A gene encoding the periplasmic a-amylase of Xanthomonas campestris K-11151 was cloned into Escherichia coli using pUC19 as a vector. An ORF of 1578 bp was deduced to be the amylase structural gene. The primary structure of the enzyme had little identity with other a-amylases, except with the enzyme from Bacillus megateriwn. The enzyme was expressed in Em coli from the lac promoter of pUC19 and was found to be transported to the periplasmic space. The expressed enzyme showed the same thermal stability, optimum temperature and substrate specificity as the enzyme from X. campestris. The enzyme formed maltotetraose, but not 6'-nor 6*-maltosyl-rnaItose, from maltose by the reverse reaction, and the tetraose was then hydrolysed to maltotriose and glucose. The addition of maltotriose enhanced the production of glucose from maltose. In addition, maltose was formed by the condensation of glucose by the enzyme. Thus, the periplasmic a-amylase of X. campestris was shown to produce glucose from maltose by hydrolysing maltotetraose and possibly higher maltooligosaccharides, which were the products of a condensation reaction, as a major pathway, and by direct hydrolysis of maltose as a minor pathway.
The ultraviolet spectrum of 1,5-anhydro-D-fructose (1,5-anhydro-D-arabino-hexo-2-ulose , 1,5-AF) showed two absorption peaks at 215 and 266 nm, and the absorption at 215 nm was higher . The molar absorptivity at 215 nm was low (34 M-1• cm-1) and was unsuitable for the assay. The conven tional methods for the determination for total sugar were unsuitable for 1,5-AF because the phenolsulfuric acid method gave only 15% value of glucose and no coloration by the anthrone-sulfuric acid method. On the other hand, 1,5-AF reduced Cu2+ (the method of Somogyi and Nelson or neocuproine assay) and Fe3+ (the method of Park and Johnson) even at 35°C. The former reaction enabled the specific determination of 1,5-AF in the presence of the other hexoses and pentoses . Neocuproine assay for reducing sugars was sensitive (0-501a g/mL) to 1,5-AF and could perform on a small scale (sample 20 ,a L) on a microtitre plate.
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