Hyper-production of an isomalto-dextranase of an Arthrobacter sp. by a proteases-deficient Bacillus subtilis: sequencing, properties, and crystallization of the recombinant enzyme

Abstract

Arthrobacter globiformis T6 is unique in that it produces an enzyme yielding only isomaltose from dextran. In the present study, the organism was re-identified and its classification as a new species of the genus Arthrobacter, A. dextranlyticum, was proposed. The high G+C gene (66.8 mol%) for the isomalto-dextranase was sequenced. The deduced amino acid sequence, with a calculated molecular mass of 65,993 Da (603 amino acids), was confirmed by nanoscale capillary liquid chromatography coupled to tandem mass spectrometry, which covered 71.1% of the amino acid residues of the entire sequence. The enzyme was grouped into glycoside hydrolase family 27, and the C-terminal domain has homology to carbohydrate-binding module family 6. Hyper-exoproduction of the recombinant enzyme was achieved at a level corresponding to approximately 4.6 g l(-1) of culture broth when proteases-deficient Bacillus subtilis cells were used as the host. The purified enzyme (65.5 kDa) had an optimal pH and temperature for activity of 3.5 and 60 degrees C, respectively. It was crystallized using the sitting-drop vapor-diffusion method at 293 K.