Gene cloning, nucleotide sequence and biochemical properties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius, reclassification of a group of enzymes

A gene encoding a cyclomaltodextrinase (neopullulanase) was cloned from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius ATCC27009 and its nucleotide sequence was determined. The encoded CdaA protein lacked an N-terminal signal sequence and aligned well with a family of bacterial proteins described as maltogenic alpha-amylases, neopullulanases or cyclomaltodextrinases. Escherichia coli cells harboring the cloned cdaA gene produced a 66-kDa protein that degraded pullulan in a sodium dodecyl sulfate-polyacrylamide gel. A. acidocaldarius cells grown on maltose, soluble starch or pullulan synthesized the same protein. Neopullulanase activity of the protein was cytoplasmic and its pH optimum of 5.5 was close to the pH value of the cytoplasm. CdaA degraded cyclomaltodextrins rapidly and pullulan (to panose) more slowly. It is proposed that CdaA functions as a cytoplasmic cyclomaltodextrinase (EC 3.2.1.54).