## 5 Application of Recombinant Lipases
#### Pharmaceutical Applications of Recombinant Lipases
phaffii_ was immobilized and used to synthesize vitamin A palmitate by transesterification of vitamin A acetate and palmitic acid in organic solvent with a conversion ability of 54.3% after 15 cycles [129]. As represented in Figure 5, vitamin A palmitate synthesis could be catalyzed by _C. antarctica_ lipase B.  
Pregabalin, a GABA (gamma-aminobutyric acid) analogue, is a drug developed for treatment of several central nervous system disorders, involving neuropathic pain, fibromyalgia, anxiety, social phobia and epilepsy. The synthesis of pregabalin involves 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester, an intermediate substance with chiral characteristic. The (S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid enantiomer is the key chiral intermediate for pregabalin synthesis. Considering the significant increase in the use of pregabalin in recent years, different routes have been explored using chemical and enzymatic approaches [171; 172]. Lipozyme TL IM(r) and Lipolase(r) (Novozymes) are commercially available lipases for the kinetic resolution of (S)-2-Carboxyethyl-3-cyano-5-methylhexanoic acid [172]. In particular, high resolution yields (45%) and enantioselectivity (98% enantiomeric excess, ee) were achieved by using commercial Lipolase, a recombinant _T. languinosus_ lipase expressed in _A. oryzae_[173]. Figure 6 illustrates an optimized route of pregabalin synthesis by enzymatic resolution and decarboxylation steps, considering the work of [PERSON] et al. [173]. Figure 5: Immobilized lipase-catalyzed scheme of vitamin A palmitate (adapted from [PERSON] et al. [129]). A mutant L206F/P207F/L259F of _T. thermophilus_ lipase was efficiently immobilized onto epoxy resin D5730 to enable the reuse (using n-heptane/water biphasic system) of the enzyme and to improve the conversion at a high concentration of 2-carboxyly3-cyano-5-methylhexanoic acid ethyl ester with 49.7% conversion and 95% \(ee_{\text{p}}\) (enantiomeric excess of the product) under the optimized conditions [172]. Diliazem hydrochloride is a typical calcium channel blocker clinically used as an antianginal and antihypertensive agent [174]. _Trans-_3-(4'-methoxyphenyl)glycidic acid methyl ester ((2R, 3S)-MPGM or (--)-MPGM) is a key chiral intermediate for its synthesis. Some approaches involving lipases have been applied in the production of this diltiazem intermediate, such as the application of the _S. marcescens_ lipase, a well-known and important lipase for industrial applications. Lipase from _S. marcescens_ ECU1010 catalyzed production of (--)-MPGM by the enzymatic resolution of _trans-_3-(4'-methoxyphenyl)glycidic acid methyl ester ((\(\pm\))-MPGM) with chitosan in an isopropyl ether water biphasic system ([PERSON] et al., 2010). Overexpression of _S. marcescens_ lipase in _E. coli_ often promotes inclusion bodies, which is one of the major problems in industrial recombinant protein production [174; 175]. A simple and efficient method for refolding with simultaneous purification of _S. marcescens_ lipase in _E. coli_ in the production of (--)-MPGM has been described with an overall yield of 41.5% and _ee_ of 99% [175]. Figure 7 shows a lipase-catalyzed scheme of the synthetic process of diltiazem. Beta-blockers belong to an important therapeutic class, which is used to treat several cardiovascular conditions [176]. Chirality is a key factor to consider for the efficacy of this class of drugs. Atenol is a synthetic, beta1-selective (cardioselective) adrenoreceptor blocking agent and it is used in the treatment of hypertension, angina pectoris due to coronary atherosclerosis, as well as acute myocardial infarction angina [177]. It is known that its b1-blocking activity is attributed to its (S)-enantiomer [178]. In a  
Figure 6: Optimized chemoenzymatic pregablalin synthesis (adapted from [PERSON] et al. [173]). Figure 7: Lipase-catalyzed scheme of the synthetic process for diltiazem (adapted from [PERSON] et al. [174]).