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"Penelitian karakterisasi produk gen sintetik lipase Thermomyces lanuginosus yang diekspresikan oleh Bacillus subtilis DB104 rekombinan K7 bertujuan untuk mengetahui pengaruh suhu, pH, dan ion logam terhadap aktivitas lipase. Bacillus subtilis DB104 promXynAQ1 non rekombinan digunakan sebagai kontrol. Lipase rekombinan optimal diproduksi pada media LB yang mengandung substrat minyak zaitun 1% selama 24 jam. Aktivitas lipase rekombinan diuji pada berbagai variasi perlakuan suhu (40°C--80°C), pH (5--10), dan penambahan ion logam menggunakan metode uji aktivitas spektrofotometri p-nitrofenil palmitat (pNPP assay). Data aktivitas spesifik lipase rekombinan dianalisis menggunakan data standar deviasi. Hasil penelitian menunjukkan bahwa lipase rekombinan aktif maksimal pada suhu 80°C dan optimal pH 8 dengan aktivitas spesifik sebesar 1,488 U/mg. Penambahan ion logam Ca2+, Mg2+, Cu2+, dan senyawa pengelat EDTA berpengaruh menghambat aktivitas enzim lipase rekombinan. ......The research of characterization of lipase Thermomyces lanuginosus synthetic gene product expressed by recombinant Bacillus subtilis DB104 had been conducted to investigate the effects of temperature, pH, and metal ions toward the enzymatic activity. Non recombinant lipase of Bacillus subtilis DB104 promXynAQ1 was used as control. Recombinant lipase was optimally produced using LB media containing 1% olive oil during 24 hours incubation time. Recombinant lipase was assayed in various treatments of temperature (40°C--80°C), pH (5--10), and metal ion addition using spectrophotometric method of p-nitrophenyl palmitate assay (pNPP assay). Specific activity of recombinant lipase data were analyzed with deviation standard. Experiment results showed that activity of recombinant lipase is maximum at temperature 80°C and optimum at pH 8 in the amount of 1,488 U/mg. The presence of metal cations Ca2+, Mg2+, Cu2+, and chelating-agent EDTA gave an inhibitory effect on recombinant lipase activity."

"Gen CSF3 merupakan gen penyandi human granulocyte-colony stimulating factor hG-CSF. Gen CSF3 yang digunakan dalam penelitian ini merupakan gen sintetik dari penelitian terdahulu disebut CSF3syn yang dikonstruksi secara in vitro. Gen tersebut mengandung kodon preferensi Escherichia coli dan telah berhasil digunakan untuk mengekspresikan protein hG-CSF rekombinan pada E. coli menggunakan plasmid ekspresi berbasis promotor T7. Gen CSF3syn disubkloning ke dalam plasmid ekspresi yang mengandung promotor rhaBAD pada penelitian ini. Promotor ini dapat terinduksi oleh rhamnose dalam media sebagai sumber karbon. Tujuan penelitian ini adalah mendapatkan konstruk plasmid rekombinan pJ804-mCSF3 sebagai sumber vektor alternatif untuk produksi protein hG-CSF rekombinan pada sistem ekspresi E. coli menggunakan plasmid berbasis promotor rhaBAD. Konstruksi plasmid dilakukan melalui subkloning gen CSF3syn ke dalam plasmid pJ804. Terdapat dua varian gen CSF3syn yang digunakan, yaitu mCSF3-ori dan mCSF3-new2. Kedua gen tersebut diisolasi masing-masing dari plasmid pJ414-mCSF3-ori dan pJ414-mCSF3-new2 setelah didigesti dengan enzim restriksi NdeI dan XhoI. Gen target dan plasmid selanjutnya diligasi dan plasmid rekombinan yang diperoleh diklon ke dalam E. coli DH5?. Sebanyak 12 dari 30 koloni transforman pJ804-mCSF3-ori dan 7 dari 44 koloni transforman pJ804-mCSF-new2, positif menunjukkan pita gen CSF3syn sebesar 558 pb setelah diverifikasi menggunakan teknik PCR koloni dan PCR plasmid. Analisis digesti plasmid menggunakan enzim NdeI dan XhoI mengonfirmasi situs ligasi dan ukuran dari plasmid rekombinan yang diperoleh 4.723 pb. Plasmid rekombinan pJ804-mCSF-ori dan pJ804-mCSF3-new2 telah berhasil dikonstruksi.

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CSF3 is a gene that encodes the human granulocyte colony stimulating factor hG CSF. The CSF3 gene used in this study was a synthetic gene from a previous study called CSFsyn that was constructed in vitro. The gene contains the Escherichia coli codon preference and has been successfully used to express the recombinant hG CSF protein in E. coli using T7 promoter based expression plasmid. The CSF3syn gene was subcloned into an expression plasmid containing rhaBAD promoter in this study. This promoter can be induced by rhamnose in the media as a carbon source. The purpose of this study was to obtain a recombinant plasmid construct of pJ804 mCSF3 as an alternative vector source for the production of recombinant hG CSF protein in an E. coli expression system using rhaBAD promoter based plasmid. The plasmid construction was performed by subcloning the CSF3syn gene into the pJ804 plasmid. There are two variants of CSF3syn genes used, mCSF3 ori and mCSF3 new2. The two genes were isolated from pJ414 mCSF3 ori and pJ414 mCSF3 new2 plasmids respectively after digestion using NdeI and XhoI restriction enzymes. The target genes and plasmid were subsequently ligated and recombinant plasmid obtained were cloned into E. coli DH5 . Twelve out of the 30 transformant colonies of pJ804 mCSF3 ori and 7 out of 44 transformant colonies pJ804 mCSF new2, positively demonstrated the presence of CSF3syn gene band of 558 bp. Those colonies have been verified using colony PCR and plasmid PCR techniques. Plasmid digestion analysis using NdeI and XhoI enzymes confirmed ligation site and size of the recombinant plasmids obtained 4.723 bp. Recombinant plasmids pJ804 mCSF ori and pJ804 mCSF3 new2 have been successfully constructed."

"Lipase merupakan salah satu enzim yang penting di industri enzim, dan banyak digunakan dalam pembuatan zat aditif makanan, kosmetik, dan industri farmasi. Penelitian sebelumnya yang dilakukan di PTB-Laptiab BPPT, pengklonaan gen sintetik T. lanuginosus lipase pada B. substilis memiliki aktivitas lipase yang rendah yaitu sebesar 1,488 U/mg. Penelitian ini bertujuan untuk mengklona gen sintetik T. lanuginosus lipase TLL ke dalam vektor ekspresi Pichia pastoris menggunakan sinyal peptida asli TTL. Gen TLL yang mengandung sinyal peptida asli diamplifikasi dengan PCR, dan disisipkan ke dalam pPICZ? A di antara situs XhoI dan XbaI, kemudian ditransformasikan ke dalam sel kompeten E. coli DH5?. Hasil transformasi dipilih dua rekombinan positif untuk dilakukan analisis sekuensing. Hasil sekuensing, kedua rekombinan mengandung gen target lipase. Plasmid yang telah dikonfirmasi kemudian dilinearisasi dan ditransformasikan ke dalam P. pastoris X-33 dengan menggunakan metoda elektroporasi. Gen T. lanuginosus lipase berhasil diintegrasi ke dalam kromosom P. pastoris X-33, yang ditunjukkan dengan terbentuknya zona bening pada media Yeast extract Peptone Dextrose Tributyrin YPD.TB agar yang mengandung zeocin. Thermomyces lanuginosus lipase memiliki daerah open reading frame ORF 916 bp yang mengkode 291 asam amino dengan massa molekul teoritis 35 kDa. Enzim rekombinan T. lanuginosus memiliki suhu optimum 80 C dan pH optimum 8. ......Lipase is one of the most important industrial enzymes, which is widely used in the preparation of food additives, cosmetics, and pharmaceutical industries. In the previous study, we have cloned synthetic Thermomyces lanuginosus lipase gene into Bacillus subtilis and Escherichia coli and resulting low expression of enzyme activity. The aim of this research was to construct the T. lanuginosus lipase TLL gene into P. pastoris vector expression with TLL original signal peptide. Thermomyces lanuginosus lipase gene was amplified by PCR and contained original signal peptide and then inserted into pPICZ A between XhoI and XbaI site, and transformed into competent cell E. coli DH5. From the transformant, two of positive recombinants were analyzed by sequencing analysis. As the result, both of two recombinant have a positive target gene which has lipase gene. The correct plasmid was linearized and then was transformed into P. pastoris X 33 by electroporation method. Thermomyces lanuginosus synthetic gene lipase has been successfully integrated into chromosome of P. pastoris X 33, which revealed by clear zones around the colony on Yeast extract Peptone Dextrose Tributyrin YPD.TB plate with zeocin. The Thermomyces lanuginosus lipase had an open reading frame of 916 bp encoding TLL of 291 amino acids with theoretical molecular mass of 35 kDa. The recombinant enzyme, T. lanuginosus lipase had optimal temperature at 80 C and optimal pH at pH 8.0."