Nunoura N, Ohdan K, Tanaka K, Tamaki H, Yano T, Inui M, Yukawa H, Yamamoto K, Kumagai H
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Biosci Biotechnol Biochem. 1996 Dec;60(12):2011-8. doi: 10.1271/bbb.60.2011.
Genomic DNA encoding a beta-D-glucosidase (EC 3.2.1.21), which has beta-D-fucosidase activity, was cloned from Bifidobacterium breve clb. We sequenced a 1.9-kbp cloned DNA fragment that contained a single open reading frame encoding 460 amino acids with a calculated molecular mass of 51,513 Da. A putative ribosome binding site was found 5 bp upstream of the initiation codon. The amino acid sequence of this beta-D-glucosidase from Bifidobacterium breve clb had 46% identity with that of beta-glucosidase from Microbispore bispore. The enzyme of Bifidobacterium breve clb was expressed in Escherichia coli. A cell-free extract prepared from the recombinant strain showed 80 to 90-fold more beta-D-glucosidase activity than that from Bifidobacterium breve clb. The recombinant enzyme was purified to homogeneity from cell-free extracts of the recombinant strain using 4 column chromatographies. The recovery of enzyme from the recombinant strain was about 138-fold-higher than that of Bifidobacterium breve clb. The enzymatic properties were similar to those of Bifidobacterium breve clb. For application of this recombinant enzyme, we attempted to synthesize a disaccharide that seemed to be specifically assimilated by Bifidobacteria using the condensation activity of the enzyme.
编码具有β-D-岩藻糖苷酶活性的β-D-葡萄糖苷酶（EC 3.2.1.21）的基因组DNA从短双歧杆菌clb中克隆得到。我们对一个1.9 kbp的克隆DNA片段进行了测序，该片段包含一个单一的开放阅读框，编码460个氨基酸，计算分子量为51,513 Da。在起始密码子上游5 bp处发现了一个假定的核糖体结合位点。来自短双歧杆菌clb的这种β-D-葡萄糖苷酶的氨基酸序列与来自双小孢子双歧杆菌的β-葡萄糖苷酶的氨基酸序列具有46%的同一性。短双歧杆菌clb的酶在大肠杆菌中表达。从重组菌株制备的无细胞提取物显示出比短双歧杆菌clb高约80至90倍的β-D-葡萄糖苷酶活性。使用4种柱色谱法从重组菌株的无细胞提取物中纯化重组酶至均一性。重组菌株中酶的回收率比短双歧杆菌clb高约138倍。酶学性质与短双歧杆菌clb的相似。为了应用这种重组酶，我们尝试利用该酶的缩合活性合成一种似乎能被双歧杆菌特异性同化的二糖。
