Identification, Characterization, and Expression of a β-Galactosidase from Arion Species (Mollusca)

β-Galactosidases (β-Gal, EC 3.2.1.23) catalyze the cleavage of terminal non-reducing β-D-galactose residues or transglycosylation reactions yielding galacto-oligosaccharides. In this study, we present the isolation and characterization of a β-galactosidase from Arion lusitanicus, and based on this, the cloning and expression of a putative β-galactosidase from Arion vulgaris (A0A0B7AQJ9) in Sf9 cells. The entire gene codes for a protein consisting of 661 amino acids, comprising a putative signal peptide and an active domain. Specificity studies show exo- and endo-cleavage activity for galactose β1,4-linkages. Both enzymes, the recombinant from A. vulgaris and the native from A. lusitanicus, display similar biochemical parameters. Both β-galactosidases are most active in acidic environments ranging from pH 3.5 to 4.5, and do not depend on metal ions. The ideal reaction temperature is 50 °C. Long-term storage is possible up to +4 °C for the A. vulgaris enzyme, and up to +20 °C for the A. lusitanicus enzyme. This is the first report of the expression and characterization of a mollusk exoglycosidase.


Introduction
N-Glycosylation of proteins is one of the most complex forms of post-translational modification. Its accuracy is important for cellular processes, such as protein folding, secretion, cell adhesion, intracellular trafficking, recognition, and signaling [1,2]. The biosynthesis of N-glycans takes place in the ER and the Golgi, and is a species-specific tightly regulated interplay of several glycosyltransferases and glycosidases [3]. Most of the degradation is located in the lysosomes, and is mediated by highly specific endo-and exoglycosidases that act synergistically [4].
While these enzymes are well studied in mammals, plants, yeast, and bacteria, the knowledge about the glycosylation machinery of mollusks is still rudimentary, even when this phylum is evolutionary very successful. For more than 500 million years, mollusk species with very heterogenous morphology (gastropods, cephalopods, bivalvia) have populated freshwater, marine, and terrestrial habitats worldwide, executing important functions in the corresponding ecosystems in terms of waste disposal and cleaning. Others are used due to their nutritional value and shells [5]. Some of them are of medical relevance, as they act as intermediate hosts in parasite life cycles.
Mollusks display a broad spectrum of glycosylation abilities shown in their heterogenous N-and O-glycan patterns. But so far, only a few of the involved glycosyltransferases and glycosidases have been described and characterized [6,7].
The best-studied β-galactosidase is certainly the one from E. coli, which is widely used in molecular biology as a reporter marker to screen gene expression through a technique called α-complementation. In E. coli, the protein is encoded by the lacZ gene, which is regulated by the lac operon in the presence/absence of lactose, and which requires Mg 2+ and Na + for maximal activity [12,13]. Further β-galactosidases, derived from other microorganisms (Kluyveromyces, Penicillium, Lactobacillus), have been investigated with regards to activity properties based on their structure [14][15][16][17]. Some of these enzymes are used on an industrial scale for the modification of oligosaccharides in food industry [18,19].
In multicellular organisms, β-galactosidases are located in the lysosomes or in the cytosol, depending on their function and substrate specificity, acting at acidic or neutral pH-optima. A change in the pH-environment has been found to play an important role in protein folding and, therefore, influences the activity properties [20,21].
Starting in the late seventies, snail extracts were found to be an interesting source of endo-and exoglycosidases. β-Galactosidases were detected and partly characterized from Biomphalaria glabrata, Achatina achatina, and Pomacea canaliculata [22][23][24][25]. However, in none of these studies were the enzymes expressed recombinantly. Similar to mammalian or plant enzymes, their function lies mainly in the degradation and modification of glycans. However, the snail glycosidases also seem to play a role in the snail immune response. In the plasma of Biomphalaria glabrata, the enzyme activity of several glycosidases, including β-galactosidase, was found to correlate with the progress of infection by Schistosoma mansoni [26].
Due to the enormous variety of glycosylation abilities of snails, and due to their continuous successful position in the ecosystem, snails are a valuable model for future investigations of glycosylation processes (for example-the interaction of parasites with their intermediate hosts). Detailed knowledge of the involved enzymes is therefore of essential importance.
In this study we present, for the first time, the cloning and expression of a mollusk exoglycosidase (in particular, a β-galactosidase from Arion vulgaris). Furthermore, this enzyme was characterized and compared to a native β-galactosidase purified from Arion lusitanicus.

Materials
Arion lusitanicus individuals were collected in September and October 2018 by the authors in their private gardens located in and around Vienna. Whole slugs were immediately frozen and stored at −80 • C until further use.
Q5/Taq DNA Polymerases, restriction enzymes, and T4 ligase were purchased from New England Biolabs (Frankfurt, Germany). All enzymes were used according to the supplier's instructions. Primers and gBlock gene fragments were synthesized commercially by Sigma-Aldrich (Vienna, Austria) and Integrated DNA Technologies (Leuven, Belgium), respectively. A pACEBac1 vector was purchased from Geneva Biotech (Genève, Switzerland).
All other chemicals and molecular biology reagents were of the highest quality available. They were purchased from Sigma-Aldrich (Vienna, Austria), Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), Honeywell (Vienna, Austria), and ThermoFisher Scientific (Bonn, Germany), unless indicated otherwise.

Mass Spectrometry of the Protein
After the reduction with DTT (15 mM in 100 mM ammonium bicarbonate pH 7.0) and carbamidomethylation with iodoacetamide (30 mM) [29], the sample was digested with trypsin (Promega, sequencing grade), followed by a C18-SPE clean-up. The sample was loaded onto a LC (Dionex UltiMate 3000 Rapid, ThermoFisher Scientific -Bonn, Germany) equipped with nano-C18 column, coupled to an Orbitrap (ThermoFisher Scientific-Bonn, Germany, performed at IMBA, Vienna). A MS/MS ion search was performed using MAS-COT. The files were searched against an in-house database ("snails") and the reviewed uniprot database.

Expression of the Full-Length β-Galactosidase Gene from Arion Vulgaris
Based on the mass spectrometry results of the native A. lusitanicus enzyme, the A. vulgaris β-galactosidase full-length gene (GenBank: A0A0B7AQJ9) was chosen and modified with a C-terminal hexahistidine-tag and a N-terminal gp64 secretion signal sequence (MVSAIVLYVLLAAAAHSAFA) [30].
The SacI and XbaI restriction sites were added to the sequence via primers (underlined in the primer sequences below). The recombinant gene was PCR amplified by using the forward primer 5 GATGATGAGCTCATGGTAAGTGCTATAGTGCTG3 and the reverse primer 5 GATGATTCTAGATTAATGGTGGTGATGATGATG3 . The purified PCR fragment was ligated to the pACEBac1 vector in a 5:1 ratio. The correct insertion and gene sequence was verified by Sanger sequencing (Microsynth-Vienna, Austria). The recombinant plasmid (pACEBac1:β-gal) was amplified in NEB 5-alpha (NEB, Frankfurt, Germany), and 1 ng of the purified plasmid was used for integration into a MultiBac genome via Tn7 transposition in DH10EMBacY cells (Geneva Biotech, Genève, Switzerland). Midiprep of DH10EMBacY was performed by modifying the NucleoSpin Plasmid EasyPure kit protocol (Macherey-Nagel, Dueren, Germany) according to [31]. 5 µg of the recombinant β-gal construct were transfected to Sf9 insect cells (2 mL of 0.9 × 10 6 cells/mL) using the FuGENE HD Transfection Reagent (Promega-Walldorf, Germany) in a 1:1.8 ratio, respectively. After 5 days, the seed stock was collected, and 100 µl was used for the production of the intermediate stock (8 × 10 6 cells in 12 mL + 0.03% FBS). After 3-4 days, 200 µl of the intermediate stock was used for the production of the working stock (2 × 10 6 cells/mL in 50 mL media + 0.04% FBS). For protein production, 100 µl of the working stock was added to 50 mL of 2 × 10 6 cells/mL and incubated for 3 days. The recombinant β-gal protein was extracted from cell pellet fraction via the I-PER Cell Protein Extraction Reagent (Ther-moFisher Scientific -Bonn, Germany), and further purified through immunoprecipitation using either protein G-plus or protein G-plus/Protein A-agarose beads (CALBIOCHEM, San Diego, CA, USA) linked to mouse anti Penta Histidine Tag:HRP monoclonal antibodies (BIORAD-Vienna, Austria) or magnetic beads (Abbkine-Wuhan, China), according to the supplier's instructions. The purified recombinant protein was analyzed by SDS-Page and Western blot using the mouse anti Penta Histidine Tag:HRP monoclonal antibodies (1:2500, BIORAD-Vienna, Austria) following alkaline phosphatase conjugated anti-mouse IgG from goat (1:4000, Sigma-Aldrich, Vienna, Austria) [32].

Determination of Protein Content
Protein concentrations were determined by Micro-BCA protein assay (Pierce, Bonn, Germany) with bovine serum albumin as the standard.

Determination of β-Galactosidase Activity
The analysis of the enzyme activity of β-galactosidase was based on a colorimetric assay using the artificial substrate 4-Nitrophenyl-β-D-galactopyranosid (pNP-β-Gal, Merck, Darmstadt, Germany). The reaction was performed in 50 µL containing 5 µL of enzyme solution, 20 µL of 0.9% (w/v) NaCl-solution, and 25 µL of pNP-β-Gal substrate (5 mM pNP-β-Gal in 0.1 M NaCitrat buffer, pH 4.5) at 37 • C for 2 h. The reaction was terminated by adding 200 µL of Glycine/NaOH (0.4 M, pH 10.4). The absorbance of the released p-nitrophenol was measured at 405 nm. For analysis of the biochemical parameters of the enzyme, the standard assay conditions using pNP-β-Gal as the substrate were modified as follows. For the determination of cation requirement, the standard assay was carried out without any cation addition, or in presence of 20 mM of EDTA, Mn 2+ , Mg 2+ , Ca 2+ , Co 2+ , Cu 2+ , Ni 2+ , or Ba 2+ . Chemical stability of the enzyme, optimal storage conditions, and pH-optimum were processed according to [28]. For storage stability in chemicals, the enzyme was incubated for approximately 16 h in 10% (v/v) or 20% (v/v) of methanol, acetonitrile, glycerol, or imidazole [50 mM or 100 mM]. For inhibition studies, the standard assay was performed in the presence of 6 mM or 12 mM monosaccharide (GlcNAc, GalNAc, Gal, Glc, Ara, Xyl, Fuc, Rha, Rib or Man). A time course was carried out by measuring the release of p-nitrophenol (up to 22 h in total). Kinetic data (K M -values) were acquired using pNP-β-Gal in a range from 0.2 mM to 12.5 mM in the standard incubation assay.
Each assay was at least performed in duplicates with appropriate controls. All quantitative values were calculated using a calibration curve of p-nitrophenol.
A test for transglycosylation activity was carried out according to [36], with 5 mM pNP-β-Gal as donor and GlcNAc or Glc labelled with 2-amino-benzoic acid as the acceptors. The reaction was incubated at 37 • C over night with the enzyme preparation, and analysed as described above.

Purification of β-Galactosidase from Arion Lusitanicus
Viscera of A. lusitanicus were used for the purification of β-galactosidase. Performing several chromatographic steps, the enzyme was purified successfully [ Table 1]. It was particularly difficult to remove other exoglycosidases (such as β-N-acetylglucosaminidase, α-mannosidase, β-mannosidase, and α-fucosidase) from the desired galactosidase. Finally, the total amount of enzyme was at about 0.10 U with a specific activity of 2.44 U/mg and a yield of 3.5%, which means a 62.83-fold purification. The purified β-galactosidase fraction was used for biochemical analysis and, after tryptic digest, also for peptide analysis.

Recombinant β-Galactosidase from A. Vulgaris Expressed as Protein in Sf9 Insect Cells
Generally, there is a lack of genetic information regarding mollusks. Few of them are fully sequenced because, for most of them, just limited data are available. While for A. vulgaris at least some genetic data are available, there is no information regarding A. lusitanicus. Searching with the obtained peptides for homologies (Table S1), one of the major hits was a predicted β-galactosidase from Arion vulgaris.
Furthermore, homology search was performed with active β-galactosidases from other organisms (Homo sapiens, C. elegans, D. melanogaster), which confirmed the identified candidate from A. vulgaris (A0A0B7AQJ9) as an interesting target [ Figure 1]. Furthermore, homology search was performed with active β-galactosidases fro other organisms (Homo sapiens, C. elegans, D. melanogaster), which confirmed the identifi candidate from A. vulgaris (A0A0B7AQJ9) as an interesting target [ Figure 1]. The putative full-length β-galactosidase sequence from A. vulgaris (A0A0B7AQJ9) was selected and further modified to express and secrete the protein using the baculovirus expression system.
The gp64 leader sequence for secretion was added to the N-terminus, and a hexahistidinetag for purification was added to the C-terminus of the sequence. The recombinant construct consisted of 688 amino acids with a molecular weight of approximately 77 kDa and a calculated isoelectric point (pI) of 5.8 [ Figure 2]. vulgaris (A0A0B7AQJ9). Yellow rhomb indicates putative N-glycosylation sites in Arion vu cording to the N-X-S/T consensus sequence. (*) marks identical amino acid residues, (:) amino acid residues, and (.) predominantly the same amino acid residues.
The putative full-length β-galactosidase sequence from A. vulgaris (A0A0B was selected and further modified to express and secrete the protein using the bacu expression system. The gp64 leader sequence for secretion was added to the N-terminus, and a h tidine-tag for purification was added to the C-terminus of the sequence. The recom construct consisted of 688 amino acids with a molecular weight of approximately and a calculated isoelectric point (pI) of 5.8 [ Figure 2]. The presence and activity of the expressed β-gal protein was analyzed in supe (secreted proteins) and lysate (soluble proteins).
β-Galactosidase activity was present in the supernatant (secreted protein), as in lysate fractions (soluble, but not secreted protein) [ Figure S1]. Purification of creted protein (supernatant) over a HisTrap TM excel column (1 mL, Cytiva-Vienn tria) was not successful, but by using immunoprecipitation of the lysate fraction, t active enzyme was obtained [ Figure S2].

Biochemical Parameters of Native and Recombinant β-Galactosidases from Two Arion cies
The optimal storage temperature for the recombinant β-galactosidase (A. v was within a temperature range of −80 to + 4 °C, while the native enzyme from A. l cus could be stored at up to + 20 °C. The activity for both enzymes declined at temperatures above room temperature until the complete loss of activity at a temp above 50 °C for A. vulgaris or 60 °C for A. lusitanicus [ Figures S3]. However, 50 °C optimal reaction temperature for both enzymes in assays up to 2 h [ Figure S4]. The of the recombinant protein from A. vulgaris was not affected by lyophilization (d shown). To investigate the storage stability in chemicals, the recombinant enzyme garis) was stored in methanol, acetonitrile, glycerol, or imidazole. The enzyme activ highly affected by 20% (v/v) acetonitrile, as it drastically reduced activity to approx 40% [ Figure S5], as well as by imidazole [100 mM] and glycerol [10% (v/v)] durin term incubation (2 h, 37 °C). No influence on enzyme activity was observed by th tion of methanol [up to 40% (v/v)]. The native β-galactosidase from A. lusitanicus, ho was already influenced by the addition of 10% (v/v) acetonitrile, with an activity re to 40% during short term incubation. Altogether, only methanol was tolerated both species [ Figure S6].
β-galactosidase activity was not dependent on divalent cations in either specie was no loss of activity in the presence of EDTA. Addition of cations (Co 2+ , Mn 2+ , Mg Ni 2+ , Ba 2+ ) had no influence, except for Cu 2+ , which drastically reduced the activity (A. lusitanicus) and 68% (A. vulgaris) [ Figure S7]. The presence and activity of the expressed β-gal protein was analyzed in supernatant (secreted proteins) and lysate (soluble proteins).
β-Galactosidase activity was present in the supernatant (secreted protein), as well as in lysate fractions (soluble, but not secreted protein) [ Figure S1]. Purification of the secreted protein (supernatant) over a HisTrap TM excel column (1 mL, Cytiva-Vienna, Austria) was not successful, but by using immunoprecipitation of the lysate fraction, the pure active enzyme was obtained [ Figure S2].

Biochemical Parameters of Native and Recombinant β-Galactosidases from Two Arion Species
The optimal storage temperature for the recombinant β-galactosidase (A. vulgaris) was within a temperature range of −80 to + 4 • C, while the native enzyme from A. lusitanicus could be stored up to + 20 • C. The activity for both enzymes declined at storage temperatures above room temperature until the complete loss of activity at a temperature above 50 • C for A. vulgaris or 60 • C for A. lusitanicus [ Figure S3]. However, 50 • C was the optimal reaction temperature for both enzymes in assays up to 2 h [ Figure S4]. The activity of the recombinant protein from A. vulgaris was not affected by lyophilization (data not shown). To investigate the storage stability in chemicals, the recombinant enzyme (A. vulgaris) was stored in methanol, acetonitrile, glycerol, or imidazole. The enzyme activity was highly affected by 20% (v/v) acetonitrile, as it drastically reduced activity to approximately 40% [ Figure S5], as well as by imidazole [100 mM] and glycerol [10% (v/v)] during short term incubation (2 h, 37 • C). No influence on enzyme activity was observed by the addition of methanol [up to 40% (v/v)]. The native β-galactosidase from A. lusitanicus, however, was already influenced by the addition of 10% (v/v) acetonitrile, with an activity reduction to 40% during short term incubation. Altogether, only methanol was tolerated well by both species [ Figure S6].
The optimal pH for the native β-galactosidase from A. lusitanicus, as well as for the recombinant A. vulgaris enzyme, was in the range of pH 3.5-4.5 using acetat or citrat as buffer salts [ Figure 3, Table S2]. The optimal pH for the native β-galactosidase from A. lusitanicus, as well as for the recombinant A. vulgaris enzyme, was in the range of pH 3.5-4.5 using acetat or citrat as buffer salts [ Figure 3, Table S2]. In consideration of inhibiting substances, the activity of the recombinant protein (A. vulgaris) was tested in the presence of monosaccharides (GlcNAc, GalNAc, Gal, Glc, Ara, Xyl, Fuc, Rha, Rib, and Man). Thereby, product inhibition by galactose was detected by the addition of 6 mM and 12 mM galactose, which resulted in an approximately 25% and 40% reduction of activity, respectively. All other monosaccharides did not show any inhibitory effects [ Figure S8].
Analysis of enzyme kinetics using the pNP-β-Gal substrate at a pH of 4.5 revealed a KM of 8.3 mM and a vmax of 0.002 μmol/min for A. lusitanicus, and a KM of 3.2 mM and a vmax of 0.0002 μmol/min for A. vulgaris.
Analysis of enzyme kinetics using the pNP-β-Gal substrate at a pH of 4.5 revealed a K M of 8.3 mM and a v max of 0.002 µmol/min for A. lusitanicus, and a K M of 3.2 mM and a v max of 0.0002 µmol/min for A. vulgaris.
Furthermore, because some β-galactosidases also catalyze a transglycosylation reaction, we tested activity with pNP-β-Gal as the donor, and GlcNAc or Glc as acceptors, according to [36]. However, no transglycosylation activity was detectable under the chosen conditions.

Discussion and Conclusions
β-Galactosidases (EC 3.2.1.23) are a large family of exoglycosidases which catalyze single cleavage reactions that remove terminal galactose residues from N-and O-glycans, hydrolyze β-galactosides into monosaccharides, or produce galacto-oligosaccharides (GOSs) by a transglycosylation reaction [10,36]. They can be found throughout the animal kingdom, as well as in plants, yeast, and bacteria [18,37]. In mollusks, several studies were conducted to analyze and characterize β-galactosidases of different species [23,[38][39][40]. However, compared to the β-galactosidases from E. coli or humans, only little is known about these enzymes in mollusks.
In search for the β-galactosidase from Arion, we started by purifying the enzyme form A. lusitanicus through several chromatographic steps. The sample was further analyzed with a LC coupled to an Orbitrap (Thermo), and blasted against the in-house "snail" databank. Based on a homology search with the fragments obtained from the purified enzyme, we successfully expressed the predicted β-galactosidase enzyme from A. vulgaris (A0A0B7AQJ9, MASCOT score: 2221.1) in Sf9 insect cells.
The activity and specificity of the full-length recombinant A. vulgaris enzyme was compared with the purified native A. lusitanicus enzyme using the artificial pNP-β-Gal and several native substrates.
In terms of pH-optimum, the β-galactosidase enzymes from A. lusitanicus and A. vulgaris were mostly active in acidic environments ranging from pH 3.5 to 4.5, which

Discussion and Conclusions
β-Galactosidases (EC 3.2.1.23) are a large family of exoglycosidases which catalyze single cleavage reactions that remove terminal galactose residues from N-and O-glycans, hydrolyze β-galactosides into monosaccharides, or produce galacto-oligosaccharides (GOSs) by a transglycosylation reaction [10,36]. They can be found throughout the animal kingdom, as well as in plants, yeast, and bacteria [18,37]. In mollusks, several studies were conducted to analyze and characterize β-galactosidases of different species [23,[38][39][40]. However, compared to the β-galactosidases from E. coli or humans, only little is known about these enzymes in mollusks.
In search for the β-galactosidase from Arion, we started by purifying the enzyme form A. lusitanicus through several chromatographic steps. The sample was further analyzed with a LC coupled to an Orbitrap (Thermo), and blasted against the in-house "snail" databank. Based on a homology search with the fragments obtained from the purified enzyme, we successfully expressed the predicted β-galactosidase enzyme from A. vulgaris (A0A0B7AQJ9, MASCOT score: 2221.1) in Sf9 insect cells.
The activity and specificity of the full-length recombinant A. vulgaris enzyme was compared with the purified native A. lusitanicus enzyme using the artificial pNP-β-Gal and several native substrates.
In terms of pH-optimum, the β-galactosidase enzymes from A. lusitanicus and A. vulgaris were mostly active in acidic environments ranging from pH 3.5 to 4.5, which correlates with other β-galactosidases from mollusks, which range from pH 3.2 to 5.6 [23,[38][39][40]. In general, β-galactosidases are known to work in a relatively broad pH range. The enzymes from fungi, on the one hand, prefer more acidic environments with a pH-value starting at 2.5 and extending to 7.0 [41]. On the other hand, bacterial β-galactosidases favor nearly neutral environments, and hence act best between pH 7.0 and 7.5 [17]. Moreover, in mammals, the specific pH of the protein depends on the enzyme's localization, e.g., lysosomal or cytosolic, and varies between pH 3.0 and 6.0 [42]. Hence, we concluded that in Arion the acid optimum of our protein might indicate its localization in lysosomes rather than in the cytoplasm.
The influence of cations on β-galactosidases from different species is very heterogenous. In the microorganism Bacillus stearothermophilus, most cations had no effect on the enzyme's activity. Heavy metals-Cu 2+ , as well as Fe 2+ , Zn 2+ , Cu 2+ , Pb 2+ , and Sn 2+ -were shown to inhibit the enzyme drastically. In contrast, the enzyme from E. coli showed the need for Mg 2+ and Mn 2+ for its activity [43,44]. In addition, kinetic studies on the β-galactosidase of E. coli showed that Mg 2+ is more important for the catalytic process, rather than for substrate binding or the formation of the quaternary structure [45]. Regarding mammalian β-galactosidase, the necessity of MgCl 2 on the enzyme in rat liver was shown [46]. Nevertheless, the influence of ions on mammalian β-galactosidase, and especially those from mollusks, has received only little attention compared to those from microorganisms.
In regards to cation additives, we identified an inhibitory effect on our snail enzymes by Cu 2+ , which might be attributed to the metal-catalyzed oxidation of some critical amino acid residues, e.g., histidine or cysteine [47]. Other ions, such as Mn 2+ , Ba 2+ , Mg 2+ , Ca 2+ , Co 2+ , and Ni 2+ , had no significant effect on the enzymes' activities from both Arion species. Hence, the β-galactosidase from A. lusitanicus and A. vulgaris were independent in terms of metal ions, supporting previous data from Achatina balteata [40].
β-Galactosidases from various sources exhibit significant differences in their temperature optimum. The fungal enzymes work in ranges from 4 to 67 • C, while most bacterial β-galactosidases have optima between 40 and 65 • C [18,19,48]. For both of our snail enzymes, we identified an optimal reaction temperature of 50 • C, which also correlates with other β-galactosidases from mollusks [23]. Moreover, the recombinant protein from A. vulgaris was active for up to 22 h at 37 • C. The addition of several chemicals lowered the activity drastically. Especially the negative effect of imidazole has to be emphasized, as it plays an essential role in the purification of His-tagged proteins. From all chemicals tested, only methanol did not influence the enzyme's activity.
Analysis of enzyme activity revealed a K M of 8.3 mM for A. lusitanicus and a K M of 3.2 mM for A. vulgaris using the pNP-β-Gal substrate. Both K M values are similar compared to other β-galactosidases using the same artificial substrate as shown for E. coli with a K M of 0.093 mM [49], the bovine β-galactosidase with a K M of 2.5 mM, or the fungi β-galactosidase with a K M of 1.0 mM [17].
Both enzymes (native and recombinant) highly favored the artificial pNP-β-Gal, but also showed low activity on pNP-α-Glc. Activity was further tested on native β1,3and β1,4substrates. Exclusively β1,4linkages were cleaved. Terminal galactose residues on biantennary glycans were clearly preferred over lactose by the exoglycosidase activity. We further detected some endoglycosidase activity cleaving 2-fucosyllactose. No transglycosylation activity could be detected.
In conclusion, for the first time an exoglycosidase from mollusk origin was recombinantly expressed, characterized, and compared to a purified native enzyme originating from the same genus (Arion). The snail enzyme was found to be specific towards β1,4linkages, and displays exo-as well as endoglycosidase activity. Our results showed that both enzymes, the recombinant from A. vulgaris and the native from A. lusitanicus, are akin in their biochemical parameters.