Biochemical Characterization of a Novel Endo-1,3-β-Glucanase from the Scallop Chlamys farreri

Endo-1,3-β-glucanases derived from marine mollusks have attracted much attention in recent years because of their unique transglycosylation activity. In this study, a novel endo-1,3-β-glucanase from the scallop Chlamys farreri, named Lcf, was biochemically characterized. Unlike in earlier studies on marine mollusk endo-1,3-β-glucanases, Lcf was expressed in vitro first. Enzymatic analysis demonstrated that Lcf preferred to hydrolyze laminarihexaose than to hydrolyze laminarin. Furthermore, Lcf was capable of catalyzing transglycosylation reactions with different kinds of glycosyl acceptors. More interestingly, the transglycosylation specificity of Lcf was different from that of other marine mollusk endo-1,3-β-glucanases, although they share a high sequence identity. This study enhanced our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-β-glucanases, which facilitated development of a unique endo-1,3-β-glucanase tool in the synthesis of novel glycosides.


Expression and Purification of Lcf
In a previous study, direct extraction of the enzyme from the digestive organs of marine mollusks was the general method used for collecting endo-1,3-β-glucanases [24][25][26]. However, the isolation of endo-1,3-β-glucanases from digestive organs is not only time-consuming, but is also limited by the source of marine mollusks. Therefore, we carried out the expression of Lcf in vitro using the Escherichia coli expression system and Bacillus brevis expression system. Although the recombinant proteins were detected in the supernatant of the cell lysis, they did not exhibit enzymatic activity, suggesting the occurrence of incorrect protein folding in the expression of Lcf in the E. coli expression system. Fortunately, recombinant Lcf with hydrolytic activity was detected successfully in the fermentation broth of Bacillus brevis. After a three-step purification, including ammonium sulfate salting out, hydrophobic chromatography, and ion exchange chromatography, about 12 mg of Lcf was extracted from 1 L of fermentation broth of Bacillus brevis. The molecular weight of Lcf was about 37 kDa, as shown in Figure 2.
Through the Bacillus brevis expression system, Lcf was expressed in vitro successfully, providing a more convenient method for the extraction of marine mollusk endo-1,3-β-glucanases. In addition, this method may facilitate performing further structural research, which will be important for understanding the unique transglycosylation activity of marine mollusk endo-1,3-β-glucanases. The identical residues are highlighted in black, and similar residues are shaded in gray. The two key catalytic residues conserved in GH16 endo-1,3-β-glucanases are marked with triangles. For β-glucanase ZgLamA GH16 , the residues involved in substrate binding are marked with closed circles and stars. The residues marked with closed circles are conserved in these endo-1,3-β-glucanases, but the two residues marked with stars are not conserved. The GeneBank accession numbers of the present endo-1,3-β-glucanases are: Additionally, sequence alignment demonstrated the key residues involved in catalytic reactions and substrate binding. A previous study had demonstrated that two conserved glutamates in the pattern EXDX(X)E function in the catalytic reaction were a nucleophile and general acid/base, respectively [31]. In L cf , the equivalent to the nucleophile was Glu150, whereas the general acid/base was Glu155 ( Figure 1). In addition, the structures of the β-glucanase ZgLamA GH16 complex with oligo substrates has previously been solved (PDB ID: 4BOW) [31], providing knowledge of key residues involved in substrate binding. The complex structures of ZgLamA GH16 identified seven residues involved in substrate binding, five of which are conserved based on the sequence alignment. In L cf , the equivalent to the conserved residues were Lys105, Trp130, Trp134, Asp152, and Trp145 ( Figure 1).

Expression and Purification of L cf
In a previous study, direct extraction of the enzyme from the digestive organs of marine mollusks was the general method used for collecting endo-1,3-β-glucanases [24][25][26]. However, the isolation of endo-1,3-β-glucanases from digestive organs is not only time-consuming, but is also limited by the source of marine mollusks. Therefore, we carried out the expression of L cf in vitro using the Escherichia coli expression system and Bacillus brevis expression system. Although the recombinant proteins were detected in the supernatant of the cell lysis, they did not exhibit enzymatic activity, suggesting the occurrence of incorrect protein folding in the expression of L cf in the E. coli expression system. Fortunately, recombinant L cf with hydrolytic activity was detected successfully in the fermentation broth of Bacillus brevis. After a three-step purification, including ammonium sulfate salting out, hydrophobic chromatography, and ion exchange chromatography, about 12 mg of L cf was extracted from 1 L of fermentation broth of Bacillus brevis. The molecular weight of L cf was about 37 kDa, as shown in Figure 2

Enzymatic Characterization of Lcf
A hydrolytic activity assay at various pH values indicated that Lcf exhibited the maximum hydrolytic activity at pH 6.0 ( Figure 3a). Additionally, the assay at different temperatures demonstrated that the optimal temperature for Lcf was 44 °C (Figure 3b). When the incubation temperature was higher than 45 °C, the hydrolytic activity of Lcf decreased significantly ( Figure 3c). The enzymatic activity of Lcf was abolished after pre-incubation of Lcf at 60 °C and 70 °C (Figure 3c). In addition, Lcf retained approximately 25% of the enzymatic activity with the addition of EDTA (Figure 3d), suggesting that metal ions might influence the enzymatic activity of Lcf, but are not essential for Lcf. Notably, the enzymatic activity of Lcf was increased by approximately three times in the presence of Mn 2+ . The enzymatic activity of Lcf was also enhanced by Fe 3+ , and the addition of Ca 2+ seemed not to affect the enzymatic activity of Lcf. However, the enzymatic activity of Lcf was inhibited in the presence of Mg 2+ , Cu 2+ , and Zn 2+ , especially in the presence of Cu 2+ (Figure 3d).
Furthermore, the kinetic parameters of Lcf towards laminarin were measured. The specific activity for Lcf was 1.67 U/mg, and its Km and Kcat values were 10.27 mg/mL and 1230.64 S −1 , respectively.  Through the Bacillus brevis expression system, L cf was expressed in vitro successfully, providing a more convenient method for the extraction of marine mollusk endo-1,3-β-glucanases. In addition, this method may facilitate performing further structural research, which will be important for understanding the unique transglycosylation activity of marine mollusk endo-1,3-β-glucanases.

Enzymatic Characterization of L cf
A hydrolytic activity assay at various pH values indicated that L cf exhibited the maximum hydrolytic activity at pH 6.0 ( Figure 3a). Additionally, the assay at different temperatures demonstrated that the optimal temperature for L cf was 44 • C ( Figure 3b). When the incubation temperature was higher than 45 • C, the hydrolytic activity of L cf decreased significantly (Figure 3c). The enzymatic activity of L cf was abolished after pre-incubation of L cf at 60 • C and 70 • C (Figure 3c). In addition, L cf retained approximately 25% of the enzymatic activity with the addition of EDTA (Figure 3d), suggesting that metal ions might influence the enzymatic activity of L cf , but are not essential for L cf . Notably, the enzymatic activity of L cf was increased by approximately three times in the presence of Mn 2+ . The enzymatic activity of L cf was also enhanced by Fe 3+ , and the addition of Ca 2+ seemed not to affect the enzymatic activity of L cf . However, the enzymatic activity of L cf was inhibited in the presence of Mg 2+ , Cu 2+ , and Zn 2+ , especially in the presence of Cu 2+ (Figure 3d).
Furthermore, the kinetic parameters of L cf towards laminarin were measured. The specific activity for L cf was 1.67 U/mg, and its K m and K cat values were 10.27 mg/mL and 1230.64 S −1 , respectively. essential for Lcf. Notably, the enzymatic activity of Lcf was increased by approximately three times in the presence of Mn 2+ . The enzymatic activity of Lcf was also enhanced by Fe 3+ , and the addition of Ca 2+ seemed not to affect the enzymatic activity of Lcf. However, the enzymatic activity of Lcf was inhibited in the presence of Mg 2+ , Cu 2+ , and Zn 2+ , especially in the presence of Cu 2+ (Figure 3d). Furthermore, the kinetic parameters of Lcf towards laminarin were measured. The specific activity for Lcf was 1.67 U/mg, and its Km and Kcat values were 10.27 mg/mL and 1230.64 S −1 , respectively.

Analyses of Hydrolytic Products and Transglycosylation Products
To test the hydrolytic products of L cf , Thin layer chromatography (TLC) analysis was carried out. As shown in Figure 4, both the laminarin and the laminarinexaose were hydrolyzed into several oligomers, confirming that L cf acted in an endo-type mode. However, different cleavage efficiencies were observed. L cf showed a more efficient cleavage rate toward laminarinexaose. After reaction for 1 min, hydrolysis products with a degree of polymerization (Dp) 1-5 were generated. These oligomeric products were further hydrolyzed into monomers and dimers as end-products ( Figure 4). In contrast, laminarin was not hydrolyzed completely ( Figure 4). Considering the fact that laminarins from Laminaria digitate have a branch degree of 7.68%, and these branches contain about 9.51% β-1,6-glycosidic bonds [33], the more complex structure and the longer chain of laminarins might lead to a lower cleavage rate for L cf .

Analyses of Hydrolytic Products and Transglycosylation Products
To test the hydrolytic products of Lcf, Thin layer chromatography (TLC) analysis was carried out. As shown in Figure 4, both the laminarin and the laminarinexaose were hydrolyzed into several oligomers, confirming that Lcf acted in an endo-type mode. However, different cleavage efficiencies were observed. Lcf showed a more efficient cleavage rate toward laminarinexaose. After reaction for 1 min, hydrolysis products with a degree of polymerization (Dp) 1-5 were generated. These oligomeric products were further hydrolyzed into monomers and dimers as end-products ( Figure 4). In contrast, laminarin was not hydrolyzed completely ( Figure 4). Considering the fact that laminarins from Laminaria digitate have a branch degree of 7.68%, and these branches contain about 9.51% β-1,6glycosidic bonds [33], the more complex structure and the longer chain of laminarins might lead to a lower cleavage rate for Lcf. To explore the transglycosylation specificity of Lcf, the transglycosylation products were analyzed by MS. Laminarin has usually been selected as the substrate for transglycosylation reactions in previous studies [16,23,24]. However, Lcf showed a more efficient cleavage rate toward laminarinexaose than laminarin, as described above. Accordingly, the substrate we used in the transglycosylation reaction was laminarinexaose. Furthermore, the acceptors contained To explore the transglycosylation specificity of L cf , the transglycosylation products were analyzed by MS. Laminarin has usually been selected as the substrate for transglycosylation reactions in previous studies [16,23,24]. However, L cf showed a more efficient cleavage rate toward laminarinexaose than laminarin, as described above. Accordingly, the substrate we used in the transglycosylation reaction was laminarinexaose. Furthermore, the acceptors contained monosaccharides (methyl α-d-glucopyranoside and methyl β-d-glucopyranoside), alcohol (ethanol), polyols (glycerol and d-sorbitol), and amino acids with hydroxyl side chains (l-serine). As shown in Figure 5a, the methylated disaccharide, trisaccharide, and tetrasaccharide were detected in the presence of methyl α-d-glucopyranoside or methyl β-d-glucopyranoside, suggesting that the donors of glycosylation were glucose, laminaribiose, and laminaritriose. Transglycosylation products were also observed in the presence of the other acceptors (Figure 5c-f). In the presence of glycerol, five transglycosylation products were generated (Figure 5c). Similar to methyl α-d-glucopyranoside or methyl β-d-glucopyranoside, three transglycosylation products were observed in the presence of d-sorbitol (Figure 5d). However, only one transglycosylation product was detected using ethanol or l-serine as the acceptor (Figure 5e,f). Besides transglycosylation products, hydrolytic products, including glucose, G2, G3, G4, and G5, were detected. The m/z of each product in ESI-MS (+) is shown in Table 1.
The transglycosylation activity of O-glycoside hydrolases can be used for the synthesis of new glycosides, which has potential for the development of novel drugs and functional foods. For example, transglycosylation may function in the synthesis of glycosylated therapeutic antibodies and glycoside-specific antibody-drug conjugates [34]. Therefore, the diverse transglycosylation specificities of endo-1,3-β-glucanases have attracted much attention. As described above, L cf exhibited transglycosylation activity towards different kinds of acceptors, which facilitated the synthesis of novel glycosides. Additionally, in the transglycosylation reaction catalyzed by the endo-1,3-β-glucanase from Mizuhopecten yessoensis, only a sorbitol-Glc 3 product was detected when using d-sorbitol as an acceptor [24]. However, three transglycosylation products linked with sorbitol were observed after the transglycosylation reaction catalyzed by L cf (Figure 5d). These findings demonstrated that the two endo-1,3-β-glucanases from mollusks exhibit different transglycosylation specificities, although they share a high sequence identity. The unique transglycosylation activity of L cf towards sorbitol facilitates the development of glycoconjugates with intestinal functions, and noncariogenic sugars [35]. In sum, this study provided insights into novel endo-1,3-β-glucanases from marine mollusks, enhancing our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-β-glucanases.  Table 1.    Table 1.

RNA Extraction and Gene Cloning
The scallops were purchased from the Nanshan seafood market in Qingdao. The hepatopancreas was dissected by hand from the scallop viscera. Then, the hepatopancreas was ground into powder in liquid nitrogen. Next, the total RNA was extracted from the powder using the Mollusc RNA Kit (Omega Bio-tek, Norcross, GA, USA). A cDNA library of scallop hepatopancreas was constructed by reverse transcription, using the extracted total RNA as a template (RevertAid First Strand cDNA Synthesis Kit, Thermo Fisher Scientific, Waltham, MA, USA). Using the cDNA library as a template, a full-length gene of L cf without the signal peptide was cloned using the following primers: a forward primer (5 -CGGGATCCGCAGGCTTCCGTGACGATTTCAC-3 ) and a reverse primer (5 -CCGCTCGAGTCAATGAGGTATCATCTCTATGTAATC-3 ). Target gene fragments were collected using the Gel Extraction Kit (Omega Bio-tek, Norcross, GA, USA), and ligated into the shuttle vector pNCMO2 (Takara, Dalian, China) using the restriction enzymes BamHI and XhoI. Then, the ligation solution was transformed into E. coli JM109. Expression plasmids were extracted using the Plasmid Mini Kit (Omega Bio-tek, Norcross, GA, USA).

Protein Expression in Brevibacillus
The Brevibacillus (Bacillus brevis) expression system (Takara, Dalian, China) is a prokaryotic expression system that is particularly effective in the production of secretory proteins [36]. The expression plasmids were transformed into Brevibacillus-competent cells using the new Tris-PEG (NTP) method, as described in the protocol. Brevibacillus transformant cells were grown in MTNm solid medium (glucose 10 g/L, polypeptone 10 g/L, beef powder 5 g/L, yeast extract 2 g/L, FeSO 4 ·7H 2 O 10 mg/L, MnSO 4 ·4H 2 O 10 mg/L, ZnSO 4 ·7H 2 O 1 mg/L, MgCl 2 ·6H 2 O 4.1 g/L, and neomycin 10 mg/L, pH 7.0) at 37 • C. DNA sequencing was performed to confirm the recombinant Brevibacillus. Then, the recombinant Brevibacillus cells were cultured in TMNm liquid medium (i.e., MTNm medium without MgCl 2 ) at 32 • C for 48 h. SDS-PAGE analysis indicated that L cf was successfully secreted into the fermentation broth.

Protein Purification
As described above, the Brevibacillus transformant cells were cultured for 48 h, then the fermentation broth was harvested by centrifuging at 6000× g rpm for 5 min. To precipitate the enzymes, ammonium sulfate was slowly added to the fermentation broth to 60% saturation (0 • C). After 10 h, the precipitate was collected by centrifugation (12,000× g, 4 • C), followed by washing using ammonium sulfate solution (60% saturation, 0 • C). The resulting protein was re-dissolved in a Tris-HCl buffer (20 mM, pH 7.5) containing 15% saturation (0 • C) ammonium sulfate. Then, the protein was loaded onto Phenyl Sepharose beads (GE Healthcare, Boston, MA, USA), which were washed and eluted using Tris-HCl buffer (20 mM, pH 7.5) containing 15-0% saturation (0 • C) ammonium sulfate. After purification by the Phenyl Sepharose beads, the resulting protein was dialyzed into a sodium acetate buffer (50 mM, pH 5.0). Next, the protein was loaded onto CM Sepharose beads (GE Healthcare), which were washed and eluted using sodium acetate buffer (50 mM, pH 5.0) containing 0-0.5 M NaCl.

Hydrolytic Activity Assay
The hydrolytic activity of L cf was determined by the modified DNS method [37]. The reaction mixture (200 µL), containing 0.1% (w/w) laminarin from Laminaria digitate dissolved in sodium acetate buffer (50 mM, pH 5.6) and 20 µL purified L cf , was incubated for 20 min at 37 • C. Then, 150 µL DNS was added to terminate the reaction. The resulting mixture was boiled for 5 min at 100 • C and measured at 520 nm. One unit (1 U) of hydrolytic activity was defined as the amount of enzyme required to produce the reducing sugar equivalent to 1 µmol of glucose per minute under the above reaction conditions.
The optimal pH of L cf was determined by measuring the hydrolytic activity at different pH conditions. The buffers (50 mM) used for this assay contained sodium acetate buffers (pH 4.5, 5.0 and 5.6), sodium phosphate buffers (pH 5.9, 6.4 and 6.9), and Tris-HCl buffer (pH 7.5 and 8.0). The effect of the temperature was measured at 15-65 • C in 50 mM sodium acetate buffer, at pH 5.6. The above enzymatic activity was tested under standard conditions. To determine the thermal stability of L cf , the residual hydrolytic activity of L cf (dissolved in sodium acetate buffer (50 mM, pH 5.6)) after incubation at different temperatures (4-70 • C) for 15 min was measured. The effect of metal ions was determined by measuring the activity with different metal ions (Mg 2+ , Ca 2+ , Mn 2+ , Cu 2+ , Zn 2+ , Fe 3+ , and EDTA) at 5 mM in 50 mM sodium acetate buffer, at pH 5.6. All the above assays were performed in triplicate.

TLC Assay
The products of L cf degradation of laminarin and laminarihexaose (Megazyme) were generated as described above. The reaction mixture (200 µL) contained 0.1% (w/w) laminarin or laminarihexose dissolved in sodium acetate buffer (50 mM, pH 5.6) and 20 µL purified L cf . At different times, 5 µL of reaction mixture were taken for TLC analysis. Samples were spotted on silica gel 60 F 254 plates (Merck, Darmatadt, Germany), and separated in a solvent of 1-butanol/acetic acid/water (2:1:1, v/v). To visualize the products, the plate was sprayed with a reagent containing 2 g of diphenylamine, 2 mL of aniline, 1 mL of HCl, 10 mL of H 3 PO 3 , and 100 mL of acetone, and was heated at 100 • C for 10 min [38].

Transglycosylation Products Assay
To explore the transglycosalating ability of L cf , several acceptors, including methyl α-d-glucopyranoside, methyl β-d-glucopyranoside, glycerol, d-sorbitol, ethanol, and L-serine, were involved in the transglycosylation reactions. Briefly, L cf (0.02 U) was added into the mixture containing laminarihexaose (2 mg/mL) and an acceptor (2 mg/mL) dissolved in sodium acetate buffer (25 mM, pH 5.6). Then, the resulting mixture (200 µL) was incubated at 37 • C for 5 min. The reaction was terminated by the addition of an equal volume of 2.5% (v/v) aqueous ammonia. After centrifugation, the supernatant was collected and detected using positive ion electrospray ionization mass spectrometry (ESI-MS) (Agilent 6460 Triple Quad, Santa Clara, CA, USA).