Isolasi dan Kloning Gen Penyandi Lipase Termo-halotoleran dari Bacillus tequilensis asal Mata Air Panas Lainea di Sulawesi Tenggara

Abstract

Kebutuhan lipase oleh berbagai bidang industri dengan aplikasi yang beragam membuat lipase perlu diproduksi secara cepat dan banyak. Namun lipase yang diproduksi saat ini belum sepenuhnya sesuai dengan industri yang memerlukan pemrosesan dengan kondisi suhu tinggi dan pelarut organik serta interaksi ion dalam larutan seperti kadar garam atau salinitas. Penelitian ini bertujuan untuk mengisolasi bakteri penghasil lipase termo-halotoleran dari sumber air panas, mengkloning dan mengekspresikan gen penyandi lipase serta karakterisasi biokimia lipase termo-halotoleran. Pengayaan dilakukan sebelum mengisolasi bakteri lipolitik dari habitat ekstrim seperti sumber air panas. Pengayaan dilakukan secara langsung pada habitat asli atau in situ enrichment. Bakteri lipolitik diisolasi dari sampel hasil pengayaan in situ dengan metode Spread Plate pada media Rhodamin B–Agar. Isolat bakteri lipolitik diidentifikasi berdasarkan gen 16S rRNA. Gen penyandi lipase diisolasi dengan teknik PCR menggunakan degenerate primer. Gen lipase diklon pada vektor pGEM-T Easy kemudian ditransformasi ke Escherichia coli DH5α untuk preservasi gen dan diklon pada vektor pET24a, ditransformasi ke E. coli BL21 (DE3) pLysS untuk diekspresikan. Ekspresi LipA_BTeq dilakukan 2 perlakuan yaitu mengikutsertakan sekuen signal peptida dan diekspresikan tanpa signal peptida (lipase mature). Analisis massa molekuler protein enzim lipase dilakukan dengan SDS-PAGE dan zimogram. Selain itu dilakukan analisis struktur 3D serta kekerabatan dalam Family lipase dianalisis menggunakan aplikasi MEGA XI. Karakterisasi lipase yang dilakukan adalah suhu optimum, pH optimum, stabilitas enzim terhadap salinitas (larutan NaCl), spesifisitas substrat, ketahanan terhadap pelarut organik, serta pengaruh ion logam. Tujuh belas isolat berhasil diisolasi dari sampel hasil pengayaan. Gen lipase dari 5 isolat bakteri lipolitik berhasil diklon dengan kode isolat HSAS, HSDS2, HSC3.1, HSC3.2 dan HSB3. Hasil identifikasi terhadap isolat HSAS, memiliki tingkat kemiripan 99.5% dengan Bacillus tequilensis strain A13 dengan panjang gen lipase (LipA_Bteq) 639 bp yang menyandikan 213 asam amino dengan 31 asam amino di awal sekuen merupakan sekuen signal peptida. Hasil ekspresi gen lipase menunjukkan perlakuan lipase mature memiliki aktivitas spesifik lebih tinggi (17 ± 1.12 U/mg) daripada yang memiliki signal peptida (8 ± 1.11 U/mg), sehingga lipase mature dipilih untuk dikarakterisasi. Hasil analisis SDS-PAGE dan Zimogram menunjukkan berat protein LipA_Bteq sebesar ~20 kDa. Hasil tersebut dikonfirmasi pula analisis MEGA XI yang membentuk klad dengan subfamily lipase I.4. LipA_Bteq memiliki suhu optimum 30 oC, dan memiliki rentang stabilitas suhu 30 oC - 60 oC dengan rata-rata aktivitas relatif 98 %. LipA_Bteq memiliki ketahanan terhadap konsentrasi NaCl 0.1M sampai 3M, dan memiliki aktivitas optimum pada konsentrasi NaCl 2 M. LipA_Bteq digolongkan sebagai lipase halotoleran karena membutuhkan NaCl untuk mencapai aktivitas optimum. Untuk parameter pH, LipA_Bteq memiliki aktivitas tertinggi pada pH 8. LipA_Bteq memiliki preferensi yang baik pada substrat C6 (rantai pendek). Kehadiran ion logam (Fe3+, Cu2+, Mg2+, Ca2+, Na+, dan Mn2+) dengan konsentrasi 100 mM dalam campuran reaksi mampu meningkatkan aktivitas LipA_Bteq. Disisi lain Zn2+ menurunkan aktivitas lipase sampai 83%. Penambahan pelarut organik 50% metanol, isopropanol dan etanol menurunkan aktivitas enzim, sedangkan kehadiran butanol dan n-heksan meningkatkan aktivitas enzim sebesar 12 % dan 20 % secara berurutan.

The need for lipase by various industrial fields with diverse applications makes lipases need to be produced quickly and in large quantities. However, the lipases produced today are not fully suitable for industries that require processing with high temperature conditions and organic solvents and ionic interactions in solution such as salt content or salinity. This study aimed to isolate thermohalotolerant lipase-producing bacteria from hot springs, clone and express lipasecoding genes and biochemical characterization of thermal-halotolerant lipases. Enrichment was carried out before isolating lipolytic bacteria from extreme habitats such as hot springs. Enrichment is carried out directly in the natural habitat or in situ enrichment. Lipolytic bacteria were isolated from in situ using the Spread Plate on Rhodamine B–Agar media. Lipolytic bacterial isolates were identified based on the 16S rRNA gene. Lipase encoding gene was isolated by PCR technique using degenerate primer. The lipase gene was cloned on the pGEM-T Easy vector and then transformed into Escherichia coli DH5α for gene preservation and cloned on the pET24a vector, transformed into E. coli BL21 (DE3) pLysS for expression. LipA_BTeq expression was carried out in 2 treatments, which included signal peptide sequences and expressed without signal peptides (mature lipase). Molecular mass analysis of lipase enzyme protein was performed using SDS-PAGE and zymogram. In addition, 3D structure analysis and family lipase relationships were analyzed using the MEGA XI application. Lipase characterization carried out were optimum temperature, optimum pH, enzyme stability to salinity (NaCl solution), substrate specificity, resistance to organic solvents, and the influence of metal ions. Seventeen isolates were isolated from the enriched samples. Lipase genes from five lipolytic bacterial isolates were successfully cloned with isolate codes HSAS, HSDS2, HSC3.1, HSC3.2 and HSB3. The results of the identification of the HSAS isolate, had a 99.5% similarity with Bacillus tequilensis strain A13 with a lipase gene length (LipA_Bteq) 639 bp encoding 213 amino acids with 31 amino acids at the beginning of the sequence being a signal peptide sequence. The result of lipase gene expression showed that mature lipase treatment had higher specific activity (17 ± 1.12 U/mg) than those with signal peptide (8 ± 1.11 U/mg), so that mature lipase was chosen for characterization. The results of SDS-PAGE and Zimogram analysis showed that the protein weight of LipA_Bteq was ~20 kDa. These results were also confirmed by MEGA XI analysis which formed a clade with the lipase I.4 subfamily. LipA_Bteq has an optimum temperature of 30 oC, and has a temperature stability range of 30 oC - 60 oC with an average relative activity of 98%. LipA_Bteq has resistance to 0.1M to 3M NaCl concentration, and has optimum activity at 2 M NaCl concentration. LipA_Bteq is classified as halotolerant lipase because it requires NaCl to achieve optimum activity. For pH parameters, LipA_Bteq has the highest activity at pH 8. LipA_Bteq has a good preference for C6 (short chain) substrates. The presence of metal ions (Fe3+, Cu2+, Mg2+, Ca2+, Na+, and Mn2+) with a concentration of 100 mM in the reaction mixture was able to increase the activity of LipA_Bteq. On the other hand, Zn2+ decreased lipase activity up to 83%. The addition of 50% organic solvents methanol, isopropanol and ethanol decreased enzyme activity, while the presence of butanol and n-hexane increased enzyme activity by 12% and 20%, respectively.