ENVIRONMENTAL CHEMISTRY

30 6 2011 6 ENVIRONMENTAL CHEMISTRY Vol 30 No 6 June 2011 * ** 266100 AChE ph 46 18 mmol L - 1 4 60 d 17% AChE 5 34 μg L - 1 acetylcholinesterase AChE organophosphorus pesticides OPs 1 AChE OPs 2-3 Tris 4 OPs ph 5-6 OPs AChE OPs AChE OPs 1 1 1 CS 96 7% AChE Sigma PBS 0 1 mol L - 1 ph = 7 5 ATChI Fluka 5 5-2- DTNB Sigma 79 2 mg 10 ml PBS 15mg NaHCO 3 0 45μm 35 ph 8 0 methy-parathion malathion chlorpyrifos 100 μg ml - 1 1 250 μg L - 1 T6 DL-5-B LRH-250 HZS-H 1 2 CS-MS AChE 100 0 ml 2 0 ml span-80 25 0 ml 2wt% CS 2% 2010 6 21 * 2001AA635130 ** Tel 0532-66782875 E-mail fanpingm@ tom com

6 1069 1 h 0 5 ml 25% GA 0 5 h CS-MS 1 000 g CS-MS 5 0 ml 0 5% V /V GA 0 25 h 0 75 ml 1 U ml - 1 Tween-20 4 24 h PBS 0 1 mol L - 1 ph7 5 Tween-204 PBS 1 3 AChE Ellman 7 3 00 ml 20 μl 30 20 min 100 μl DTNB 20 mmol L - 1 20 μl ATChI 0 1 mol L - 1 412 nm 3 min 0 5 min 3 U μmol min - 1 ml - 1 = V A / v k L V V = 3 14 ml A min - 1 v v = 0 02 ml k k = 1 36L mmol - 1 mm - 1 L L = 10 mm Ellman 7 8 3 00 ml DTNB ATChI 3 min 412 nm U μmol min - 1 g - 1 = V A / m k L m g 1 4 0 015g 20 μl 3 00 ml 1 3 5 40 20 min 5 35 ph ph 7 0 9 0 1 5 K m 1 750 mmol L - 1 ATChI 1 3 A 412 υlineweaver-burk 1 K m 1 = K m 1 v v m S + 1 v m S υ υ m K m 1 6 50 30 1 3 4 0 1 mol L - 1 PBS ph = 7 5 5 d 1 3 1 7 AChE 3 OPs 3 OPs 0 015 g 100 μg L - 1 3 ml 1 3 5 10 20 30 45 60 min OPs 0 015 g 1 250 μg L - 1 3 ml 1 3 OPs I % = I 0 - I C 100% I 0 I C I 0 1 8 SPSS13 0 one-way ANOVA Tukey

1070 30 P < 0 05 2 2 1 2 1 1 1 30 40 92 92% 100% 57 34% 100% Tripathi 9 α- 65 75 10 29 641 kj mol - 1 40 150 kj mol - 1 1 3 ± p < 0 05 Fig 1 s i Effect of temperature on the activity of soluble and immobilized AChE 2 1 2 2 25 35 Pfeifer 11 Mytilus sp AChE Tomlinson 12 Electrophorus electricus AChE Na + Mg 2 + Ca 2 + AChE 2 1 3 ph 3 ph ph 7 0 9 0 ph 7 5 8 5 ph 7 0 8 5 ph 9 0 ph 7 5 8 5 1 CNBr Sepharose4B AChE ph 7 5 8 0 ph Ye 13 ph ph -ph 14 ph ph

6 1071 ph 2 3 ph Fig 2 Effect of salinity on the activity of soluble and immobilized AChE Fig 3 Effect of ph on the activity of soluble and immobilized AChE 2 2 K m K m S υ A 412 Lineweaver-Burk 4 K m 8 157 mmol L - 1 46 18 mmol L - 1 K m Andreesy 5 AChEK m 0 32 mmol L - 1 0 45 mmol L - 1 0 1 mmol L - 1 15 16 K m 17 4 Fig 4 Lineweaver-Burk The kinetic property and Lineweaver-Burk plots of soluble and immobilized enzyme

1072 30 2 3 2 3 1 5 50 0 25 h 60% 1 h 1 h 29% 5 h 25% 18 S- 19-20 2 3 2 6 4 0 1 mol L - 1 ph 7 5 PBS 60 d 83% 60 d 68% 6 9 16 14 1 2 Fig 5 5 6 Thermal stability of soluble and immobilized AChE Fig 6 Storage stability of soluble and immobilized AChE 2 4 AChE 3 OPs OPs 100 μg L - 1 5 60 min 3 OPs 7 5 20 min 30 min 15 43% 20 min 30 min 20 min 20 min Fig 7 7 3 OPs AChE Effect of reaction time on the inhibition rate of three OPs to the immobilized AChE in the seawater

6 1073 OPs -OPs 8 OPs - OPs R 2 = 0 923 R 2 = 0 9403 R 2 = 0 8992 5% OPs 3 OPs 5 34 μg L - 1 5 75 μg L - 1 5 95 μg L - 1 Fig 8 8 OPs Relation curves between inhibition rate of immobilized AChE and concentration of OPs 3 1 AChE ph 7 5 8 5 K m 2 50 1 h 70% 4 60 d 83% 68% 3-3 OPs 5 34 μg L - 1 5 75 μg L - 1 5 95 μg L - 1 1 D 2004 2 Pogacnik L Franko M Optimization of FIA system for detection of organophosphorus and carbamate pesticides based on cholinesterase inhibition J Talanta 2001 54 4 631-641 3 Jeanty G Ghommidh C Marty J L Automated detection of chlorpyrifos and its metabolites by a continuous flow system-based enzyme sensor J Analytica Chimica Acta 2001 436 1 119-128 4 Andrei F Badea M Marty J L et al Flow analysis for determination of paraoxon with use of immobilized acetylcholinesterase reactor and new type of chemiluminescent reaction J Biopolymers 2000 57 1 37-42 5 Andreesy S Barthelthebs L Marty J L Immobilization of acetylcholinesterase on screen- printed electrodes comparative study between three immobilization methods and applications to the detection of organophosporus insecticides J Analytica Chimica Acta 2001 464 2 171-180 6 Chiou S H Wu W T Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups J Biomaterials 2004 25 2 197-204 7 Ellman G L A new and rapid colorimetric determination of acetylcholinesterease activity J Biochem Pharmaeol 1961 7 2 88-95 8 J 2006 35 6 721-724 9 Tripathi P Kumari A Rath P et al Immobilization of α-amylase from mung beans Vigna radiata on Amberlite MB 150 and chitosan beads A comparative study J Journal of Molecular Catalysis B Enzymatic 2007 49 1 /4 69-74 10 D 2009 11 Pfeifer S Schiedek D Dippner J W Effect of temperature and salinity on acetylcholin-esterase activity a common pollution biomarker in Mytilus sp from the south-western Baltic Sea J Journal of Experimental Marine Biology and Ecology 2005 320 1 93-103

1074 30 12 Tomlinson G Mutus B McLennan I Activation and inactivation of acetylcholinesterase by metal ions J Can J Biochem 1981 59 9 728-735 13 Ye P Xu Z K Che A F et al Chitosan-tethered poly acrylonitrile-co-maleic acid hollow fiber membrane for lipase immobilization J Biomaterials 2005 26 32 6394-6403 14 M 2005 93 15 J 2009 20 2 328-331 16 Yang Y M Wang J W Tan R X Immobilization of glucose oxidase on chitosan-sio 2 gel J Enzyme and Microbial Technology 2004 34 2 126-131 17 Yong Y Bai Y X Li Y F et al Characterization of Candida rugosa lipase immobilized onto magnetic microspheres with hydrophilicity J Process Biochemistry 2008 43 11 1179-1185 18 - S- J 2006 25 5 576-579 19 Tang Z X Qian J Q Shi L E Characterizations of immobilized neutral lipase on chitosan nano-particles J Materials Letters 2007 61 1 37-40 20 Ye P Jiang J Xu Z K Adsorption and activity of lipase from Candida rugosa on the chitosan -modified poly acrylonitrile-co-maleic acid membrane surface J Colloids and surfaces B Biointerfaces 2007 60 1 62-67 CHARACTERIZATION OF ACETYLCHOLINESTERASE IMMOBILIZED ON CHITOSAN MICROSPHERES ZHANG Aijing MENG Fanping YANG Feifei Key Laboratory of Marine Environment and Ecology Ministry of Education Ocean University of China Qingdao 266100 China ABSTRACT In order to evaluate the feasibility of using immobilized acetylcholinesterase AChE on chitosan microspheres to monitor organophosphorus pesticides OPs in seawater basic properties of the immobilized enzyme were investigated such as environmental adaptability dynamic property stability response characteristics to OPs The results indicated that the immobilized enzyme was more adaptive to the temperature salinity ph of seawater compared with the solution enzyme The Michaelis constant of the immobilized AChE increased to 46 18 mmol L - 1 which may be caused by the steric hindrance between the immobilized enzyme and its substrate Thermal stability and storage stability were also enhanced after immobilization The immobilized AChE lost only 17% of its initial activity after 60 days storage at 4 Moreover there was a good linear correlation between the activity inhibition rate of immobilized AChE and the logarithmic value of OPs concentration The immobilized enzyme was most sensitive to methyl-parathion with a detection limit of 5 34 μg L - 1 The results of this research suggested that the immobilized enzyme have a good prospect in monitoring OPs in seawater due to its good performance and sensitive response Keywords chitosan immobilization acetylcholinesterase AChE property seawater