Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine‐plus‐cytosine contents

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Abstract
An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and chgaracterized, weh have cloned two additional genes, amyB, and amyC, coding for amylases of this thermophile, into Escherichia coil and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coil promoters. Almost all activity was detected in the intracellular fraction in the E. coil cells.The molecular mass and NH2‐terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coil transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59000). The optimal temperatures for AmyB and AmyC activities on soluble strach were 80oC and 70oC, respectively. Both AmyB and AmyC showed a pH optimum of 5.5 AmyB and AmyC showed a different pattern of strach hydrolysis when examined by thinlayer chromatography. Some homology in the amino sequences with the functional regions of Taka‐amylase A was found in both AmyB and AmyC.The condon usage in the amyA., amyB and amyC genes was highly biased, which reflects the fact that the guanine‐plus‐cytosine (G+C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the condons was non‐random; the G+C content of the first position of condons is significantly high, whereas that of the third position is somewhat low. In addition, condons consisiting only of A and T were preferntially used in this thermophile.