# Enzymatic Synthesis of Biobased Polyesters and Polyamides
## 5 Lipases
After that, the hydroxyl residue of the serine attacks the carbonyl group of the substrate (carboxylic ester or carboxylic acid), forming the first tetrahedral intermediate with a negative charge on the oxygen of the carbonyl group. The oxyanion hole is formed by hydrogen bonding between the amide groups of the amino acid residuals of the enzyme and the carbonyl group oxygen of the substrate. By the formation of at least two hydrogen bonds in the oxyanion hole, the charge distribution is stabilized and the state energy of the tetrahedral intermediate is reduced. Then the alcohol component (R\({}_{1}\)-OH) is released from the bond with the intermediate, while the \"acidic component\" of the substrate remains covalently bound to the serine residue in the acyl-enzyme intermediate. When the enzyme is attacked by a nucleophile (R\({}_{2}\)-OH), the deacylation step occurs. The product (a new carboxylic ester or carboxylic acid) is then released, while the enzyme is regenerated. This nucleophile (R\({}_{2}\)-OH) can be water (hydrolysis) or an alcohol (alcoholysis). To increase the stability towards organic solvents and to facilitate the recycling and reusing, lipases are normally used in their immobilized forms [178; 179; 180; 181; 182; 183; 184; 185]. The immobilized lipases may show improved catalytic activity, specificity or selectivity. Similar to other enzymes, lipases can be generally immobilized via three strategies [178]: (1) chemical or physical adsorptions onto an  
Figure 9: Secondary structure diagram of the \(\alpha/\beta\) hydrolase fold and the location of catalytic triad amino acid residues in lipases. Ser: serine residue; Asp or Glu: aspartic or glutamic acid residue; His: histidine residue; helixes indicate \(\alpha\)-helixes; arrows indicate \(\beta\)-sheets [177]. inert matrix; (2) entrapment within an inert matrix; and (3) immobilized as water-insoluble particles: cross-linked enzyme aggregates (CLEAs), cross-linked enzyme crystals (CLECs), and protein-coated microcrystals (PCMC). Due to the broad substrate specificity, high selectivity, and high thermal stability and catalytic reactivity, _Candida antarctica_ lipase b (CALB), which was reclassified as _Pseudozyma antarctica_ lipase b (PALB) more recently [186], is the most popular biocatalyst which is extensively used in biocatalytic synthesis of small molecules and polymers. CALB is a globular protein that is composed of 317 amino acids (Scheme 11), having a molecular weight of 33 kDa. Similar to other lipases, CALB possesses a Ser-His-Asp catalytic triad (Ser105, Asp187 and His224) in its active site and two oxyanion holes (Thr40 and Gln106) [187], and the catalytic mechanism of CALB is the same as other lipases. However, the presence of the lid structure and the interfacial activation of CALB are still under debate. Some literature suggested that the two \(\alpha\)-helixes (\(\alpha\)5 and \(\alpha\)10) surrounding the active center of CALB, the most mobile part of the structure, could work as the lid [188; 189; 190; 191], and CALB is an interfacial activated enzyme. A recent study indicated the hydrophobicity of the interface and the overall size of the substrate determine the interfacial activation of CALB [190], Others suggested that CALB has no lid covering the entrance of the active site [187] and displays no interfacial activation [192]. In addition, CALB has a very limited available space in the pocket of active site compared to other lipases and this explains its high selectivity [193]. CALB shows improved thermal stability and more stable performance in its immobilized form. At present, several immobilized CALB formulations are commercially available, including Novozym(r) 435 (N435, Novozymes A/S, Copenhagen, Denmark), Chirazyme(r) L-2 (Roche Molecular Biochemical, Mannheim, Germany), LCAHNHE and LCAME (SPRIN S.p.A, Milano, Italy), and CalB immo Plus(tm) (c-LEcta, Leipzig, Germany, and Purolite, Bala Cynwyd, PA, USA) [194].