Characterization of ML-005, a Novel Metaproteomics-Derived Esterase

Abstract

A novel gene encoding for a lipolytic enzyme, designated ML-005, was recently identified using a functional metaproteomics approach. We heterologously expressed this protein in Escherichia coli and biochemically characterized it. ML-005 exhibited lipolytic activity toward short-chained substrates with the preferred substrate being p-nitrophenyl-butyrate, suggesting that ML-005 is an esterase. According to homology analysis and site-directed mutagenesis, the catalytic triad of the enzyme was identified as Ser-99, Asp-164, and His-191. Its optimal pH was determined to be at pH 8. Optimal activity was observed at 45°C. It also exhibited temperature, pH and salt tolerance. Residual relative activity after incubating at 50-60°C for 360 min was above 80% of its initial activity. It showed tolerance over a broad range of pH (5-12) and retained most of its initial activity. Furthermore, incubating ML-005 in 1 - 5M NaCl solution had negligible effect on its activity. DTT, EDTA, and ß-mercaptoethanol had no significant effect on ML-005's activity. However, addition of PMSF led to almost complete inactivation consistent with ML-005 being a serine hydrolase. ML-005 remains stable in the presence of a range of metal ions, but addition of Cu²⁺ significantly reduces its relative activity. Organic solvents have an inhibitory effect on ML-005, but it retained 21% of activity in 10% methanol. SDS had the most pronounced inhibitory effect on ML-005 among all detergents tested and completely inactivated it. Furthermore, the V_max of ML-005 was determined to be 59.8 μM/min along with a K_m of 137.9 μM. The k_cat of ML-005 is 26 s^-1 and k_cat/K_m is 1.88 × 10⁵ M^-1 s^-1.

Keywords: biocatalysis; esterase; lipase; metagenomics; metaproteomics.

FIGURE 1

Functional Metaproteomics workflow used to discover ML-005. Grease-contaminated soil sample was used for bacterial enrichment with olive oil as the sole carbon source. Community DNA was extracted, sequenced and annotated. Community protein was separated in a 2D gel and 2D PAGE zymography was carried out to visualize protein spots harboring lipolytic activity. Protein spots were identified using mass-spectrometry, searching against a protein database derived from the metagenomic DNA sequences. ML-005 was selected for this study for further characterization. Functional Metaproteomics has the immediacy of an activity-based approach, while still retaining the comprehensive information of the metagenome (Sukul et al., 2017).

FIGURE 2

(A) Sequence alignment of ML-005 with the uncharacterized esterase YdeN from Bacillus subtilis (UniProt accession number P96671.1) showed a sequence identity of 28.44% (highlighted in black, similar amino acids in shades of gray). Residues forming the catalytic triad i.e., serine, (99), aspartic acid (164), and histidine (191) are highlighted in yellow. These residues form an integral part of the active site. The pentapeptide Ala – His – Ser – Leu – Gly motif is highlighted in green. This represents the nucleophilic elbow and is a conserved structure found within lipolytic enzymes. Residues 1–20 form a signal peptide (red) which was processed in E. coli (see panel C). (B) Three dimensional structure of ML-005 was modeled using the Phyre² server (blue ribbon; http://www.sbg.bio.ic.ac.uk/phyre/). Potential catalytic triad residues (His-191, Asp-164, and Ser-99) were predicted to be in close proximity to each other. The structural alignment with YdeN from B. subtilis (green ribbon) shows substantial similarity (C) ML-005 was cloned into a pET22b vector with a T7 promoter system and a C-terminal His₆-tag und purified to homogeneity. The purified ML-005 band showed a size that approximates 23.4 kDa, consistent with the calculated mass of His₆-tagged ML-005 with a removed signal peptide.

FIGURE 3

(A) Substrate specificity of ML-005 showed clear preference for p-nitrophenyl butyrate (C4). Substrate with a chain length of C8 retained approximately 66% of the maximum activity, whereas activity dropped significantly with longer chains. (B) Serine (99), aspartic acid (164), and histidine (191) constituting the catalytic triad were mutated and negligible activity was observed for any of the mutants. (C) Temperature optimum was observed to be at 45°C. (D) pH optimum was observed to be at pH 8, however, activity of ML-005 was largely stable between pH 7.5 and 9. At pH 7, still approximately 64% of maximum activity was retained. pH 9.5 resulted in a drastic loss of activity.

FIGURE 4

Phylogenetic comparison of ML-005 and 5 other well-characterized bacterial esterases. Tree of lipase sequences with 35% identity to ML-005 and to esterases EstA from P. aeruginosa, EstB from B. gladioli, EstC from S. coelicolor, EstD from T. maritima and Carboxylesterase NP from B. subtilis. ML-005 is member of an as of yet uncharacterized family of esterases, which also contains YdeN from B. subtilis.

FIGURE 5

(A) Michaelis-Menten kinetics were observed for ML-005 with pNP-butyrate. V_max of ML-005 was determined to be 59.8 μM/min along with a K_m of 137.9 μM. The k_cat of ML-005 is 26 s^-1 and k_cat/K_m is 1.88 × 10⁵ M^-1 s^-1. (B) ML-005 showed temperature tolerance from 20 to 60°C. (C) ML-005 showed tolerance over a broad range of pH (5–12) and retained most of its activity. At pH 4 it retained approximately 50% of its activity while pH 13 almost completely deactivated ML-005. (D) ML-005 showed halotolerance when incubated in increasing NaCl concentrations. After 7 days of incubation at close to saturated NaCl solution (5M), ML-005 still retained most of its activity.

FIGURE 6

(A) Metal ions showed negligible effect on ML-005 at a concentration of 1 mM, with copper showing the most drastic effect by inhibiting ML-005 by approximately 50%. (B) Organic solvents had an overall inhibiting effect on ML-005 without exception, but ML-005 was moderately stable in the presence of even 10% Methanol. (C) All inhibitors showed moderate inhibiting effect at a concentration of 1 mM, with relative activity staying at around 80%. Only PMSF showed almost complete inhibition of ML-005, consistent with an active-site serine. (D) Detergents at 1% were also found to have an overall inhibiting effect. CHAPS showed the least effect with 66% relative activity and SDS inactivating ML-005 completely with negligible remaining activity.