Characterization and Immobilization of a Novel SGNH Family Esterase (LaSGNH1) from Lactobacillus acidophilus NCFM

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.


Introduction
Lipolytic enzymes such as (phospho)lipases or esterases, which are present throughout three domains of life (Eukarya, Bacteria, and Archaea), are generally involved in the hydrolysis of lipids or their derivatives [1][2][3]. They share similar structural and catalytic features, including a highly conserved catalytic triad (Ser-Asp/Glu-His), an α/β hydrolase fold, broad substrate specificity, and an absence of cofactors [4,5]. Among them, enzymes of microbial origin have been extensively used in a wide variety of applications, such as pharmaceutical, fine chemical, and food industries. They displayed excellent stability, high efficiency, and strong stereoselectivity [6,7]. Recently, SGNH family esterases have attracted interest because they are highly useful for the preparation of aromas, flavors, drug intermediates, and pharmaceutical products [8][9][10]. They are characterized by four conserved sequence blocks of I-III and V in their primary sequences [8,9]. In these enzymes, the catalytic serine is located in the highly conserved motif of Gly-Asp-Ser (GDS) in the N-terminal region. In addition, Gly and Asn in motif II and III are responsible for the formation of a tetrahedral intermediate and an oxyanion hole. The Asp-x-x-His tetrapeptide in motif V constitutes the catalytic machinery of these enzymes. To date, a number of SGNH family esterases have been identified and characterized from several microorganisms [11][12][13][14][15][16][17][18][19], but there are very few reports in lactic acid bacteria.
Lactobacillus acidophilus is one of the most widely used industrial microorganisms in the bioprocessing of dairy products, fermented food, and nutritional and dietary supplements [20][21][22]. In addition, L. acidophilus can produce a number of antimicrobial peptides, organic metabolites and acids, and vitamins through diverse metabolic processes. The production of these molecules is largely responsible for the stimulation of inherent immune systems and the reduction of pathological inflammations [23,24]. Therefore, this bacterium could be used as a rich and unique source for the identification of a large variety of enzymes with novel functions or characteristics.
Although several esterases have been described in L. acidophilus, no studies have been reported regarding SGNH family esterases [25,26]. Here, characterization and immobilization of a novel SGNH family esterase (NCBI Reference Sequence: WP_125978798, LaSGNH1) from L. acidophilus NCFM were investigated. To our knowledge, this study is the first report on the SGNH family esterase from L. acidophilus.

Bioinformatic Analysis of LaSGNH1
In the chromosome of L. acidophilus, a gene encoding a novel SGNH family esterase (LaSGNH1, locus tag: AZN77234, 561 bp) was identified using in silico bioinformatic analysis. Sequence analysis revealed that LaSGNH1 had a molecular mass of~21 kDa with a single polypeptide chain of 188 amino acids, with a pI of 5.93. For phylogenetic tree analysis, eight bacterial lipases/esterases families (I-VIII) were investigated ( Figure 1A). LaSGNH1 was shown to be a member of family II lipases/esterases, which is further subdivided into clade I and clade II subfamilies [25]. More specifically, as shown in Figure 1B, LaSGNH1 was clustered in the clade I subfamily with a lipase/acylhydrolase from Enterococcus faecalis (AAO80043, 30.4% sequence identity).
As shown in Figure 1C, four blocks (I, II, III, and V) are highly conserved of LaSGNH1 in clade I and II of family II lipases/esterases. The catalytic Ser 10 is shown to be located in a GDS motif in block I, while a DXXH motif was localized in block V. Conserved residues in block II and III were shown to be involved in the formation of an oxyanion hole [8,25].
Genomic cluster analysis revealed that LaSGNH1 has homologues in other Lactobacillus species, including Lactobacillus amylovorus, which implies the invariant and important roles of these enzymes in Lactobacillus species (Figure 2). To date, there are no reports on these proteins, and their functional properties are largely unknown. The molar percentage (30.7%) of four hydrophobic amino acid residues (Alanine (Ala), Valine (Val), Isoleucine (Ile), and Leucine (Leu)) in LaSGNH1, which was shown to be important for protein stability [26], is comparable to that of an SGNH hydrolase (LI22) from Listeria innocua [18] or a SGNH hydrolase (Est24) from Sinorhizobium meliloti [19]. locus tag: AZN77234, 561 bp) was identified using in silico bioinformatic analysis. Sequence analysis revealed that LaSGNH1 had a molecular mass of ~21 kDa with a single polypeptide chain of 188 amino acids, with a pI of 5.93. For phylogenetic tree analysis, eight bacterial lipases/esterases families (I-VIII) were investigated ( Figure 1A). LaSGNH1 was shown to be a member of family II lipases/esterases, which is further subdivided into clade I and clade II subfamilies [25]. More specifically, as shown in Figure 1B, LaSGNH1 was clustered in the clade I subfamily with a lipase/acylhydrolase from Enterococcus faecalis (AAO80043, 30.4% sequence identity).  As shown in Figure 1C, four blocks (I, II, III, and V) are highly conserved of LaSGNH1 in clade I and II of family II lipases/esterases. The catalytic Ser 10 is shown to be located in a GDS motif in block I, while a DXXH motif was localized in block V. Conserved residues in block II and III were shown to be involved in the formation of an oxyanion hole [8,25].
Genomic cluster analysis revealed that LaSGNH1 has homologues in other Lactobacillus species, including Lactobacillus amylovorus, which implies the invariant and important roles of these enzymes in Lactobacillus species (Figure 2). To date, there are no reports on these proteins, and their functional properties are largely unknown. The molar percentage (30.7%) of four hydrophobic amino acid residues (Alanine (Ala), Valine (Val), Isoleucine (Ile), and Leucine (Leu)) in LaSGNH1, which was shown to be important for protein stability [26], is comparable to that of an SGNH hydrolase (LI22) from Listeria innocua [18] or a SGNH hydrolase (Est24) from Sinorhizobium meliloti [19].

Characterizations of LaSGNH1
Recombinant LaSGNH1 protein was purified by an immobilized Ni 2+ -affinity column to near homogeneity ( Figure 3A). The molecular mass of LaSGNH1 is similar to those of other SGNH family
As shown in Figure 4C, LaSGNH1 retained ~65% of its initial activity in the presence of 10% ethanol and ~40% of its activity in the presence of 0.1% Tween 20. In contrast, the addition of 1.0% Triton X-100 resulted in less than 10% of its original activity. In the presence of 30% ethanol, LaSGNH1 retained only 10% of its initial activity ( Figure 4C). The chemical stability of LaSGNH1 against urea was investigated by monitoring the intrinsic fluorescence changes. In the native state, LaSGNH1 exhibited a λmax at 330 nm, indicating that the tryptophan residues of LaSGNH1 were located in the hydrophobic interior ( Figure 4D,E). However, a red shift of λmax to 344 nm was observed with a noteworthy increase of fluorescence intensity at 5 M urea. In contrast, the addition of 2.0 M urea resulted in almost complete loss of LaSGNH1 activity ( Figure 4F).

Homology Modeling and Substrate Analysis of LaSGNH1
A structural model of LaSGNH1 was constructed based on the crystal structure of lipase/acylhydrolase from Enterococcus faecalis (PDB I.D.: 1YZF). The putative catalytic triad of Ser 10 , Asp 161 , and His 164 are positioned close to the outer solvent available surfaces ( Figure 5A). Three amino acids, Gly 45 , Gly 70 , and Asn 72 , were identified to control the entrance of substrates toward the catalytic triad via noncovalent interactions ( Figure 5B). These resides are also highly conserved in SGNH family esterases (see also Figure 1C). In molecular docking analysis, Asn 72 , Tyr 118 , and Gln 163 were In addition, LaSGNH1 displayed its maximal activity at pH 8.0, whereas~30% of this maximal activity was observed at pH 7.0 ( Figure 4B). This optimum pH is similar to other SGNH family esterases, such as an esterase gene (Tlip) from Thauera sp. [14] or an SGNH hydrolase (LI22) from Listeria innocua [19]. Furthermore, other esterases from L. acidophilus showed the optimum pH of 7.0-8.0 such as a cinnamoyl esterase [30] or LaAcE [31].
As shown in Figure 4C, LaSGNH1 retained~65% of its initial activity in the presence of 10% ethanol and~40% of its activity in the presence of 0.1% Tween 20. In contrast, the addition of 1.0% Triton X-100 resulted in less than 10% of its original activity. In the presence of 30% ethanol, LaSGNH1 retained only 10% of its initial activity ( Figure 4C). The chemical stability of LaSGNH1 against urea was investigated by monitoring the intrinsic fluorescence changes. In the native state, LaSGNH1 exhibited a λ max at 330 nm, indicating that the tryptophan residues of LaSGNH1 were located in the hydrophobic interior ( Figure 4D,E). However, a red shift of λ max to 344 nm was observed with a noteworthy increase of fluorescence intensity at 5 M urea. In contrast, the addition of 2.0 M urea resulted in almost complete loss of LaSGNH1 activity ( Figure 4F).

Homology Modeling and Substrate Analysis of LaSGNH1
A structural model of LaSGNH1 was constructed based on the crystal structure of lipase/acylhydrolase from Enterococcus faecalis (PDB I.D.: 1YZF). The putative catalytic triad of Ser 10 , Asp 161 , and His 164 are positioned close to the outer solvent available surfaces ( Figure 5A). Three amino acids, Gly 45 , Gly 70 , and Asn 72 , were identified to control the entrance of substrates toward the catalytic triad via noncovalent interactions ( Figure 5B). These resides are also highly conserved in SGNH family esterases (see also Figure 1C). In molecular docking analysis, Asn 72 , Tyr 118 , and Gln 163 were shown to stabilize the p-nitrophenol ring ( Figure 5C,D). In addition, the backbone nitrogen of Gly 163 is involved in the stabilization of an oxyanion hole. shown to stabilize the p-nitrophenol ring ( Figure 5C,D). In addition, the backbone nitrogen of Gly 163 is involved in the stabilization of an oxyanion hole. The hydrolytic properties of LaSGNH1 towards a wide range of substrates were studied using a colorimetric assay [33,34]. The ability of LaSGNH1 to hydrolyze tertiary alcohol esters (TAEs) was investigated using tert-butyl acetate, α-terpinyl acetate, and linalyl acetate. As shown in Figure 6A, LaSGNH1 was able to effectively hydrolyze tert-butyl acetate, but not α-terpinyl acetate nor linalyl acetate. Additionally, significant hydrolytic activity of LaSGNH1 was only detected for glyceryl tributyrate, which was indicated by the yellow color of the solution ( Figure 6B). In addition, strong hydrolytic activity of LaSGNH1 for glucose pentaacetate was observed, although very little activity was observed in the presence of cellulose acetate or N-acetylglucosamine ( Figure 6C). The preference of LaSGNH1 for small-size substrates could be explained by the restricted dimensions of the substrate-binding pocket [35]. The hydrolytic properties of LaSGNH1 towards a wide range of substrates were studied using a colorimetric assay [33,34]. The ability of LaSGNH1 to hydrolyze tertiary alcohol esters (TAEs) was investigated using tert-butyl acetate, α-terpinyl acetate, and linalyl acetate. As shown in Figure 6A, LaSGNH1 was able to effectively hydrolyze tert-butyl acetate, but not α-terpinyl acetate nor linalyl acetate. Additionally, significant hydrolytic activity of LaSGNH1 was only detected for glyceryl tributyrate, which was indicated by the yellow color of the solution ( Figure 6B). In addition, strong hydrolytic activity of LaSGNH1 for glucose pentaacetate was observed, although very little activity was observed in the presence of cellulose acetate or N-acetylglucosamine ( Figure 6C). The preference of LaSGNH1 for small-size substrates could be explained by the restricted dimensions of the substrate-binding pocket [35].
colorimetric assay [33,34]. The ability of LaSGNH1 to hydrolyze tertiary alcohol esters (TAEs) was investigated using tert-butyl acetate, α-terpinyl acetate, and linalyl acetate. As shown in Figure 6A, LaSGNH1 was able to effectively hydrolyze tert-butyl acetate, but not α-terpinyl acetate nor linalyl acetate. Additionally, significant hydrolytic activity of LaSGNH1 was only detected for glyceryl tributyrate, which was indicated by the yellow color of the solution ( Figure 6B). In addition, strong hydrolytic activity of LaSGNH1 for glucose pentaacetate was observed, although very little activity was observed in the presence of cellulose acetate or N-acetylglucosamine ( Figure 6C). The preference of LaSGNH1 for small-size substrates could be explained by the restricted dimensions of the substrate-binding pocket [35].

Immobilization of LaSGNH1
Enzyme immobilization, which could provide low cost, fast recovery, and high product yields, is widely used in industrial applications [36,37]. In previous reports, immobilized SGNH family esterases were shown to have better thermal stability, chemical stability, and recycling ability than free enzymes [12,18,19,29,35]. Specifically, cross-linked enzyme aggregates of LpSGNH1 displayed higher recycling ability and thermal stability than soluble LpSGNH1 [12]. In addition, enhanced thermal and chemical stability as well as good durability were observed in the crosslinked forms of LI22 [18] and Est24 [19]. Based on these studies, we immobilized LaSGNH1 via chemical crosslinking. First, LaSGNH1-crosslinked enzyme aggregates (CLEA) were prepared by precipitating LaSGNH1 with ammonium sulfate and glutaraldehyde. In addition, arginine (Arg) was also included in the preparation of LaSGNH1-Arg-CLEA, which was shown to be effective for the stability of immobilized enzymes [31,38]. Similarly, LaSGNH1 was co-precipitated with magnetite Fe 3 O 4 nanoparticles, and crosslinked using glutaraldehyde to obtain magnetic LaSGNH1-CLEA (mCLEA-LaSGNH1). Enzyme immobilization using magnetite Fe 3 O 4 nanoparticles could be used for fast separation [39]. Among these four different immobilization approaches (LaSGNH1-CLEA, LaSGNH1-Arg-CLEA, mCLEA-LaSGNH1, and mCLEA-Arg-LaSGNH1), LaSGNH1-Arg-CLEA showed the highest immobilization efficiency, which was comparable to that of free LaSGNH1 ( Figure 7A).
Next, the thermal stability of LaSGNH1-Arg-CLEA was investigated for 1-h of incubation at 37 • C. As shown in Figure 7B, immobilized LaSGNH1-Arg-CLEA retained~70% of its original activity after 30 min, while the free LaSGNH1 showed only 31% of its activity. Furthermore, the reusability of LaSGNH1-Arg-CLEA was studied over 10 cycles. After each cycle, the LaSGNH1-Arg-CLEAs were separated by centrifugation and washed for the next cycle. As shown in Figure 7C, LaSGNH1-Arg-CLEA showed good recycling ability, retaining about 60% of the original activity even after the 10th cycle. Therefore, LaSGNH1-Arg-CLEA showed good immobilization efficiency, enhanced thermal stability, and high reusability, which could be exploited to facilitate the applications of LaSGNH1. enzymes [31,38]. Similarly, LaSGNH1 was co-precipitated with magnetite Fe3O4 nanoparticles, and crosslinked using glutaraldehyde to obtain magnetic LaSGNH1-CLEA (mCLEA-LaSGNH1). Enzyme immobilization using magnetite Fe3O4 nanoparticles could be used for fast separation [39]. Among these four different immobilization approaches (LaSGNH1-CLEA, LaSGNH1-Arg-CLEA, mCLEA-LaSGNH1, and mCLEA-Arg-LaSGNH1), LaSGNH1-Arg-CLEA showed the highest immobilization efficiency, which was comparable to that of free LaSGNH1 ( Figure 7A). Next, the thermal stability of LaSGNH1-Arg-CLEA was investigated for 1-h of incubation at 37 °C. As shown in Figure 7B, immobilized LaSGNH1-Arg-CLEA retained ~70% of its original activity after 30 min, while the free LaSGNH1 showed only 31% of its activity. Furthermore, the reusability of LaSGNH1-Arg-CLEA was studied over 10 cycles. After each cycle, the LaSGNH1-Arg-CLEAs were separated by centrifugation and washed for the next cycle. As shown in Figure 7C, LaSGNH1-Arg-CLEA showed good recycling ability, retaining about 60% of the original activity even after the 10th cycle. Therefore, LaSGNH1-Arg-CLEA showed good immobilization efficiency, enhanced thermal stability, and high reusability, which could be exploited to facilitate the applications of LaSGNH1.

Reagents
DNA-modifying enzymes were obtained from New England BioLabs (Ipswich, MA, USA). DNA purification kits were obtained from Qiagen Korea (Daejon, Korea), and protein purification columns were purchased from GE Healthcare (Seoul, Korea). All other reagents were of analytical grade and were purchased from Sigma-Aldrich Korea (Yongin, Korea).

Bioinformatic Analysis
The primary sequences of LaSGNH1 and related proteins were retrieved from the NCBI database. Multiple sequence alignments and sequence comparisons were carried out using Clustal Omega [40] and ESPript [41]. A phylogenetic tree was constructed by MEGA v. 7.0 using the neighbor-joining method with 2000 iterations [42]. A structural model of LaSGNH1 was constructed based on the crystal structure of lipase/acylhydrolase from Enterococcus faecalis (PDB I.D.: 1YZF) using the SWISS-MODEL server. Molecular docking analysis was performed using flexible side chain methods and AutoDock Vina [43].

Cloning and Purification
L. acidophilus NCFM (KCTC 3145; Korean Collection for Type Cultures) were cultured in MRS medium (BD Difco, NJ, USA) and chromosomal DNA was purified using a DNeasy Tissue and Blood Kit (Qiagen, USA). The open reading frame of the LaSGNH1 gene was amplified by polymerase chain reaction (PCR), and the PCR product was cloned into pQE-30 plasmid using BamHI and XhoI. After DNA sequencing, the recombinant plasmid (pET-LaSGNH1) was transformed into Escherichia coli cells for protein expression of LaSGNH1. E. coli cells were grown until the optical density (OD 600nm ) reached 0.6-0.8. After 1 mM isopropyl-β-D-1-thiogalactoside induction for 4 h at 37 • C, cells were centrifuged and resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA). After keeping on ice for 15 min, the cellular membrane was disrupted using a microtip (1-s pulse, 3-s pause, and 51% amplitude) in a Q500 sonicator (Terra Universal, Fullerton, CA, USA). After sonication, the supernatants were loaded onto a HisTrap HP column using an AKTA Prime Plus (GE healthcare, Chicago, IL, USA). The recombinant LaSGNH1 protein was eluted with an imidazole gradient from 50 to 300 mM. After a washing process, the pooled fractions were desalted with a lysis buffer. Protein concentration was determined using a Biorad Protein assay kit (Bio-rad Laboratories, Chicago, IL, USA) and purified LaSGNH1 was stored at −20 • C.
The thermostability and pH stability of LaSGNH1 were investigated at different temperatures ranging from 25 to 60 • C and across a pH range of 3.0 to 10.0. Effects of chemicals (10% ethanol, 30% ethanol, 30% iso-propanol, 0.1% Tween 20, 0.1% SDS, 1.0% Triton X-100, 1 Mm PMSF, and urea (from 0 to 5 M)) on the activity of LaSGNH1 were investigated after 1-h incubation using p-nitrophenyl butyrate (p-NB) as a substrate, and the enzyme activity of LaSGNH1 in buffer alone was defined as 100%. For intrinsic fluorescence spectra, the emission spectra from 300 to 400 nm were measured after excitation at 295 nm. All spectra were measured with a scan speed of 500 nm·min −1 and a 2 nm bandwidth using a Jasco FP-8200 spectrofluorometer.

Immobilization of LaSGNH1
For the preparation of cross-linked enzyme aggregates (CLEA), 0.5 mg·mL −1 of LaSGNH1 was co-precipitated with 70% ammonium sulfate with glutaraldehyde, incubated overnight, and centrifuged. The pellet (LaSGNH1-CLEA) was resuspended and washed extensively until no significant enzyme activity was detected in the supernatant. Addition of Arg and Fe 3 O 4 magnetic nanoparticles for the preparation of LaSGNH1-Arg-CLEA, mCLEA-LaSGNH1, and mCLEA-Arg-LaSGNH1 was carried out as described previously [31,45]. For thermal stability, LaSGNH1-Arg-CLEA and free LaSGNH1 were incubated at 37 • C for 1-h. For the reusability experiments, LaSGNH1-Arg-CLEA was reused after extensive washing in subsequent cycles.

Conclusions
Although SGNH family esterases have attracted interest due to their potential applications, there remains little information about this family from lactic acid bacteria. Here, a novel SGNH family esterase (LaSGNH1) from L. acidophilus NCFM was identified, characterized, and immobilized. The novel properties of LaSGNH1 could make it a promising candidate for the food, cosmetics, pharmaceutical, and biofuel industries. In addition, this study could help us to better understand the SGNH family esterases, although the physiological role of LaSGNH1 has not yet been revealed. Further studies on LaSGNH1, including mutagenesis of key residues, structural determination, formation of the enzyme-substrate complex, will be necessary to further our understanding of this LaSGNH1 enzyme.