EXPRESSION OF PERIPLASMIC ALPHA-AMYLASE OF XANTHOMONAS-CAMPESTRIS K-11151 IN ESCHERICHIA-COLI AND ITS ACTION ON MALTOSE

A gene encoding the periplasmic alpha-amylase of Xanthomonas campestri s 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 pri mary structure of the enzyme had little identity with other alpha-amyl ases, except with the enzyme from Bacillus megaterium. The enzyme was expressed in E. coli from the lac promoter of pUC19 and was found to b e transported to the periplasmic space. The expressed enzyme showed th e same thermal stability, optimum temperature and substrate specificit y as the enzyme from X. campestris. The enzyme formed maltotetraose, b ut not 6(1)- nor 6(2)-maltosyl-maltose, from maltose by the reverse re action, and the tetraose was then hydrolysed to maltotriose and glucos e. The addition of maltotriose enhanced the production of glucose from maltose. In addition, maltose was formed by the condensation of gluco se by the enzyme. Thus, the periplasmic alpha-amylase of X. campestris was shown to produce glucose from maltose by hydrolysing maltotetraos e and possibly higher maltooligosaccharides, which were the products o f a condensation reaction, as a major pathway, and by direct hydrolysi s of maltose as a minor pathway.