Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea.
J Microbiol Biotechnol. 2013;23(7):913-22. doi: 10.4014/jmb.1209.09009.
An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The β-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to β-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant β-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at 55oC and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.
从天然海水琼脂平板上分离到一株从红海草（Gelidium amansii）中分离到的琼脂降解细菌，并鉴定为 Saccharophagus sp. AG21。通过长而准确（LA）-PCR 筛选出 Saccharophagus sp. AG21 的β-琼脂酶基因（agy1）。预测序列具有 1908bp 的开放阅读框，编码 636 个氨基酸（aa），包括糖苷水解酶家族 16（GH16）β-琼脂酶模块和两个家族 6 的碳水化合物结合模块（CBM6）。推导的 aa 序列与 Saccharophagus degradans 和 Microbulbifer agarilyticus 的β-琼脂酶分别具有 93.7%和 84.9%的相似性。成熟的 agy1 被克隆并在大肠杆菌中作为 His 标记的重组β-琼脂酶（rAgy1）过表达，预测分子量为 69kDa，等电点为 4.5。rAgy1 在 55°C 和 pH 7.6 下表现出最佳活性，比活为 85U/mg。与对照相比，FeSO4（40%）、KCl（34%）和 NaCl（34%）增强了 rAgy1 的活性。新鉴定的 rAgy1 是一种β-琼脂酶，可将琼脂糖降解为 neoagarotetraose（NA4）和 neoagarohexaose（NA6），可能在化妆品、食品、生物乙醇和试剂工业中有应用价值。
