Ekspresi Gen Lipase T1.2RQ dari Geobacillus stearothermophilus T1.2 pada Cereibacter sphaeroides 2.4.1 Menggunakan Promotor Terinduksi Anaerobiosis

Abstract

Lipase (EC 3.1.1.3) is a hydrolase enzyme that is an important commodity in the food industry, oil industry, wastewater treatment, cosmetics industry, leather industry, and detergents. Demand for microbial lipase is expected to experience significant growth in the near future, due to the variety of food processing applications, making lipase one of the most needed enzymes today. One of the microorganisms that produces lipase is bacteria. Geobacillus stearothermophilus T1.2 isolated from Seram Island, Maluku. The T1.2RQ lipase gene has been successfully expressed in the Escherichia coli and Bacillus subtilis host systems. However, lipase production uses a host E. coli and B. subtilis has certain challenges, such as the high cost of organic carbon substrates. An attractive alternative host cell is to use photosynthetic bacteria, such as Cereibacter sphaeroides 2.4.1 due to its ability to photosynthesize. The use of photosynthetic hosts can offer a more cost-effective production system by utilizing light as an energy source for growth and reducing reliance on expensive organic carbon sources as the primary substrate. Cereibacter sphaeroides 2.4.1, which belongs to the non-sulfur purple bacteria, is a promising host organism due to its versatile metabolism, allowing growth under both aerobic and anaerobic conditions. Its unique ability for anaerobic photoheterotrophic culture—harnessing light energy in the absence of oxygen—offers potential for sustainable biotechnological applications. Lipase T1.2RQ can be expressed heterologously in C. sphaeroides 2.4.1. However, the host system of C. sphaeroides 2.4.1 needs to be developed and optimized. One possible development is optimizing the expression of T1.2RQ lipase in the pRK415 vector using the indigenous promoter from C. sphaeroides namely puf. This research was conducted to test the feasibility of C. Sphaeroides 2.4.1 as a host for heterologous expression of the lipase gene T1.2RQ from G. stearothermophilus T1.2 using the pRK415 expression vector. The T1.2RQ lipase gene was cloned and expressed under the control of the puf (photosynthetic unit fixed) promoter, which can be induced under anaerobic and aerobic photosynthetic conditions, and compared with conventional lac promoter. The first method used was the construction of the pRK415-puf expression vector which begins with the isolation of genomic DNA of C. sphaeroides 2.4.1, followed by amplification of puf promoter (459 bp) via PCR. This PCR product was then cloned into the pGEMT-Easy vector using the TA-Cloning method; successful insertion was verified through colony PCR and restriction EcoRI. Next, the promoter fragment of puf was restricted from pGEMT-puf using enzymes of EcoRI and ligated into the pRK415 vector which has been restricted with the same enzyme (EcoRI) and dephosphorylated to prevent religation. The result of this ligation, the plasmid pRK415-puf, transformed to E. coli DH5a and its orientation was confirmed using PCR. In parallel, the gene encoding the T1.2RQ lipase was amplified from the template plasmid by PCR and cloned first into the pGEMT-Easy vector using the TA-Cloning method. After successful insertion was verified, the next step was to restrict the T1.2RQ lipase gene from pGEMT-T1.2RQ using the restriction enzyme of NotI. The T1.2RQ lipase fragment was then ligated into the pSL301 vector which had also been restricted with NotI, resulting in plasmid pSL301-T1.2RQ. Integration of the lipase gene into the expression vector (pRK415-puf) is a crucial step in the success of the construction. For this purpose, the T1.2RQ lipase fragment was restricted from pSL301-T1.2RQ by double restriction using XbaI and HindIII. Simultaneously, plasmid pRK415-puf was restricted with the same enzyme. The T1.2RQ lipase gene fragment was then ligated into pRK415-puf linearly to produce the final recombinant plasmid: pRK415-puf-T1.2RQ. This plasmid was verified by double restriction and then the plasmid was transferred into the host cell of C. sphaeroides2.4.1 through the triparental mating conjugation, using E. coli HB101_pRK2013 as a helper, with antibiotic selection (rifampicin and tetracycline). Recombinant lipase production was carried out under two different cultivation conditions to compare host efficiency: aerobic heterotrophs and anaerobic photoheterotrophs. Protein was extracted intracellularly by ultrasonic cell disruption. Enzyme characterization was performed qualitatively and quantitatively; SDS-PAGE was used to confirm the presence of protein, Native-PAGE zymogram with tributyrin substrate was used to verify functional lipolytic activity, and spectrophotometric assay with p-nitrophenyl palmitate substrate was used to measure specific lipase activity quantitatively. Recombinant plasmid (pRK415-puf-T1.2RQ) has been successfully transferred to the host C. sphaeroides 2.4.1 via conjugation with an efficiency of 2.9 x 10-6. Qualitative test (spot test) shows that C. sphaeroides (pRK415-puf-T1.2RQ) and positive control (C. sphaeroides which expresses the lipase gene T1.2RQ under the lac promoter) formed a clear zone in LA+tributyrin media after 24 hours, confirming successful lipolytic activity. In contrast, the negative control (C. sphaeroides without the lipase gene T1.2RQ) did not form a clear zone. Quantitative results showed that the lipase activity controlled by the puf promoter under anaerobic conditions (4.37 ± 1.34 U/mL) was significantly higher than the expression regulated by the lac promoter (1.77 ± 0.16 U/mL). In contrast, under aerobic conditions, the lac promoter resulted in significantly higher expression (10.16 ± 0.06 U/mL) when compared to the puf promoter (4.81 ± 0.11 U/mL). Protein analysis using SDS–PAGE and zymogram confirmed the expression of T1.2RQ lipase with a consistent molecular mass of approximately 43 kDa. Overall, this study indicates that the puf promoter can effectively induce heterologous gene expression in C. sphaeroides, highlighting its potential as a photoinducible regulatory system for the development of anaerobic biocatalytic processes and other light-based biosynthesis applications.