Biochemical characterization of an esterase from Thermobifida fusca YX with acetyl xylan esterase activity.

BACKGROUND: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. METHODS AND RESULTS: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.

The role of CE16 exo-deacetylases in hemicellulolytic enzyme mixtures revealed by the biochemical and structural study of the novel TtCE16B esterase.

Acetyl esterases belonging to the carbohydrate esterase family 16 (CE16) is a growing group of enzymes, with exceptional diversity regarding substrate specificity and regioselectivity. However, further insight into the CE16 specificity is required for their efficient biotechnological exploitation. In this work, exo-deacetylase TtCE16B from Thermothelomyces thermophila was heterologously expressed and biochemically characterized. The esterase targets positions O-3 and O-4 of singly and doubly acetylated non-reducing-end xylopyranosyl residues, provided the presence of a free vicinal hydroxyl group at position O-4 and O-3, respectively. Crystal structure of TtCE16B, the first representative among the CE16 enzymes, in apo- and product-bound form, allowed the identification of residues forming the catalytic triad and oxyanion hole, as well as the structural elements related to the enzyme preference for oligomers. The role of TtCE16B in hemicellulose degradation was investigated on acetylated xylan from birchwood and pre-treated beechwood biomass. TtCE16B exhibited complementary activity to commercially available OCE6 acetylxylan esterase. Moreover, it showed synergistic effects with SrXyl43 ß-xylosidase. Overall, supplementation of xylan-targeting enzymatic mixtures with both TtCE16B and OCE6 esterases led to a 3-fold or 4-fold increase in xylose release, when using TmXyn10 and TtXyn30A xylanases respectively.

Functional analysis of the N-terminal region of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis.

Acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866) has an N-terminal region (NTR; residues 23-135) between the signal sequence (residues 1-22) and the catalytic domain (residues 136-324), which is of unknown function. Our previous study revealed the crystal structure of the wild-type (WT) enzyme containing the NTR and the catalytic domain. Although the structure of the catalytic domain was successfully determined, that of the NTR was undetermined, as its electron density was unclear. In this study, we investigated the role of the NTR through functional and structural analyses of NTR truncation mutants. Based on sequence and secondary structure analyses, NTR was confirmed to be an intrinsically disordered region. The truncation of NTR significantly decreased the solubility of the proteins at low salt concentrations compared with that of the WT. The NTR-truncated mutant easily crystallized in a conventional buffer solution. The crystal exhibited crystallographic properties comparable with those of the WT crystals suitable for structural determination. These results suggest that NTR plays a role in maintaining the solubility and inhibiting the crystallization of the catalytic domain.

The structure of Phocaeicola vulgatus sialic acid acetylesterase.

Sialic acids terminate many N- and O-glycans and are widely distributed on cell surfaces. There are a diverse range of enzymes which interact with these sugars throughout the tree of life. They can act as receptors for influenza and specific betacoronaviruses in viral binding and their cleavage is important in virion release. Sialic acids are also exploited by both commensal and pathogenic bacteria for nutrient acquisition. A common modification of sialic acid is 9-O-acetylation, which can limit the action of sialidases. Some bacteria, including human endosymbionts, employ esterases to overcome this modification. However, few bacterial sialic acid 9-O-acetylesterases (9-O-SAEs) have been structurally characterized. Here, the crystal structure of a 9-O-SAE from Phocaeicola vulgatus (PvSAE) is reported. The structure of PvSAE was determined to resolutions of 1.44 and 2.06 Šusing crystals from two different crystallization conditions. Structural characterization revealed PvSAE to be a dimer with an SGNH fold, named after the conserved sequence motif of this family, and a Ser-His-Asp catalytic triad. These structures also reveal flexibility in the most N-terminal α-helix, which provides a barrier to active-site accessibility. Biochemical assays also show that PvSAE deacetylates both mucin and the acetylated chromophore para-nitrophenyl acetate. This structural and biochemical characterization of PvSAE furthers the understanding of 9-O-SAEs and may aid in the discovery of small molecules targeting this class of enzyme.

Identification of an acetyl esterase in the supernatant of the environmental strain Bacillus sp. HR21-6.

Bacillus sp. HR21-6 is capable of the chemo- and regioselective synthesis of lipophilic partially acetylated phenolic compounds derived from olive polyphenols, which are powerful antioxidants important in the formulation of functional foods. In this work, an acetyl esterase was identified in the secretome of this strain by non-targeted proteomics, and classified in the GDSL family (superfamily SGNH). The recombinant protein was expressed and purified from Escherichia coli in the soluble form, and biochemically characterized. Site-directed mutagenesis was performed to understand the role of different amino acids that are conserved among GDSL superfamily of esterases. Mutation of Ser-10, Gly-45 or His-185 abolished the enzyme activity, while mutation of Asn-77 or Thr-184 altered the substrate specificity of the enzyme. This new enzyme is able to perform chemoselective conversions of olive phenolic compounds with great interest in the food industry, such as hydroxytyrosol, 3,4-dihydroxyphenylglycol, and oleuropein.

Screening of novel fungal Carbohydrate Esterase family 1 enzymes identifies three novel dual feruloyl/acetyl xylan esterases.

Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate-Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation.

Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis.

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1-22), an N-terminal region (NTR; residues 23-135) and a catalytic domain (residues 136-324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23-324) was successfully determined. The crystal diffracted to 1.9 Šresolution and belonged to space group I212121. The catalytic domain (residues 136-321) exhibited a (ß/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23-135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

Active site architecture of an acetyl xylan esterase indicates a novel cold adaptation strategy.

SGNH-type acetyl xylan esterases (AcXEs) play important roles in marine and terrestrial xylan degradation, which are necessary for removing acetyl side groups from xylan. However, only a few cold-adapted AcXEs have been reported, and the underlying mechanisms for their cold adaptation are still unknown because of the lack of structural information. Here, a cold-adapted AcXE, AlAXEase, from the Arctic marine bacterium Arcticibacterium luteifluviistationis SM1504T was characterized. AlAXEase could deacetylate xylooligosaccharides and xylan, which, together with its homologs, indicates a novel SGNH-type carbohydrate esterase family. AlAXEase showed the highest activity at 30 °C and retained over 70% activity at 0 °C but had unusual thermostability with a Tm value of 56 °C. To explain the cold adaption mechanism of AlAXEase, we next solved its crystal structure. AlAXEase has similar noncovalent stabilizing interactions to its mesophilic counterpart at the monomer level and forms stable tetramers in solutions, which may explain its high thermostability. However, a long loop containing the catalytic residues Asp200 and His203 in AlAXEase was found to be flexible because of the reduced stabilizing hydrophobic interactions and increased destabilizing asparagine and lysine residues, leading to a highly flexible active site. Structural and enzyme kinetic analyses combined with molecular dynamics simulations at different temperatures revealed that the flexible catalytic loop contributes to the cold adaptation of AlAXEase by modulating the distance between the catalytic His203 in this loop and the nucleophilic Ser32. This study reveals a new cold adaption strategy adopted by the thermostable AlAXEase, shedding light on the cold adaption mechanisms of AcXEs.

The sialyl-O-acetylesterase NanS of Tannerella forsythia encompasses two catalytic modules with different regiospecificity for O7 and O9 of sialic acid.

The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O-acetylated sialyl residues susceptible to the action of its sialidase and sialic acid uptake system, Tannerella produces NanS, an O-acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine-histidine-aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl-O-acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7-O-acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl-O-acetylesterases and fills the current gap in tools targeting 7-O-acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di-O-acetylated sialoglycans by monitoring the gain in 9-O-acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9-O-acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9-O-acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9-O-acetylation proved advantageous over de-9-O-acetylation as the additional removal of the 7-O-acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di-O-acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O-acetylation.

Functional Expression and Characterization of Acetyl Xylan Esterases CE Family 7 from Lactobacillus antri and Bacillus halodurans.

Acetyl xylan esterase (AXE; E.C. 3.1.1.72) is one of the accessory enzymes for xylan degradation, which can remove the terminal acetate residues from xylan polymers. In this study, two genes encoding putative AXEs (LaAXE and BhAXE) were cloned from Lactobacillus antri DSM 16041 and Bacillus halodurans C-125, and constitutively expressed in Escherichia coli. They possess considerable activities towards various substrates such as p-nitrophenyl acetate, 4-methylumbelliferyl acetate, glucose pentaacetate, and 7-amino cephalosporanic acid. LaAXE and BhAXE showed the highest activities at pH 7.0 and 8.0 at 50°C, respectively. These enzymes are AXE members of carbohydrate esterase (CE) family 7 with the cephalosporine-C deacetylase activity for the production of antibiotics precursors. The simultaneous treatment of LaAXE with Thermotoga neapolitana ß-xylanase showed 1.44-fold higher synergistic degradation of beechwood xylan than the single treatment of xylanase, whereas BhAXE showed no significant synergism. It was suggested that LaAXE can deacetylate beechwood xylan and enhance the successive accessibility of xylanase towards the resulting substrates. The novel LaAXE originated from a lactic acid bacterium will be utilized for the enzymatic production of D-xylose and xylooligosaccharides.

Optogenetic control of cofilin and αTAT in living cells using Z-lock.

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.

Designer bioemulsifiers based on combinations of different polysaccharides with the novel emulsifying esterase AXE from Bacillus subtilis CICC 20034.

BACKGROUND: Bioemulsifiers are surface-active compounds, which exhibit advantages including low toxicity, higher biodegradability and biocompatibility over synthetic chemical surfactants. Despite their potential benefits, some obstacles impede the practical applications of bioemulsifiers, including low yields and high purification costs. Here, we aimed to exploit a novel protein bioemulsifier with efficient emulsifying activity and low-production cost, as well as proposed a design-bioemulsifier system that meets different requirements of industrial emulsification in the most economical way. RESULTS: The esterase AXE was first reported for its efficient emulsifying activity and had been studied for possible application as a protein bioemulsifier. AXE showed an excellent emulsification effect with different hydrophobic substrates, especially short-chain aliphatic and benzene derivatives, as well as excellent stability under extreme conditions such as high temperature (85 °C) and acidic conditions. AXE also exhibited good stability over a range of NaCl, MgSO4, and CaCl2 concentrations from 0 to 1000 mM, and the emulsifying activity even showed a slight increase at salt concentrations over 500 mM. A design-bioemulsifier system was proposed that uses AXE in combination with a variety of polysaccharides to form efficient bioemulsifier, which enhanced the emulsifying activity and further lowered the concentration of AXE needed in the complex. CONCLUSIONS: AXE showed a great application potential as a novel bioemulsifier with excellent emulsifying ability. The AXE-based-designer bioemulsifier could be obtained in the most economical way and open broad new fields for low-cost, environmentally friendly bioemulsifiers.

Characterization, immobilization, and mutagenesis of a novel cold-active acetylesterase (EaAcE) from Exiguobacterium antarcticum B7.

Cold-active enzymes with distinctive properties from a psychrophilic Exiguobacterium antarcticum B7 could be excellent biocatalysts in industrial and biotechnological processes. Here, the characterization, immobilization, and site-directed mutagenesis of a novel cold-active acetylesterase (EaAcE) from E. antarcticum B7 is reported. EaAcE does not belong to any currently known lipase/esterase family, although there are some sequence similarities with family III and V members. Biochemical characterization of EaAcE was carried out using activity staining, mass spectrometry analysis, circular dichroism spectra, freeze-thaw experiments, kinetic analysis, acetic acid release assays, and enantioselectivity determination. Furthermore, immobilization of EaAcE using four different approaches was explored to enhance its thermal stability and recyclability. Based on a homology model of EaAcE, four mutations (F45A, S118A, S141A, and T216A) within the substrate-binding pocket were investigated to elucidate their roles in EaAcE catalysis and substrate specificity. This work has provided invaluable information on the properties of EaAcE, which can now be used to understand the acetylesterase enzyme family.

Two Novel Acetylesterases from Pantoea dispersa: Recombinant Expression, Purification, and Characterization.

Two novel acetylesterases from Pantoea dispersa, with low amino acid sequence identity between them, were expressed in Escherichia coli with a carboxyl-His6 tail given by the expression plasmid, purified, and characterized. The purified proteins, named Est-1 and Est-2, had a molecular mass of 33 kDa and 37 kDa, respectively. Both proteins presented a modeled structure of homodimers with monomers presenting the α/ß-hydrolase fold, with the catalytic triad Ser-Asp-His present in the active site. The KM for p-nitrophenyl acetate and Vmax values found for Est-1 were of 1.4 ± 0.2 mM and 8.66 ± 0.59 µmol/min and for Est-2 were of 0.36 ± 0.077 mM and 6.13 ± 0.56 µmol/min, respectively. Both enzymes presented an optimum pH of 7.0. The optimum temperature for Est-1 was 40 °C and for Est-2 was 50 °C. The temperatures in which the enzymes Est-1 and Est-2 lost half of their activity (T50) were 44.1 and 58.9 °C, respectively. SDS, EDTA, and PMSF significantly inhibited the enzymes. The two purified enzymes also presented activity against triacetin and were able to deacetylate the carbohydrates pectin and xylan, with higher activity against pectin. Thus, they could be considered as carbohydrate esterases.

Crystal structure and functional characterization of a cold-active acetyl xylan esterase (PbAcE) from psychrophilic soil microbe Paenibacillus sp.

Cold-active acetyl xylan esterases allow for reduced bioreactor heating costs in bioenergy production. Here, we isolated and characterized a cold-active acetyl xylan esterase (PbAcE) from the psychrophilic soil microbe Paenibacillus sp. R4. The enzyme hydrolyzes glucose penta-acetate and xylan acetate, reversibly producing acetyl xylan from xylan, and it shows higher activity at 4°C than at 25°C. We solved the crystal structure of PbAcE at 2.1-Å resolution to investigate its active site and the reason for its low-temperature activity. Structural analysis showed that PbAcE forms a hexamer with a central substrate binding tunnel, and the inter-subunit interactions are relatively weak compared with those of its mesophilic and thermophilic homologs. PbAcE also has a shorter loop and different residue composition in the ß4-α3 and ß5-α4 regions near the substrate binding site. Flexible subunit movements and different active site loop conformations may enable the strong low-temperature activity and broad substrate specificity of PbAcE. In addition, PbAcE was found to have strong activity against antibiotic compound substrates, such as cefotaxime and 7-amino cephalosporanic acid (7-ACA). In conclusion, the PbAcE structure and our biochemical results provide the first example of a cold-active acetyl xylan esterase and a starting template for structure-based protein engineering.

Degradation studies of modified inulin as potential encapsulation material for colon targeting and release of mesalamine.

Due to the potential to treat colon specific diseases with reduced side effects, colon targeting has become of high interest over the last decades. Chemical modified inulin was investigated for its potential as encapsulation material regarding its enzymatic degradability and its drug release behavior. Different degrees of acetylated inulin (degree of substitution, DS, 0.3-2.1) were synthesized. The chemical modification leads to a reduction in enzymatic degradability by inulinase and esterase, enzymes which can be expressed by the colon microbiota. Acetylated inulin was only hydrolyzed to fructose units up to DS of 1.3. Microparticles made of native inulin and acetylated inulin (DS 1.8) were loaded with the colon-specific drug mesalamine by spray drying. Compared to the burst release of mesalamine by inulin particles within 6 h, acetylated inulin particles showed less burst release followed by a continuous drug release phase caused by diffusion up to 30% mesalamine after 52 h.

Bifunctional properties and characterization of a novel sialidase with esterase activity from Bifidobacterium bifidum.

Sialidases catalyze the removal of terminal sialic acid from various complex carbohydrates. In the gastrointestinal tract, sialic acid is commonly found in the sugar chain of mucin, and many enteric commensals use mucin as a nutrient source. We previously identified two different sialidase genes in Bifidobacterium bifidum, and one was cloned and expressed as an extracellular protein designated as exo-α-sialidase SiaBb2. The other exo-α-sialidase gene (siabb1) from the same bifidobacterium encodes an extracellular protein (SiaBb1) consisting of 1795 amino acids with a molecular mass of 189 kDa. SiaBb1 possesses a catalytic domain that classifies this enzyme as a glycoside hydrolase family 33 member. SiaBb1 preferentially hydrolyzes α2,3-linked sialic acid over α2,6-linked sialic acid from sialoglycan, which is the same as SiaBb2. However, SiaBb1 has an SGNH hydrolase domain with sialate-O-acetylesterase activity and an N-terminal signal sequence and C-terminal transmembrane region. SiaBb1 is the first bifunctional sialidase identified with esterase activity. Abbreviations: GalNAc: N-acetyl-D-galactosamine; Fuc: L-fucose; Gal: D-galactose.

Characterising N-acetylglucosaminylphosphatidylinositol de-N-acetylase (CaGpi12), the enzyme that catalyses the second step of GPI biosynthesis in Candida albicans.

Candida albicans N-acetylglucosaminylphosphatidylinositol de-N-acetylase (CaGpi12) recognises N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) from Saccharomyces cerevisiae and is able to complement ScGPI12 function. Both N- and C-terminal ends of CaGpi12 are important for its function. CaGpi12 was biochemically characterised using rough endoplasmic reticulum microsomes prepared from BWP17 strain of C. albicans. CaGpi12 is optimally active at 30°C and pH 7.5. It is a metal-dependent enzyme that is stimulated by divalent cations but shows no preference for Zn2+ unlike the mammalian homologue. It irreversibly loses activity upon incubation with a metal chelator. Two conserved motifs, HPDDE and HXXH, are both important for its function in the cell. CaGPI12 is essential for growth and viability of C. albicans. Its loss causes reduction of GlcNAc-PI de-N-acetylase activity, cell wall defects and filamentation defects. The filamentation defects could be specifically correlated to an upregulation of the HOG1 pathway.

Structural Characterization and Directed Evolution of a Novel Acetyl Xylan Esterase Reveals Thermostability Determinants of the Carbohydrate Esterase 7 Family.

A hot desert hypolith metagenomic DNA sequence data set was screened in silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded an ∼36-kDa protein (Axe1NaM1). The synthesized gene was cloned and expressed, and the resulting protein was purified. NaM1 was optimally active at pH 8.5 and 30°C and functionally stable at salt concentrations of up to 5 M. The specific activity and catalytic efficiency were 488.9 U mg-1 and 3.26 × 106 M-1 s-1, respectively. The crystal structure of wild-type NaM1 was solved at a resolution of 2.03 Å, and a comparison with the structures and models of more thermostable carbohydrate esterase 7 (CE7) family enzymes and variants of NaM1 from a directed evolution experiment suggests that reduced side-chain volume of protein core residues is relevant to the thermal stability of NaM1. Surprisingly, a single point mutation (N96S) not only resulted in a simultaneous improvement in thermal stability and catalytic efficiency but also increased the acyl moiety substrate range of NaM1.IMPORTANCE AcXEs belong to nine carbohydrate esterase families (CE1 to CE7, CE12, and CE16), of which CE7 enzymes possess a unique and narrow specificity for acetylated substrates. All structurally characterized members of this family are moderately to highly thermostable. The crystal structure of a novel, mesophilic CE7 AcXE (Axe1NaM1), from a soil metagenome, provides a basis for comparisons with thermostable CE7 enzymes. Using error-prone PCR and site-directed mutagenesis, we enhanced both the stability and activity of the mesophilic AcXE. With comparative structural analyses, we have also identified possible thermal stability determinants. These are valuable for understanding the thermal stability of enzymes within this family and as a guide for future protein engineering of CE7 and other α/ß hydrolase enzymes.

Identification, characterization, immobilization, and mutational analysis of a novel acetylesterase with industrial potential (LaAcE) from Lactobacillus acidophilus.

Lactic acid bacteria, which are involved in the fermentation of vegetables, meats, and dairy products, are widely used for the productions of small organic molecules and bioactive peptides. Here, a novel acetylesterase (LaAcE) from Lactobacillus acidophilus NCFM was identified, functionally characterized, immobilized, and subjected to site-directed mutagenesis for biotechnological applications. The enzymatic properties of LaAcE were investigated using biochemical and biophysical methods including native polyacrylamide gel electrophoresis, acetic acid release, biochemical assays, enzyme kinetics, and spectroscopic methods. Interestingly, LaAcE exhibited the ability to act on a broad range of substrates including glucose pentaacetate, glyceryl tributyrate, fish oil, and fermentation-related compounds. Furthermore, immobilization of LaAcE showed good recycling ability and high thermal stability compared with free LaAcE. A structural model of LaAcE was used to guide mutational analysis of hydrophobic substrate-binding region, which was composed of Leu156, Phe164, and Val204. Five mutants (L156A, F164A, V204A, L156A/F164A, and L156A/V204A) were generated and investigated to elucidate the roles of these hydrophobic residues in substrate specificity. This work provided valuable insights into the properties of LaAcE, and demonstrated that LaAcE could be used as a model enzyme of acetylesterase in lactic acid bacteria, making LaAcE a great candidate for industrial applications.