Characterization and Immobilization of a Novel SGNH Family Esterase (La SGNH1) from Lactobacillus acidophilus NCFM

Abstract

The SGNH family esterases are highly effective biocatalysts due to their strong catalytic efficiencies, great stabilities, relatively small sizes, and ease of immobilization. Here, a novel SGNH family esterase (LaSGNH1) from Lactobacillus acidophilus NCFM, which has homologues in many Lactobacillus species, was identified, characterized, and immobilized. LaSGNH1 is highly active towards acetate- or butyrate-containing compounds, such as p-nitrophenyl acetate or 1-naphthyl acetate. Enzymatic properties of LaSGNH1, including thermal stability, optimum pH, chemical stability, and urea stability, were investigated. Interestingly, LaSGNH1 displayed a wide range of substrate specificity that included glyceryl tributyrate, tert-butyl acetate, and glucose pentaacetate. Furthermore, immobilization of LaSGNH1 by crosslinked enzyme aggregates (CLEAs) showed enhanced thermal stability and efficient recycling property. In summary, this work paves the way for molecular understandings and industrial applications of a novel SGNH family esterase (LaSGNH1) from Lactobacillus acidophilus.

Keywords: LaSGNH1; Lactobacillus acidophilus; SGNH family esterases; crosslinked enzyme aggregates; immobilization.

Figure 1

Phylogenetic tree and sequence analysis of LaSGNH1. (A) Bacterial lipases/esterases family I–VIII, and (B) clade I and II of family II are shown. A red box in each panel indicates the location of LaSGNH1. The phylogenetic trees were constructed with MEGA v. 7.0 using the neighbor-joining method, and all sequences were retrieved from the NCBI database. (C) Sequence alignments of four conserved blocks (Block I, II, III, and V) are shown, and highly conserved residues are highlighted in red. Sequences are aligned with Clustal Omega and ESPript. Highly conserved catalytic triad, glycine, and asparagine are shown as red or yellow triangles. Four sequences of the clade I subfamily are shown in the upper region, while three sequences of the clade II subfamily are shown in the bottom region. Highly important amino acids for catalysis are shown as red and yellow triangles.

Figure 2

Gene cluster analysis of LaSGNH1. Similar gene clusters were found in Lactobacillus species including in Lactobacillus acidophilus La-1, L. acidophilus NCFM, Lactobacillus amylovorus, Lactobacillus helveticus, Lactobacillus crispatus AB70, and Lactobacillus kefiranofaciens ZW3. EF: elongation factor, PK: type I pantothenate kinase, AT: acetyltransferase, TR: amino acid ABC transporter. Homologous proteins of LaSGNH1 are shown in the red box.

Figure 3

Characterization of LaSGNH1. (A) Sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified LaSGNH1. (B) Native-polyacrylamide gel electrophoresis (PAGE) analysis. (C) Substrate specificity of LaSGNH1 using p-nitrophenyl (p-NP) esters. The hydrolase activities are shown relative to the activity toward p-NB. (D) Regioselectivity of LaSGNH1 was studied using 1-naphthyl acetate (1-NA), 1-naphthyl butyrate (1-NB), and 2-naphthyl acetate (2-NA). The hydrolase activities are shown relative to the activity toward 1-NA. (E,F) Detection of fluorescence due to the formation of 4-methylumbelliferone (4-MU) by LaSGNH1. All experiments were performed at least in triplicate.

Figure 4

Stability of LaSGNH1. (A) Thermal stability of LaSGNH1. The residual activity of LaSGNH1 was measured during incubation for 1-h. (B) The pH stability of LaSGNH1 was studied at a pH from 3 to 10. (C) Chemical stability of LaSGNH1 was studied against various chemicals. (D,E) Urea-induced unfolding of LaSGNH1. Fluorescence was monitored after 1-h of incubation in from 1 to 5 M urea. A red-shift of λ_max from 330 to 344 nm was detected. (F) Activity of LaSGNH1 in the different concentrations of urea. All experiments were performed at least in triplicate.

Figure 5

Homology modeling of LaSGNH1. (A) Ribbon representation of LaSGNH1. The substrate binding pocket is also shown in the square and important residues for catalysis are shown as sticks. (B) Electrostatic potential diagram of substrate-binding regions of LaSGNH1. (C) Modeling of p-nitrophenyl acetate (pNA, cyan) in the substrate-binding pocket of LaSGNH1. The amino acid residues interacting with pNA are shown as sticks (green). (D) LigPlot analysis of p-nitrophenyl acetate in the substrate-binding pocket of LaSGNH1.

Figure 6

Hydrolysis of various substrates by LaSGNH1. A pH shift assay was performed for (A) tertiary alcohol esters (TAEs), including tert-butyl acetate, α-terpinyl acetate, and linalyl acetate, (B) glyceryl esters, including glyceryl tributyrate (GTB) and glyceryl trioleate (GTO), and oils, including olive oil (O.O.) and fish oil (F.O.), and (C) acetylated carbohydrates, including glucose pentaacetate, cellulose acetate, and N-acetyl-glucosamine. The hydrolysis reaction changed the color of the solution from red to yellow.

Figure 7

Immobilization of LaSGNH1. (A) Immobilization efficiency of free LaSGNH1, LaSGNH1- crosslinked enzyme aggregates (CLEA), mCLEA-LaSGNH1, LaSGNH1-Arg-CLEA, and mCLEA-Arg-LaSGNH1. (B) Thermal stability of free LaSGNH1 and LaSGNH1-Arg-CLEA. (C) Reusability of LaSGNH1-Arg-CLEA. The reaction was repeated for 10 cycles after each washing step. All assays were performed at least in triplicate.