Electronic Theses and Dissertation

Lipase merupakan enzim hidrolase yang mengkatalisis reaksi hidrolisis triasilgliserol menghasilkan gliserol dan asam lemak. Lipase memiliki potensial di berbagai bidang industri seperti deterjen, pembuatan kertas, kosmetik, kulit dan makanan. Tujuan penelitian ini adalah melakukan amplifikasi gen lipase PLS 80 dan PLS A, menentukan vektor yang paling efektif dan ukuran gen lipase PLS 80 pada proses kloning, memprediksi struktur gen lipase dari clone PLS 80 pada vektor terbaik dan PLS A. Kloning dilakukan menggunakan dua vektor yaitu pJET1.2/blunt dan pGEMT Easy serta E. coli TOP 10 sebagai sel inang. Hasil amplifikasi gen lipase PLS 80 dan PLS A menggunakan primer Flip2-Rlip2 berukuran 1246 bp dan 1188 bp. Amplikon gen lipase PLS 80 selanjutnya disisipkan ke dalam vektor pJET1.2/blunt dan pGEMT Easy menggunakan enzim T4 DNA ligase. Plasmid rekombinan PLS 80 selanjutnya ditransformasi pada E. coli TOP 10 menggunakan metode heat shock. Efisiensi transformasi pada vektor pJET1.2/blunt sebesar 2,74 x 10 cfu/μg, sedangkan pGEMT Easy sebesar 2,94 x 10 4 cfu/μg. Ukuran gen lipase clone PLS 80 pada vektor pJET1.2/blunt dan pGEM-T Easy masing-masing sebesar 1307 bp dan 1502 bp. Hasil menunjukkan clone PLS 80 pada vektor pGEM-T Easy memiliki ukuran yang lebih besar sehingga dilakukan prediksi struktur protein. Analisis struktur gen lipase clone PLS 80 pada vektor pGEM-T Easy memiliki kemiripan terdekat dengan Geobacillus sp. SBS-4S, sementara struktur gen lipase PLS A memiliki kemiripan terdekat dengan Geobacillus zalihae strain T1. Hasil yang diperoleh menyarankan kloning gen lipase PLS 80 lebih efektif menggunakan vektor pGEM-T Easy dibandingkan dengan vektor pJET1.2/blunt.
Kata kunci: kloning, lipase, termo-halofilik, rekombinan

Lipase is a hydrolase enzyme that catalyzes the hydrolysis reaction of triacylglycerol to produce glycerol and fatty acids. Lipase has potential in various industrial fields such as detergents, papermaking, cosmetics, leather, and food. The purpose of this study was to amplify the PLS 80 and PLS A lipase genes, determine the most effective vector and the size of the PLS 80 lipase gene in the cloning process, and predict the lipase gene structure of the PLS 80 clone on the best vector and PLS A. Cloning was performed using two vectors, namely pJET1.2/blunt and pGEM-T Easy and E. coli TOP 10 as host cells. The amplification results of PLS 80 and A lipase genes using Flip2-Rlip2 primers were 1246 bp and 1188 bp. The amplicon of the PLS 80 lipase gene was then inserted into pJET1.2/blunt and pGEM-T Easy vectors using the T4 DNA ligase enzyme. The recombinant plasmid PLS 80 was then transformed into E. coli TOP 10 using the heat shock method. The transformation efficiency of the pJET1.2/blunt vector was 2.74 x 10 cfu/μg. The size of the lipase gene clone PLS 80 in pJET1.2/blunt and pGEM-T Easy vectors is 1307 bp and 1502 bp, respectively. The results show that clone PLS 80 in the pGEM-T Easy vector has a larger size so that protein structure prediction is carried out. Analysis of the structure of the lipase gene clone PLS 80 in pGEM-T Easy vector has the closest similarity with Geobacillus sp. SBS-4S, while the structure of the PLS A lipase gene has the closest similarity with Geobacillus zalihae strain T1. The results obtained suggest that cloning the PLS 80 lipase gene is more effective using the pGEM-T Easy vector than the pJET1.2/blunt vector. Keywords: cloning, lipase, thermo-halophilic, recombinant