Acta Entomologica Sinica, August 2005, 48 (4) : 498-502 ISSN 045426296 N2 2 2D2 1, 2, 1, 2, 1 3 (1.,, 350002 ; 2.,, 361005) : Helicoverpa armigera, Sephadex G2200 DEAE232, N2 2 2D2 2 678179 UΠmg 2 N2 2 2D2 (pnp2 2D2GlcNAc), : ph 5163, 55 ph 4 8, ph > 8 ; 50 30 min,, 50,, K m 0116 mmolπl, V m 10173 mol L - 1 min - 1 pnp2 2D2GlcNAc 66124 kjπmol : ; N2 2 2D2 ; ; ; : Q965 : A : 045426296 (2005) 0420498205 Isolation, purification and partial characterization of the 2N2acetyl2D2 glucosaminidase from the pupae of Helicoverpa armigera HUANG Xiao2Hong 1, CHEN Qing2Xi 2, YOU Min2Sheng 1, WANG Jun 2, GUAN Xiong 1 3 ( 11 Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China ; 21 Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China) Abstract: 2N2acetyl2D2glucosaminidase ( EC31211152) was purified from the pupae of ammonium sulfate fractionation and chromatography on Sephadex G2200 and DEAE2cellulose. Helicoverpa armigera by The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis. It was found that the specific activity of the enzyme was 2 678179 UΠmg. The optimal ph value was 5163 and the optimal temperature 55. The enzyme was stable in the ph ranges of 4 to 8 under 37. The enzyme follows typical Michaelis2Menten kinetics for the hydrolysis of pnp2 2D2GlcNAc and the K m and V m values were 0116 mmolπl and 10173 mol L - 1 min - 1, respectively. The activation energy of the enzyme for the hydrolysis of pnp2 2D2GlcNAc was 66124 kjπmol. Key words : Helicoverpa armigera ; 2N2acetyl2D2glucosaminidase ; isolation and purification ; kinetics ; stability N2 2 2D2 ( 2N2acetyl2D2 glucosaminidase, NAG, EC 31211152) 3, ( Koga et al., 1987 ; Spindler and Buchholz,1988 ;Joshi et al., 1989 ; Kono et al., 1990 ; Broadway et al., 1995), NAG 21,42 N2 2D2,, (,2001),, NAG, : (2002AA245011) ; (2002N004) :,,1966 12,,,,,E2mail : xhhuang @vip. sina. com 3 Author for correspondence,e2mail : guanxfafu @1261com Received : 2004210209 ; Accepted : 2005202203
4 : N2 2 2D2 499,, ; (,2000 ;,2001) Helicoverpa armigera, 20 90,,,, NAG,,,, 1 111, 2N2 2 2D2 (pnp2 2D2GlcNAc) ;Sephadex G2200 Pharmacia ;DEAE2cellulose232 Whatman ; ; 112 11211 : NAG, 30 % 70 %, Sephadex G2200, 0101 molπl ph 618 ( 012 molπl NaCl), 216 cm 60 cm, 8 mlπ h,, 210 mlπ DEAE2 32, NaCl (0 112 molπl) 0101 molπl ph 618 210 cm 30 cm, 12 mlπh,,310 mlπ, 11212 NAG : Lin (2003) pnp2 2D2GlcNAc, 2 ml ( 011 molπl ph 5163,012 mmolπl ), 37 20 L, 10 min, 2 ml 015 molπl NaOH Backman DU2650 405 nm (OD 405 nm ) (pnp), 818 10 3 L mol - 1 cm - 1 (U) :, 1 mol pnp 11213 : Folin2 (Lowry et al., 1951), 11214 : 11215 pnp2 2D2GlcNAc :,,, Lineweaver2Burk, ( V m ), V m, Arrhenius, (lgv m ) 1ΠT,, pnp2 2D2GlcNAc 2 211 NAG 1 Sephadex G2 200 80 110 DEAE232 28 32 ( 1), 2 678179 UΠmg ( 2) 212 ph 37, ph, ph pnp2 2D2 GlcNAc ( 3) ph, ph 5163 213 ph, 30 min, 20 L ph 5163, ph, ( 3) ph 4 8, ph 4 ph 8
500 Acta Entomologica Sinica 48 1 N2 2 2D2 Sephadex G2200 (A) DEAE232 (B) Fig. 1 Purification chromatography of the 2N2acetyl2D2glucosaminidase on Sephadex G2200 (A) and on DEAE2cellulose (B) 1 N2 2 2D2 Table 1 Purification of the 2N2acetyl2D2glucosaminidase from Helicoverpa armigera (ml) (mg) (U) (UΠmg) ( %) Purification Total volume Total protein Total activity Specific activity Recovery Purification factor Crude preparation 50 645 166 761136 258154 100 110 30 % 70 % 30 % - 70 %(NH 4 ) 2 SO 4 15 34015 103 50715 303199 6211 1118 Sephadex G2200 60 3916 63 750 1 60918 6116 6123 DEAE2cellulose 28 4162 12 376 2 678179 1914 10136 2 N2 2 2D2 Fig. 2 SDS2PAGE of the purified 2N2 acetyl2d2glucosaminidase 214 ph 5163 011 molπl, 20 L ( 015 mgπml), ( 4),, 55 215 (25 75 ) 30 min 3 ph N2 2 2D2 ph Fig. 3 The effect of ph on the activity and ph stability of 2N2acetyl2D2glucosaminidase 4 N2 2 2D2 Fig. 4 The effect of temperature on the activity and temperature stability of 2N2acetyl2D2glucosaminidase
4 : N2 2 2D2 501,, 20 L (ph 5163,37 ), ( 4) : 45 30 min,, 50 216 pnp2 2D2GlcNAc 2 ml ph 5163,, 37,, Michaelis2Menten ( 5 ) Lineweaver2Burk, pnp2 2D2GlcNAc K m V m 0116 mmolπl 10173 mol L - 1 min - 1 ( 5) 5 N2 2 2D2pNP 2 2D2GlcNAc Lineweaver2Burk Fig. 5 The Lineweaver2Burk plot of 2N2acetyl2D2 glucosaminidase for the hydrolysis of pnp2 2D2GlcNAc The inset shows the relationship between the initial rate and the substrate concentration. 217 pnp2 2D2GlcNAc pnp2 2D2GlcNAc V m Arrhenius, (lgv m ) 1ΠT, ( 6), pnp2 2D2GlcNAc 66124 kjπmol 3,,, 6 pnp2 2D2GlcNAc Fig. 6 Determination of the activation energy of 2N2acetyl2D2glucosaminidase for the hydrolysis of pnp2 2D2GlcNAc, N2 2 2D2 Sephadex G2200 DEAE232,N2 2 2D2 10136,, 8016 % Koga (1986) DEAE Bio2Gel Sephacryl S2200 Bombyx mori N2 2 2D2 64177, 96123 % Koga,,,,,, NAG ph 5163,, ph 4 8 ( Koga et al.,1986),, ph (, 2004) Ohtakara ( 1979) Vibrio sp. ph 9 11,,,
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