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Applied and Environmental Microbiology, February 2006, p. 1065-1071, Vol. 72, No. 2
0099-2240/06/$08.00+0 doi:10.1128/AEM.72.2.1065-1071.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Purification, Characterization, and Gene Cloning of a Novel Maltosyltransferase from an Arthrobacter globiformis Strain That Produces an Alternating 
-1,4- and -1,6-Cyclic Tetrasaccharide from Starch
Kazuhisa Mukai,*
Hikaru Watanabe,
Michio Kubota,
Hiroto Chaen,
Shigeharu Fukuda, and
Masashi Kurimoto
Amase Institute, Hayashibara Biochemical Laboratories, Inc., 7-7 Amase minami-machi, Okayama 700-0834, Japan
Received 7 July 2005/ Accepted 12 November 2005
A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{
6)-
-D-Glcp(14)--D-Glcp(16)--D-Glcp(14)--D-Glcp(1}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50°C and was stable from pH 5.0 to 9.0 and up to 30°C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 45°C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of 
3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose 64-O--maltosyl-maltotetraose + maltose and (ii) 64-O--maltosyl-maltotetraose CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular -1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to -amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the -amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.
* Corresponding author. Mailing address: Amase Institute, Hayashibara Biochemical Laboratories, Inc., 7-7 Amase minami-machi, Okayama 700-0834, Japan. Phone: 81-86-231-6731. Fax: 81-86-231-6738. E-mail: k-mukai{at}hayashibara.co.jp.
Applied and Environmental Microbiology, February 2006, p. 1065-1071, Vol. 72, No. 2
0099-2240/06/$08.00+0 doi:10.1128/AEM.72.2.1065-1071.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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