## 5 Application of Recombinant Lipases
#### Pharmaceutical Applications of Recombinant Lipases  
Profens (2-arylpropionic acids) are drugs commonly used in the treatment of pain and inflammation associated with tissue injury. The anti-inflammatory activity of ketoprofen, flurbiprofen, ibuprofen, and naproxen is mainly related to their (S)-enantiomer. For these drugs, (R)-enantiomers contribute weakly to cyclooxygenase (COX) inhibition and have been related to side effects--e.g., gastrointestinal pain [163; 164]. In recent years, the kinetic resolution of 2-arylpropionic acids by enantioselective esterification has been widely studied. Lipases have been applied in this process and their relevant characteristics demonstrated in many papers; the promising results point to the future applications of these enzymes [5; 6; 164; 165]. For instance, lipases from _C. antarctica_, _C. rugosa_[164; 166] and _A. niger_[6] have been applied in the kinetic resolution of profens, making it possible to obtain drugs with high enantiomeric purity--e.g., (R)-flurbiprofen, ee\({}_{\text{p}}\) = 96.3%; E = 90.5%; C = 35.7%), therefore, demonstrating their great potential as biocatalysts in pharmaceutical synthesis. Figure 4 illustrates the kinetic resolution of racemic profens using lipases. Figure 4: Lipase-catalyzed scheme of (RS)-ethyl ester racemetame of naproxen, ketoprofen and ibuprofen by an enantioselective lipase (adapted from [PERSON] et al. [167]). R (Radical) represents the different ethyl profens (ethyl-ibuprofen, ethyl- naproxen and ethyl-ketoprofen). A lipase from the _S. marcescens_ ECU1010 strain produced in _E. coli_ catalyzed the hydrolysis of racemic ketoprofen esters into (S)-ketoprofen, with the highest ee (enantiomeric excess) (91.6%) and yield (48.2%) [168]. A lipase from _C. rugosa_ produced in _K. phaffii_ was used for the kinetic resolution of racemic ibuprofen by means of enantioselective esterification with 74% ee [130]. Commercial lipase from _C. antarctica_ produced in _A. niger_ and immobilized onto polymethylmethacrylate (Novozym 435) has been efficiently used for the enantioselective esterification of (R,S)-flurbiprofen to obtain the (R)- flurbiprofen methyl ester in 96% ee [166]. A lipase gene from _S. marcescens_ ES-2 was reinserted in the same strain for lipase homologous production and was applied in the enantioselective hydrolysis of (R,S)-flurbiprofen ethyl ester for the production of the optically active form (S)-flurbiprofen [169]. Taking into consideration that the lipase Lip2p from _Y. lipolytica_ has higher catalytic activity than _C. rugosa_ lipases to hydrolyze profen ethyl esters, though its selectivity is not sufficient, [PERSON] et al. [167] improved Lip2p enantioselectivity by site-directed mutagenesis. The authors identified two amino acid residues (232 and 235) in the lipase hydrophobic substrate binding site as crucial for enantiomer discrimination and enzyme activity. Their results demonstrated the high potential of rational engineering to create biocatalysts suitable for industrial applications. Chemical synthesis is the common process for obtaining vitamin A derivatives. However, the process presents some issues, such as synthesis steps, which could destroy raw materials (use of acid and alkali), create difficulties in the separation of the final product due to the instability of intermediate products, as well as considerable energy consumption and pollution. Accordingly, a green and environmentally friendly manufacturing method has become the current trend in obtaining vitamin A derivatives. To conveniently prepare the immobilized enzyme for the green synthesis of vitamin A palmitate, _T. languinosus_ lipase (TLL) was assembled with apatite derived from calcium phosphate in solution to form TLL@apatite hybrid nanoflowers (hNFs) by mimetic biomineralization. Under the optimized conditions, when vitamin A acetate and palmitic acid were applied as substrates, the yield of the product was up to 90.4% [170]. Recombinant CALB produced by _K.