STUDI PROTEOMIK ENZIM ALKALIN PROTEASE Bacillus cereus LS2B SEBAGAI KANDIDAT POTENSIAL PADA PROSES BUANG BULU RAMAH LINGKUNGAN

YENDRI JUNAIDI, Nanung Agus Fitriyanto, S.Pt.,M.Sc.,Ph.D dan Ir. Ambar Pertiwiningrum, M.Sc.,Ph.D

2017 | Tesis | S2 Ilmu Peternakan

Analisis protein secara menyeluruh dari enzim alkalin protease Bacillus cereus LS2B telah berhasil dilakukan, terkait dengan pemurnian dan karakter enzim. Tahapan pemurnian meliputi penentuan konsentrasi ammonium sulfat, penentuan konsentrasi enzim, dialisis, fraksinasi menggunakan hitrap ion exchange kromatografi kolom dengan matriks DEAE sepharose kolom flow rate 1,5 ml min-1, serta penentuan pH dan suhu optimum enzim yang sudah murni. Tahap karakterisasi meliputi penentuan total protein dengan menggunakan tyrosin sebagai standar dan penetuan aktivitas spesifik dengan BSA sebagai standar protein. Berat molekul band protein dari masing-masing tahapan pemurnian diidentifikasi dengan metode SDS-PAGE, untuk keperluan sekuensing band protein ditransfer pada PVDF membrane menggunakan metode western blotting, kemudian N-Terminal asam amino dianalisis dengan metode Edman degradation. Hasil dari tiap tahap pengerjaan menyangkut konsentrasi ammonium sulfat terbaik adalah dengan konsentrasi 80% serta konsentrasi enzim terbaik adalah 100%. Terdapat 35 fraksi enzim dari hasil hitrap ion exchange dan aktivitas spesifik enzim tertinggi didapatkan pada fraksi ke 15 (64,4 U/mg). Total protein dari pemurnian bertahap menyangkut crude enzim (235.80 mg/ml), presipitasi ammonium sulftat (80%) (211.40 mg/ml), dialisis (16.10 mg/ml) dan hitrap ion exchange kromatografi (6.60 mg/ml). Aktivitas spesifik dari crude enzim (0,4 U/mg), ammonium sulfat (0,5 U/mg), dialisis (1,8 U/mg) dan hitrap ion exchange (7,2 U/mg). Analisis profil band protein dengan SDS-PAGE didapatkan 2 molekul protein dominan dengan berat 20 kDa dan 13 kDa. Suhu dan pH optimum dari enzim alkalin protease yang memiliki aktivitas spesifik tertinggi hasil hitrap ion exchange kromatografi didapatkan pada suhu 40oC dan pH 8. Hasil sekuensing N-Terminal asam amino dari band protein tahap hitrap ion exchange kromatografi yaitu dengan urutan LIVTQTMKG yang teridentifikasi sebagai Beta-Lactoglobulin.

Protein analysis of the enzyme alkaline protease Bacillus cereus LS2B has been successfully carried out, and the characters associated with the purification of the enzyme. Stages of purification involve determining the concentration of ammonium sulfate, the determination of enzyme concentration, dialysis, fractionation using HiTrap ion exchange chromatography column with the matrix DEAE Sepharose column flow rate of 1.5 ml min-1, and the determination of the optimum pH and temperature of the enzyme that has been pure. Phase characterization includes determining the total protein by using tyrosine as standard and determination of specific activity with BSA as a standard protein. The molecular weight of the protein bands each stage of purification was identified using SDS-PAGE, for the purposes of sequencing the protein band transferred to PVDF membrane using western blotting method, then the N-terminal amino acid was analyzed by Edman degradation. The results of each stage of the work concern the concentration of ammonium sulfate was best with was concentration of 80% as well as the best enzyme concentration are 100%. There is 35 factions enzyme from the HiTrap ion exchange and high specific activity of the enzyme was obtained at a fraction to 15 (64.4 U/mg). Protein total of the gradual purification involves crude enzyme (235.80 mg / ml), precipitation of ammonium sulfate (80%) (211.40 mg/ml), dialysis (16.10 mg / ml) and HiTrap ion exchange chromatography (6.60 mg/ml). The specific activity of crude enzyme (0.4 U/mg), ammonium sulfate (0.5 U/mg), dialysis (1.8 U/mg) and HiTrap ion exchange (7.2 U/mg). Analysis of protein band profiles by SDS-PAGE obtained two dominant protein molecule with a weight of 20 kDa and 13 kDa. The optimum temperature and pH of the enzyme alkaline protease which has the highest specific activity of ion exchange chromatography HiTrap results obtained at a temperature of 40oC and pH 8. Sequencing N-terminal amino acids of the protein band HiTrap stage ion exchange chromatography in the order LIVTQTMKG identified as Beta-Lactoglobulin.

Kata Kunci : Bacillus cereus LS2B, alkaline protease, protein purification, characterization