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|Title: ||Evaluation of HPSEC-ELSD method for precise measurement of β-agarase kinetics|
|Authors: ||Chorng-Liang Pan;Katarzyna Kazlowska;Bartosz Kazłowski|
|Contributors: ||NTOU:Department of Food Science|
|Issue Date: ||2011-10-21T02:25:08Z
|Abstract: ||Abstract:Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics the reaction rate is measured and the effects of varying the conditions of the reaction investigated. For the inspection of -agarase enzyme kinetics, the high performance size exclusion chromatography (HPSEC) coupled with evaporative light scattering detector (ELSD) was utilized. Results obtained by this method were compared with that from traditional reducing sugar content - ferricyanide method. For precise measurement of enzyme kinetics for -agarase, the fixed amount of enzyme (1 unit) and reaction time (15 min) were chosen in order to limit substrate utilization below 5%. The measurement of Km (the substrate concentration needed to achieve a half-maximum enzyme velocity; mg/mL) and the Vmax (maximum enzyme velocity; µg/mL × min) by HPSEC-ELSD and ferricyanide showed distinct difference between them, amounted to 0.56, 0.88 and 1.83, 3.06, respectively. It is apparent that HPSEC-ELSD displayed precise results, while those monitored by ferricyanide method were underestimated. Inaccuracy of ferricyanide method is connected with short reaction time causing the agarose (substrate) was not digested completely to the end product (oligosaccharides with degree of polymerization 4 and 6; N4 and N6). Therefore the reducing sugar content of the product was distinctly differing from the reducing sugar content of the same amount of N6 (used as a standard in this experiment). Also, the measurement of product with concentration below 10 g/mL for ferricyanide method was insufficient for analysis with precision. Therefore, HPSEC-ELSD system was apparently most appropriate for agarase kinetics.|
|Relation: ||壁報論文 B12|
|Appears in Collections:||[食品科學系] 演講及研討會|
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