ENANTIOSELECTIVITY OF ISOLATE LIPASE FROM THE METAGENOM APPROACH TO (R,S)- MANDELIC ACID IN TRANSESTERIFICATION REACTIONS

• Main Author: Fatmawati, Fenti
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/75030
• Institution: Institut Teknologi Bandung

LK1, LK2, and LK3 are lipase genes obtained from the metagenome of compost samples. The gene that encodes this lipase has been stored in GenBank with access numbers KP204883, KP204884, and KP204885 respectively. Lipase gene expression LK1, LK2, and LK3 in the pET30a(+) expression vector in Eschericia coli hosts induced by IPTG was carried out to obtain recombinant protein lipase. Mandelic acid enantiomers and their derivatives have been considered important substances due to their wide use for synthetic purposes. (R)-mandelic acid is used to make semi-synthetic antibiotics such as cephalosporins and penicillins. (S)-mandelic acid is used to make anti- inflammatory drugs such as deracoxib and celecoxib. The advantages of a single enantiomer are greater selectivity for its biological target, better therapeutic index, better pharmacokinetics than a mixture of enantiomers, and reduced side effects due to unwanted enantiomer activity. This study aims to obtain lipase which acts as an enantioselective biocatalyst for (RS-S)-mandelic acid The enantioselectivities of lipases LK1, LK2, and LK3 were measured using a chiral column HPLC where optimization of the hexane mobile phase had previously been carried out: isopropanol, flow rate of 1 mL/min, column temperature of 30oC and addition of 4% TFA and also validation of the method to ensure that the test to be carried out complies with the test requirements. The method validation results showed that the percent recovery was 100.06 for the R enantiomer and 100.10 for the S enantiomer with a % standard deviation relative to each of 0.482% and 0.318%. This shows that the analytical method used provides data that is close to the actual value. Separation of the racemic (R-S) Mandelat acid mixture under these conditions produced two separate peaks with the first peak appearing to be the enantiomer (S)-Mandelat acid peak and the second peak being the (R)-Mandelat acid enantiomer peak. This is in line with his computational studies which show that the affinity of column complexes with R ligands is more stable than column complexes with S ligands. The transesterification reaction between vinyl acetate and (R-S)-Mandelic acid catalyzed by the three lipases above was incubated at a temperature range of 45-65°C. Enantioselectivity is followed by calculating the enantiomeric excess (ee), which shows the ratio of the dominant enantiomer to the racemic mixture. Observational data showed graphical profiles between typical temperatures. From this pattern, it is observed that there are 2 important points obtained, namely the optimum temperature and minimum temperature. At the optimum temperature, the maximum excess enantiomeric value is obtained. The optimum temperature at LK1 is at 45 oC with an excess enantiomeric value of 10.2%, LK2 is at 65 oC with an excess enantiomeric of 12.3% and LK3 is present at 60 oC with an excess enantiomeric of 13.3%. Whereas at the minimum temperature, LK1 is present at 55 oC (excess 3.4% enantiomeric), LK2 is present at 55oC (3.2% enantiomeric excess) and LK3 is present at 55 oC (0.6% enantiomeric). This measurement was carried out using a chiral column HPLC with optimized parameter results. From the results of the computational docking approach with Autodock Vina and MD, it was found that the (R)-mandelic acid enantiomer interacted more strongly than the (S)-mandelic acid enantiomer. It can be seen from the enantiomer binding energy value which is lower than the S enantiomer for lipase LK1, LK2, and LK3. In the LK1-R enzyme complex, active site residues (Ser109, Asp255, His277) bind to ligands. In the LK1-S enzyme complex, the active site residue does not bind to the ligand. This allows the R enantiomer to react with vinyl acetate. The LK2-R complex has residues of active sites (Ser109, Asp255, His277) that bind to ligands, while the LK2- S enzyme complex of active site residues does not bind to ligands. In the LK3 lipase, the LK3-R enzyme complex has active site residues (Ser109, Asp255, His277) that bind to the ligand while the LK3-S ligand enzyme complex only binds to His277 and Asp 255. This is in line with experimental results which show that the enantiomer converted to product is seen from the reduction of the R enantiomer substrate. There are more hydrogen bonds in the R enantiomer in Lk1, Lk2, and Lk3 than in the S enantiomer. At the optimum temperature, lipase local isolates LK1, LK2, and LK3 had better enantiomeric separation, this was due to the influence of the specificity residue of each isolate. In LK1 it is affected by the specificity residues of Ala217 and Leu220. In LK2 it is affected by the specificity residues of Leu205 and Asn207. Meanwhile, LK3 is affected by the specificity residues of Leu205 and Met210. The experimental results are in line with computational studies. Whereas at the minimum temperature, it can be said that the enzyme cannot distinguish between the R and S enantiomers. The specificity residues involved in the reaction at the optimum temperature are not found at the minimum temperature.