Cloning, Sequencing and Expression of the Genes Encoding Cyclic .ALPHA.-Maltosyl-(1.RAR.6)-maltose Hydrolase and .ALPHA.-Glucosidase from an Arthrobacter globiformis Strain

Abstract

The gene encoding for a novel hydrolase, cyclic α-maltosyl-(1→6)-maltose [CMM, cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}] hydrolase (CMMase), was cloned from the genomic library of Arthrobacter globiformis M6 and designated cmmF. The gene consisted of 1,353 bp encoding a protein of 450 amino-acids with a calculated molecular mass of 49,344 Da. The deduced amino-acid sequence showed similarities to cyclodextrinase, maltogenic amylase and neopullulanase. On the other hand, the complete sequence of the α-glucosidase gene (cmmB), which encodes an enzyme involved in the degradation of CMM, revealed that the gene consisted of 1,704 bp encoding a protein of 567 amino-acids with a calculated molecular mass of 63,014 Da. The four conserved regions common in the α-amylase family enzymes were also found in CMMase and α-glucosidase, indicating that these enzymes should be assigned to this family. The DNA sequence of 5,675 bp analyzed in this study contained another four open reading frames (ORFs), designated cmmC, cmmD, cmmE and cmmG, downstream of cmmB. CmmC, cmmD and cmmE were expected to encode proteins concerned with incorporation of CMM via cell membrane. CmmG was expected to encode a transcriptional regulator protein. CMMase gene, α-glucosidase gene and another four ORFs formed a gene cluster together with 6-α-maltosyltrasferase gene (cmmA) which encodes a CMM-forming enzyme, namely cmmABCDEFG. The results of gene analysis suggested that A. globiformis M6 has a unique starch utilization pathway via CMM.