A Continuous Method for Enzymatic Assay of Sucrose Synthase in the Synthetic Direction

• Published in: Journal of agricultural and food chemistry Vol. 47; no. 7; pp. 2746 - 2750
• Main Authors: Huang, Y. H, Picha, David H, Kilili, Anthony W
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.07.1999
• Subjects: assays; Biological and medical sciences; Biotechnology; Enzyme engineering; enzyme preparations; Food industries; Fundamental and applied biological sciences. Psychology; General aspects; Glucosyltransferases - analysis; L-Lactate Dehydrogenase - chemistry; Methods of analysis, processing and quality control, regulation, standards; Methods. Procedures. Technologies; Miscellaneous; Oxidation-Reduction; Pyruvate Kinase - chemistry; Reproducibility of Results; sucrose synthase; Biochemical analysis; Purification; Pyruvate kinase; Enzyme; Extraction process; Transferases; Glycosyltransferases; Experimental study; L-Lactate dehydrogenase; Microfiltration; Analysis method; Synthesis; Enzymatic activity; Hexosyltransferases; Continuous measurement; Oxidoreductases; Sucrose synthase
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf981264w

An appropriate method was developed for the continuous assay of sucrose synthase (SS) (EC 2.4.1.13) by spectrophotometry. The uridine 5‘-diphosphate derived from sucrose synthesis was stoichiometrically coupled to oxidation of β-nicotinamide adenine dinucleotide by the enzymes nucleoside-5‘-diphosphate kinase (NDPK), pyruvate kinase, and lactate dehydrogenase. Utilization of crude extracts led to a complete masking of SS assay by adenylate kinase, adenosine 5‘-triphosphatase (ATPase), and phosphoenolpyruvate phosphatase found in the crude extracts. These interfering enzymes were mostly removed from the crude extracts by using a combination of gel filtration, centrifugation through a selectively permeable membrane (Biomax-100 Ultrafree centrifugal device), and inhibition by the addition of K2HPO4 to the assay buffer. Sensitivity of the SS assay was significantly increased by the inclusion of NDPK and ATP, which are essential to the reaction in the coupling system. Keywords: Sugars; enzyme assay; microfiltration; coupling reactions