Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity.
A Butyrivibrio fibrisolvens H17c glgB gene, was isolated by direct selection for colonies that produced clearing on starch azure plates. The gene was expressed in Escherichia coli from its own promoter. The glgB gene consisted of an open reading frame of 1,920 bp encoding a protein of 639 amino acids (calculated Mr, 73,875) with 46 to 50% sequence homology with other branching enzymes. A limited region of 12 amino acids showed sequence similarity to amylases and glucanotransferases. The B. fibrisolvens branching enzyme was not able to hydrolyze starch but stimulated phosphorylase alpha-mediated incorporation of glucose into alpha-1,4-glucan polymer 13.4-fold. The branching enzyme was purified to homogeneity by a simple two-step procedure; N-terminal sequence and amino acid composition determinations confirmed the deduced translational start and amino acid sequence of the open reading frame. The enzymatic properties of the purified enzyme were investigated. The enzyme transferred chains of 5 to 10 (optimum, 7) glucose units, using amylose and amylopetin as substrates, to produce a highly branched polymer.
Department of Microbiology, University of Cape Town, South Africa.
E
E
Rumbak
D E
DE
Rawlings
G G
GG
Lindsey
D R
DR
Woods
D015061:1,4-alpha-Glucan Branching Enzyme / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001438:Bacteroidaceae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002848:Chromatography, DEAE-Cellulose; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D005936:Glucans / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012417:Rumen / Q000382:microbiology*; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism*
D004269:DNA, Bacterial; D005936:Glucans; D013213:Starch; D015061:1,4-alpha-Glucan Branching Enzyme