Lipases (EC 3.1.1.3) are enzymes that play important roles in various biological systems by hydrolyzing or synthesizing ester bonds in fats and other lipid compounds [1]. Due to their substrate specificity and regio- and enantio-selectivity, these enzymes have received a lot of attention for various industrial applications [2], [3], [4]. They are used to synthesize flavors, oils, and fats in the food industry [5], to synthesize some anti-cancer and anti-inflammatory drugs in the pharmaceutical industry [6], to remove oily stain in the detergent industry [7], and to synthesize fragrances, moisturizers, and emulsifiers in the cosmetic industry [8].

Since lipases are widely used in various industrial fields and is an environmentally friendly catalyst, researchers are conducting many studies to find enzymes with useful functionality as well as high activity and stability [9]. In particular, psychrophilic lipase shows high activity at low temperatures, which can minimize loss of taste, aroma, and nutrients that can occur in high-temperature processes in the food industry [10]. By maintaining lipase activity during refrigeration and transport, the productivity and efficiency of the enzyme can be increased [11]. Psychrophilic lipases are also used in environmental remediation because they can break down contaminants in low-temperature environments [12].

LipCA, a lipase from Croceibacter atlanticus isolated from the Antarctic Ross Sea, is known to have a moderately high optimum reaction temperature (40 °C) and a low activity at low temperatures, despite being found in Antarctica [13]. According to previous studies, recombinant LipCA expression is significantly increased in E. coli through molecular evolution techniques [14]. Therefore, if we technically mutate LipCA to have high activity even at low temperatures, this enzyme can be used as a psychrophilic enzyme catalyst in the food industry [15].

A variety of mutant enzyme libraries can be generated by randomly changing nucleotide sequences of enzyme genes using molecular evolution techniques. Among them, error-prone PCR (ep-PCR) is a robust and efficient method for generating lipase mutant libraries with diverse amino acid substitutions [16]. By applying an effective screening method, lipases with improved activities and stabilities at low temperatures can be found [17], [18].

In this study, ep-PCR was used to generate mutants with high activity at low temperatures from LipCA lipase. Afterwards, 3D structures of selected mutant enzymes were analyzed and lipase activity was compared to that of the wild-type (WT) enzyme [19]. Back mutation was induced to analyze effects of mutated amino acid residues on enzyme activity. We then performed synthesis of octyl butyrate, a flavor compound widely used in the flavor and fragrance industry, to explore the utility of LipCA mutant enzyme in food and cosmetic industries [20].