Pengaruh Tipe Media Kultivasi pada Transforman Escherichia coli BL21 (DE3) dalam Ekspresi Gen Sintetik Endoxilanase GH-10

Putri, Aisyah Virgina (2023) Pengaruh Tipe Media Kultivasi pada Transforman Escherichia coli BL21 (DE3) dalam Ekspresi Gen Sintetik Endoxilanase GH-10. Other thesis, Institut Teknologi Sepuluh Nopember.

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Abstract

Xilanase memiliki nilai pemanfaatan tinggi karena dapat memecah ikatan β-1,4-glikosida pada xilan sehingga dapat menghasilkan xilosa dan berbagai macam xilooligosakarida (XOS). Pemanfaatannya yang tinggi ini juga berdampak pada adanya peningkatan permintaan xilanase komersil. Peningkatan produksi melalui regulasi ekspresi gen dapat dilakukan untuk meningkatkan ekspresi xilanase, dan kloning gen tertentu dianggap dapat meningkatkan produktivitas serta spesifitas produksi xilanase. Ekspresi serta produksi xilanase dapat dipengaruhi oleh pemilihan media yang tepat. Melalui transformasi pada proses kloning gen maka memungkinkan terjadinya implifikasi gen target pada host atau inang yang dipilih. Pada penelitian ini digunakan gen sintetik endoxilanase GH-10 dari Kitasatospora sp. yang ditransformasikan pada Escherichia coli BL21 (DE3). Performa hasil transformasi dianalisis melalui PCR koloni dan media agar berlapis ganda xilan untuk melihat keberadaan gen target serta potensi koloni. Koloni transforman Escherichia coli BL21 (DE3) terbaik lalu dikultivasi pada media umum, media diperkaya, dan media diperkaya yang dimodifikasi yang kemudian diinduksi dengan IPTG (Isopropyl β- d-1-thiogalactopyranoside). Pengaruh media kultivasi diukur secara spektrofotometeri pada jam ke-20 dengan panjang gelombang 600 nm. Efisiensi hasil ekspresi kemudian dianalisis melalui SDS-PAGE dan zymogram serta aktivitas enzim dari kultur 16, 24, dan 48 jam. Analisis data hasil penelitian dilakukan secara deskriptif kualitatif dan kuantitatif. Hasil dari penelitian menunjukkan hasil ekspresi pada pembentukan pita protein dari analisis SDS-PAGE dan zymogram menunjukkan terbentuknya pita aktivitas enzim spesifik hasil degradasi xilanase terhadap substrat pada kisaran 101 dan 56,2 kDa. Hasil zymogram menunjukkan adanya pita aktivitas spesifik dengan ketebalan terbaik pada sampel media diperkaya yang dimodifikasi di jam ke 24. Media kultivasi berpengaruh terhadap aktivitas enzim, ditunjukkan dengan adanya perbedaan nilai aktivitas dan terdapat satu nilai aktivitas terbaik yaitu senilai 0,0018 U/mL pada sampel media diperkaya dimodifikasi di jam ke 24.
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Xylanase has a high utilization value because it can break the β-1,4-glycoside bond in xylan so that it can produce xylose and various kinds of xylooligosaccharides (XOS). This high utilization also has an impact on the increase in demand for commercial xylanase. Increased production through regulation of gene expression can be done to increase xylanase expression, and cloning of certain genes is thought to increase productivity and specificity of xylanase production. Expression and production of xylanase can be influenced by the selection of appropriate media. Through transformation in the gene cloning process, it is possible to implant the target gene in the selected host. In this study, the endoxylanase GH-10 synthetic gene from Kitasatospora sp. was transformed into Escherichia coli BL21 (DE3). The transformation performance was analyzed through colony PCR and xylan double-layered agar media to see the presence of target genes and colony potential. The best Escherichia coli BL21 (DE3) transformant colonies were then cultivated on general media, enriched media, and modified enriched media which were then induced with IPTG (Isopropyl β- d-1-thiogalactopyranoside). The effect of the cultivation medium was measured spectrophotometerically at the 20th hour with a wavelength of 600 nm. The expression efficiency was then analyzed through SDS-PAGE and zymogram as well as enzyme activity from 16, 24, and 48 hours of culture. Data analysis of the results of the study was carried out descriptively qualitative and quantitative. The results of the study showed the results of expression on the formation of protein bands from SDS-PAGE and zymogram analysis showed the formation of specific enzyme activity bands from the degradation of xylanase to the substrate in the range of 101 and 56.2 kDa. The zymogram results showed a specific activity band with the best thickness in the modified enriched media sample at 24 hours. Cultivation media influenced enzyme activity, indicated by differences in activity values ​​and there was one best activity value of 0.0018 U/mL in modified enriched media samples at 24 hours.

Item Type:	Thesis (Other)
Uncontrolled Keywords:	Escherichia coli BL21 (DE3), Gen Sintetik Endoxilanase GH-10, Media Kultivasi, Transforman, Xilanase Cultivation Media, Esvherichia coli BL21 (DE3), GH-10 Endoxylanase Synthetic Gene, Transformants, Xylanase
Subjects:	Q Science Q Science > QH Biology > QH301 Biology Q Science > QH Biology > QH426 Genetics Q Science > QR Microbiology > QR74.8 Bacteria
Divisions:	Faculty of Science and Data Analytics (SCIENTICS) > Biology > 46201-(S1) Undergraduate Thesis
Depositing User:	Aisyah Virgina Putri
Date Deposited:	10 Aug 2023 07:39
Last Modified:	10 Aug 2023 07:39
URI:	http://repository.its.ac.id/id/eprint/104280

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