## 1 Introduction  
Lipidvic enzymes, such as esterase (EC 3.1.1.1) and lipase (EC3.1.1.3), are ubiquitous and hydrolyze small ester-containing molecules and insoluble long-chain triglycerides, respectively, yielding hydroxyl and carboxylate groups [1]. Lipases hydrolyze ester bonds in long-chain fatty acids (>10 carbons), whereas esterases hydrolyze ester bonds in short-chain fatty acids (<10 carbons). The application of esterases and lipases have been of interest in several industries, such as food, detergent, oil and fat, pulp and paper, leather, cosmetics, biodiesel production, textiles, and pharmaceuticals [2; 3]. In addition, esterase usage for ester prodrugs has been used for cleaving and reactivation [4].  
Due to their diversity, bacterial esterases and lipases have been widely studied, including their purification and biochemical properties [5; 6]. The first classification of bacterial esterases and lipases proposed included only eight families [1], whereas recently, the 19 th family was reported with a family XIX lipase from _Stentrorophomonas multiphilia_Psi-1 [7]. All esterases and lipases contain an \(\alpha/\beta\) hydrolase fold [8]. However, diversity of the enzymes is variegated, dependent on their sequence identity and biochemical properties.  
Bacterial esterase and lipase sources have been expanded using metagenomes. A metagenome refers to the collective genomes of individual microorganisms in an environment without cultured microflora [9]. Microbial DNA has been directly extracted from sediments [10]. Metagenomes have also been called environmental DNA [11; 12]. Metagenomics offers great advantages in biotechnology due to wider gene sources including unculturable microorganisms. Recently, it has been suggested that metagenomics is a powerful tool to obtain lipases for biocatalysts [13]. In practice, many types of esterases and lipases have been cloned from various ecosystems, such as lotus pond sludge [14], hypersaline lakes [15], mangrove sediments [16], paper mill wastewater sediments [17; 18], soil [19; 20; 21; 22; 23], frozen soil [24], rumen [25], compost [26; 27], and anaerobic ecosystems, such as rumens of dairy cows, large intestines of horses, freshwater sediments, and forest toposils [28], and their properties have been extensively investigated and improved.  
In our previous study, metagenomes were constructed from mud flats contaminated by oil spills, esterase and lipase clones were isolated from the metagenomic library, and the molecular properties of family IV Est3K and family I.3 Lip3K were reported [29]. During further analysis of the library, two novel positive clones were identified with different structures and properties from the two esterases.  
In this paper, analyses of the two positive clones from the library and the molecular characteristics of two novel genes coding CzcO-containing family IV Est2L and family V Est4L with broad substrate specificity are described in detail. CzcO is associated with the cation diffusion facilitator CzcD, which is related to efflux Zn\({}^{2+}\), Co\({}^{2+}\), and Cd\({}^{2+}\)[30; 31]. The CzcO-containing family IV esterase was matched in the gene database; however, no information about its molecular characterization is available to date. Est4L has a low similarity to other Family V esterases whose molecular properties have been reported. Therefore, this work provides novel information about natural fusion esterase and diverse substrate specificity.