ENVIRONMENTAL CHEMISTRY

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Άγνη Αναγνωστάκης
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1 ENVIRONMENTAL CHEMISTRY Vol 30 No 6 June 2011 * ** AChE ph mmol L 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 CS 96 7% AChE Sigma PBS 0 1 mol L - 1 ph = 7 5 ATChI Fluka DTNB Sigma 79 2 mg 10 ml PBS 15mg NaHCO μm 35 ph 8 0 methy-parathion malathion chlorpyrifos 100 μg ml μg L - 1 T6 DL-5-B LRH-250 HZS-H 1 2 CS-MS AChE ml 2 0 ml span ml 2wt% CS 2% * 2001AA ** Tel fanpingm@ tom com

2 h 0 5 ml 25% GA 0 5 h CS-MS g CS-MS 5 0 ml 0 5% V /V GA 0 25 h 0 75 ml 1 U ml - 1 Tween h PBS 0 1 mol L - 1 ph7 5 Tween-204 PBS 1 3 AChE Ellman ml 20 μl min 100 μl DTNB 20 mmol L μl ATChI 0 1 mol L 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 ml DTNB ATChI 3 min 412 nm U μmol min - 1 g - 1 = V A / m k L m g g 20 μl 3 00 ml min 5 35 ph ph K m 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 mol L - 1 PBS ph = d AChE 3 OPs 3 OPs g 100 μg L ml min OPs g μg L ml 1 3 OPs I % = I 0 - I C 100% I 0 I C I SPSS13 0 one-way ANOVA Tukey

3 P < % 100% 57 34% 100% Tripathi 9 α kj mol kj mol ± p < 0 05 Fig 1 s i Effect of temperature on the activity of soluble and immobilized AChE Pfeifer 11 Mytilus sp AChE Tomlinson 12 Electrophorus electricus AChE Na + Mg 2 + Ca 2 + AChE ph 3 ph ph ph ph ph 9 0 ph CNBr Sepharose4B AChE ph ph Ye 13 ph ph -ph 14 ph ph

4 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 mmol L mmol L - 1 K m Andreesy 5 AChEK m 0 32 mmol L mmol L mmol L K m 17 4 Fig 4 Lineweaver-Burk The kinetic property and Lineweaver-Burk plots of soluble and immobilized enzyme

5 h 60% 1 h 1 h 29% 5 h 25% 18 S mol L - 1 ph 7 5 PBS 60 d 83% 60 d 68% Fig 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 min 3 OPs min 30 min 15 43% 20 min 30 min 20 min 20 min Fig OPs AChE Effect of reaction time on the inhibition rate of three OPs to the immobilized AChE in the seawater

6 OPs -OPs 8 OPs - OPs R 2 = R 2 = R 2 = % OPs 3 OPs 5 34 μg L μg L μg L - 1 Fig 8 8 OPs Relation curves between inhibition rate of immobilized AChE and concentration of OPs 3 1 AChE ph K m h 70% 4 60 d 83% 68% 3-3 OPs 5 34 μg L μg L μg L D Pogacnik L Franko M Optimization of FIA system for detection of organophosphorus and carbamate pesticides based on cholinesterase inhibition J Talanta 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 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 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 Chiou S H Wu W T Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups J Biomaterials Ellman G L A new and rapid colorimetric determination of acetylcholinesterease activity J Biochem Pharmaeol J 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 / D 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

7 Tomlinson G Mutus B McLennan I Activation and inactivation of acetylcholinesterase by metal ions J Can J Biochem 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 M J Yang Y M Wang J W Tan R X Immobilization of glucose oxidase on chitosan-sio 2 gel J Enzyme and Microbial Technology Yong Y Bai Y X Li Y F et al Characterization of Candida rugosa lipase immobilized onto magnetic microspheres with hydrophilicity J Process Biochemistry S- J Tang Z X Qian J Q Shi L E Characterizations of immobilized neutral lipase on chitosan nano-particles J Materials Letters 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 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 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 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

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