Abstract
Publisher Summary This chapter describes the assay method, the purification procedure, and properties of sucrose-6-phosphate hydrolase from Streptococcus mutans . The continuous spectrophotometric assay is based upon the observation that when glucose-6-phosphate dehydrogenase is present in excess, the rate of sucrose 6-phosphate hydrolysis is proportional to the rate of NADP reduction, which is measured as an increase in absorbance at 340 nm. The purification process involves: growth of the organism, preparation of cell extracts, diethylaminoethyl (DEAE)-cellulose chromatography, and ultrogel AcA-54 chromatography. Sucrose 6-phosphate hydrolase is found in S . mutans cells cultured on a number of sugars; however, the best yield is obtained from sucrose-grown cells. Sucrose 6-phosphate and sucrose are the only known substrates of the enzyme. Among the buffers tested, 2-( N -Morpholino)ethanesulfonic acid (MES) is superior to N -2-hydroxyethylpiperazine- N' -2-ethanesulfonic acid (HEPES) and Tris and the pH optimum is 7.1. The purified enzyme is stable for days at room temperature, weeks at 4 ° C, and indefinitely, when frozen in 30% glycerol at –20 ° C.
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