PENINGKATAN AKTIVITAS DAN EKSPRESI PROTEIN TOTAL ENZIM SELULASE REKOMBINAN SERTA POTENSI APLIKASINYA PADA PAKAN IKAN
Abstract
Meskipun enzim selulase rekombinan banyak dikembangkan untuk meningkatkan efisiensi pemecahan serat, informasi mengenai bagaimana modifikasi seperti mutasi dan penambahan tag SKIK (Ser-Lys-Ile-Lys) dapat memengaruhi aktivitas enzim dan produksi protein total masih terbatas. Oleh karena itu, penelitian ini bertujuan mengevaluasi pengaruh mutasi dan penambahan tag SKIK terhadap aktivitas dan protein total enzim selulase rekombinan yang diproduksi oleh Escherichia coli BL21 (DE3). Penelitian dilakukan secara eksperimental menggunakan rancangan acak lengkap (RAL) dengan empat perlakuan, yaitu P1= wild type (WT), P2 = WT + SKIK, P3 = mutan, dan P4 = mutan + SKIK, masing-masing dengan empat ulangan. Produksi enzim dilakukan melalui kultur E. coli rekombinan yang membawa plasmid pET-22b-CellE dengan induksi laktosa monohidrat, kemudian dianalisis menggunakan uji aktivitas selulase metode 3,5-dinitrosalisilat (DNS) dan pengukuran protein total dengan metode Bradford. Data dianalisis menggunakan ANOVA satu arah dan uji lanjut Duncan pada tingkat signifikansi 5% (p < 0,05). Hasil penelitian menunjukkan bahwa perlakuan mutan + SKIK menghasilkan nilai tertinggi baik pada aktivitas enzim (10,66 U mL-1) maupun protein total (0,85 mg mL-1). Penambahan SKIK meningkatkan ekspresi dan kelarutan protein, sedangkan mutasi memperbaiki efisiensi katalitik enzim. Kombinasi keduanya memberikan efek sinergis dalam meningkatkan perfoma enzim selulase rekombinan. Penelitian ini merupakan yang pertama mengombinasikan penambahan tag SKIK dengan mutagenesis untuk meningkatkan ekspresi dan aktivitas katalitik enzim selulase pada E. coli. Temuan ini tidak hanya relevan untuk peningkatan produksi enzim pada industri bioteknologi, tetapi juga berpotensi diaplikasikan dalam formulasi pakan ikan.
Although recombinant cellulase enzymes have been widely developed to enhance fiber-degradation efficiency, information regarding how modifications such as mutagenesis and the addition of the SKIK (Ser-Lys-Ile-Lys) tag influence enzymatic activity and total protein production remains limited. Therefore, this study aimed to evaluate the effects of mutagenesis and SKIK tagging on the activity and total protein yield of recombinant cellulase expressed in Escherichia coli BL21 (DE3). The research was conducted experimentally using a completely randomized design (CRD) with four treatments: P1 = wild type (WT), P2 = WT + SKIK, P3 = mutant, and P4 = mutant + SKIK, each consisting of four replications. Enzyme production was carried out using recombinant E. coli harboring the pET-22b-CellE plasmid induced with lactose monohydrate, and the resulting enzyme was analyzed for cellulase activity using the 3,5-dinitrosalicylic acid (DNS) method and total protein concentration using the Bradford assay. Data were analyzed using one-way ANOVA followed by Duncan’s post-hoc test at a 5% significance level (p < 0.05). The results showed that the mutant + SKIK treatment produced the highest values for both enzymatic activity (10.66 U mL-1) and total protein (0.85 mg mL-1). The addition of the SKIK tag enhanced protein expression and solubility, whereas mutagenesis improved catalytic efficiency. The combination of both modifications produced a synergistic effect, resulting in superior performance of the recombinant cellulase enzyme. This study is the first to combine SKIK tagging with mutagenesis to enhance the expression and catalytic activity of cellulase in E. coli. These findings are not only relevant for improving enzyme production in biotechnology industries but also hold potential applications in fish feed formulation.
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DOI: http://dx.doi.org/10.15578/jra.20.3.2025.257-267
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