Supplementary Material for: Secreted Expression of a Hyperthermophilic α-Amylase Gene from <b><i>Thermococcus</i></b> sp. HJ21 in <b><i>Bacillus subtilis</i></b>

The hyperthermophilic α-amylase from <i>Thermococcus</i> sp. HJ21 possesses unique traits (Ca²⁺-independent thermostability and optimal temperature of 95°C) that make it a great potential candidate for use in the food industry. However, this Archaea isolated from a deep-sea thermal vent requires strict control of culture conditions and produces only small amounts of α-amylase. To solve these problems, the α-amylase gene was cloned and expressed in <i>Bacillus subtilis</i>, which is an ideal food-grade host for heterologous protein expression. To express high levels of this α-amylase in <i>B. subtilis</i>, the promoters P_<i>grac</i>, P_<i>xylA</i>, P43, and P_<i>hag</i> were used to construct four different expression vectors for testing. The vector containing the P_<i>xylA</i> promoter was found to have the highest transcriptional activity and produce the highest amylase activity (19.6 U/ml). To test the secretion efficiency of signal peptides in <i>B. subtilis</i>, three signal peptides were cloned and fused to the α-amylase gene (lacking its native signal peptide). The optimal signal peptide was S_<i>amyQ</i>, with a secretion efficiency of approximately 90%. These results indicate that the promoter P_<i>xylA</i> and signal peptide S_<i>amyQ</i> tested in this study may be useful for the expression and secretion of archaeal proteins in <i>B. subtilis</i>.

来自嗜热球菌属（Thermococcus）菌株HJ21的超嗜热α-淀粉酶（hyperthermophilic α-amylase）具备独特特性：不依赖钙离子的热稳定性以及95℃的最适反应温度，使其成为食品工业极具应用潜力的候选酶制剂。然而，该分离自深海热液喷口的古菌对培养条件要求严苛，且仅能合成少量α-淀粉酶。为解决上述问题，研究人员将该α-淀粉酶基因克隆至枯草芽孢杆菌（Bacillus subtilis）中进行异源表达——该菌是理想的食品级异源蛋白表达宿主。为在枯草芽孢杆菌中高效表达该α-淀粉酶，研究人员分别采用启动子P_grac、P_xylA、P43与P_hag构建了四种不同的表达载体开展测试。结果显示，携带P_xylA启动子的载体具有最高的转录活性，可获得最高的淀粉酶活性（19.6 U/ml）。为探究枯草芽孢杆菌中信号肽的分泌效率，研究人员克隆了三种信号肽，并将其与去除自身信号肽的α-淀粉酶基因相融合。其中最优信号肽为S_amyQ，其分泌效率约为90%。上述结果表明，本研究测试的P_xylA启动子与S_amyQ信号肽，可有效用于古菌蛋白在枯草芽孢杆菌中的表达与分泌。

搜集汇总

背景与挑战

背景概述

该数据集是关于超热稳定α-淀粉酶基因从Thermococcus sp. HJ21在Bacillus subtilis中分泌表达的补充材料，主要包含实验数据和方法。关键特点包括：该α-淀粉酶具有Ca2+非依赖的热稳定性和95°C的最适温度，在食品工业中有应用潜力；研究通过克隆基因并在B. subtilis中表达，解决了原始来源培养条件苛刻和产量低的问题；测试了多种启动子和信号肽，发现启动子P_xylA能产生最高淀粉酶活性（19.6 U/ml），信号肽S_amyQ分泌效率约为90%，为古菌蛋白在B. subtilis中的表达和分泌提供了有效策略。