Affinity purification, identification, and biochemical characterization of Gamma-glutamyl transpeptidase, a membrane anchored enzyme of Gigantocotyle explanatum

Gamma-glutamyl transpeptidase is an enzyme that facilitates the transfer of glutamyl groups from glutamyl peptides to other peptides or water. Additionally, it also participates in important processes such as amino acid transport, cellular redox control, drug detoxification, apoptosis, and DNA fragmentation in a various organism. In the present study, GGT activity in Gigantocotyle explanatum was examined in order to characterize the enzyme in the helminth system. GGT is isolated using membrane solubilization and purified through affinity column chromatography (Con-A Sepharose column). Km and Vmax values, as well as the optimal pH, optimal temperature, and incubation period, are also determined using enzyme kinetics. The hetero-dimeric property of the enzyme is demonstrated by the purified GGT, which yielded two subunits of 65.5 and 55 kDa. The optimal pH and temperature are found to be 8.0 and 37 °C, respectively. While assessing the optimal incubation time of the enzyme, it was observed that the purified GGT not only retained its functional integrity up to 15 min but also reflected considerable thermostability at higher temperatures, by retaining 78% and 25% of its initial activities at 50 °C and 60 °C, respectively. One millimolar concentration of 6-Diazo-5-Oxo Nor-isoleucine (DON), a specific inhibitor of GGT, completely abolished GGT activity. These results suggest that GGT in these worms is a catalytically active enzyme with distinguishing characteristics that can be used for further study to comprehend its function in amphistome biology and in host-parasite relationships, especially since the potential therapeutic candidacy of the GGT enzyme has already been indicated in these groups of organisms.


Introduction
Gamma-glutamyl transpeptidase (GGT; EC 2.3.2.2) catalyzes the transfer of the gamma glutamyl group from the glutathione (GSH) molecule to an acceptor molecule. It is an N-hydrolase superfamily auto-proteolytic enzyme that usually consists of two subunits with respective molecular weights of 20 to 66 kDa and 38 to 72 kDa (Castellano and Merlino 2012). GGT regulates the glutathione equilibrium of the cell by breaking the glutamyl moiety of the extracellular GSH molecule to produce gamma glutamyl amino acids. The reduced glutathione is used for a number of purposes, including, but not limited to, surface protein thiolation, phase II detoxification, cell proliferation, apoptosis, and defense against oxidative stress (Castellano and Merlino 2012). Furthermore, the role of the GGT enzyme in a variety of biochemical and physiological processes, such as DNA fragmentation, apoptosis control, redox regulation, cysteine transport, and xenobiotic detoxification, has been amply demonstrated (Griffith et al. 1979;Orlowski and Meister 1970;Gabriele and Riccardo 1995;Graber and Losa 1995;Sweiry et al. 1995;Drozdz et al. 1998;Dominici et al. 1999Dominici et al. , 2003Paolicchi et al. 2002;Carlisle et al. 2003;Uetrecht and Trager 2007). In recent years, both humans and microorganisms have been the subject of substantial GGT enzyme research, although helminthic parasites have gotten far less attention.
Abidi and Nizami speculate that GGT activity, which was previously found in the biliary amphistome of the Gigantocotyle explanatum, may be involved in the intake and transport of amino acids (Abidi and Nizami 1995). Parasitic nematodes such A. suum, S. cervi, B. malayi, and W. bancrofti have been demonstrated a remarkable GGT activity (Dass and Donahue 1986;Gupta et al. 2005a, b;Lobos et al. 1996). It is concerning that helminths have received such little attention over the past two decades, especially since it is still possible that the GGT enzyme could be used as a therapeutic agent in these types of organisms. Another key worry that requires immediate attention is the increasing drug resistance against the few anthelmintics that are now accessible in helminthic flukes. In the current situation, finding an alternative therapeutic target in these helminths could prove to be beneficial. The current study was designed to investigate at the GGT enzyme of Gigantocotyle explanatum (GeGGT), a biliary amphistome parasite that infects the bile duct tissues of Indian water buffaloes, Bubalus bubalis, and results in amphistomosis. As the situation worsens, migratory larvae damage the tissues of the liver, and adult worms cause bile duct tissue to deteriorate, hyperplasia, and hypertrophy (Swarup et al. 1987;Haque et al. 2011). The agrarian economy on the Indian subcontinent is severely harmed by the disease prevalence rate, which can reach 60% (DAHD Annual report 2017, 2018). Despite the lack of a precise calculation of economic losses, millions of rupees are being lost due to lower milk and meat yield and high production costs as a result of widespread usage of relatively less effective and repetitive anthelmintic medications.

Somatic homogenate preparation
Infected livers from freshly slaughtered buffaloes were utilized to collect adult liver amphistome parasites, Gigantocotyle explanatum, in a nearby slaughterhouse run by the municipal corporation of Aligarh, India. The worms were homogenized in 100 mM phosphate-buffered saline, pH 7.4, and centrifuged at 10,000 g for 20 min at 4 °C after being immediately washed three to four times in Hank's balanced salt solution (HBSS), pH 7.4, held at 37 °C and regurgitated 20-30 min.

Isolation and solubilization of tegumental surface plasma membrane
The freshly retrieved adult G. explanatum worms were rinsed 3-4 times in Hanks' balanced salt solution (HBSS), pH 7.4, maintained at 37 ± 1 °C followed by a quick rinse in HBSS containing 0.1% antibiotics (Penicillin-Streptomycin) and incubated for regurgitation for 20-30 min at 37 ± 1 °C. Afterwards, the teguments of G. explanatum worms were isolated using the membrane disrupting solution (1 g of Quillaja Bark Saponin in 100 ml of Hanks' balanced salt solution (HBSS), pH 7.8). A total 200 G. explanatum worms were taken in membrane disrupting solution and agitated on ice for 20 min and then vortexed vigorously for about 2-3 min. The filtrate is centrifuged at 15,000 g for 15 min, after which the supernatant is collected and centrifuged once more for 20 min. The resulting pellet is centrifuged after being re-suspended in a 100 mM Tris-HCl solution with a pH of 7.0 in order to collect as many membrane pellets as feasible (Rahman et al. 1981).
For the solubilization of isolated surface plasma membrane, the final precipitated membrane fractions were dissolved in a solution containing 100 mM Tris-HCl (pH 7.0) and then disintegrated using an ultrasonic disintegrator with a 5-mm probe for intervals of 10 s (Ralsonic India RR120). The sonicated samples were centrifuged at 10,000 g for 20 min after being treated with 1% Triton X-100 (v/v) at 37 °C for 3 h with gentle stirring (Sener and Yardimci 2000).

Protein estimation
Protein estimation of somatic homogenate and Triton X-100 solubilized membrane pellets were carried out by the dye binding method using bovine serum albumin as standard (Spector 1978).

Scanning electron microscopy
To assess the degree of membrane isolation, scanning electron microscopy of control and membrane isolated worms was carried out according to the method described by Shareef et al. (2014). Briefly, saponin-treated and saponin-untreated worms were fixed in 4% (w/v) glutaraldehyde in Na-cacodylate buffer for 4 h at 4 °C and washed 2-3 times in 0.1 M Na-cacodylate buffer containing 3% (w/v) sucrose followed by dehydration in the ascending grades of ethanol. The worms were mounted on stubs, sputter coated with gold, and viewed on scanning electron microscope (JEOL, JSM 6510LV) operating at 15 keV at the University Sophisticated Instrumentation Facility, Aligarh Muslim University, Aligarh (India).

Native polyacrylamide gel electrophoresis
The Davis (1964) method was used to electrophorese the solubilized membrane samples in 6% polyacrylamide gel, followed by staining. Gels were pre-incubated in a 16 mM glycylglycine solution (pH 7.4) before being moved to a substrate solution containing 0.5 mM L-glutamic acid -4-methoxy-naphthylamide (GMNA), 1 M glycylglycine, and 8 mg/ml Fast Blue RR salt at room temperature for staining. Until being photographed, gels were kept at 4 °C in the dark after being fixed in a 100 mM CuSO4 solution (Bluvshtein et al. 1999).

Purification of GGT from Gigantocotyle explanatum
The isolated membrane pellets were suspended in 7 volumes of 0.01 M Tris-HCl buffer containing 0.15 M NaCl and 0.2% Lubrol WX, homogenized at 25 °C for 2 h, and then centrifuged at 16,000 g for 60 min. Ammonium sulfate was used to achieve 50% saturation in the collected supernatant. The ammonium sulfate-precipitated pellet was suspended in 0.1 M Tris-HCl buffer, combined with an equal volume of acetone (precooled at -20 °C), and left for 20 min at 4 °C. The mixture was then centrifuged at 16,000 g for 30 min, and pellets are collected. The collected pellet was suspended in 2 volumes of 0.1 M Tris-HCl buffer containing 1% sodium deoxycholate, homogenized with constant stirring at 4 °C for 20 h, and then centrifuged at 16,000 g for 60 min. To achieve 70% saturation, the recovered supernatant was once more treated with ammonium sulfate. The resulting pellet was dialyzed in 0.01 M Tris-HCl buffer with 0.15 M NaCl, 0.2 MnCl 2 , 0.2 M CaCl 2 , and 0.2% Lubrol WX (column buffer). In order to prepare columns for affinity chromatography on Con-A Sepharose, binding buffer (column buffer containing 50 mg/ml α-methyl glucopyranoside) was diluted until it reaches a pH of 8.0. Dialyzed sample was loaded onto the column and eluted with a linear gradient of 0.05-0.2 M α-methyl glucopyranoside in column buffer at a flow rate of 0.6 ml/min (Tate and Meister 1975).

GGT assay
The GGT activity was recorded in the assay mixture, which contains the enzyme solution at a final concentration of 1 ml of 2.5 mM L-glutamyl-p-nitroanilide (GpNA), 50 mM glycylglycine, and 50 mM Tris-HCl (pH 8.0) buffer (Meister et al. 1981;Tate and Meister 1985). The assay solution was warmed to 37 °C before the enzyme is added to start the reaction. When the reaction was stopped after 10 min at 37 °C by adding 2 ml of 1.5 N acetic acid, the release of p-nitro aniline is measured at 410 nm. The enzyme activity was calculated using the p-nitroaniline molar absorption value of 8800 M −1 cm −1 at 410 nm. The amount of enzyme that releases 1 mol of p-nitroaniline each minute is considered one unit of enzyme.

SDS-PAGE
The purified samples were mixed with Laemmli's sample buffer in the ratio of 1:1 and resolved onto a 10% SDS-PAGE (Mini-PROTEAN Tetra Cell-BioRad) for 90 min at room temperature using a continuous voltage of 100 V. (Laemmli 1970). Soon after the electrophoresis, gels were rinsed 2-3 times in distilled water and immersed in staining solution (colloidal Coomassie R-250) (Dyballa and Metzger 2009) and left overnight by keeping the gel on a gel rocker system. After staining, the gel was transferred to destaining solution (10% v/v ethanol, 2% orthophosphoric acid) which was changed 1-2 times until the protein bands become clear. Gel documentation system (Bio-Rad) was used for imaging, and Image Lab 6.0.1 is employed to analyze and estimate molecular weight using a standard molecular weight marker (Bio-Rad Precision Plus).

Liquid chromatography-mass spectrometry (LC-MS)
Briefly, 50 μg of purified GeGGT was mixed with an equal volume of SDS sample buffer, resolved on an 8% SDS-polyacrylamide gel, and stained with colloidal CBBG-250 dye (Dyballa and Metzger 2009). The required band was extracted from the gels in a sterile environment before being added to a solution of 5% glacial acetic acid. Before being sent to the Central Instrumentation Facilities (CIF), University of Delhi South Campus, New Delhi, for additional processing, the sample tubes were packaged with the pre-cooled ice packs at -80 °C and sealed with Parafilm. The identified peptide sequences were BLAST compared to the previously known GGTs submitted to the protein databases (https:// www. unipr ot. org/). The discovered peptide sequences were BLAST compared to the previously known GGTs submitted to the protein databases (UniProt, NCBI).

Enzyme localization
G. explanatum adult worms were rinsed in HBSS, pH 7.4, and 0.1% antibiotics (Penicillin-Streptomycin) solution. Then, the mounted worms were immediately frozen at -20 °C, followed by sectioning (10 m) using a Leica Cryostat (Model No. CM1950). Cryostat sections were placed on glass slides, let to air dry, and then incubated for 2 h at room temperature in GMNA (2.5 mg/ml) substrate solution. All that was left out of the staining solution for control sections was the substrate (GMNA). Sections were rinsed in 0.85% saline after incubation, then fixed in 0.1 M CuSO4 solution, and rinsed once more in 0.85% saline. They were then air dried and mounted in Apathy's media (Rutenburg et al. 1969).

Kinetic analysis
In a pre-warmed assay mixture with varying concentrations of the substrate, GpNA, ranging from 1 to 6 mM, fixed concentrations of the purified GeGGT were added under the prescribed conditions and incubated at 37 °C for 15 min.
A 2 ml addition of 1.5 N glacial acetic acid stopped the process, and the GeGGT activity was measured at 410 nm. In MS Excel, the Lineweaver-Burk plot (1934) was used to plot the data after the kinetic parameters were derived using the Michaelis-Menten equation.

Determination of pH and temperature
A fixed concentration of the enzyme was incubated at 37 °C in a Tris-HCl buffer solution with a pH range of 6 to 10 to establish the pH optimal range for GeGGT.
The purified enzyme was incubated between 30 and 70 °C to achieve the optimum temperature for GeGGT. The preheated assay mixtures were added with the enzyme already incubated, and after 15 min, the GGT assay was performed.
The enzyme was incubated in a pre-warmed assay mixture at 37 °C for 10, 20, 30, and 40 min independently, followed by an enzyme assay, to assess the thermal stability of the purified enzyme at optimum temperature (37 °C).

Inhibitor treatment
One millimolar of 6-Diazo-5-Oxo Nor-isoleucine (DON) was used to treat purified GeGGT, and different time intervals between 10 and 60 min are used for incubation. The assay solution was pre-warmed after the enzyme has been incubated, and then, the activity of the enzyme was measured. In order to ensure the validity of the findings, separate controls (without DON) were run in parallel for each experiment.

Molecular modeling simulations
Using the fundamental methodology outlined by Khan et al. (2008), in silico molecule docking simulations were performed. The molecular docking simulations of the interaction between GGT and DON were carried out using the Auto Dock Vina 1.1.2 programme (Trott and Olson 2010). The RCSB Protein Data Bank was used to download the GGT crystal structure (PDB ID: 2DBU). DON's threedimensional (3D) structure was retrieved from PubChem (pubchem.ncbi.nlm.nih.gov). Using the structures of GGT and DON, a genetic algorithm was built in Auto Dock Vina to predict the probable conformations of the inhibitor that binds to the protein molecule. During the docking phase, binding, up to 8 different conformations were taken into consideration. The conformer with the lowest binding free energy was chosen for additional analysis. The tool included with Auto Dock Vina 1.1.2 was used to calculate the binding energy of docked complexes as well as the amino acid residues forming hydrogen bonds, hydrophobic interactions, and electrostatic interactions. In Discover Studio visualizer 2021, the protein-ligand interaction and its structures (2D/3D) were illustrated.

GGT activity assay
G. explanatum worm somatic fractions exhibited 0.33 U/ mg protein, but isolated tegumental membrane fractions displayed 25.35 U/mg protein GGT activity (Fig. 1). In the isolated tegumental fractions of G. explanatum, a 76.7-fold enhanced GGT activity is observed.

Isolation of surface plasma membrane
The surface plasma membrane of adult G. explanatum worms is isolated in order to study the GGT activity in its tegumental layer because whole homogenate samples of these worms exhibited very little GGT activity. To determine the level of tegumental layer isolation, the membrane isolated worms and their corresponding controls are examined under a scanning electron microscope. It is evident from the scanning electron micrographs that the control worms showed a prominent oral sucker (Fig. 2a), smoothen tegumental layers around ventral sucker (Fig. 2f), and intact tegumental infoldings with evenly distributed dome shaped papillae (Figs. 2b and 2e), whereas in membrane isolated worms, shrinkage of oral sucker (Fig. 2c) as well as shrinkage of tegumental layers around acetabulum (Fig. 2h) and the complete erosion of surface papillae from all over the surface (Figs. 2d and 2g, h) can easily be observed. Thus, the purity of membrane isolation is confirmed through scanning electron microscopy which showed fine stripping off of tegumental layer without any deep lesions or without any parenchymal damages.

Native polyacrylamide gel electrophoresis
Maximum solubilized protein was obtained by solubilizing membrane fractions with 1 percent Triton X-100 at 37 °C for 3 h, and as a result, a prominent GGT band of about 120 KDa appeared on a 6% native-PAGE (Fig. 3).

somaƟc membrane
Ge GGT acƟvity u/mg protein Fig. 1 GGT activity in total somatic homogenate and isolated membrane fractions of G. explanatum

Purification of GGT from G. explanatum
The initial GGT activity of the crude somatic extract of G. explanatum worms was 43.16 ± 2.12 U, and the specific activity was 0.33 ± 0.016 U/mg protein. Both the initial and specific activity of the enzyme are found to be greatly boosted after membrane isolation (Table 1). When the enriched membrane fractions are applied to Con-A Sepharose affinity columns and eluted using a linear gradient substrate concentration (0.05 M-0.2 M gluco pyranoside), an increasing GGT activity is observed in the subsequent eluted fractions along with the increasing substrate concentration in the elution buffer up to 0.2 M concentration (Fig. 4). The Con-A Sepharose purified GGT active fractions had a specific activity of 62.48 ± 0.79 U/mg protein and an initial activity of 31.24 ± 0.39 U. GeGGT was purified 76.81-fold with a yield of 193.8% via surface plasma membrane isolation, and 189.3-fold with a yield of 72.4% from subsequent purification on an affinity column (Table 1).

SDS-PAGE
Two polypeptide bands of 65.5 kDa and 55 kDa are visible when the purified eluent containing the GeGGT is exposed to SDS-PAGE on a 10% linear gel and overstained with colloidal Coomassie Brilliant Blue G-250 dye for protein staining. These bands represent the two subunits of the enzyme (Fig. 5).

Liquid chromatography-mass spectrometry (LC-MS)
The LCMS data of GeGGT is compared with that of B. malayi GGT, which is available in the database, because of the close phyletic distribution of G. explanatum and B. malayi. GeGGT showed sequence homologies of 26.33 and 58.33% with the light and heavy subunits of B. malayi GGT, respectively (Fig. 5).

GeGGT localization
The pattern of GGT enzyme distribution throughout the entire G. explanatum worm population was revealed by histo-enzymological analysis of GGT activity in the 10-micron cryo-cut sections. In the experimental sections, where GMNA was present, the by-product of the substrate bound with the azo dye (Fast blue B salt) and produced deep orange color in the regions of GGT activity, in contrast to the control sections where the specific substrate of GGT (GMNA) was absent, which did not produce any color when stained for GGT activity. Both the tegumental areas and the inside of the acetabulum exhibit extremely strong GGT activity. Amphistome parasite's gonadal region also exhibits some activity in addition to these areas (Fig. 6).

Kinetic analysis
The Michaelis constant (Km) and maximal velocity (Vmax) of GeGGT is estimated to be 2.43 ± 0.059 mM and 11.6 ± 0.19 U, respectively, when glycylglycine is used as an acceptor amino acid and γ-glutamyl-p-nitroanilide as substrate under standard conditions. The turn over number (Kcat) of the enzyme is calculated to be 23.78 ± 0.39 s −1 (Fig. 7).

pH optima
The optimal pH is observed at 8; however, significant enzyme activity is also recorded at pH 9 (Fig. 7), showing that the alkaline environment is favorable for the enzyme activity, a possible reflection of the adaptability to the microenvironment of the parasite inhabiting the buffalo liver.

Temperature optima
The optimum temperature is found to be 37 °C. The enzyme showed retention of 78% and 25% residual activity at 50 °C and 60 °C, respectively, showing considerable thermo-stability at higher temperatures (Fig. 7).

Incubation time
The GeGGT activity assayed for different time periods at 37 °C in order to find out the optimal incubation period for maximal enzyme activity. It is observed that the enzyme activity reached to a maximum at 15-min incubation and thereafter significantly declined within 25 min. Therefore, 15 min time period is considered optimum for GeGGT assays (Fig. 7).

Effect of inhibitor
The results of inhibitor treatment showed that 6-Diazo-5-Oxo-l-Nor isoleucine (DON) at 1 mM concentration did not inhibit GeGGT up to 10 min, while 67.6% and 91.7% inhibition is recorded at 20 min and 30 min of incubation, respectively. The complete inhibition is observed only when the enzyme is incubated up to 40 min (Fig. 8).

Molecular docking simulation
The results of the in silico analyses confirmed the binding mode of DON with GGT according to the anticipated amino acid residues (Fig. 9), where Tyr444, Asn411, Gly483, Thr391, Gly484, and Thr413 can form hydrogen bonds with the DON molecule. Given that there is an Asp190 residue close by the binding ligands, the interaction between GGT and the tested molecules is not solely hydrophilic in nature and instead plays a significant part in stabilizing them through electrostatic interaction. As a result, the hydrogenbonding serves as an anchor, strongly defining the position of the tested molecules in the binding pocket in three dimensions and promoting the electrostatic interaction of the chemical rings with the side chains of proteins.

Discussion
The present study investigates the GGT enzyme in parasitic helminth, Gigantocotyle explanatum (GeGGT), which may shed light on a previously unexplored area of tegumental biology pertaining to the GGT enzyme. The GGT enzyme from adult G. explanatum worms was successfully isolated and purified, and some of its biochemical properties, particularly the kinetic parameters, have been studied. Adult G. explanatum worms were subjected to a saponin-containing membrane disrupting solution, which resulted in the tegumental layer being delicately stripped off. Scanning electron micrographs revealed the purity of the plasma membrane separation, which was free of any lesions or physical damage. The GGT enzyme is evidently membrane-bound in this amphistome parasite as indicated by the enrichment of GGT activity by several folds observed following isolation of the surface plasma membrane. The enriched enzyme activity in the vicinity of the tegumental areas of these amphistomes was further supported by the histo-enzymological localization of GeGGT. GeGGT needs to be membrane solubilized in order to be evaluated for activity because it is a membrane-anchored enzyme. To achieve the necessary solubilization procedure, a variety of detergents and salts are being used. One percent Triton X-100 produced the most solubilized protein with the highest enzyme activity when the isolated membrane pellets were solubilized in different detergents like CHAPS, Tween-20, and Triton X-100 for various times (results not shown). It was also noticed that the rate of solubilization also affected the rate at which the enzyme penetrated the polyacrylamide gels. The Triton X-100 solubilized membrane samples readily penetrated the 6% native gels, resulting in a sharp band of about 120 kDa, while the insolubilized membrane samples did not. This may be because of the high hydrophobicity and aggregative properties of the insolubilized membrane samples, which are a typical characteristic of membrane proteins.
The amphistome enzyme GeGGT was effectively purified to an apparent homogeneous form and bound to the Con-A Sepharose. As determined by SDS-PAGE, the purified enzyme appeared to be dimeric and produced two subunits of 65.5 kDa and 55 kDa. This finding explains the typical property of the GGT enzyme, which is the dissociation of enzyme in its different subunits in the presence of SDS, heavy subunit with higher molecular weight, and light subunit with lower molecular weight. The molecular weights of the two subunits supported the 120 kDa molecular weight of the native form. The molecular weights of the two subunits were found to be different in comparison to mammalian GGT subunits (bovine liver 68 kDa and 27 kDa, rat kidney 46 kDa and 22 kDa), but fairly similar to those of A. suum GGT subunits, which were reported to be of 43 and 30 kDa (Goldberg 1980;Furukawa et al. 1983;Hussein and Walter 1996). It has been noted that the size heterogeneity of the GeGGT subunits closely resembles that of the GGT of the worm parasite A. suum. The GeGGT described in this study exhibited a difference of just 11.5 kDa, which was found to be close to A. suum GGT where a difference of 13 kDa was identified between the two subunits. Normally most GGTs published show a size heterogeneity of a minimum of 20 kDa between their two subunits. In this regard, some bacterial GGTs, such as those from E. coli and B. pumilus, only show 15 kDa and 16-17 kDa variances between their subunits, respectively (Suzuki et al. 1986;Moallic et al. 2006 According to biochemical analyses, GeGGT that had been affinity purified had the highest specific activity (62.48 U/mg protein) in comparison to somatic extract (0.33 U/mg protein), showing 189.33-fold increase in enzyme enrichment after con-A Sepharose affinity purification. GGT has also been purified from a variety of organisms, including certain helminth species like A. suum (Hussein and Walter 1996), B. malayi (Lobos et al. 1996), and S. cervi (Gupta et al. 2005a, b), while the bacterial and human GGT has been described up to its crystalline state (Suzuki et al. 1986;Okada et al. 2006;Castellano et al. 2010;West et al. 2013). The con-A purified GGT from B. subtilis (Ogawa et al. 1991) and A. suum (Hussein and Walter 1996) similarly shown somewhat comparable specific activity, 65 U/mg protein and 62.9 U/mg protein, respectively. It was shown that the specific enzyme activity dramatically increased when the concentration of the substrate, α-methyl glucopyranoside, increased up to a maximum concentration of 0.2 M in the elution buffer, during the affinity purification process of GeGGT. Shaw et al. (1978) used a substrate concentration of 0.2 M to successfully elute human liver GGT, while Furukawa et al. (1983) used a substrate concentration of 0.1 M to successfully elute bovine liver GGT.
The Michaelis constant, Km, of purified GeGGT is calculated to 2.43 ± 0.059 mM. The Km value of GeGGT is consistent with the GGTs of A. suum GGT (1.28 mM), bovine liver (0.8 mM), human kidney (0.8 mM), and B. subtilis SK 11.004 (1.73 mM) (Hussein and Walter 1996;Miller et al. 1976;Furukawa et al. 1983;Shuai et al. 2011). The pH is one of the most important parameters influencing the GGT activity. The GeGGT is pH-optimized at 8, but there is still significant enzyme activity (73%) at pH 9; nevertheless, additional increases in pH considerably (p < 0.001) reduced the enzyme activity. The enzyme activity was strongly impacted by both the acidic range and the alkaline range above optimal pH. The pH optimal of GeGGT observed in the current work is equivalent to the pH optimum of GGTs observed in various human tissues, rabbit liver, E. coli, B. pumilus, B. subtilis TAM-4, and A. suum, which were observed between the pH 8 and pH 9 (Miller et al. 1976;Shaw et al. 1978;Bagrel et al. 1981;Suzuki et al. 1986b;Hussein and Walter 1996;Sener and Yardimci 2005;Moallic et al. 2006;Abe et al. 2009).
Other biochemical characterizations revealed the optimal GeGGT activity at temperature 37 °C and incubation time of 15 min. Given that 78% and 25%, respectively, of the enzyme activity were observed after incubation at 50 °C and 60 °C, the GeGGT seems to be a remarkably thermo stable enzyme. GeGGT's thermostability is also found to be time-dependent, as the enzyme loses up to 80-90% of its initial activity after 20 min at a temperature of 37 °C. An earlier study on A. suum GGT indicated that the enzyme performed best at a temperature of 37 °C and that its activity was linear for the first 30 min of incubation (Hussein and Walter 1996). The most popular inhibitor, DON, permanently binds at the acceptor site of the enzyme and completely suppresses the GeGGT activity after about 40 min of incubation at ambient temperature. These outcomes are consistent with earlier studies on GGT inhibition, where Moallic et al. (2006) used a variety of inhibitors, including 1 mM DON for 1 h, which completely inhibited the GGT activity of B. pumilus. One millimolar DON exhibited a 67% enzyme inhibition for the suppression of human kidney GGT after 15 min of incubation (Tate and Ross 1977). The molecular docking studies also confirmed the high binding affinity of DON towards the GGT molecule.

Conclusion
The enzyme gamma-glutamyl transferase in the biliary amphistome parasite, Gigantocotyle explanatum, is a membrane-bound enzyme with significant activity. In order to deal with the enzyme, some of its hitherto unreported characteristics have also been investigated. The tegument of these parasites is a metabolically active surface, and since GeGGT is a tegumental enzyme, it may be a potential alternative pharmaceutical target (Farhat et al. 2022) in view of the increased need for alternative anthelmintic drugs or vaccine targets, as the majority of the chosen soluble proteins failed to demonstrate appreciable efficacy in field tests for the development of vaccines against these parasitic helminths.