Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli
Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli
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DOI：
10.1016/j.enzmictec.2008.05.006
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发表时间：
2008-10-06
影响因子：
3.4
通讯作者：
Aoki, Kenji
中科院分区：
文献类型：
--
作者：
Murakami, Shuichiro;Nagasaki, Kenji;Aoki, Kenji
一株新分离的耐盐芽孢杆菌（Bacillus halodurans）菌株MS-2-5产5种碱性和耐热淀粉酶。将这五种淀粉酶（命名为淀粉酶A-E）彼此分离，并纯化至均一。这些淀粉酶的分子量彼此不同，估计为90、85、70、65和58 kDa。这些淀粉酶在60-65 ℃和pH 10.5-11时显示最大活性。每个酶反应的主要产物是麦芽四糖。通过对反应产物的异头形式分析，这些淀粉酶被分类为α-淀粉酶。纯化的酶的内部氨基酸序列分析表明，这些酶是从一个单一的多肽通过蛋白水解降解。将该基因命名为amyA，克隆并在大肠杆菌的T7启动子系统中表达。为了提高重组酶的产量和生产力，对培养条件进行了研究。当E.将携带amyA的大肠杆菌大肠杆菌在25 ℃下在补充有1.0%D-葡萄糖、1.0%D-山梨糖醇、0.1%MgSO4中心点7 H（2）O和2.0%酵母提取物的Luria-Bertani培养基中培养。与出发菌株MS-2-5相比，该菌株的产量提高了104倍。(c)2008年爱思唯尔公司All rights reserved.
A newly isolated strain, MS-2-5, identified as Bacillus halodurans, produced five alkaline and thermotolerant amylases. The five amylases, named amylases A-E, were separated from each other, and purified to homogeneity. The molecular masses of these amylases were different from each other, and estimated to be 90, 85, 70, 65, and 58 kDa. These amylases showed the maximal activities at 60-65 degrees C and pH 10.5-11. A predominant product by each enzyme reaction was maltotetraose. These amylases were classified as an alpha-amylase by anomeric form analysis of the reaction products. Internal amino acid sequence analyses of the purified enzymes suggested that these enzymes were produced from a single polypeptide by proteolytic degradation. The gene, named amyA, was cloned and expressed in the T7 promoter systems of Escherichia coli. To increase yield and productivity of recombinant enzyme, cultivation conditions were examined. The maximal amount of enzyme was produced when an E. coli transformant carrying amyA was cultivated at 25 degrees C in Luria-Bertani medium supplemented with 1.0% D-glucose, 1.0% D-sorbitol, 0.1% MgSO4 center dot 7H(2)O, and 2.0% yeast extract. The yield of the transformant increased 104-fold as, compared with that of the parent strain MS-2-5. (c) 2008 Elsevier Inc. All rights reserved.