Lipase's thermostability and organic solvent tolerance are two crucial properties that enable it to function as a biocatalyst. This study examined the characteristics of two recombinant thermostable lipases (Lk2, Lk3) based on transesterification activity. Conversion of C12-C18 methyl ester with paranitrophenol was investigated in various organic solvent. Both lipases exhibited activity on difference carbon chain length (C12 – C18, C18:1, C18:2) of substrates. The activity of Lk2 was higher in each of substrate compared to that the Lk3. Experimental findings showed that the best substrates for Lk2 and Lk3 are C18:1 and C18:2 respectively, in agreement with the computational analysis. The activity of both enzymes prefers on nonpolar solvent. On nonpolar solvent the enzymes are able to keep its native folding shown by the value of radius gyration, solvent-enzyme interaction and orientation of triad catalytic residues. Lk3 appeared to be more thermostable, with maximum activity at 55°C. The presence of Fe3+ increased the activity of Lk2 and Lk3. However, the activity of both enzymes were dramatically decreased by the present of Ca2+ despite of the enzymes belong to family I.1 lipase known as calcium dependent enzyme. Molecular analysis on His loop of Lk2 and Lk3 on the present of Ca2+ showed that there were shifting on the orientation of catalytic triad residues. All the data suggest that Lk2 and Lk3 are novel lipase on the family I.1 and both lipase available as a biocatalyst candidate.

Lk2 and Lk3 were thermostable recombinant lipase and highly expressed in Escherichia coli BL21 (DE3). However, Lk2 and Lk3 accumulated as an inclusion body. To further characterize both recombinant lipases, the soluble enzyme must be obtained first. This study aimed to optimize the disruption of the cell membrane in order to obtain soluble and active lipases. The effects of temperature lysis, pH, and SDS concentration on lipolytic activity Lk2 and Lk3 were investigated using a three-factor Box-Behnken design response surface methods. The optimum condition for the temperature variables at 50°C, pH 8, and 0.34% SDS which gave a lipolytic activity of 0.9 U for Lk2. Meanwhile, Lk3 lipolytic activity of 0.9 U obtained at the temperature of 50°C, pH 8, and 0.1% SDS. This result showed efficient one-step membrane disruption methods using thermolysis with addition of a low concentration of detergent at pH 8. The methods used were effective and applicable in the production of active and soluble thermostable recombinant lipase.

<p>Dioscorea spp. merupakan tanaman umbi-umbian yang termasuk dalam kategori pangan fungsional karena mengandung senyawa bioaktif, di antaranya ialah diosgenin. Senyawa ini merupakan senyawa golongan steroid saponin digunakan sebagai bahan baku dalam sintesis obat golongan steroid. Sumber utama senyawa diosgenin adalah Dioscorea spp. Indonesia sebagai salah satu center of origin Dioscorea berpeluang untuk menjadi produsen diosgenin dunia karena mempunyai beragam plasma nutfah Dioscorea. Oleh sebab itu, perlu dikaji potensi diosgenin dari Dioscorea spp. lokal Indonesia. Dalam penelitian ini dilakukan karakterisasi kuantitatif senyawa diosgenin menggunakan metode spektrofotometri Ultra Violet-Visible (UV-Vis) pada 26 aksesi Dioscorea spp. lokal Indonesia, terdiri atas 10 aksesi D. alata, 7 aksesi D. esculenta, dan 9 aksesi D. hispida. Validasi metode spektrofotometri UV-Vis ditentukan berdasarkan nilai korelasi linear, LOD, dan LOQ. Ekstraksi diosgenin dilakukan dengan refluks dan<br />hidrolisis pada saat yang bersamaan dengan pelarut isopropanol 70% dan H2SO4 20%. Metode UV-Vis yang digunakan dalam penelitian ini cukup sensitif dan akurat dengan nilai korelasi linear 0,996, LOD 2,604 μg, dan LOQ 7,890 μg. Kandungan diosgenin tertinggi diperoleh dari aksesi D. hispida dengan kode II sebesar 0,294%. D. hispida dapat dimanfaatkan sebagai kandidat sumber potensial senyawa diosgenin.</p>

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