Lipolytic enzymes, including esterases (3.1.1.1) and lipases (3.1.1.3), are members of the α/β hydrolase superfamily, which contain a catalytic triad (Ser-His-Asp/Glu)^[1]. With the development of industrial biotechnology, lipolytic enzymes are important catalysts in the biological manufacturing processes, such as food, bioenergy, detergent, pharmaceutical and advanced chemical manufacturing^[2-3]. Lipolytic enzymes can catalyze the cleavage and formation of ester bonds, and the catalytic mechanisms for esterases and lipases are similar^[4]. In organic media, both esterases and lipases catalyze various reactions, such as esterification, transesterification, aminolysis and interesterification^[5]. The increasing demand for novel biocatalysts has prompted the development of new methods which are used to screen for new genes, such as genome sequencing.

Stenotrophomonas maltophilia is a Gram-negative bacterium which, in our previous research, has been demonstrated to secrete esterases and lipases to prepare free astaxanthin efficiently^[6]. This result suggests that S. maltophilia OUC_Est10 is a good microbial resource for novel lipolytic enzymes. However, there are only few genome sequences available for S. maltophilia^[7-8]. They are all focused on the drug resistance of S. maltophilia, and to the best of our knowledge, there has been no study on the diversity of lipolytic enzymes. Since lipolytic enzymes have wide application in industry, it is necessary to investigate the diversity of relevant genes in the genome of S. maltophilia in order to discover more encoding genes for the biocatalysts.

In this study, the lipolytic enzymes of S. maltophilia OUC_Est10 (China General Microbiological Culture Collection Center (CGMCC), 10672) were induced in the fermentation medium (KH₂PO₄, 0.025%; MgSO₄·7H₂O, 0.025%; FeSO₄·7H₂O, 0.001%; beef extract, 0.1%; peptone, 1.0%; cholesterol oleate, 0.1%; Tween-80, 1.0%; H₂O, 100 mL; pH 7.0). Esterase activity and lipase activity were determined spectrophotometrically at 405 nm. Fermentation liquid of OUC_Est10 was centrifuged at 5439×g for 10 min, and the supernatant was subjected to ammonium sulfate precipitation. The resulting crude enzyme was put onto a DEAE-Sepharose Fast Flow column, which was previously equilibrated with buffer A (20 mmol/L Tris-HCl, pH 8.0). The unbound proteins were washed with buffer A until the absorbance at 280 nm reached the baseline. Furthermore, the bound proteins were eluted by a gradient of 0.1–0.7 mol/L NaCl in buffer A (Figure 1). The fractions resulted in a peak were analyzed in terms of esterase activity (p-nitrophenyl butyrate (pNPB) as substrate) and lipase activity (p-nitrophenyl palmitate (pNPP) as substrate).

The result showed that esterase activities and lipase activities were both determined in 8 peaks (Table 1), and it was finally proven that OUC_Est10 could secrete a wide range of lipolytic enzymes. S. maltophilia OUC_Est10 was isolated from the slaughterhouse soil in Qingdao, China. The slaughterhouse soil was rich in lipids, which might explain the wide range of lipolytic enzymes in OUC_Est10. The feature of multiple lipolytic enzymes in S. maltophilia OUC_Est10 contributes to the efficient hydrolysis of lipids in the slaughterhouse soil.

To investigate the diversity of lipolytic enzyme genes, the genomic DNA of OUC_Est10 was extracted using Puregene Yeast/Bact. Kit B (QIAGEN, Maryland, USA) and sent to Tianjin Biochip Corporation (Tianjin, China) to sequence. The whole genomic DNA was sequenced on the single-molecule real-time (SRMT) sequencing platform PacBio RS Ⅱ (Pacific Biosciences, USA). The genomic sequence was obtained after the reads were de novo assembled using the RS Hierarchical Genome Assembly Process (HGAP) assembly protocol version 3.0 in SMRT Analysis version 2.3.0 (Pacific Biosciences, USA). The protein coding sequences (CDSs) were predicted using Glimmer 3.0^[9]. The procedures of tRNA and rRNA prediction were conducted using tRNAscan-SE^[10] and RNAmmer^[11], respectively. Functional annotation and metabolic pathway analysis were performed on the Integrated Microbial Genomes-Expert Review (IMG-ER) pipeline^[12].

The features of the complete genome sequence of S. maltophilia OUC_Est10 are listed in Table 2. The complete genome sequence consists of a single chromosome of 4668743 bp with a GC content of 66.25% (Figure 2), 3315 genes were identified in OUC_Est10, and the average number of genes in sequenced S. maltophilia was classified to functional categories according to clusters of orthologous genes (COG) designation (Table 3). Through genome searching, it was found that S. maltophilia was rich in lipolytic enzyme genes, which was one of the factors contributing to the virulence in S. maltophilia^[7]. After detailed analysis, 33 proteins with putative lipolytic enzyme activities are found in the genome sequence of OUC_Est10 (Table 4), and the locations of these genes were also marked in Figure 2. This indicates that OUC_Est10 may be a good candidate used for hydrolyzing lipids.

To prove the diversity and the potential use of lipolytic enzymes in S. maltophilia OUC_Est10, 16 pairs of primers were designed. The result of nucleic acid electrophoresis is shown in Supplementary material Figure S1. After being ligated to the pET-28a (+) vector, LEn4, LEn6, LEn12, LEn27 and LEn30 were successfully expressed in BL21 (DE3). Substrate specificity determination was carried out using p-nitrophenyl (pNP) esters with different acyl chain length. The results showed that they had different preferences for the length of fatty acid. LEn4, LEn12 and LEn27 had a preference for short-chain fatty acids, while LEn6 and LEn30 had a preference for medium-chain fatty acids (data not shown). It was worth noting that LEn27 was able to hydrolyze pNP esters with acyl chain length from 4 to 16, which indicated that LEn27 could be widely used for hydrolysis or synthesis of esters with different acyl chain lengths.

The organic solvents tolerance experiment showed that LEn4, LEn6, LEn12 and LEn27 had good organic solvent resistant properties, while LEn30 was highly denatured by organic solvents. LEn4, LEn6, LEn12 and LEn27 could all be used to synthesize ethyl esters (such as cinnamyl acetate) in a non-aqueous system (Figure 3). The determination of cinnamyl acetate was carried out using an HP-5 capillary column (30 m×0.25 mm×0.25 mm).

These experiments indicated that lipolytic enzymes in OUC_Est10 had varied catalytic properties, which could broaden the application range of OUC_Est10. Our further studies will focus on expressing these putative lipolytic enzymes, and they will be used in many fields according to their catalytic properties.

Nucleotide sequence accession number

The complete genome sequence of S. maltophilia OUC_Est10 is available at the IMG database under the accession number Ga0114270 and the GenBank database under the accession number CP015612.