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Kloning Gen Penyandi Protease dariBacillus haloduransCM1 padaEscherichia coli DH5α

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ABSTRAK

Aktivitas enzim protease terdeteksi dari kultur koleksi Bacillus halodurans strain CM1 milik BPPT yang berasal dari pemandian air panas Cimanggu, Bandung. Enzim protease yang dihasilkan bersifat alkalotermofilik, sehingga unggul dalam segi stabilitas di suhu tinggi. Potensi enzim protease alkalotermofilik tersebut dapat digali untuk kepentingan bioindustri, misalkan produk detergen yang mengandung bahan aditif enzim. Tujuan dari penelitian ini adalah untuk memperoleh dan memperbanyak gen penyandi protease dari Bacillus halodurans CM1 menggunakan teknik kloning gen. Penelitian ini diawali dengan ekstraksi DNA genom B. halodurans CM1. Gen penyandi protease diamplifikasi dari DNA genom hasil ekstaksi dengan 2 pasang primer, yaitu (1) ProteaseCM1-up-fwd dan Protease-ORF-rev serta (2) Protease-ORF-fwd dan Protease-ORF-rev. Fragmen gen penyandi protease diligasi dalam vektor plasmid pGEM T-Easy kemudian dikloning pada Escherichia coli DH5α melalui proses transformasi. Hasil amplifikasi dari primer ProteaseCM1-up-fwd adalah fragmen DNA berukuran 1.411 bp, sementara dengan primer Protease-ORF-fwd adalah fragmen DNA berukuran 1.086 bp. Analisis hasil sekuensing menggunakan metode NCBI BLAST menyatakan bahwa kedua fragmen DNA identik 99% dengan gen penyandi protease alkali termostabil dari Bacillus halodurans. Gen penyandi protease dalam Escherichia coli DH5α transforman diuji ekspresinya melalui uji kualititatif dalam medium kalsium kaseinat agar. Hasil uji kualitatif ialah timbul zona bening di sekitar koloni Escherichia coli DH5α transforman. Zona bening menandakan protein susu kasar (kasein) telah diuraikan menjadi asam amino akibat aktivitas proteolitik. Kloning gen penyandi protease dari Bacillus halodurans CM1 menghasilkan gen penyandi protease aktif yang mampu diekspresikan menjadi enzim protease fungsional.

Kata kunci: Protease alkalotermofilik, Bacillus halodurans, kloning gen

ABSTRACT

The activity of protease enzyme was detected from the culture collection of Bacillus halodurans strain CM1 from BPPT from Cimanggu Hot Spring, Bandung. Protease enzyme that produced tend to be alkalothermophilic and it makes the enzyme has high stability in extreme temperature. The potential of alkalothermophilic protease enzyme could be investigated for bioindustrial purpose, for example, the detergent product which has enzyme as additive substance. The reason of this research is to produce and multiply the gene encoding protease that came from B. halodurans CM1 using gene cloning technique. This research was conducted by the extraction of B. halodurans CM1 genome DNA. The gene encoding protease is amplified from the genome DNA using two pairs of primer such as (1) ProteaseCM1-up-fwd and Protease-ORF-rev then (2) Protease-ORF-fwd and Protease-ORF-rev. The DNA fragments encoding protease were ligated inside of the vector plasmid pGEM T-Easy, then DNA fragments were cloned into Escherichia coli DH5α. The research result using ProteaseCM1-up-fwd primer was the 1.411 bp DNA fragment, meanwhile with Protease-ORF-fwd primer was the 1.086 bp. Both of the DNA fragments from BLAST method were showed 99% identical percentage with the gene encoding thermostable alkaline protease from B. halodurans. The expression of gene encoding protease inside the E. coli DH5α transforman’s was tested with the qualitative assay in the calcium caseinat agar. The clear zone which appeared in around E. coli DH5α transforman colony informs that the raw milk’s protein (casein) has been degradated into amino acid as the result of proteolytic process. Cloning gene technique from this research found an active gene encoding protease that able to be expressed as functional protease enzyme.

Keyword: Alkalothermophilic protease, Bacillus halodurans, gene cloning

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Ketersediaan

1157B17IV

1157 B 17-iv

Perpustakaan FSM Undip (Referensi)

Tersedia

Informasi Detail

Judul Seri

BIOLOGI

No. Panggil

1157 B 17-iv

Penerbit

: ., 2017

Deskripsi Fisik

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Bahasa

Indonesia

ISBN/ISSN

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Klasifikasi

1633

Tipe Isi

-

Tipe Media

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Tipe Pembawa

-

Edisi

-

Subjek

-

Info Detail Spesifik

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Pernyataan Tanggungjawab

Natasha Furgeva

Versi lain/terkait

Lampiran Berkas

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