α-淀粉酶AmyP中淀粉结合结构域的鉴定

Identification of starch-binding domain in α-amylase AmyP

摘要:

【目的】检测具有生淀粉降解活性的新型α-淀粉酶AmyP是否具有淀粉结合结构域(SBD)。【方法】通过结构域预测和序列分析, 推测AmyP的C端是一个SBD。将这段序列克隆、表达和重组蛋白纯化后, 采用亲和电泳和生淀粉吸附两种方法对重组表达的蛋白进行研究。【结果】AmyP的C端序列是一个新型的SBD, 根据序列特征可以将其划分在碳水化合物结合结构域(CBM) 20家族。该SBD与生大米淀粉的吸附能力最强, 生玉米淀粉次之, 不能与生小麦淀粉、生马铃薯淀粉和生绿豆淀粉吸附。【结论】α-淀粉酶AmyP在蛋白C端具有一个SBD, 有助于理解AmyP快速偏好性降解生淀粉的能力。

Abstract:

[Objective] To explore whether a starch-binding domain (SBD) locates in the newly identified α-amylase (AmyP) that can degrade raw starches. [Methods] Based on sequence alignment and protein domain prediction, a putative SBD was found in the C-terminal sequence of AmyP. The sequence was cloned, expressed and purified, and the recombinant protein was identified by affinity gel electrophoresis and adsorption on raw starch. [Results] The sequence was defined as an SBD that belongs to the carbohydrate-binding module family 20 (CBM20) based on sequenced similarity. SBD binds with higher affinity to raw rice starch compared to raw corn starch, and is unable to adsorb to raw wheat starch, raw potato starch or raw mungbean starch. [Conclusion] The C-terminal region of AmyP was identified as a novel SBD, which may contribute to the specific raw starch-degarding ability of AmyP.