## 1 Introduction
CALB has open, large, and medium substrate-binding pockets, which imparts structural flexibility and broad substrate specificity [89]. The flexible structure of CALB allows the efficient catalysis of various industrially important reactions. CALB catalyzes the epoxidation of \(\alpha\),\(\beta\)-unsaturated aldehydes with hydrogen peroxide to give \(\alpha\),\(\beta\)-epoxy aldehydes (Figure 1A) [90]. Aldol addition of ethanethiol to \(\alpha\),\(\beta\)-unsaturated hexanal gives diastereomeric addition products 2-methyl-3-(ethylthio)pentanal by CALB mutant (Ser105 Ala) (Figure 1B) [91]. CALB also efficiently catalyzes aldol addition reaction between tricyclic diketone to give bis-product within 4 h (Figure 1C) [92]. Under polar and nonpolar solvents, CALB catalyzes different reactions [93]. For example, CALB catalyzes the amidation reaction between \(\alpha\),\(\beta\)-unsaturated ester and amine to give \(\alpha\),\(\beta\)-unsaturated amide under a polar solvent, whereas Michael addition is favored under a non-polar solvent to give \(\beta\)-amino ester (Figure 1D) [93]. The Baeyer-Villiger oxidation reaction of \(\alpha\)-alkyl cyclic ketone to give ethyl 5-acetoxypentanoate can also be catalyzed by CALB (Figure 1E) [94]. These results imply that CALB could accommodate various substrates, which could be useful for the catalysis of industrially important reactions. Similar to other acylaminoacyl peptidases (AAPs), the cold-active AAP (_Sp_AAP) from psychrophilic bacterium _Sporosarcina psychrophilia_ possesses promiscuous ester hydrolysis activity [85; 95]. The _Sp_AAP exhibited hydrolytic activity on native \(N\)-acylated amino acids \(p\)-nitroanilide substrates, and on several promiscuous \(p\)-nitrophenyl esters of fatty acids of different chain length (C4-C18). Similar characteristics in the primary sequence were found in the primary sequence _Sp_AAP with a lack of disulfide bonds, which probably confers structural flexibility for broader substrate specificity [95]. Contrary to hyperthermophilic AAP from _Aeropyrum pernix_ (_Ap_AAP) [96; 97; 84], _Sp_AAP had a lower \(K_{m}\) and \(k_{cat}\) for \(N\)-acyl amino acids, but, interestingly, higher \(K_{m}\) and \(k_{cat}\) for short-chain fatty acids esters. This discrepancy was supported by finding that the diameter of a tunnel in the \(\beta\)-propeller domain to the active site is wider in _Sp_AAP than in mesophilic and thermophilic orthologs [98]. The cold-active acetyl xylan esterase (_Pb_AcE) from psychrophilic soil microbe _Paenibacillus_ sp. had a broad substrate specificity (Figure 2A) [32]. Structural analysis of _Pb_AcE suggested that flexible subunit movements and different active site loop conformations might allow _Pb_AcE to have these characteristics different from other mesophilic and thermophilic homologs. _Pb_AcE forms hexamer in solution and each monomer has \(\alpha\)/\(\beta\) hydrolase fold with conserved catalytic triad residues of Ser185, Asp274, and His303. Activity assay data showed that _Pb_AcE has typical properties of cold-active enzymes with strong low-temperature activity and broad substrate specificity. _Pb_AcE has moderate and strong deacetylation activity for glucose penta-acetate and xylan acetate as well as reversible acetylation activity for xylan. In addition, _Pb_AcE exhibited strong deacetylation activity for lipids and tertiary alcohol esters. These results proposed that _Pb_AcE can accommodate various substrates and it could be useful for deacetylation biocatalyst of other xenobiotic ligands. For example, acetyl group of beta-lactam antibiotics such as cefotaxime, 7-Aminocephalosporic acid (7-ACA), and cephalospororus C were successfully removed by _Pb_AcE (Figure 2B). The enzymatic modification of antibiotics is of significant importance to overcome the antibiotic resistance issue by increasing the chemical diversity. Furthermore, the possibility of commercial application of _Pb_AcE was assessed by ensuring that the activity was maintained more than 80% even after immobilization followed by repetitive use of 18 times.