Cloning and Expression of a β-Galactosidase Gene ofBacillus circulans

The Discoidin Domain ofBacillus circulansβ-Galactosidase Plays an Essential Role in Repressing Galactooligosaccharide Production

“…AB605256). 24) Our results indicate that the enzyme has a multiple domain architecture. The polypeptide, designated BgaD, is comprised of a 35-amino acid (a.a.)-long signal peptide, a LacZ domain containing a Glyco hydro 2 N domain (a sugar-binding domain), a Glyco hydro 2 domain, and a Glyco hydro 2 C domain (a TIM barrel domain), a BID 1 domain (a bacterial Ig-like domain, group 1), four repeats of the Big 4 domain (a bacterial Ig-like domain, group 4), and a F5/8 type C domain, known as a discoidin (DS) domain, 27) from the amino to the carboxyl terminus, as shown in Fig.…”

“…The primers used in this study are shown in Table 1. Deletion mutagenesis was performed as follows: Expression plasmids harboring DNA fragments encoding BgaD-A, deleting 80 a.a. residues (pColdII-bgaD-AÁC80) that correspond to the peptide sequence from the C-terminus to the amino acid residue just behind the C-terminus of the DS domain, and that deleting 247 a.a. residues corresponding to the peptide sequence from the C-terminus to the N-terminus of the DS domain (pColdIIbgaD-AÁC247), were constructed, by performing PCR using pColdIIbgaD-A 24) as template and primers 1-3 ( Table 1). The amino acid sequence of BgaD-A starts at the 36th a.a. residue of BgaD.…”

“…-Galactosidase activity was assayed using 8 mM o-NPG and 143 mM lactose as substrates in the assay buffer at 40 C by a method used in a previous study. 12,24) The final enzyme concentration was 2-4 mg/mL, unless otherwise noted. One unit with o-NPG as substrate (U o-NPG ) was defined as the amount of protein required to produce 1 mmol of o-NP per minute at 40 C at pH 6, and 1 unit with lactose as substrate (U lactose ), as the amount of protein required to produce 1 mmol of D-glucose under the same conditions, as reported previously.…”

“…One unit with o-NPG as substrate (U o-NPG ) was defined as the amount of protein required to produce 1 mmol of o-NP per minute at 40 C at pH 6, and 1 unit with lactose as substrate (U lactose ), as the amount of protein required to produce 1 mmol of D-glucose under the same conditions, as reported previously. 12,24) Protein concentrations were determined by the BCA method using a BCA Protein Assay Reagent Kit (Pierce Chemical, Rockford, IL) with BSA as standard.…”

“…[1][2][3][4] Hence a number of attempts to isolate -galactosidases with high transgalactosylation activities from various microorganisms have been reported, [5][6][7][8][9][10][11][12][13] and genes that encode -galactosidases, [13][14][15][16][17][18][19][20][21][22][23][24] including those of B. circulans ATCC 31382, 13,24) have been cloned. However, the lack of a crystal structure as well as a lack of knowledge of the multimeric and monomeric structures of a number of high molecular weight -galactosidases, except for Escherichia coli -galactosidase, 25) has been a drawback to developing an understanding of the mechanism of transgalactosylation.…”