enzymatic characterizations and activity regulations of n-acetyl-β-d-glucosaminidase from the...

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Journal of Bioscience and BioengineeringVOL. xx No. xx, 1e5, 2013

Enzymatic characterizations and activity regulations of N-acetyl-b-D-glucosaminidase from the spermary of Nile tilapia (Oreochromis niloticus)

Wei-Ni Zhang,1,z Ding-Ping Bai,1,z Yi-Fan Huang,1 Chong-Wei Hu,1 Qing-Xi Chen,2 andXiao-Hong Huang1,*

University Key Lab for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fujian Agriculture and Forestry University, Fuzhou350002, China1 and School of Life Sciences, Xiamen University, Xiamen 361005, China2

Received 11 May 2013; accepted 29 July 2013Available online xxx

* CorrespondE-mail add

z The first twAbbreviatio

MichaeliseMeacrylamide gelpNP-NAG, p-n

1389-1723/$http://dx.doi

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N-Acetyl-b-D-glucosaminidase (NAGase) is proved to be correlated with reproduction of male animals. In this study,enzymatic characterizations of NAGase from spermary of Nile tilapia (Oreochromis niloticus) were investigated in orderto further study its reproductive function in fish. Tilapia NAGase was purified to be PAGE homogeneous by the followingtechniques: (NH4)2SO4 fractionation (40e55%), DEAE-cellulose (DE-32) ion exchange chromatography, Sephadex G-200gel filtration and DEAE-Sephadex (A-50). The specific activity of the purified enzyme was 4100 U/mg. The enzyme mo-lecular weight was estimated as 118.0 kD. Kinetic studies showed that the hydrolysis of p-nitrophenyl-N-acetyl-b-D-glucosaminide (pNP-NAG) by the enzyme followed MichaeliseMenten kinetics. The MichaeliseMenten constant (Km)and maximum velocity (Vm) were determined to be 0.67 mM and 23.26 mM/min, respectively. The optimum pH andoptimum temperature of the enzyme for hydrolysis of pNP-NAG was to be at pH 5.7 and 55�C, respectively. The enzymewas stable in a pH range from 3.3 to 8.1 at 37�C, and inactive at temperature above 45�C. The enzyme activity wasregulated by the following ions in decreasing order: Hg2D > Zn2D > Cu2D > Pb2D > Mn2D. The IC50 of Cu2D, Zn2D and Hg2D

was 1.23, 0.28, and 0.0027 mM, respectively. However, the ions LiD, NaD, KD, Mg2D and Ca2D had almost no influence onenzyme activity. In conclusion, the enzymatic characterizations of NAGase from tilapia were special to the other animals,which were correlated with its living habit; besides, CuSO4 and ZnSO4 should used very carefully as insecticides in tilapiacultivation since they both had strong regulations on the enzyme.

� 2013, The Society for Biotechnology, Japan. All rights reserved.

[Key words: N-Acetyl-b-D-glucosaminidase; Kinetics; Activity regulation; Tilapia; Reproduction]

N-Acetyl-b-D-glucosaminidases (EC3.2.1.52, NAGases), belongingto glycosyl hydrolase families, cleave terminal N-acetyl-b-D-glucos-amine (NAG) residues from the nonreducing end of N-acetyl-b-D-glucosaminidases (1,2). NAGases arewidely distributed amongmosttypes of living organisms and have numerous biological functions.Bacterial NAGase has a crucial physiological role in cell wall recy-cling, even in altering the function of the host cell (3). In fungi, it is akey enzyme in the chitinolytic system in fungi growth (4) andbiocontrol against some plant pathogens (5). In plants, NAGase isproved to be employed in the seeds germination (6) and implicatedin fruits ripening (7). In animal field, numerous physiological func-tions of NAGase have been identified. For example, N-acetyl-b-D-hexosaminidases A existing in humans, plays a role in the brain andspinal cord. Lacking of this enzyme activity, leads to neurons disease

ing author. Tel.: þ86 591 83851490; fax: þ86 591 83758852.ress: [email protected] (X.-H. Huang).o authors contributed equally to this work.ns: IC50, inhibitor concentrations leading to 50% activity loss; Km,nten constant; NAGase, N-acetyl-b-D-glucosaminidase; PAGE, poly-electrophoresis; PBS, sodium phosphate buffer; pNP, p-nitrophenyl;

itrophenyl-N-acetyl-b-D-glucosaminide; Vm, maximum velocity.

e see front matter � 2013, The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2013.07.014

this article in press as: Zhang, W.-N., et al., Enzymatic characermary of Nile tilapia (Oreochromis niloticus), J. Biosci. Bioeng

(8). In insects, the enzyme is important in the turnover of the chitinexoskeleton (9). However, some identified functions of NAGase fromanimals are correlated with reproduction of male animals. SpermNAGase from human (10) and Drosophila melanogaster (11) partici-pates in the penetration of zona pellucid, and is involved in spermprimary binding to the egg extracellular envelope. The enzymereleased from ascidian egg during fertilization, is thought to blockpolyspermy (12).

Nile tilapia (Oreochromis niloticus) is one of the most importantfarmed fish in China. Catches of tilapia were about 1332 kilo tons in2010, accounting for 52% of the world’s production (FAO, 2010).NewGIFT Nile tilapia is a new strain selected fromGIFT (GeneticallyImproved Farmed Tilapia) tilapia by Shanghai Fishery University(China). It has been certified as a desirable strain by the NationalCertification Committee ofWild and Bred Varieties in January 2006,and announced by the Ministry of Agriculture to expand in Chinabecause of its good growth performance (13). However, there arevery few reports about NAGase from this fish, and its function re-mains unclear. In order to further study the reproduction functionof NAGase in fish, we choose New GIFT Nile tilapia as the material.In this article, we describe the enzymatic characterization and itsactivity regulation of NAGase from the spermary of New GIFT Niletilapia.

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terizations and activity regulations of N-acetyl-b-D-glucosaminidase., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.07.014

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http://dx.doi.org/10.1016/j.jbiosc.2013.07.014




2 ZHANG ET AL. J. BIOSCI. BIOENG.,

MATERIALS AND METHODS

Biological materials New GIFT Nile tilapia with an average body mass of0.6 kg and length of 0.24 m were purchased from the local market at Fuzhou City,China. The fish were kept in ice and transported to the research laboratory within1 h. The fish were eviscerated, and the spermaries were separated and cleaned withcold deionized water.

Reagents p-Nitrophenyl-N-acetyl-b-D-glucosaminide (pNP-NAG) was pur-chased from the Biochemistry Laboratory of Shanghai Medicine Industry Academy(China). p-Nitrophenyl (pNP) was made in England. DEAE-cellulose (DE-32) wasfrom Whatman. Sephadex G-200 and DEAE-Sephadex (A-50) were Pharmacia(Sweden) products. All other reagents were local products of analytical grade. Thewater used was re-distilled and ion free.

Purification of crude extract NAGase was prepared from spermary of NewGIFT tilapia by extractionwith 0.01M TriseHCl buffer (pH 7.5) containing 0.2MNaClat 4�C for 3 h. The mixture was centrifuged at 30,000 �g for 30 min at 4�C using aBeckman refrigerated centrifuge (USA) to remove the tissue debris. The supernatantwas collected and referred to as the crude extract.

NAGase purification and determination of enzyme homogeneity Thecrude enzyme extract was subjected to ammonium sulfate fraction, and the pre-cipitate in the 40e55% saturation rangewas collected by centrifugation at 30,000�gfor 30 min at 4�C. The precipitate was dissolved in a minimal volume of 0.01 MTriseHCl buffer (pH 7.5) and dialyzed at 4�C against this buffer. The enzyme dialy-zate was then chromatographed on a DEAE-cellulose (DE-32) column (2.5 � 30 cm)pre-equilibratedwith 0.01M TriseHCl buffer (pH 7.5). The columnwas elutedwith alinear gradient of 0e1 M NaCl at a flow rate of 0.2 mL/min and fraction volume of4 mL per tube. Protein concentration and enzyme activity were measured, respec-tively. Fractions showing NAGase activities were pooled. Active fractions werepooled and then applied onto a Sephadex G-200 column (2.5 � 60 cm), which waspre-equilibrated with 0.01 M TriseHCl buffer (pH 7.5) containing 0.2 M NaCl. Thecolumn was eluted with the same buffer with flow rate of 0.2 mL/min and fractionvolume of 4 mL per tube. The active fractions were pooled and dialyzed against0.01 M TriseHCl buffer (pH 7.5) and were condensed by the polyethylene glycol20,000. The condensed sample was loaded onto DEAE-Sephadex (A-50) column(2.5� 20 cm) pre-equilibratedwith 0.01M TriseHCl buffer (pH 7.5). The columnwaseluted with a linear gradient of 0e0.5 M NaCl at a flow rate of 0.2 mL/min andfractions of 4 mL were collected. Fractions showing NAGase activities were pooled.Pooled fraction were all dialyzed against 0.01 M TriseHCl buffer (pH 7.5) and thenstored at �20�C for further research. All purified processes were performed in achromatographic ice cuber at temperature of 4�C. The final preparation was deter-mined to be homogeneous by polyacrylamide gel electrophoresis (PAGE).

Determination of the protein concentration and assay of enzymeactivity The protein concentration was measured by the method of Lowryet al. (14) using bovine serum albumin (BSA) as a standard. Enzyme activity wasdetermined at 37�C by following the increase in optical density at 405 nmaccompanying the hydrolysis of the substrate pNP-NAG with the molar absorptioncoefficient of 1.73 � 104 M�1 cm�1 (15). A portion of 10 mL of enzyme solutions(8 mg/mL) was added to the reaction medium (2.0 mL) containing 0.2 mM pNP-NAG in 0.1 M NaAceHAc buffer (pH 5.7). After reaction for 10 min at 37�C, 2 mLof 0.5 M NaOH was added into the mixture to stop the reaction. Absorption wasrecorded using a Beckman UV-650 spectrophotometer (Beckman, USA). One unit(U) of enzymatic activity was defined as the amount of enzyme catalyzing thehydrolysis of pNP-NAG to form 1 mM of pNP per min at 37�C.

Molecular weight determination The molecular weight of the nativeenzyme was determined using gel filtration on Sephadex G-200. A Sephadex G-200gel filtration column (2.5 � 60 cm) was pre-equilibrated and eluted with 0.01 MTriseHCl buffer (pH 7.5) containing 0.2 M NaCl. Chymotrypsin (Chy, 25 kD), oval-bumin (OA, 44.3 kD), bovine serum albumin (BSA, 66.4 kD), bovine intestine alkalinephosphatase (BIALP, 115 kD), and bovine gamma globulin (BGG, 169 kD) were usedas standard proteins.

Optimum pH and pH stability The optimum pH was determined bymeasuring activities as described above at different pH values (range of pH valuesfrom 3.6 to 10.6) at 37�C. The pH stability of the enzyme was monitored by incu-bating the enzyme in different pH buffers (range of pH values from 2.1 to 10.6) for24 h at 4�C before enzyme activity was measured. An amount of 10 mL of enzymesolutions (8 mg/mL) was used for activity assay.

TABLE 1. Summary of the purifica

Purification steps Total activity (U) Total protein (mg)

Filtrate 421540 16754(NH4)2SO4 (40e55%) 324164 1508DEAE-cellulose (DE-32) 249973 278Sephadex G-200 150490 113DEAE-Sephadex (A-50) 45105 11

Please cite this article in press as: Zhang, W.-N., et al., Enzymatic characfrom the spermary of Nile tilapia (Oreochromis niloticus), J. Biosci. Bioeng

Optimum temperature and thermal stability The optimum temperature ofthe enzyme was determined bymeasuring the activity as described above at varioustemperatures (range of temperatures from 5�C to 80�C) at pH 5.7. The thermalstability of the enzyme was monitored by incubating the enzyme at different tem-peratures (range of temperatures from 5�C to 80�C) for 30 min before the enzymeactivity was measured. A 10 mL aliquot of the enzyme was used for the activity assayas described above.

Regulations of some metal ions on enzyme activity Experiments wereperformed in the standard assay system described earlier with various metal ioncompounds, such as LiCl, NaCl, KCl, MgCl2, CaCl2, MnCl2, Pb(NO3)2, CuSO4, ZnSO4,HgCl2 with final concentrations of 0.2, 2, 20 mM at 37�C for 10 min. Enzyme activitywas measured and expressed as percentage of the activity without added cationsunder the same conditions.

Assay of kinetic parameters of enzyme The activity was assayed withdifferent final concentrations of pNP-NAG ranging from 0.1 to 0.4 mM at pH 5.7 and37�C. The final enzyme concentration for the assay was 8 mg/mL. The respectivekinetic parameters including the MichaeliseMenten constant (Km) and maximumvelocity (Vm) were evaluated by plotting the data on a LineweavereBurk double-reciprocal graph.

RESULTS AND DISCUSSION

Purification of NAGase Purification of NAGase from sper-mary of New GIFT Nile tilapia was summarized in Table 1.(NH4)2SO4 precipitation was usually an initial step to removeother proteins in the crude extract. Wang et al. (16) found that(NH4)2SO4 precipitation (30e70%) of trypsin from hybrid tilapiaintestine resulted in a 2.7-fold increase in specific activity withthe yield of 71.4%. In purification of NAGase from the spermary ofNew GIFT Nile tilapia, an increase of 8.6-fold was obtained withthe yield of 76.9% by (NH4)2SO4 precipitation (40e55%),suggesting the removal of more other proteins in the crudeextract. Then, the protein was subjected to gel filtration on aDEAE-cellulose (DE-32) column (Fig. 1A), leading to an increase inpurity by 36.0-fold with a recovery of 59.3%. Pooled active DE-32fractions were further purified using a Sephadex G-200 column(Fig. 1B), leading to an increase in purity by 53.3-fold with arecovery of 35.7%. Then pooled active fractions of Sephadex G-200 were further purified with a DEAE-Sephadex (A-50) column(Fig. 1C). This step resulted in a considerable increase in thespecific activity by 164.0-fold with a recovery of 10.7%, and aspecific activity of 4100 U/mg protein was obtained. The finalpreparation was determined to be homogeneous by PAGE (Fig. 2).Therefore purification to homogeneity of NAGase from thespermary of New GIFT Nile tilapia was achieved by (NH4)2SO4fractionation (40e55%), DEAE-cellulose (DE-32) ion exchangechromatography, Sephadex G-200 gel filtration, and DEAE-Sephadex (A-50) chromatography.

Molecular weight of enzyme Molecular weight of tilapiaNAGase was determined by gel filtration on a Sephadex G-200column (Fig. 3). And it was estimated to be 118.0 kD, which wassimilar to that of NAGase from carp blood (122.0 kD) (17).Molecular weight of NAGase from prawn (Penaeus vannamei),green crab (Scylla Serrata), scallop (Patinopecten yessoensis),cabbage butterfly (Pieris rapae) was 105.0 kD (18), 132.0 kD (19),56.0 kD (20), 106.0 kD (21), respectively. The relationshipbetween molecular weight of this enzyme from differentorganisms and its property and function deserve further research.

tion of NAGase from tilapia.

Specific activity (U/mg) Purification (fold) Yield (%)

25 1 100215 8.6 76.9899 36.0 59.3

1332 53.3 35.74100 164.0 10.7


FIG. 1. Column chromatography of NAGase from tilapia on DEAE-cellulose (DE-32) (A),Sephadex G-200 (B) and DEAE-Sephadex (A-50) (C). Solid circles, enzyme activity;hollow circles, protein concentration.

FIG. 3. Measurement of molecular weight of tilapia NAGase by Sephadex G-200 gelfiltration. Hollow circles, standard proteins (BGG, bovine gamma globulin; BIALP,bovine intestine alkaline phosphatase; BSA, bovine serum albumin; OA, ovalbumin;CHy, chymotrypsin); solid circle, NAGase.

FIG. 2. Electrophoresis of purified NAGase from tilapia determined by PAGE.

VOL. xx, 2013 CHARACTERIZATIONS OF NAGASE FROM NILE TILAPIA 3


Optimum pH and pH stability The effect of pH on enzymeactivity was determined over a pH range of 3.6e10.6. And themaximal activity of the enzymewas observed at pH 5.7 (Fig. 4). Theoptimum pH of NAGase from tilapia was similar to those NAGasefrom prawn (pH 5.2) (18), green crab (pH 5.6) (19), and river crab(pH 5.5) (22). When the test pH value was higher or lower thanthe optimum pH, enzyme activity decreased rapidly (Fig. 4). Thereason is that tilapia NAGase suffers irreversible denaturation invery acidic or alkaline conditions just like most enzymes.

For pH stability, tilapia NAGase was very stable in a broad pHrange of 3.3e8.1. A sharp decrease of enzyme activity was foundwhen the test pH value was higher or lower than the stable pH(Fig. 4). After incubating at pH 10.0, the NAGase lost all the enzymeactivity. NAGase from tilapia was active at both acidic and alkalinepH, while NAGase from carp blood performed an alkaline enzyme

FIG. 4. Optimum pH and pH stability of the purified tilapia NAGase (0.1 M citricacideeNa2HPO4 buffer for pH values 2.1e3.6, 0.1 M NaAceHAc buffer for pH values3.6e5.7, 0.1 M PBS buffer for pH values 5.7e7.8, 0.1 M TriseHCl buffer for pH values7.8e9, 0.1 M Na2CO3eNaHCO3 buffer for pH values 9e10.6). Hollow circles, pH opti-mum; solid circles, pH stability.


FIG. 5. Optimum temperature and thermal stability of the purified tilapia NAGase.Hollow circles, temperature optimum; solid circles, thermal stability.

FIG. 6. Effects of Mn2þ, Cu2þ, Zn2þ, Hg2þ on the activity of NAGase from tilapia.

4 ZHANG ET AL. J. BIOSCI. BIOENG.,

(7.0e11.0) (17), NAGase from scallop performed an acidic enzyme(3.5e5.5) (20). This may be correlated with the wider adaptabilityof tilapia to the environment.

Optimum temperature and thermal stability The effect oftemperature on enzyme activity was determined by assayingenzyme activity at different temperatures (Fig. 5). NAGase fromtilapia was active at temperatures from 40�C to 65�C and had anoptimum at 55�C. Tilapia NAGase almost lost all the enzymeactivity above 70�C, possibly due to thermal denaturation. Theoptimum temperature of NAGase from tilapia was higher thanthose from other aquatic animals: prawn (45�C) (18), scallop(45�C) (20), river crab (45�C) (22), and green crab (50�C) (19),what coincided with the higher optimum habitat temperature oftilapia. For thermal stability, the purified NAGase was highlystable below 45�C, and the activity sharply decreased above 45�C(Fig. 5).

Regulation of some metal ions on enzyme activity Activityregulation of metal ions on NAGase from tilapia was shown inTable 2. It had been testified that the acid radicals (SO4

2�, Cl�,NO3

�) did not influence the enzyme activity, so the effects ofthese salts were due to the metal ions. Enzyme activity was notaffected by the presence of Liþ, Naþ, Kþ and Mg2þ ions up toconcentrations of 20 mM, which indicated that these metal ionsshowed almost no influence on NAGase from tilapia. Therefore,the enzyme was fairly salt tolerant. Similar results have beenreported from green crab (19), and river crab (22). While slightinhibition was observed by Ca2þ at the concentration of 20 mM,which was different from its influence on NAGase from prawn(18) and green crab (19). It could be summarized that even the

TABLE 2. Regulations of various metal ions on the activity of purified NAGase fromtilapia.

Metal ions Relative activity (%)

0.2 mM 2 mM 20 mM

Liþ 101 100 97Naþ 101 99 89Kþ 100 101 89Mg2þ 100 102 99Ca2þ 103 99 82Mn2þ 92 88 50Pb2þ 99 69 6Cu2þ 91 29 0Zn2þ 58 13 0Hg2þ 0 0 0


same ion had different influence on the same enzyme fromdifferent organisms.

However, some bivalent metal ions present in the same con-centration range (0.2e20 mM) had a relatively moderate (e.g.,Mn2þ and Pb2þ) to quite strong regulation (Cu2þ, Zn2þ, Hg2þ). Cu2þ,Zn2þ, Hg2þ inhibited enzyme activity by 9%, 42%, and 100%,respectively, at a concentration as lowas 0.2mM. The IC50 (inhibitorconcentrations leading to 50% activity loss) of Cu2þ, Zn2þ and Hg2þ

was 1.23, 0.28, and 0.0027 mM, respectively (Fig. 6). These threemetal ions also strongly inhibited activity of NAGase from cabbagebutterfly, the IC50 of Cu2þ, Zn2þ and Hg2þ was 0.8, 2.0, and0.015 mM (21). Heavy metal ions generally inhibit enzymes, theyoften make enzymes denaturation. Among heavy metal ions Hg2þ

is the strongest inhibitor. CuSO4 and ZnSO4 are often used to killprotozoon parasite in aquaculture. Since they both could stronglyinhibit enzymes of aquatic animals (16,18,19,22), they should usedvery carefully as insecticides. Inhibitory kinetics of CuSO4 andZnSO4 on tilapia NAGase deserve further research.

FIG. 7. LineweavereBurk plot for the determination of Km and Vm for NAGase fromtilapia on the hydrolysis of pNP-NAG. Conditions were 0.1 M NaAceHAc buffer (pH 5.7)with different concentrations of pNP-NAG at 37�C. The concentration of enzyme was8 mg/mL.


VOL. xx, 2013 CHARACTERIZATIONS OF NAGASE FROM NILE TILAPIA 5

Kinetic studies NAGase from tilapia hydrolyzed pNP-NAGobeying MichaeliseMenten equation. Kinetic constants for pNP-NAG hydrolysis by the enzyme were determined using Line-weavereBurk double-reciprocal graph (Fig. 7). Km and Vm of theNAGase were calculated to be 0.67 mM and 23.26 mM/min,respectively.

ACKNOWLEDGMENTS

The present investigation was supported by Science and Tech-nology Major Project of Fujian Province (no. 2012N0002).

References

1. Nguyen, H.-A., Nguyen, T.-H., K�ren, V., Eijsink, V.-G.-H., Haltrich, D., andPeterbauer, C.-K.: Heterologous expression and characterization of an N-acetyl-b-D-hexosaminidase from Lactococcus lactis ssp. lactis IL1403, J. Agric.Food Chem., 60, 3275e3281 (2012).

2. Slamova, K., Bojarova, P., Petraskova, L., and Kren, V.: b-N-acetylhex-osaminidase: what’s in a name.? Biotechnol. Adv., 28, 682e693 (2010).

3. Sheldon, W.-L., Macauley, M.-S., Taylor, E.-J., Robinson, C.-E., Charnock, S.-J.,Davies, G.-J., Vocadlo, D.-J., and Black, G.-W.: Functional analysis of a group Astreptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a b-N-acetylglucosaminidase and not a hyaluronidase, Biochem. J., 399, 241e247(2006).

4. Rast, D.-M., Horsch, M., Furter, R., and Gooday, G.-W.: A complex chitinolyticsystem in exponentially growing mycelium of Mucor rouxii: properties andfunction, J. Gen. Microbiol., 137, 2797e2810 (1991).

5. Mamarabadi, M., Jensen, D.-F., and Lübeck, M.: An N-acetyl-b-D-glucosami-nidase gene, cr-nag1, from the biocontrol agent Clonostachys rosea is up-regulated in antagonistic interactions with Fusarium culmorum, Mycol. Res.,113, 33e43 (2009).

6. Oikawa, A., Itoh, E., Ishihar, A., and Iwamura, H.: Purification and charac-terization of b-N-acetylhexosaminidase from maize seedlings, J. Plant Physiol.,160, 991e999 (2003).

7. Jagadeesh, B.-H., Prabha, T.-N., and Srinivasan, K.: Activities of glycosidasesduring fruit development and ripening of tomato (Lycopersicum esculantum L.):implication in fruit ripening, Plant Sci., 166, 1451e1459 (2004).

8. Lemieux, M.-J., Mark, B.-L., Cherney, M.-M., Withers, S.-G., Mahuran, D.-J.,and James, M.-N.: Crystallographic structure of human b-hexosaminidase A:interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis,J. Mol. Biol., 359, 913e929 (2006).


9. Hogenkamp, D.-G., Arakane, Y., Kramer, K.-J., Mutukrishnan, S., andBeeman, R.-W.: Characterization and expression of the b-N-acetylhex-osaminidase gene family of Tribolium castaneum, Insect Biochem. Mol. Biol., 38,478e489 (2008).

10. Zitta, K., Wertheimer, E.-V., and Miranda, P.-V.: Sperm N-acetylglucosami-nidase is involved in primary binding to the zona pellucida, Mol. Hum. Reprod.,12, 557e563 (2006).

11. Cattaneo, F., Pasini, M.-E., Intra, J., Matsumoto, M., Briani, F., Hoshi, M., andPerotti, M.-E.: Identification and expression analysis of Drosophila mela-nogaster genes encoding b-hexosaminidases of the sperm plasma membrane,Glycobiology, 16, 786e800 (2006).

12. Lambert, C., Goudeau, H., Franchet, C., Lambert, G., and Goudeau, M.:Ascidian eggs block polyspermy by two independent mechanisms: one at theegg plasma membrane, the other involving the follicle cells, Mol. Reprod. Dev.,48, 137e143 (1997).

13. Li, S.-F., He, X.-J., Hu, G.-C., Cai, W.-Q., Deng, X.-W., and Zhou, P.-Y.:Improving growth performance and caudal fin stripe pattern in selected F6-F8generations of GIFT Nile tilapia (Oreochromis niloticus L.) using mass selection,Aquaculture Res., 37, 1165e1171 (2006).

14. Lowry, O.-H., Rosebrough, N.-J., Farr, A.-L., and Randall, R.-J.: Protein mea-surement with the folin phenol reagent, J. Biol. Chem., 193, 256e275 (1951).

15. Chen, Q.-X., Zhang, W., Zheng, W.-Z., Zhao, H., Yan, S.-X., Wang, H.-R., andZhou, H.-M.: Kinetics of inhibition of alkaline phosphatase from green crab(Scylla serrata) by N-bromosuccinimide, J. Protein Chem., 15, 345e350 (1996).

16. Wang, Q., Gao, Z.-X., Zhang, N., Shi, Y., Xie, X.-L., and Chen, Q.-X.: Purificationand characterization of trypsin from the intestine of hybrid tilapia (Oreochro-mis niloticus � O. aureus), J. Agric. Food Chem., 58, 655e659 (2010).

17. Ueno, R. and Yuan, C.-S.: Purification and properties of neutral beta-N-ace-tylglucosaminidase from carp blood, Biochim. Biophys. Acta, 1074, 79e84(1991).

18. Xie, X.-L., Chen, Q.-X., Lin, J.-C., and Wang, Y.: Purification and some prop-erties of b-N-acetyl-D-glucosaminidase from prawn (Penaeus vannamei), Mar.Biol., 146, 143e148 (2004).

19. Zhang, J.-P., Chen, Q.-X., Wang, Q., and Xie, J.-J.: Purification and someproperties of b-N-acetyl-D-glucosaminidase from viscera of green crab (Scyllaserrata), Biochemistry (Moscow), 71, S55eS59 (2006).

20. Sakai, T., Nakanishi, Y., and Kato, I.: Purification and characterization of beta-N-acetyl-D-hexosaminidase from the mid-gut gland of scallop (Patinopectenyessoensis), Biosci. Biotechnol. Biochem., 57, 965e968 (1993).

21. Shi, Y., Jiang, Z., Han, P., Zheng, G.-X., Song, K.-K., and Chen, Q.-X.: Purifi-cation and some properties of b-N-acetyl-D-glucosaminidase from the cabbagebutterfly (Pieris rapae), Biochimie, 89, 347e354 (2007).

22. Huang, X.-H., Chen, H.-H., and Huang, Y.-F.: Preliminary study on isolation,purification and some properties of the b-N-acetyl-D-glucosaminidase fromEriocheir sinensis, Acta Hydrobiol. Sin., 31, 563e569 (2007).


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