title: Multimeric immobilization of alcohol oxidase on electrospun fibers for valid tests of alcoholic saliva.
abstract: An accurate quantitation of ethanol is of great importance in clinical and forensic analyses. In the current study, alcohol oxidase (AOX) from Pichia pastoris, a multimeric enzyme consisting of eight identical subunits, was immobilized on electrospun polystyrene-co-maleic anhydride (PSMA) fibers for valid tests of alcoholic saliva. Branched polyethyleneimine (PEI) was grafted on PSMA fibers with a density of 0.15 nmol/cm(2) as tethers to allow multipoint covalent binding of enzyme molecules through glutaraldehyde activation, and the secondary and tertiary amino groups of PEI could intensify the interactions with AOX subunits to stabilize the quaternary structure. PSMA-PEI-AOX fibers were less sensitive than free AOX to the incubation temperature and pH, and indicated no detectable subunit release from the immobilized AOX after boiling in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol. Color strips were established on PSMA-PEI-AOX fibrous mats dyed with indigo Carmine after incubation into ethanol solutions of different concentrations. The color fading ratio remained no significant change after repeat tests for 9 cycles after immersion in 0.2 and 0.8 mg/mL of alcoholic saliva. It was indicated that multipoint immobilization of the multimeric enzyme was essential to improve the enzyme stability by stabilizing both the quaternary structure of the enzyme and the structure of each individual subunit.
authors: Key Laboratory of Advanced Technologies of Materials, Ministry of Education of China, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, People's Republic of China.
authors: Long
authors: L
authors: Zhao
authors: Qingjie
authors: Q
authors: Liu
authors: Shili
authors: S
authors: Yan
authors: Zhoujiang
authors: Z
authors: Chen
authors: Jianmei
authors: J
authors: Chen
authors: Xiaohong
authors: X
authors: Li
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D000438:Alcohols / Q000032:analysis* / Q000378:metabolism; D004563:Electrochemistry; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D012463:Saliva / Q000737:chemistry*
chemical_list: D000438:Alcohols; D004800:Enzymes, Immobilized; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase
keywords: Alcohol oxidase; Alcoholic saliva; Electrospun fiber; Multimeric enzyme; Multipoint immobilization