Hyper-production of an isomalto-dextranase of an Arthrobacter sp. by a proteases-deficient Bacillus subtilis: sequencing, properties, and crystallization of the recombinant enzyme.
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.
Hatada
Y
Y
Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, 237-0061, Yokosuka.
Hidaka
Y
Y
Nogi
Y
Y
Uchimura
K
K
Katayama
K
K
Li
Z
Z
Akita
M
M
Ohta
Y
Y
Goda
S
S
Ito
H
H
Matsui
H
H
Ito
S
S
Horikoshi
K
K
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification; D004795:Enzyme Stability; D017353:Gene Deletion; D015870:Gene Expression; D005798:Genes, Bacterial; D020459:Genes, rRNA / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000235:genetics*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C091051:glucan 1,6-alpha-isomaltosidase; D010447:Peptide Hydrolases