抄録
Xanthomonas campestris K-11151 produced a novel a-amylase in periplasm. The enzyme purified to homogeneity by several criteria showed almost the same activities on α-, β-, γ-cyclodextrins, soluble starch and amylose. Moreover, it was active on branched cyclodextrins, and pullulan but had very little activity on glycogen. This substrate specificity suggests that this enzyme has the combined activities of α-amylase, cyclodextrinase, and neopullulanase. The gene which codes for this enzyme was cloned into Escherichia coli JM109. An open reading frame of 1578 by was deduced as the structural gene. The enzyme was expressed in E. coli by the lac promoter of pUC plasmid and transported to its periplasmic space. The expressed enzyme showed the same N-terminal sequence, thermal stability, optimum temperature and substrate specificity on various substrates as those of the enzyme from Xanthomonas. The primary structure deduced from its nucleotide sequence showed low and high homology to those of X. campestris extracelluar and Bacillus megaterium α-amylases, respectively. The enzyme has been suggested to have the (α/β)8 barrel structure common to the α- amylase family but to have the shorter 2nd and longer 4th and 6th loops than other α-amylases.
