Isolasi dan identifikasi bakteri termotoleran yang mampu menghidrolisa protein dari limbah udang

JUMERI, Dr.drh. Widya Asmara, SU

2005 | Tesis | S2 Bioteknologi

Khitin, sebuah homopolimer dari residu N-acetyl-D-glucosamine (Glc-Nac) dan derivatnya berperan penting dalam berbagai aplikasi bioteknologi. Limbah kulit udang kaya dengan khitin, selain juga protein dan kalsium karbonat. Selama pengolahan limbah kulit udang menjadi khitin dan khitosan, baik jaringan protein maupun mineral diekstraksi. Proses deproteinisasi dan demineralisasi secara konvensional menggunakan asam atau basa kuat, telah menimbulkan banyak masalah. Untuk mengatasi hal ini, beberapa studi telah dilakukan dengan menggunakan mikroorganisme atau enzim proteolitik. Protease sejauh ini telah banyak digunakan dalam berbagai bidang dan telah menunjukkan kemampuannya dalam degradasi limbah berprotein menjadi biomassa berguna. Penelitian ini bertujuan untuk melakukan isolasi dan identifikasi isolat bakteri thermotoleran penghasil enzim protease dari limbah kulit udang, mengetahui kemampuan enzim protease isolat dalam menghidrolisa protein limbah udang, dan hubungan kekerabatan diantara isolat tersebut. Sampel penelitian berupa limbah kulit udang dan isolasi mikroorganisme dilakukan dengan media kalsium kaseinat dan LB medium. Pengujian aktivitas protease dilakukan dengan media shrimp shells solution dan pengujian kandungan protein yang tertinggal dalam limbah dengan persamaan kurva standar dan metode Lowry. Isolasi DNA genom dengan metode ekstraksi phenol/chloroform dan amplifikasi 16S rDNA dengan Polymerase Chain Reaction (PCR). Sekuensing 16S rDNA dilakukan oleh GATC Biotech AG, analisis sekuen 16S rDNA dilakukan dengan software Ribosomal Database Project (RDP II) dan ClustalW. Hasil penelitian menunjukkan bahwa berdasarkan pengecatan gram dan pengamatan mikroskopi telah diperoleh tiga isolat bakteri thermotoleran gram positif berbentuk batang dengan aktivitas protease tinggi. Aktivitas protease tertinggi dicapai oleh isolat bakteri 1 (102,41 U/ml) setelah inkubasi 24 jam. Isolat bakteri 1 dengan aktivitas protease tertinggi dapat memisahkan sebagian besar protein dari limbah udang, sehingga protein yang tersisa dalam limbah udang hanya 2,96 %. Berdasarkan analisis sekuen 16S rDNA dengan software RDP II dan ClustalW, isolat bakteri 1, 2, dan 3 mempunyai homologi yang tinggi dengan genus Bacillus, terutama spesies Bacillus licheniformis

Chitin, a homopolymer of N--acetyl-D-glucosamine (Glc-Nac) residues and its derivatives, have attracted significant interests in biotechnological application. Shrimp shells waste are very rich in chitin, beside protein and calsium carbonat. During the processing of shrimp shells waste into khitin and khitosan, both mineral and tissue protein are chemically extracted. Conventionally, deproteinization and demineralization process using strong acid or bases have caused many problem. To overcome the sortage, several studies have been conducted by using microorganisms or proteolytic enzymes. Proteases are by far used in many areas of applications and has shown its capability to degrade proteinaceous waste into useful biomass. The aim of this research were to isolate and identify proteases producing thermotolerant bacteria from shrimp shells waste, to find out its proteases capability to hydrolyze proteinaceous shrimp shell waste, and homology among these isolates. Sample of the research was shrimp shell wastes and microorganisms isolation was conducted by using calcium caseinat and LB medium. Protease activity was assayed by using shrimp shells solution medium and protein left in the shrimp shell waste was assayed based on Lowry method. Isolation of genome DNA was carried out by phenol/chloroform extraction methods and amplification of 16S rDNA by Polymerase Chain Reaction (PCR) methods. Sequencing of 16S rDNA was carried out by GATC Biotech AG, and 16S rDNA sequences analysis were conducted by using software Ribosomal Database Project (RDP II) and clustalW. The result of the research has shown that according of the gram staining and microscopic observation, three isolated stem shape’s thermotolerant bacteria with high activity of protease were revealed. The highest activity of protease was reached by bacteria isolate 1 (102,41 U/ml) during incubation of 24 hours. Bacteria isolate 1 with highest of protease activity can remove almost protein from shrimp shell waste, hence protein left in the shrimp shells was lowest (2,96 %). Based on the analysis of 16S rDNA sequence obtained by software Ribosomal Database Project (RDP II) and ClustalW, all three isolates of bacteria showed high homology with Bacillus genus, especially Bacillus licheniformis species.

Kata Kunci : Bioteknologi, Kithin dan Khitosan, Bakteri Thermotoleran, Shrimp shell waste, chitin and chitosan, proteolytic bacteria/proteases, and 16S rDNA.