OPTIMASI PROSES UNTUK EKSPRESI GEN ENDOGLUKANASE DARI Bacillus sp. RP1 OLEH Escherichia coli BL21 (DE3)/egc
Article Sidebar
Main Article Content
Abstract
Selulase adalah salah satu enzim yang banyak dimanfaatkan dalam berbagai industri. Sebagai upaya untuk memenuhi kebutuhan, 50 tahun terakhir dikembangkan beberapa strategi untuk meningkatkan produksi selulase yang mencakup rekayasa genetika dan optimasi proses. Karena itu, dilakukan kloning gen egc dan RBS yang berasal dari Bacillus sp. RP1 yang diisolasi dari sumber air panas ke dalam vektor pGEM-T Easy. E. coli BL21 (DE3) ditransformasikan dengan vektor yang mengandung gen egc tersebut. Setelah kloning, optimasi proses berupa desain medium turut dilakukan untuk mengoptimalkan ekspresi gen egc. Desain medium diawali dengan seleksi komposisi medium menggunakan metode Plackett-Burman. Komponen medium yang diuji adalah kulit beras, molase, amonium klorida, urea dan tepung ikan. Kulit beras dan molase diperoleh sebagai bahan yang paling berpengaruh terhadap aktivitas enzim dan berat kering sel. Tahap selanjutnya melibatkan metode statistik Box-Behnken dan metodologi respons permukaan yang bertujuan mengoptimalkan respons aktivitas enzim dan berat kering sel terhadap konsentrasi molase, konsentrasi kulit beras dan lama fermentasi. Konsentrasi yang diuji adalah 1%, 5,5% dan 10%, sedangkan lama fermentasi yang diuji adalah 24, 36 dan 48 jam. Konsentrasi optimal molase adalah 7,45% dan konsentrasi optimal kulit beras adalah 6,45% dengan lama fermentasi optimal 39,52 jam.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
Authors who publish with this journal agree to the following terms:
a). Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Attribution-NonCommercial-ShareAlike 4.0 International that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
b). Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
c). Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
References
Acharya S, Chaudhary A (2012) Optimization of fermentation conditions for cellulases production by Bacillus licheniformis MVS1 and Bacillus sp. MVS3 isolated from Indian hot spring. Braz Arch Biol Technol 55: 497-503. doi: 10.1590/S1516-89132012000400003
Figueira R, Brown DR, Ferreira D, Eldridge MG, Burchell L, Pan Z, Helaine S, Wignesweraraj S (2015) Adaptation to sustained nitrogen starvation by Escherichia coli requires the eukaryote-like serine/threonine kinase YeaG. Sci Rep 5: 1-14. doi: 10.1038/srep17524
Gaur R, Tiwari S (2015) Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07. BMC Biotechnol 15:19. doi: 10.1186/s12896-015-0129-9
Gohel HR, Contractor CN, Ghosh SK, Braganza VJ (2014) A comparative study of various staining techniques for determination of extra cellular cellulase activity on Carboxy Methyl Cellulose (CMC) agar plates. Int J Curr Microbiol App Sci 3: 261-266
Kim MH, Kang DU, Lee JW (2016) Construction of a recombinant Escherichia coli JM109/A-68 for production of carboxymethylcellulase and comparison of its production with its wild type, Bacillus velezensis A-68 in a pilot-scale bioreactor. Biotechnol Bioprocess Eng 21: 601-611. doi: 10.1007/s12257-016-0468-y
Kuhad RC, Deswal D, Sharma S, Bhattacharya A, Jain KK, Kaur A, Pletshcke BI, Singh A, Karp M (2016) Revisiting cellulase production and redeï¬ning current strategies based on major challenges. Renew Sust Energ Rev 55: 249-272. doi: 10.1016/j.rser.2015.10.132
Kuhad RC, Gupta R, Singh A (2011) Microbial cellulases and their industrial applications. Enzyme Res 1-10. doi: 10.4061/2011/280696
Lee YJ, Kim HJ, Gao W, Chung CH, Lee JW (2012) Statistical optimization for production of carboxymethylcellulase of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 from rice bran using response surface method. Biotechnol Bioproc Eng 17: 227-235. doi: 10.1007/s12257-011-0258-5
Maki M, Leung KT, Qin W (2009) The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int J Biol Sci 5: 500-516. doi: 10.7150/ijbs.5.500
Mattanovich D, Kramer W, Luttich C, Weik R, Bayer K, Katinger H (1998) Rational design of an improved induction scheme for recombinant Escherichia coli. Biotechnol Bioeng 58: 296-298. doi: 10.1002/(SICI)1097-0290(199804 20)58:2/3<296::AID-BIT26>3.0.CO;2-9
Moeis MR, Natalia D, Ningrum RW, Dwijayanti A (2014) Cloning and expression of endoglucanase gene from thermophilic bacteria Bacillus sp. RP1. Microbiol Indones 8: 170-176. doi: 10.5454/mi.8.4.4
Muñoz C, Hidalgo C, Zapata M, Jeison D, Riquelme C, Rivas C (2014) Use of cellulolytic marine bacteria for enzymatic pretreatment in microalgal biogas production. Appl Environ Microbiol 80: 4199-4206. doi: 10.1128/AEM.00827-14
Parekh S, Vinci VA, Strobel RJ (2000). Improvement of microbial strains and fermentation process. Appl Microbiol Biotechnol 54: 287-301. doi: 10.1007/s002530000403
Puspitasari IN, Moeis MR (2008) Screening of seven cellulase and xylanase producing Bacillus species and analysis of the cellulase and xylanase expression pattern of the selected isolate. Pp 194-201. Proceedings of the Second International Conference on Mathematics and Natural Sciences (ICMNS) 28-30 October, Bandung
Sadhu S, Maiti TK (2013) Cellulase production by bacteria: A review. Br Microbiol Res J 3: 235-258. doi: 10.5281/zenodo.8687
Singh V, Haque S, Niwas R, Srivastava A, Pasupuleti M, Tripathi CKM (2017). Strategies for fermentation medium optimization: An in-depth review. Front Microbiol 7: 2087. doi: 10.3389/fmicb.2016.02087
Smith A (1995) Gene Expression in Recombinant Microorganisms. Marcel Dekker Inc, New York
Studier FW, Daegelen P, Lenski RE, Maslov S, Kim JF (2009) Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K12 genomes. J Mol Biol 394: 653-680. doi: 10.1016/j.jmb.2009.09.021
Xu J, Banerjee A, Pan SH, Li ZJ (2012) Galactose can be an inducer for production of therapeutic proteins by auto-induction using E. coli BL21 strains. Protein Expr Purif 83: 30-36. doi: 10.1016/j.pep.2012.02.014