Molecular Characterization of a Novel Family VIII Esterase with β-Lactamase Activity (PsEstA) from Paenibacillus sp.

Molecular information about family VIII esterases, which have similarities with class C β-lactamases and penicillin-binding proteins, remains largely unknown. In this study, a novel family VIII esterase with β-lactamase activity (PsEstA) from Paenibacillus sp. was characterized using several biochemical and biophysical methods. PsEstA was effective on a broad range of substrates including tertiary butyl acetate, glyceryl tributyrate, glucose pentaacetate, olive oil, and p-nitrophenyl esters. Additionally, PsEstA hydrolyzed nitrocefin, cefotaxime, and 7-aminocephalosporanic acid. Interestingly, two forms of immobilized PsEstA (CLEAs-PsEstA and mCLEAs-PsEstA) showed high recycling property and enhanced stability, but hybrid nanoflowers (hNFs) of PsEstA require improvement. This study provides a molecular understanding of substrate specificities, catalytic regulation, and immobilization of PsEstA, which can be efficiently used in biotechnological applications.


Introduction
Bacterial β-lactamases hydrolyze chemical compounds containing a β-lactam ring and are the primary cause of bacterial resistance against different classes of β-lactam antibiotics. Therefore, the production of one or more types of β-lactamase is the most effective strategy through which clinically important gram-negative bacteria effectively hydrolyze the β-lactam rings of antibiotics, such as penicillins, cephalosporins, or carbapenems [1][2][3]. Based on amino acid motifs, protein sequence, molecular size, and 3D structure, β-lactamases are classified into four molecular classes termed A, B, C, and D [4]. Interestingly, classes A, C, and D hydrolyze their substrates through the formation of an acyl-enzyme with a catalytic serine, whereas class B utilizes a zinc ion to facilitate a hydrolytic reaction. Among β-lactamases, class C enzymes are of high importance, because they pose significant threats for antibiotic treatments due to their occurrence in many gram-negative pathogens, such as Enterobacteriaceae and Pseudomonas spp. [5,6]. Class C β-lactamases play a role in the resistance of these pathogens to cephalosporins, cephamycins, and carbapenems, and are not inhibited by clavulanic acid [7,8]. The molecular structure of class C β-lactamases consists of two domains of different sizes. The bigger domain contains a central antiparallel beta sheet flanked by alpha helices at each side, was performed in the positive ion mode on a Voyager™ BioSpectrometry™. Urea-induced unfolding was performed following 1 h incubation of PsEstA with urea at 25 • C. The emission spectra from 300 nm to 400 nm were recorded after excitation at 295 nm. Thermal unfolding was monitored from 15 • C to 90 • C at 222 nm using circular dichroism (CD) signals with a thermostatic cell holder.

β-Lactamase Assay
The β-lactamase activity of PsEstA was determined using the chromogenic β-lactam substrate nitrocefin [17]. PsEstA (0.1 mg/mL) was reacted with a 484 µM nitrocefin solution in 20 mM Tris-HCl pH 8.5 at 25 • C, and the color change in the reaction mixture was observed. In addition, pH indicator-based colorimetric assay was performed to investigate the activity of PsEstA on 1 mM cefotaxime (CTX) and 7-aminocephalosporanic acid (7-ACA) at 25 • C for 1 h.

Immobilization of PsEstA
For preparing crosslinked enzyme aggregates (CLEAs), 0.5 mg·mL −1 of PsEstA was coprecipitated by 80% (w/v) ammonium sulfate at 4 • C. Crosslinking was then performed by the dropwise addition of glutaraldehyde to the final concentration of 25 mM at room temperature. After an overnight incubation followed by centrifugation at 15,000 g for 10 min, the pellet (CLEA-PsEstA) was resuspended and repeatedly washed until no activity was observed in the supernatant. The preparation and addition of the magnetic Fe 3 O 4 nanoparticles were carried out as previously described [27,31]. Nanoparticles were mixed with PsEstA solution containing ammonium sulfate and glutaraldehyde. After gentle agitation for 12 h, magnetic CLEAs of PsEstA (mCLEA-PsEstA) were precipitated by centrifugation at 15,000× g for 10 min and stored in 50 mM sodium phosphate buffer (pH 7.5) at 4 • C. Scanning electron microscope images were obtained at various magnifications (50,000-100,000×) using a Carl Zeiss SUPRA 55VP microscope. The chemical stabilities of the three forms of PsEstA (free PsEstA, CLEA-PsEstA, and mCLEA-PsEstA) were determined by measuring the residual activity toward p-nitrophenyl butyrate (p-NB) after 1 h incubation in the presence of the chemical compounds [ethanol, iso-propanol, sodium dodecyl sulfate (SDS), urea] at 25 • C. For the recycling process, the two immobilized forms of PsEstA (CLEA-PsEstA and mCLEA-PsEstA) were extensively washed and then reused in the next cycle. For preparing enzyme-inorganic hybrid nanoflower (hNF), PsEstA (0.05, 0.1, and 0.5 mg·mL −1 ) was added into 800 µM of the Cu 2+ metal solutions for a total volume of 3 mL [32]. The resulting mixtures were incubated at 25 • C and centrifuged at 15,000× g for 10 min. The pellets were resuspended and transferred to 1.5 mL microtubes. In the reusability tests, hNF-PsEstA was recovered by centrifugation and extensively washed before being reused in the next cycle. Then, fresh substrate of 1 mM p-nitrophenyl butyrate (p-NB) was reacted for another cycle and activity was measured. In these experiments, the enzyme activity of free PsEstA was defined as 100%.

Bioinformatic Analysis
For phylogenetic tree analysis of PsEstA, the 26 representative bacterial lipases/esterases sequences were analyzed. As shown in Figure 1, PsEstA belongs to family VIII esterases, which include EstC [12], Est-Y29 [33], EstB [34], and EstU1 [35]. solutions for a total volume of 3 mL [32]. The resulting mixtures were incubated at 25 °C and centrifuged at 15,000× g for 10 min. The pellets were resuspended and transferred to 1.5 mL microtubes. In the reusability tests, hNF-PsEstA was recovered by centrifugation and extensively washed before being reused in the next cycle. Then, fresh substrate of 1 mM p-nitrophenyl butyrate (p-NB) was reacted for another cycle and activity was measured. In these experiments, the enzyme activity of free PsEstA was defined as 100%.

Bioinformatic Analysis
For phylogenetic tree analysis of PsEstA, the 26 representative bacterial lipases/esterases sequences were analyzed. As shown in Figure 1, PsEstA belongs to family VIII esterases, which include EstC [12], Est-Y29 [33], EstB [34], and EstU1 [35]. As shown in Figure 2A, three motifs were found to be highly conserved among class C βlactamases based on multiple sequence alignment. These three motifs (motif I, II, and III) have also been identified in family VIII esterases. In motif I, a nucleophilic Ser 58 is located in the characteristic S 58 -x-x-K 61 tetrapeptide, and the alkoxide ion formation at Ser 58 is believed to be enhanced by Lys 61 . The Tyr 172 residue in motif II is involved in the substrate recognition and specificity regulation. Although motif III of class C β-lactamases is composed of the highly conserved K-T-G sequence, a W-x-G motif is observed instead in PsEstA and other family VIII esterases. The third residue of motif III has to be glycine because otherwise, other amino acids would be sterically unfavorable for the binding of the substrate. In PsEstA, the W-S-G sequence was observed in motif III. In sequence analysis, PsEstA was found to contain a higher percentage of acidic (Asp and Glu, 12.0%) than basic (Lys and Arg, 9.3%) amino acids. As shown in Figure 2A, three motifs were found to be highly conserved among class C β-lactamases based on multiple sequence alignment. These three motifs (motif I, II, and III) have also been identified in family VIII esterases. In motif I, a nucleophilic Ser 58 is located in the characteristic S 58 -x-x-K 61 tetrapeptide, and the alkoxide ion formation at Ser 58 is believed to be enhanced by Lys 61 . The Tyr 172 residue in motif II is involved in the substrate recognition and specificity regulation. Although motif III of class C β-lactamases is composed of the highly conserved K-T-G sequence, a W-x-G motif is observed instead in PsEstA and other family VIII esterases. The third residue of motif III has to be glycine because otherwise, other amino acids would be sterically unfavorable for the binding of the substrate. In PsEstA, the W-S-G sequence was observed in motif III. In sequence analysis, PsEstA was found to contain a higher percentage of acidic (Asp and Glu, 12.0%) than basic (Lys and Arg, 9.3%) amino acids. The structural model of PsEstA consisted of a large α/β domain and a small α-helical domain, and these features are frequently observed in other β-lactamases and family VIII esterases [14,17,34,35]. The nucleophilic serine was found to be located near the surface of the tunnel formed The structural model of PsEstA consisted of a large α/β domain and a small α-helical domain, and these features are frequently observed in other β-lactamases and family VIII esterases [14,17,34,35]. The nucleophilic serine was found to be located near the surface of the tunnel formed by two domains ( Figure 2B). The putative catalytic triad made of Ser 58 , Lys 61 , and Tyr 169 is positioned within a catalytic pocket close to the surface ( Figure 2B, rectangular region). The substrate-binding pocket is mainly delineated by the aromatic amino acids Tyr 127 , Tyr 169 , and Trp 333 , presumably regulating the entrance of substrates via hydrophobic interactions. These residues are also highly conserved in most family VIII esterases. In molecular docking analysis, Tyr 169 and Trp 333 were shown to stabilize the p-nitrophenol ring, while side chains of Asp 302 and Ser 334 formed hydrogen bonds with the nitro moiety (-NO 2 ) of p-nitrophenyl butyrate ( Figure 2C). In addition, backbone nitrogen of Ala 336 is involved in the formation of an oxyanion hole. This model analysis suggested a number of active-site residues seem to be involved in hydrogen bonding networks among other conserved residues or substrate [34]. In addition to conservation of primary sequences, spatial orientation of catalytic residues between class C β-lactamases and family VIII esterases are very similar. As shown in Figure 3, catalytic residues of PsEstA have comparable conformation to those of AmpC [36] and blaMOX-1 [37], as well as EstU1 and Est-Y29.
Biomolecules 2019, 9, x FOR PEER REVIEW 6 of 15 by two domains ( Figure 2B). The putative catalytic triad made of Ser 58 , Lys 61 , and Tyr 169 is positioned within a catalytic pocket close to the surface ( Figure 2B, rectangular region). The substrate-binding pocket is mainly delineated by the aromatic amino acids Tyr 127 , Tyr 169 , and Trp 333 , presumably regulating the entrance of substrates via hydrophobic interactions. These residues are also highly conserved in most family VIII esterases. In molecular docking analysis, Tyr 169 and Trp 333 were shown to stabilize the p-nitrophenol ring, while side chains of Asp 302 and Ser 334 formed hydrogen bonds with the nitro moiety (-NO2) of p-nitrophenyl butyrate ( Figure 2C). In addition, backbone nitrogen of Ala 336 is involved in the formation of an oxyanion hole. This model analysis suggested a number of activesite residues seem to be involved in hydrogen bonding networks among other conserved residues or substrate [34]. In addition to conservation of primary sequences, spatial orientation of catalytic residues between class C β-lactamases and family VIII esterases are very similar. As shown in Figure  3, catalytic residues of PsEstA have comparable conformation to those of AmpC [36] and blaMOX-1 [37], as well as EstU1 and Est-Y29.

Characterization of PsEstA
The recombinant PsEstA was highly purified using an immobilized metal-affinity column, and a single band was observed ( Figure 4A). The molecular mass of PsEstA was estimated to be approximately 45 kDa by SDS-PAGE, and this value is similar to those of Est-Y29 [33] and EstB [34], but smaller than EstU1 [35]. An overlay activity assay showed high fluorescence at the position where purified PsEstA was located in native PAGE ( Figure 4B). Furthermore, strong fluorescence intensity was observed for 4-MU acetate and PsEstA, but not for 4-MU phosphate and PsEstA ( Figure 4C,D). In native PAGE, the molecular mass of active PsEstA was found to be substantially greater than 200 kDa, implying that PsEstA is in an oligomeric conformation. A similar behavior indicating an oligomeric state of PsEstA was also observed in gel filtration chromatography ( Figure 4E), which has been reported for other family VIII esterases [38][39][40]. The mass spectrometric analysis showed a major peak (m/z) at 47.7 kDa with all additional amino acids including the His-tag ( Figure 4F). was observed for 4-MU acetate and PsEstA, but not for 4-MU phosphate and PsEstA ( Figure 4C,D). In native PAGE, the molecular mass of active PsEstA was found to be substantially greater than 200 kDa, implying that PsEstA is in an oligomeric conformation. A similar behavior indicating an oligomeric state of PsEstA was also observed in gel filtration chromatography ( Figure 4E), which has been reported for other family VIII esterases [38][39][40]. The mass spectrometric analysis showed a major peak (m/z) at 47.7 kDa with all additional amino acids including the His-tag ( Figure 4F).

Biochemical Assay
The hydrolytic activity of PsEstA was analyzed using p-nitrophenyl (p-NP) esters with acyl chain lengths of different length [27][28][29]41]. As shown in Figure 5A, PsEstA had a strong substrate preference for p-NA or p-NB. However, almost no enzyme activity was observed for the long-chain substrates p-NO and p-ND. Similarly, other members of family VIII esterases such as Est22 [13], EstU1 [42], or EstM-N1 [43] showed substrate preference for p-NB. When naphthyl esters were used as the substrates, the highest enzymatic activities were observed with 1-naphthyl acetate (1-NA), followed by 1-naphthyl butyrate (1-NB) and 2-naphthyl acetate (2-NA). However, mutation of Ser 58 residue abolished most of the hydrolytic activity ( Figure 5B). As shown in Figure 5B, PsEstA exhibited

Biochemical Assay
The hydrolytic activity of PsEstA was analyzed using p-nitrophenyl (p-NP) esters with acyl chain lengths of different length [27][28][29]41]. As shown in Figure 5A, PsEstA had a strong substrate preference for p-NA or p-NB. However, almost no enzyme activity was observed for the long-chain substrates p-NO and p-ND. Similarly, other members of family VIII esterases such as Est22 [13], EstU1 [42], or EstM-N1 [43] showed substrate preference for p-NB. When naphthyl esters were used as the substrates, the highest enzymatic activities were observed with 1-naphthyl acetate (1-NA), followed by 1-naphthyl butyrate (1-NB) and 2-naphthyl acetate (2-NA). However, mutation of Ser 58 residue abolished most of the hydrolytic activity ( Figure 5B). As shown in Figure 5B, PsEstA exhibited approximately 75% activity on 2-NA. In contrast, it showed almost no activity on 1-naphthyl phosphate (1-NP). PsEstA was also shown to be highly stable in the presence of NaCl and glycerol. Specifically, it was shown to retain approximately 100% and 70% of its enzymatic activity in the presence of 3.0 M and 4.0 M NaCl, respectively ( Figure 5C). In addition, PsEstA exhibited strong tolerance to glycerol with almost no loss of enzymatic activity after incubating with up to 30% glycerol.
The chemical stability of PsEstA was investigated by monitoring the intrinsic fluorescence spectra. In its native form, PsEstA exhibited a λ max at 334 nm, implying that all the tryptophan residues of PsEstA were located in the hydrophobic interior. However, a red shift of λ max to 344 nm was observed with a remarkable increase in fluorescence intensity in the presence of 5 M urea, suggesting that the tryptophan residues were mostly exposed to the solvent ( Figure 5D). The chemical stability of PsEstA in urea was also investigated by measuring the enzymatic activity in the presence of increasing concentrations of urea. At 4.0 M,~50% of the initial activity was retained ( Figure 5E). Next, the thermal stability of PsEstA was investigated by monitoring its thermal denaturation from 15 to 90 • C using far-UV CD at 222 nm. PsEstA showed only minor changes up to 50 • C, and its melting temperature was determined as 63 • C ( Figure 5F). approximately 75% activity on 2-NA. In contrast, it showed almost no activity on 1-naphthyl phosphate (1-NP). PsEstA was also shown to be highly stable in the presence of NaCl and glycerol. Specifically, it was shown to retain approximately 100% and 70% of its enzymatic activity in the presence of 3.0 M and 4.0 M NaCl, respectively ( Figure 5C). In addition, PsEstA exhibited strong tolerance to glycerol with almost no loss of enzymatic activity after incubating with up to 30% glycerol. The chemical stability of PsEstA was investigated by monitoring the intrinsic fluorescence spectra. In its native form, PsEstA exhibited a λmax at 334 nm, implying that all the tryptophan residues of PsEstA were located in the hydrophobic interior. However, a red shift of λmax to 344 nm was observed with a remarkable increase in fluorescence intensity in the presence of 5 M urea, suggesting that the tryptophan residues were mostly exposed to the solvent ( Figure 5D). The chemical stability of PsEstA in urea was also investigated by measuring the enzymatic activity in the presence of increasing concentrations of urea. At 4.0 M, ~50% of the initial activity was retained ( Figure 5E). Next, the thermal stability of PsEstA was investigated by monitoring its thermal denaturation from 15 to 90 °C using far-UV CD at 222 nm. PsEstA showed only minor changes up to 50 °C, and its melting temperature was determined as 63 °C ( Figure 5F).

Substrate Analysis
The hydrolytic properties of PsEstA for acetylated carbohydrates, tertiary alcohol esters, and lipids were evaluated using a colorimetric assay [28,29,41]. The tertiary alcohol esters tert-butyl acetate, α-terpinyl acetate, and linalyl acetate were used. As shown in Figure 6A, PsEstA could effectively hydrolyze tert-butyl acetate, but not linalyl acetate or α-terpinyl acetate. Additionally, a significant hydrolytic activity of PsEstA was detected only for glyceryl tributyrate based on the yellow color of the solution ( Figure 6B). Furthermore, PsEstA displayed a high activity for glucose pentaacetate, although no significant activity was detected for cellulose acetate or glucosamine acetate ( Figure 6C). In addition, a high level of enzymatic activity of PsEstA was observed toward olive oil, judging from the fluorescence spectra ( Figure 6D,E). For enantioselectivity analysis, (R)-and (S)-methyl-β-hydroxyisobutyrate were used [27,32]. Following incubation with PsEstA, only the reaction mixture containing the (S)-enantiomer turned yellow, showing the (S)-selectivity of PsEstA ( Figure 6F). This indiscriminate nature of PsEstA could be useful for industrial applications as a biocatalyst. acetate ( Figure 6C). In addition, a high level of enzymatic activity of PsEstA was observed toward olive oil, judging from the fluorescence spectra ( Figure 6D,E). For enantioselectivity analysis, (R)-and (S)-methyl-β-hydroxyisobutyrate were used [27,32]. Following incubation with PsEstA, only the reaction mixture containing the (S)-enantiomer turned yellow, showing the (S)-selectivity of PsEstA ( Figure 6F). This indiscriminate nature of PsEstA could be useful for industrial applications as a biocatalyst.

β-Lactamase Activity of PsEstA
To investigate the β-lactamase activity of PsEstA, a chromogenic β-lactamase substrate (nitrocefin) was used as a substrate [12,17]. As shown in Figure 7, PsEstA apparently showed β-lactamase activity toward nitrocefin. The β-lactamase activity of PsEstA was further investigated using 7-ACA and CTX. PsEstA showed significant catalytic activities toward cefotaxime (CTX) and 7-aminocephalosporanic acid (7-ACA) in a pH shift assay ( Figure 7E,F). In accordance with PsEstA, Est22 and EstU1 were shown to possess a noteworthy β-lactam hydrolytic activity [13,35]. In contrast, EstC showed a significant hydrolyzing activity for nitrocefin, but none of these esterases displayed any activity for other β-lactam substrates [12]. EstC showed a significant hydrolyzing activity for nitrocefin, but none of these esterases displayed any activity for other β-lactam substrates [12].

Immobilization of PsEstA
Enzyme immobilization has been explored in a large variety of industrial applications [44,45]. Crosslinked enzyme aggregates (CLEAs) constitute one of the highly studied methods to generate highly efficient immobilized biocatalysts [46]. Toward this end, CLEAs-PsEstA was prepared by precipitating PsEstA with ammonium sulfate and glutaraldehyde. The SEM images of CLEAs-PsEstA showed formation of globular structures with a diameter of 1-2 nm ( Figure 8A). The stabilities of both free PsEstA and CLEAs-PsEstA in the presence of several chemical compounds were compared ( Figure 8B). For 5 M urea and 0.1% (v/v) SDS, there were no significant differences between these two forms. However, for alcohols such as ethanol and iso-propanol, CLEAs-PsEstA was highly active compared with the free form of the enzyme. Specifically, with 30% ethanol, free PsEstA retained only ~3% of its original activity, whereas CLEAs-PsEstA showed ~35% of the original activity. The operational stability of CLEAs-PsEstA was studied up to 20 cycles. As shown in Figure 8C, CLEAs-PsEstA was highly stable for 20 cycles, retaining at the end ~75% of the original activity.
Furthermore, PsEstA was immobilized as CLEAs on magnetite nanoparticles for an efficient separation during industrial applications [27,32]. To obtain magnetic CLEA form of PsEstA (mCLEA-PsEstA), PsEstA was coaggregated with nanoparticles, and then chemically crosslinked using glutaraldehyde. Transmission electron microscopy showed that the magnetic nanoparticles had a diameter of 5-8 nm ( Figure 8D). The effects of the chemical compounds on the activities of mCLEA-

Immobilization of PsEstA
Enzyme immobilization has been explored in a large variety of industrial applications [44,45]. Crosslinked enzyme aggregates (CLEAs) constitute one of the highly studied methods to generate highly efficient immobilized biocatalysts [46]. Toward this end, CLEAs-PsEstA was prepared by precipitating PsEstA with ammonium sulfate and glutaraldehyde. The SEM images of CLEAs-PsEstA showed formation of globular structures with a diameter of 1-2 nm ( Figure 8A). The stabilities of both free PsEstA and CLEAs-PsEstA in the presence of several chemical compounds were compared ( Figure 8B). For 5 M urea and 0.1% (v/v) SDS, there were no significant differences between these two forms. However, for alcohols such as ethanol and iso-propanol, CLEAs-PsEstA was highly active compared with the free form of the enzyme. Specifically, with 30% ethanol, free PsEstA retained only~3% of its original activity, whereas CLEAs-PsEstA showed~35% of the original activity. The operational stability of CLEAs-PsEstA was studied up to 20 cycles. As shown in Figure 8C, CLEAs-PsEstA was highly stable for 20 cycles, retaining at the end~75% of the original activity.
Furthermore, PsEstA was immobilized as CLEAs on magnetite nanoparticles for an efficient separation during industrial applications [27,32]. To obtain magnetic CLEA form of PsEstA (mCLEA-PsEstA), PsEstA was coaggregated with nanoparticles, and then chemically crosslinked using glutaraldehyde. Transmission electron microscopy showed that the magnetic nanoparticles had a diameter of 5-8 nm ( Figure 8D). The effects of the chemical compounds on the activities of mCLEA-PsEstA and free PsEstA were investigated by measuring the residual activities in the presence of each chemical. As shown in Figure 8E, the enzymatic activities of mCLEA-PsEstA were largely remarkably higher than those of the free form of PsEstA in the presence of the alcohols tested. Specifically, free PsEstA showed a relative activity of 21% in the presence of 10% EtOH, whereas mCLEA-PsEstA retained 47% of the initial activity. Moreover, although the free PsEstA retained less than 5% of its original activity in the presence of 30% EtOH, mCLEA-PsEstA showed 62% activity at the end. However, even 0.1% (v/v) SDS was sufficient to inactivate both free PsEstA and mCLEA-PsEstA almost completely. Similar behavior was also observed in the presence of 5 M urea. In reusability analysis, mCLEA-PsEstA retained~75% of its original activity after the 4th cycle, but lost a substantial level of activity at the 5th cycle ( Figure 8F). Taken together, immobilization of PsEstA (CLEAs-PsEstA and mCLEAs-PsEstA) was effectively performed, and these immobilized forms could function more effectively than the free form of PsEstA.
Biomolecules 2019, 9, x FOR PEER REVIEW 11 of 15 PsEstA and free PsEstA were investigated by measuring the residual activities in the presence of each chemical. As shown in Figure 8E, the enzymatic activities of mCLEA-PsEstA were largely remarkably higher than those of the free form of PsEstA in the presence of the alcohols tested. Specifically, free PsEstA showed a relative activity of 21% in the presence of 10% EtOH, whereas mCLEA-PsEstA retained 47% of the initial activity. Moreover, although the free PsEstA retained less than 5% of its original activity in the presence of 30% EtOH, mCLEA-PsEstA showed 62% activity at the end. However, even 0.1% (v/v) SDS was sufficient to inactivate both free PsEstA and mCLEA-PsEstA almost completely. Similar behavior was also observed in the presence of 5 M urea. In reusability analysis, mCLEA-PsEstA retained ~75% of its original activity after the 4th cycle, but lost a substantial level of activity at the 5th cycle ( Figure 8F). Taken together, immobilization of PsEstA (CLEAs-PsEstA and mCLEAs-PsEstA) was effectively performed, and these immobilized forms could function more effectively than the free form of PsEstA.

Formation of Organic-Inorganic Hybrid PsEstA Nanoflower
Recently, syntheses of organic-inorganic hybrid nanoflowers (hNFs) with greatly enhanced catalytic activities and stabilities have been reported [47,48]. Herein, the formation and catalytic activity of hybrid nanoflowers containing PsEstA were pursued. Scanning electron microscopy showed that the hybrid nanoflowers of PsEstA were hierarchical peony-like structures assembled from interlaced nanoplates ( Figure 9A). Reusability tests were also carried out for 10 cycles showing that the residual activity was~25% by the 3rd cycle ( Figure 9B).
Recently, syntheses of organic-inorganic hybrid nanoflowers (hNFs) with greatly enhanced catalytic activities and stabilities have been reported [47,48]. Herein, the formation and catalytic activity of hybrid nanoflowers containing PsEstA were pursued. Scanning electron microscopy showed that the hybrid nanoflowers of PsEstA were hierarchical peony-like structures assembled from interlaced nanoplates ( Figure 9A). Reusability tests were also carried out for 10 cycles showing that the residual activity was ~25% by the 3rd cycle ( Figure 9B).

Conclusions
Although family VIII esterases with β-lactamase activity have attracted considerable research interest, there is still limited information available regarding this enzyme family. Here, a novel family VIII esterase with β-lactamase activity (PsEstA) from Paenibacillus sp. was characterized using molecular modeling, spectroscopic methods, biochemical assays such as β-lactamase assay, and immobilization strategies. This structural and functional characterization of PsEstA is expected to provide a molecular platform for the comprehensive understanding of family VIII esterases as well as β-lactamase, although the physiological role of PsEstA still needs to be explored. In addition, considering the fact that very few enzymes of this family have been biochemically characterized and immobilized for biotechnological applications, PsEstA could be a promising target for biotechnological applications [49][50][51]. Further studies on PsEstA including mutagenesis of the key residues, in-depth kinetic analysis, and evaluation of enzyme-substrate complex formation will be necessary for a thorough understanding of this enzyme at the molecular level.

Conflicts of Interest:
The authors declare no conflict of interest.

Conclusions
Although family VIII esterases with β-lactamase activity have attracted considerable research interest, there is still limited information available regarding this enzyme family. Here, a novel family VIII esterase with β-lactamase activity (PsEstA) from Paenibacillus sp. was characterized using molecular modeling, spectroscopic methods, biochemical assays such as β-lactamase assay, and immobilization strategies. This structural and functional characterization of PsEstA is expected to provide a molecular platform for the comprehensive understanding of family VIII esterases as well as β-lactamase, although the physiological role of PsEstA still needs to be explored. In addition, considering the fact that very few enzymes of this family have been biochemically characterized and immobilized for biotechnological applications, PsEstA could be a promising target for biotechnological applications [49][50][51]. Further studies on PsEstA including mutagenesis of the key residues, in-depth kinetic analysis, and evaluation of enzyme-substrate complex formation will be necessary for a thorough understanding of this enzyme at the molecular level.