIN). The purpose of this study was to determine the effect of starch substrate concentration on pullulanase production using indigenous fermented glutinous bacteria with enzyme specific activity parameters and their characteristics such as protein content, protein molecular weight and isoelectric point. Variation of starch substrate concentration was carried out on pullulanase production consisting of 3 levels, the addition of starch with a concentration of 1%, 2% and 3%. Then crude pullulanase which showed the highest activity results (Uv-Vis spectrophotometer λ = 600 nm) was purified by ammonium sulfate precipitation at 40%, 60% and 80% fractions and continued purification by dialysis. The purified Pullulanase was re-characterized based on protein content using BCA (Bicinchoninic acid) reagent, and measurement of protein molecular weight with SDS PAGE and determination of its isoelectric point which indicated the pH of the amino acids was in the amphoteric form (zwitter ion). The data obtained were analyzed descriptively. The results showed that variations in starch substrate concentration using indigenous fermented glutinous bacteria (P. pentosaceus) had an effect on pullulanase production. The best concentration in producing pullulanase was starch concentration of 2% with a pullulanase specific activity value of 23,56 U/mg and protein content of 2,1 mg/ml. The specific activity of crude pullulanase showed an increase after purification by precipitation and also dialysis of 1478,84 U/mg and protein content of 0,38 mg/ml. The molecular weight of the purified pullulanase (Pull) subunit was found to be 42.88 kDa with the isoelectric point indicating precipitation at pH 5.

Item Type:	Thesis (Other)
Uncontrolled Keywords:	Dialisis, Hidrolisis, Pati, Pullulanase; Dialysis, Hydrolysis, Indigenous Bacteria, Pullulanase, Starch, Bakteri Indigenous,
Subjects:	Q Science Q Science > QH Biology Q Science > QH Biology > QH301 Biology Q Science > QR Microbiology Q Science > QR Microbiology > QR74.8 Bacteria
Divisions:	Faculty of Science and Data Analytics (SCIENTICS) > Biology > 46201-(S1) Undergraduate Thesis
Depositing User:	Dina Revon Dilan
Date Deposited:	04 Oct 2023 01:26
Last Modified:	04 Oct 2023 01:26
URI:	http://repository.its.ac.id/id/eprint/103292

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