A biosensor for the determination of amylase activity.
A new biosensing flow injection method for the determination of alpha-amylase activity has been introduced. The method is based on the analysis of maltose produced during the hydrolysis of starch in the presence of alpha-amylase. Maltose determination in the flow system was allowed by the application of peroxide electrode equipped with an enzyme membrane. The membrane was obtained by immobilisation of glucose oxidase, alpha-glucosidase and optionally mutarotase on a cellophane, co-crosslinked by gelatin-glutaraldehyde together with bovine serum albumine. alpha-Glucosidase hydrolyses maltose to alpha-D-glucose, which is converted to beta-D-glucose by mutarotase. beta-D-Glucose is then determined via glucose oxidase. The new biosensor has the limit of detection of 50 nmol l(-1) maltose, which means 2 nkat ml(-1) in alpha-amylase activity units, when the reaction time of amylase was 5 min (determined with respect to a signal-to-noise ratio 3:1). When the reaction time of alpha-amylase was 30 min, the limit of detection was 0.5 nkat ml(-1). A linear range of current response was 0.1-3 mmol l(-1) maltose, with a response time of 35s. The biosensor was stable at least two months and retained 70% of its original activity (with mutarotase the stability is decreased to 3 weeks). When the enzyme membrane was stored in a dry state at 4 degrees C in a refrigerator, the lifetime was approximately 6 months (with mutarotase only 3 months).
Zajoncová
Ludmila
L
Department of Biochemistry, Faculty of Science, Palacký University, Slechtitelů 11, 783 71 Olomouc-Holice, Czech Republic. zajoncov@prfholnt.upol.cz
Jílek
Milan
M
Beranová
Veronika
V
Pec
Pavel
P
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002238:Carbohydrate Epimerases / Q000737:chemistry; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation* / Q000379:methods; D005949:Glucose Oxidase / Q000737:chemistry; D008320:Maltose / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000516:alpha-Amylases / Q000032:analysis* / Q000737:chemistry*; D000520:alpha-Glucosidases / Q000737:chemistry
D004800:Enzymes, Immobilized; D008320:Maltose; D005949:Glucose Oxidase; D000516:alpha-Amylases; D000520:alpha-Glucosidases; D002238:Carbohydrate Epimerases; C019646:aldose 1-epimerase