## 1 Introduction
[PERSON] et al. reported that the recombinant _Ha_GSH1-type lipase from _Halocymithibacter articus_ catalyzed the synthesis of biodiesel and flavor compounds [31]. _Ha_GSH1 had a sandwich structure containing five central parallel \(\beta\)-sheets between two layers of four \(\alpha\)-helices per layer. The catalytic triad of S18, D171, and H174 was embedded near the surface within a catalytic pocket. The solvent-exposed substrate-binding pocket offered the substrate flexibility to _Ha_GSH1. The substrate-binding pocket encircled with five amino acids, Asn83, Met86, Arg87, Phe131, and Ile173, regulated the entry of substrates via noncovalent interactions. _Ha_GSH1 was applied for the hydrolysis of tert-butyl esters,carbohydrate esters, and p-nitrophenyl esters. _Ha_GGNH1 specifically hydrolyzed butyl esters such as _p_-nitrophenyl butyrate, 1-naphthyl acetate, _tert_-butyl acetate, and glyceryl tributyrate. To improve the catalytic activity of _Ha_GGNH1 six mutants, Asn83 Leu (1), Met86 Glu (2), Met86 Arg (3), Arg87 Leu (4), Phe131 Ala (5), and Ile173 Phe (6) were generated using site-directed mutagenesis. Only four showed 120% enhanced hydrolysis of _p_-nitrophenyl butyrate than wild-type _Ha_GGNH1. Two showed higher activity towards carbohydrate acetates, while three showed for larger substrates like p-nitrophenyl hexanoate. The bulky phenyl side chain of six preferred bindings to short-chain fatty esters like p-nitrophenyl acetate. The immobilized _Ha_GGNH1-CELA was applied for the synthesis of petroleum, food, and cosmetic products (butyl and oleic esters) in a nonpolar solvent. The successful synthesis of butyl acetate, butyl butyrate, and oleic acid butyl ester was confirmed by gas chromatography analysis. Figure 1: Reactions catalyzed by _Candida antarctica_ lipase B. (**A**) Epoxidation of \(\alpha\),\(\beta\)-unsaturated aldehydes with hydrogen peroxide, (**B**) aldol condensation (a and b) and [PERSON] addition (c), (**C**) formation of bis-product, (**D**) formation of [PERSON] addition and aminolysis product, and (**E**) Baeyer–Villiger oxidation reaction. Another example is Cytochrome P450 monooxygenases (CYPs). CYPs are heme-containing enzymes that catalyze the hydroxylation of a wide variety of substrate molecules [99]. Bacterial steroid hydroxylase CYPs are particularly useful for effectively introducing hydroxyl groups to a wide range of steroids in the pharmaceutical industry. Thus, these CYPs have received increasing attention in recent years for industrial applications. [PERSON] et al. (2017) reported a newly identified CYP steroid hydroxylase (_Ba_CYP106A2) from the bacterium _Bacillus_ sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, and its crystal structure was determined at a resolution of 2.7 A. Furthermore, structural comparisons with other CYPs showed that the \(\alpha\)8-\(\alpha\)9 loop region of _Ba_CYP106A2 was intrinsically mobile and might be important for broad substrate specificity. The hydroxylase activity of _Ba_CYP106A2 was examined with two different kinds of steroid substrates, 4-androstenedione and nandrolone, and the reaction was analyzed with HPLC and LC-MS [99]. CYPs oxidized 4-androstenedione and nandrolone to 158-hydroxyandrostenedione and 158-hydroxyandrolone, respectively. Unlike the mesophilic CYP orthologs from _Bacillus meagerium_[100], _Ba_CYP106A2 seemed to mainly monohydroylate steroid substrates with high conversion yields (Figure 3). The specific modification of pharmaceutically important compounds by the cold-active CYP sheds light on drug development and production. In conclusion, structural flexibility of cold-active enzymes could enhance or give rise to enzyme promiscuity. Consequently, cold-active enzymes have merits for the enzymatic modification and semi-synthesis of fine chemicals and medicines by virtue of their broad substrate specificity on top of their existing merits as industrial catalysts.