Gusman, Ery and Prof. Dr. Ir. Happy Nursyam, MS,, MS, and Dr. Ir. Yahya, MP,, MP, and Dr. Ir. Titik Dwi Sulistiyati,, MP (2024) Karakterisasi Enzim Protease Bakteri Non-Patogen Yang Diisolasi Dari Sedimen Mangrove. Doktor thesis, Universitas Brawijaya.
Abstract
Terdapat berbagai berbagai jenis mikroba yang hidup berasosiasi dengan eksistensi mangrove. Mereka beperan dalam menjaga dan merawat ekosistem mangrove, yaitu dalam proses dekomposisi dan mineralisasi material organik. Sejauh ini eksplorasi mengenai potensi sumberdaya mangrove hanya hanya terbatas pada unsur makro yang terdapat di sekitar mangrove tersebut, sedangkan komponen mikrobiotik masih kurang dilakukan. Enzim yang dihasilkan mikroba laut memiliki karakteristik khusus yaitu bersifat toleran pada kondisi garam tinggi (alkali), thermostable, barophilic, mampu beradaptasi pada suhu rendah, dan mudah diproduksi dalam jumlah banyak. Penelitian ini mengekplorasi mengenai spesies bakteri proteolitik sedimen mangrove sebagai kandidat sumber enzim protease. Penelitian dimulai dengan isolasi bakteri dari sedimen mangrove, lalu melakukan seleksi bakteri yang bersifat non patogen dan bersifat proteolitik. Selanjutnya dilakukan identifikasi morfologi, biokimia dan genetika isolat kandidat, serta melakukan karakterisasi sifat dan jenis enzim protease bakteri proteolitik yang sudah dipilih, yaitu dengan memproduksi dan pemurnian crude enzim tersebut. Adapun parameter karakter protease yang dianalisis adalah berat molekul, gugus fungsi senyawa dan jenis asam amino. Selain itu dilakukan pengujian pengaruh suhu dan pH yang berbeda, pengaruh penambahan ion-ion logam dan inhibitor protease, serta analanisis kinetika enzim untuk mengetahui kondisi optimal enzim protease yang dihasilkan dalam menghidrolisis substatnya. Berdasarkan hasil seleksi didapatkan 8 isolat bakteri dari kelompok non patogen, dan dari 8 tersebut, 4 isolat merupakan kelompok bakteri proteolitik. Terdapat 4 variabel lingkungan pada penelitian ini yang memiliki hubungan positif dengan keberadaan bakteri proteolitik non patogen tersebut, yaitu: kerapatan mangrove, jenis mangrove avicennia, jumlah komposisi tanah liat dan kehadiran jenis mangrove lainnya. Hasil identifikasi 4 isolat bakteri, 1 isolat homologue dengan spesies B. weidmannii dan B. cereus, 2 isolat homologue dengan spesies B. cereus, dan 1 isolat homologue Vibrio parahaemolyticus. Hasil uji Indeks hidrolisis Protein (IHP), 1 isolat SdC14 (homologue dengan Vibrio parahaemolyticus) menghasilkan nilai tertinggi, sehingga dipilih sebagai kandidat bakteri dianalisis karakter proteasenya. Hasil pemurnian dan pengujian pengaruh dari beberapa parameter, di ketahui karakteristik enzim protease bakteri proteolitik, diantaranya: berat molekul enzim protease sebesar 74 kDa, aktivitas protease bekerja optimum pada pH 8 dan suhu 50 °C, dan memiliki nilai Vmax 0,17448 U/ml dan nilai KM 0,19227%. Enzim protease bakteri ini termasuk dalam kelompok thermo active protease (moderate) dan alkaline protease dan dari hasil uji pengaruh penambahan ion logam dan inhibitor EDTA, protease tersebut termasuk dalam kelompok zinc metalloprotease. Berdasarkan analisis gugus fungsi, jenis asam amino yang hasilkan, serta berat molekul, protease tersebut termasuk dalam kelompok zinc metalloprotease family M4, dengan kode EC 3.4.24.27, yaitu protease thermolysin.
English Abstract
There are various types of microorganisms that exist along with mangroves, involved in the breakdown and mineralization of organic matter, to keeps mangrove ecosystem alive. The macro components have been the exclusive focus of study into the potential of mangrove resources so far, while the microbiotic component rarely studied. Marine microbes protease enzymes has unique qualities by their ability to tolerate high salinity (alkali) environments, thermostability, barophilic, low temperature tolerance, and ease of large-scale production. Proteolytic bacterial species found in mangrove sediments are investigated in this study as potential sources of protease enzymes. Bacteria from mangrove sediments were isolated, and only non-pathogenic proteolytic bacteria were selected for further study. Determining the characteristics and type of the selected proteolytic bacterial protease enzyme by producing and purifying the crude enzyme, and also the morphology, biochemistry, and genetics of the isolates were analysed to identify the species of bacteria. Amino acid type, molecular weight, and chemical functional group were the protease character factors were also examined. In order to determine the optimum conditions for the protease enzyme synthesized in hydrolysing its substrate, studies were also conducted on the effects of varying pH and temperature, the impact of adding metal ions and protease inhibitors, and the evaluation of enzyme kinetics. 8 bacterial isolates have been isolated from the non-pathogenic category based on the selection results; 4 of these isolates belong to the proteolytic bacterial group. The presence of non-pathogenic proteolytic bacteria has a positive correlation with four environmental variables, that were the density of mangroves, the type of Avicennia mangrove, the amount of clay composition, and the presence of other mangrove species. 4 bacterial isolates had been identified: 2 isolates similar to B. cereus, 1 isolate similar to B. weidmannii, and 1 isolate similar to Vibrio parahaemolyticus. One isolate (SdC14) by previous identification similar of Vibrio parahaemolyticus, show highest number in the Protein Hydrolysis Index (IHP) test, and it’s selected to analyse for protease characteristic. The results of purification and testing the influence of several parameters show the characteristics of the proteolytic bacterial protease enzyme, including: the molecular weight is 74 kDa; optimally at pH 8 and a temperature of 50 °C; Vmax value 0.17448 U/ml; and KM value 0.19227%.The protease were considerate as thermoactive (moderate) and alkaline protease, and from effects of adding metal ions and EDTA inhibitors, considerate as zinc metalloprotease. Based on functional group analysis, molecular weight, and the type of amino acid produced, this protease belongs to the zinc metalloprotease family M4 group, the thermolysin protease (EC code 3.4.24.27).
| Item Type: | Thesis (Doktor) |
|---|---|
| Identification Number: | 062308 |
| Uncontrolled Keywords: | Sedimen mangrove, bakteri proteolitik, non patogen, karakteristik, protease |
| Divisions: | S2/S3 > Doktor Ilmu Perikanan dan Kelautan, Fakultas Perikanan dan Ilmu Kelautan |
| Depositing User: | Unnamed user with username nova |
| Date Deposited: | 04 Mar 2024 07:16 |
| Last Modified: | 04 Mar 2024 07:16 |
| URI: | http://repository.ub.ac.id/id/eprint/216804 |
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