Cloning, Expression, and Characterization of GDSL-Type Lipolytic Enzyme Genes from Epidermidibacterium keratini EPI-7 Isolated from Human Skin

Abstract

This study investigated seven putative lipolytic enzymes (EstEk01-07) from the skin microbiome bacterium Epidermidibacterium keratini EPI-7, focusing on their properties relevant to industrial applications. Sequence analysis revealed conserved GDSL motifs and four conserved blocks, characteristic of the GDSL/SGNH superfamily, with predicted α/β/α folds consistent with these enzymes. Significant variations in the number of α-helices and β-sheets among the EstEk enzymes suggested diverse substrate specificities and catalytic efficiencies. The enzymes exhibited a strong preference for short-chain fatty acids (C2-C4), classifying them as carboxylesterases, a novel finding within the skin microbiome. Optimal enzyme activity was observed at alkaline pH (8.0-9.0) and thermophilic condition (50-60°C), with substantial thermostability retained after heating at 50°C for three hours. Metal ion analysis revealed a significant stimulatory effect of Ca²⁺ and Fe³⁺, while other transition metals were inhibitory. The enzymes were stable in a range of non-ionic detergents, but sensitive to SDS. Moreover, they exhibited notable tolerance to various organic solvents, particularly methanol and isopropanol, suggesting potential applications in cosmetics and pharmaceutical industries. This study identifies a novel library of thermostable, alkaline carboxylesterases from the skin microbiome, highlighting their potential for industrial biocatalysis and further investigation into their role in skin lipid metabolism.

Keywords: Epidermidibacterium keratini; GDSL-type esterase/lipase; enzyme characterization; gene cloning; heterologous expression; skin microbiome.

Fig. 1. Lipolytic activity of E. keratini EPI-7.

Clear zones were formed around E. keratini EPI-7 colonies on LB agar containing 1% tributyrin.

Fig. 2. Multiple amino acid sequence alignment (A) and phylogenetic analysis (B) of EstEk01-07 from E. keratini EPI-7.

(A) The purple bars represent α-helix structure, the yellow arrows represent β-strand, and the blue arrows represent Turn. Four conserved blocks of I, II, III, and V are boxed. The black and gray blocks show the identical and similar sequences, respectively. Closed triangles and closed inverted triangles indicate amino acid residues belonging to oxyanion hole, and the catalytic triad, respectively. (B) Phylogenetic tree of EstEk01-07 from E. keratini generated using Geneious Prime 24.0.3. Sequence with the following accession numbers were obtained from NCBI/GenBank: E. keratini EPI-7 (CP047156). EstEk01: GDSL-type esterase/lipase family protein of E. keratini EPI-7 (Accession No. WP_159546105), EstEk02-07: SGNH/GDSL hydrolase family protein of E. keratini EPI-7 (Accession No. WP_225983817, 225983862, 159544411, 159544506, 159546432, and 159544982, respectively).

Fig. 3. 3D Structure Modeling of EstEk01-07.

(A) The α-helix, β-sheet, and random coil are shown in cartoon. (B) The catalytic triad are zoomed in.

Fig. 4. SDS-PAGE and Western blot analysis of recombinant EstEk01-07 proteins from E. keratini EPI-7.

(A) SDS-PAGE analysis of the purified EstEk01-07. (B) Western blotting was performed using an anti-His antibody to detect EstEk01-07. Lane M, molecular size marker; lane 1, purified EstEk01; lane 2, purified EstEk02; lane 3, purified EstEk03; lane 4, purified EstEk04; lane 5, purified EstEk05; lane 6, purified EstEk06; lane 7, purified EstEk07.

Fig. 5. Substrate specificity of recombinant EstEk01-07.

The lipase activity of the purified recombinant enzymes toward various chain lengths of p-NP esters was assayed at 40°C with final substrate concentrations of 10 mM in 50 mM Tris- HCl buffer, pH 8.0. The highest level of activity with the substrate was taken as 100%. The error bars represent the means ± SD (n = 3). C2, pNP-C2; C4, pNP-C4; C6, pNP-C6; C8, pNP-C8; C10, pNP-C10; C12, pNP-C12; C14, pNP-C14; C16, pNP-C16.

Fig. 6. Effect of pH on recombinant EstEk01-07 activity and stability.

For the determination of optimal pH, the activity was measured by hydrolyzing p-NP-C2 at 40°C for 15 min in different buffers ranging from pH 5 to 10 (solid line). The highest level of activity with the substrate was taken as 100%. The pH stability of enzymes was assayed by pre-incubating the enzymes at different buffers of pH 5.0-10.0 for 3 h at room temperature (dashed line). Sodium acetate buffer (▼, pH 5.0-6.0), sodium phosphate buffer (●, pH 6.0-8.0), Tris-HCl buffer (□, pH 8.0-9.0), and glycine/NaOH buffer (■, pH 9.0-10.0). The activity at time 0 for each pH condition was considered 100%. The error bars represent the means ± SD (n = 3).

Fig. 7. Effect of temperature on recombinant EstEk01-07 activity and thermostability.

For the determination of optimal temperature, the activity was measured in Tris-HCl buffer (pH 8.0) at different temperatures ranging from 0°C to 80°C (solid line). The highest activity observed was defined as 100%. The thermostability of enzymes was assayed by pre-incubating enzymes at different temperatures (10-80°C) for 3h and measured under standard conditions (dashed line). The relative activity of the enzyme maintained at 10°C was taken as 100%. The error bars represent the means ± SD (n = 3).

Fig. 8. Effect of metal ions, inhibitors, and detergents on recombinant EstEk01-07.

The enzymes were preincubated with metal ions, (1 mM, and 10 mM), inhibitors (1 mM, and 10 mM), and detergents (1%) at room temperature for 1 h, respectively. Residual activity was then assayed under standard conditions. The activity of the enzyme without additives was defined as 100%. The error bars represent the means ± SD (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ^* or ^# indicates p < 0.001 compared with the control group.