Purification and properties of N-acetyl-β-D-glucosaminidase from bovine testicle

N-Acetyl-β-D-glucosaminidase (NAGase) from bovine testicle was purified by ammonium sulfate fractionation followed by diethylaminoethyl (DEAE)-cellulose (DEAE-32) and Sephacryl S-300 chromatography. The enzyme was purified to homogeneity as analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing gel electrophoresis (IFGE). The specific activity of the purified enzyme was 658.21 U/mg. The enzyme was a single subunit with molecular weight of 68.3 kDa and contained 3.03% sugar. The pI value was calculated to be 5.54 using IFGE. The optimal pH and temperature of the enzyme for hydrolysis of p-Nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were found to be pH 5.6 and 50°C, respectively. The kinetics results showed that the enzyme hydrolyzed pNPNAG following Michaelis-Menten with Km of 0.71 mM and Vm of 16.72 M/min at pH 5.6 and 37°C. The enzyme was stable at pH values ranging from 2 to 6.5 and at temperatures below 60°C. The activation energy was determined to be 64.19 kJ/mol.


INTRODUCTION
N-Acetyl-β-D-glucosaminidase (NAGase, EC 3.2.1.52),an intracellular lysosomal hydrolase, is widely distributed in nature (Liu et al., 2009;Keyhani et al., 1996).The enzyme not only is involved in glycosylation of glycolprotein and glycolipid biosynthesis but also plays an important role in oligosaccharide and polysaccharide chain formation and degradation.Therefore, an in-depth study of NAGase can provide a basis for life science research.Research reports have shown that NAGase has been investigated more in microbial systems and has been the focus of a large number of gene cloning and expression studies (Mamarabadi et al., 2009).NAGase has been purified from the human spleen (Robinson et al., 1968), bovine liver (Bernard, 1964) and spleen (Vehpoorte et al., 1972), and cortical granules of Xenopus eggs (Prody et al., 1985).The enzymatic characteristic of NAGase, which was obtained from rat spleen cytoplasm, has been described in detail (Dong and Hart, 1994).Using muscovy duck testicles as a source, NAGase was extracted, and some of its enzymatic properties and the effect of metal ions on enzyme activity have been determined (Wang et al., 2007).Moreover, high NAGase activity is present in rabbit semen (Farooqui and Srivastava, 1980), human sperm (Perez et al., 2007), and ascidian eggs (Koyanagi and Honegger, 2003).The activity of NAGase changes regularly in female buffalo saliva in the telocinesia of estrus, which can be used to determine ovulation time and the optimal time for artificial *Corresponding author.E-mail: zjhyfang138@aliyun.com.
Abbreviations: NAGase, N-Acetyl-β-D-glucosaminidase; pNP, p-Nitrophenyl; pNP-NAG, p-Nitrophenyl-N-acetyl-β-Dglucosaminide; pI, isoelectric point; PAGE, polyacrylamide gel electrophoresis; IFGE, isoelectric focusing gel electrophoresis; SDS, sodium dodecyl sulfate insemination to improve calving rates (Feng et al., 1997).Mouse egg and sperm lost combination capacity after the eggs were treated with NAGase (Miller et al., 1993).Additionally, NAGase can help the sperm penetrate the eggs' zona pellucida (Shur, 1982).Therefore, NAGase is relevant to the reproductive function of animals.However, there are very few studies of animal NAGase at the molecular level.In the present research, we purified NAGase from bovine testicle and studied the enzymatic characteristics to provide further details on the physiological functions of NAGase in bovines.

MATERIALS AND METHODS
Bovine testicle was purchased from Fuzhou in China.p-Nitrophenyl (pNP) was made in England.p-Nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) was purchased from the Biochemistry Lab of Shanghai Medicine Industry Academy (Shanghai China).Diethylaminoethyl (DEAE)-cellulose (DE-32) was purchased from Whatman.Sephadex G-200 was purchased from Pharmacia.Sephacryl S-300, Con A-Sepharose and standard marker proteins were purchased from Amersham.All other reagents were local products of analytical grade.The water used was re-distillated and ion-free.

Enzyme activity and protein concentration assays
Enzyme activity was determined at 37°C by following the increase in absorbance at 405 nm accompanying the hydrolysis of pNP-NAG as described by Xie et al. (2004).A 10 l aliquot of the enzyme solution was added to the reaction media (2 ml) containing 0.5 mM pNP-NAG in 0.15 M NaAc-HAc buffer (pH 5.2).After incubation for 10 min at 37°C, 2 ml of 0.5 M NaOH was added to the reaction mixture to stop the reaction.Absorption was recorded using a Beckman UV-650 spectrophotometer.The molar absorption coefficient of the product pNP is 1.73 × 10 4 M -1 cm -1 (Xie et al., 2004).One unit (U) of enzymatic activity was defined as the amount of enzyme catalyzing the formation of 1 μM pNP-NAG at the conditions denoted above.The protein concentration was measured by the method of Follin phenol with bovine serum albumin as the standard or by measuring the absorbance at 280 nm.

Enzyme purification and determination of homogeneity
Unless otherwise noted, all operations were performed at 4°C.One hundred grams of frozen bovine testicle tissue was homogenized in 250 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl and incubated for 2 h.The homogenate was centrifuged at 10, 000 g for 30 min.The supernatant was fractionated with ammonium sulfate.The fraction between 30 and 50% saturation ammonium sulfate was collected by centrifugation, dissolved in a small volume of 50 mM Tris-HCl buffer (pH 7.5), and then dialyzed against this buffer until sulfate could not be detected.
The crude preparation was concentrated by ultrafiltration and loaded onto a DEAE-cellulose column (2.5 × 20 cm) pre-equilibrated with the same buffer.The elution was carried out using a linear gradient of 0 to 1.5 M NaCl at a flow rate of 20 ml/h, and the eluate was collected at 2.5 ml per tube.The fractions with enzyme activity were pooled.The collected enzyme solution was further chromatographed on a Sephacryl S-300 gel filtration column (2.5 × 60 cm) pre-equilibrated with the same buffer containing 0.2 M NaCl.The elution was carried out at a flow rate of 20 ml/h, and eluate was collected at 2 ml per tube.The fractions with enzyme activity were pooled.The enzyme homogeneity was assessed using the methods Huang et al. 5355 of polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Determination of enzyme molecular weight and subunit number
The molecular weight was determined using gel filtration and SDS-PAGE.The gel filtration was performed on a Sephadex G-200 (2.5 × 70 cm) column with 0.01 M Tris-HCl buffer, pH 7.5, containing 0.2 M NaCl.Pepsin (PEP, 35 kDa), ovalbumin (OA, 45 kDa), bovine serum albumin (BSA, 68 kDa), rabbit phosphorylase B (RPR, 97.2 kDa), and bovine gamma globulin (BGG, 140 kDa) were used as protein standards.SDS-PAGE was performed on a discontinuous horizontal thick-layer with a stacking gel of 3% acrylamide in Tris-HCl buffer (pH 6.7) at 10 mA current and a separating gel of 12% acrylamide in Tris-HCl buffer (pH 8.9) 0.1% SDS at 20 mA current.

Determination of isoelectric point (pI)
The pI value of the enzyme was determined using isoelectric focusing electrophoresis.The electrophoresis was run on a continuous disc (5 × 100 mm) with 7% acrylamide containing ampholyte (pH 3 to 10) at 150 V for 4 h.After electrophoresis, the gel column was immersed into a 10% trichloroacetic acid (TCA) solution, and the enzyme protein band was visualized.The other gel column was sliced into 5 mm pieces.Each gel piece was immersed into 1 ml distilled water for 8 h, and the pH value was measured.

Glycoprotein assay
Concanavalin A (con A)-sepharose column was used to assess whether the enzyme was a glycoprotein.The sulfate-phenol methods introduced by Xu et al. (2005) were used to test the saccharide contents of the purified enzyme preparation.

Determination of the kinetic parameters of NAGase
The enzyme hydrolysis of pNP-NAG was studied using various concentrations of pNP-NAG.The Lineweaver-Burk plot was used to determine the kinetic parameters of the K m and V m values.

Optimal pH and stability assays
The optimal pH was determined by measuring the enzyme activities as described above at different pH values at 37°C.The pH-stability of the enzyme was monitored by incubating the enzyme in buffers with different pH values for 20 h at 4°C.A 10 l aliquot of the mixture was used for the activity assay.

Optimal temperature and thermal stability assays
The optimal temperature of the enzyme was determined by measuring the activity as described above at various temperatures at pH 5.6.The thermal stability of the enzyme was monitored by incubating the enzyme at different temperatures for 30 min.A 10 l aliquot of the mixture was used for the activity assay as described above.

RUSULTS Purification of NAGase from bovine testicle
NAGase (EC3.2.1.52)was first prepared from bovine testicle according to the method of Xie et al. (2004) until the ammonium sulfate fractionation step; the crude preparation was chromatographed by ion-exchange with DEAE-cellulose (DE-32).The elution profile is shown in Figure 1.Most of the enzymes eluted at above 0.5 M of NaCl.The activity peak (from tubes 175 to 190) overlapped with the protein peak.The active fractions were pooled and chromatographed by gel filtration with a Sephacryl S-300 column for further purification.The active fractions were pooled.The final preparation was homogeneous as determined by PAGE (Figure 2A) and SDS-PAGE (Figure 2B).The specific activity of the purified enzyme was determined to be 658 U/mg.

Determination of enzyme molecular weight and subunit number
The molecular weight of bovine testicle NAGase was determined by gel filtration on a Sephadex G-200 column, and the result is shown in Figure 3.The molecular weight of the enzyme was estimated to be 68.5 kDa.In addition, the enzyme showed a single band on SDS-PAGE (Figure 2B).The molecular mass of the enzyme subunit was estimated to be 68.3 kDa.From these results, we concluded that the enzyme is composed of a single polypeptide chain.

Determination of the isoelectric point of the enzyme
The isoelectric point of the enzyme was determined by IFGE.The result shows that the pI value of the enzyme was approximately 5.54, which is close to that of porcine semen acidic NAGase (Huang et al., 2009), and same as papaya NAGase (Chen et al., 2011).However, this value was different from that of the Bombyx mori alimentary canal (pI 4.86) (Koga et al., 1986) and Penaeus vannamei (pI 4.8) NAGases.

Glycoprotein assay
A concanavalin A (Con A) column was used to determine whether the enzyme is a glycoprotein.In this study, we found that the purified NAGase is bound to the Con A column.These results show that this enzyme contains oligosaccharides and is a glycoprotein.

Determination of the kinetic parameters of NAGase
The kinetic behavior of the enzyme during hydrolysis of pNP-NAG was studied.Under the conditions employed in the present investigation, the hydrolysis of pNP-NAG by the enzyme followed Michaelis-Menten kinetics (Figure 4).The kinetic parameters for the enzyme obtained from a Lineweaver-Burk plot showed that K m and V m were 0.71 mM and 16.72 M/min, respectively.

Effects of pH on the enzyme activity and stability
The effect of pH on the enzyme activity for the hydrolysis of pNP-NAG was determined at 37°C.The results demonstrated that the optimal catalysis reaction occurred at pH 5.6.The enzyme activity decreased rapidly at pH values either lower or higher than the optimal pH (Figure 5a).In addition, when the enzyme was incubated at different pH values for 20 h at 4°C (Figure 5b), the enzyme was stable in the pH range between 2 and 6.5.The optimal pH of bovine testicle NAGase was at pH 5.6, and this enzyme was stable at pH 3.0 to 6.0.The optimal pH of hetian chicken testicle NAGase was 5.6, and the enzyme was active at pH 3.0 to 9.2.The optimal pH of avian broilers chicken testicle NAGase was pH 5.7, and the enzyme maintained its activity at pH 4.0 to 9.2 (Jin et al., 2009).The optimal pH of muscovy duck testicle NAGase was 5.6, and the enzyme maintained its activity at pH 3.0 to 8.0 (Wang et al., 2007).The results described above indicate that the optimal pH of NAGase from the same tissue of different varieties is similar, although the tolerance to acidic and basic conditions is different.The optimal pH of human liver NAGase was 4.5, and it was stable at pH 6.5 to 8.5 (Sasaki et al., 1991).The optimal pH of crustacean NAGase was at pH 5.0 to 6.0 (Zhang et al., 2006;Huang et al., 2005).

Effects of temperature on the enzyme activity and thermal stability
The enzyme activities were measured at various temperatures to determine the effect of temperature on the enzyme activity.The results indicate that the optimal temperature was 45°C, while the activity decreased rapidly at temperatures higher or lower than the optimal temperature (Figure 6a).The enzyme was stable below 60°C and unstable above 60°C after incubation for 30 min (Figure 6a).The optimal temperature of bovine testicle NAGase was 45°C.The enzyme maintained activity at a temperature of 60°C when preincubated for 30 min.The results show that bovine testicle NAGase can be stable under the normal temperature of a bull, but it did not have full activity.It has been reported that the optimal temperature of boar semen alkaline NAGase is 50°C and that the enzyme activity remains stable at 10 to 45°C (Huang et al., 2009).The optimal temperature of hetian chicken testicle NAGase is 55°C, and it is stable at 10 to 60°C (Jin et al., 2009).The optimal temperature of papaya NAGase is 50°C, and it is stable at 30 to 45°C; however, its enzyme activity rapidly decreases at temperatures higher than 50°C (Chen et al., 2011).2), the concentration of substrate was 0.5 mM.(b) pH stability: the enzyme (15.6 g/ml) was incubated at different pH buffers of Gly-HCl (pH 2.5 to 4.0), NaAc-HAc (pH 4.0 to 5.8), Na 2 HPO 4 -NaH 2 PO 4 (pH 5.8 to 8.0) and Gly-NaOH (pH 8.0 to 10.6) for 20 h at 4°C.A 10-l aliquot of the mixture was used for the activity assay.

Enzyme activation energy
The activation energy (E a ), which is the energy produced when reactants (initial state) change into intermediate products (transition state), can reflect the degree of an  15.6 g/ml) was incubated in 0.15 M NaAc-HAc buffer (pH 5.2) at different temperature for 30 min.A 10 l-aliquot of the mixture was used for the activity assay at 37°C.enzymatic reaction.The E a was assayed by measuring the maximum velocity of the enzymatic hydrolysis of pNP-NAG, and the value of E a was calculated from the plot of log V m versus 1/T to be 64.19 kJ/mol, which is different for a variety of organisms.It has been reported that the E a values of NAGase from pig semen alkaline (Huang et al., 2008), pig semen acid (Huang et al., 2009), muscovy duck testicle (Wang et al., 2007), hetian chicken testicle and avian broilers testicle (Jin et al., 2009) are 88.73, 41.70, 69.05, 85.74 and 73.47 kJ/mol, respectively.The degree of catalytic reaction was as follows: pig semen alkaline > hetian chicken testicle > avian broilers testicle > muscovy duck testicle > bovine bull testicle > pig semen acid.

DISCUSSION
N-Acetyl-β-D-glucosaminidase, belonging to the glycolside hydrolase family, has the function of degrading N-Acetyl-β-D-glucosamine, which is connected with β-1,4glycosidic non-restored glycoconjugates in organisms.We found that bovine testicle has a high activity of NAGase, which is directly related to the reproductive activity of male cattle.Therefore, it is of significant value in theory and application to study the enzyme.Bovine testicle NAGase is a sugar protein composed of a single polypeptide chain that weighs 68.3 kD.Most bacterial NAGases are monomeric enzymes with molecular weights greater than 150 kD.However, most of the fungal NAGases are homodi- dimers with molecular weights between 110 and 150 kD.Most animal NAGases are less than 150 kD, such as the krill epidermal NAGase, which is a single subunit enzyme with a molecular weight of 120 kD, and the krill visceral NAGase is a 150 kD single subunit enzyme (Peters et al., 1998).Scylla serrata NAGase is composed of two homotypic subunits, and its molecular weight is 132 kD (Zhang et al., 2006).Bombyx mori digestive tract NAGase is composed of two special subunits with molecular weights of 67.5 and 57.5 kD (Koga et al., 1986).Manduca sexta NAGase has a molecular weight of 62 kD (Zen et al., 1996).Helicoverpa armigera pupal NAGase has a molecular weight of 89.3 kD (Huang et al., 2005).It was recently found that there are three types of lysosomal NAGases (HexA, HexB, and HexS).All three are dimers composed of α-and β-subunits.HexA is a heterotypic dimer, while HexB is homodimer (Mark et al., 2003).The molecular weight of bovine liver NAGase is 50 kD (Bernard, 1964).NAGase A and B are purified from bovine spleen, with NAGase B having only one subunit with a molecular weight of 140 kD (Vehpoorte, 1972).Pig semen alkaline NAGase has only one subunit, and its molecular weight is 58.03 kD (Huang et al., 2008).
NAGase is a glycoprotein in bovine testicle.It has been shown that Antarctic krill NAGase has two types; one is a glycoprotein and the other is not (Peters et al., 1998).
NAGases have different biological functions in different organisms.The lysosomes of vertebrate contain NAGase, which participates in the hydrolysis of glycoprotein, ganglioside, and osamine.It is also related to the differentiation, recognition, and adhesion of somatic and germ cells (Slamova et al., 2010).Bacterial and insect NAGases are mainly involved in chitin degradation (Liu et al., 2009;Keyhani et al., 1996;Mamarabadi et al., 2009).Crustacean NAGase is related to chitin metabolism, and it participates in the molting process (Xie et al., 2004).NAGase in plants is related to glycan metabolism and defense functions (Chen et al., 2011).NAGase in mammals is mainly related to the synthesis and degradation of glycan in glycoproteins and is involved in the physiological process of sperm and egg binding (Miller et al., 1993;Shur and Hall, 1982).Through the study of NAGase characteristics from bovine testicle, we provided a theoretical basis to further study the enzyme's physiological function in bull reproduction.This study also provided a theoretical foundation and technical reference for related enzyme research on mammalian fertilization processes.

Figure 3 .
Figure 3. Measurement of the molecular weight of the enzyme on a Sephadex G-200 gel filtration.•, Standard proteins; ○, purified NAGase.

Figure 6 .
Figure 6.Effects of temperature on bovine testicle NAGase activity.(a) Optimal temperature, (b) thermal stability of the enzyme.The enzyme (15.6 g/ml) was incubated in 0.15 M NaAc-HAc buffer (pH 5.2) at different temperature for 30 min.A 10 l-aliquot of the mixture was used for the activity assay at 37°C.

Figure 4 .Figure 5 .Figure 6 .
Figure 4. Michaelis-Menten kinetics for the hydrolysis of pNP-NAG by bovine testicle NAGase.Conditions were 0.15 M NaAc-HAc buffer (pH 5.2) and different concentrations of pNP-NAG at 37 °C.The enzyme concentration was 15.6 g/ml.The inset is the Lineweaver-Burk plot used to determine the kinetic parameters of K m and V m values.