Department of Biotechnology, Hankyong National University, Anseong 17579, Republic of Korea.
HK Ginseng Research Center, Hankyong National University, Anseong 17579, Republic of Korea.
J Microbiol Biotechnol. 2020 Mar 28;30(3):391-397. doi: 10.4014/jmb.1910.10065.
In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenoside-converting that was cloned and expressed in BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb into Rd. The gene, termed , consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb2 under optimal conditions (pH 7.0 and 40°;C). Kinetic parameters for α-Larabinopyranosidase showed apparent K and V values of 0.078 ± 0.0002 micrometer and 1.4 ± 0.1 μmol/min/mg of protein against -nitrophenyl-α-L-arabinopyranoside. Using a purified AbpBs (1 μg/ml), 0.1% of ginsenoside Rb was completely converted to ginsenoside Rd within 1 h. The recombinant AbpBs could be useful for high-yield, rapid, and low-cost preparation of ginsenoside Rd from Rb.
在这项研究中，我们使用了一种从转化人参皂苷的新型α-L-阿拉伯吡喃糖苷酶（AbpBs），该酶在 BL21（DE3）中克隆和表达，并将其应用于人参皂苷 Rb 向 Rd 的生物转化。该基因被命名为 ，由 2406 个核苷酸（801 个氨基酸残基）组成，预测翻译的蛋白质分子量为 86.4 kDa，被克隆到 pGEX4T-1 载体中。使用 AbpBs 氨基酸序列进行的 BLAST 搜索显示与家族 2 糖苷水解酶（GH2）具有显著同源性。在 BL21（DE3）中过表达的重组 AbpBs 在最佳条件（pH 7.0 和 40°C）下催化人参皂苷 Rb2 上 C-20 位置连接的阿拉伯吡喃糖部分的水解。α-L-阿拉伯吡喃糖苷酶的动力学参数显示出明显的 K 和 V 值，分别为 0.078±0.0002 微米和 1.4±0.1 微摩尔/分钟/毫克蛋白，对 -硝基苯-α-L-阿拉伯吡喃糖苷。使用纯化的 AbpBs（1μg/ml），0.1%的人参皂苷 Rb 在 1 小时内完全转化为人参皂苷 Rd。重组 AbpBs 可用于从 Rb 中高效、快速、低成本制备人参皂苷 Rd。
