Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians
Salt-dependent thermo-reversible α-amylase: cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians
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DOI：
10.1007/s00253-010-2882-y
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发表时间：
2011-02
影响因子：
5
通讯作者：
Rui Yamaguchi;H. Tokunaga;M. Ishibashi;T. Arakawa;M. Tokunaga
中科院分区：
文献类型：
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作者：
Rui Yamaguchi;H. Tokunaga;M. Ishibashi;T. Arakawa;M. Tokunaga
一株中度嗜盐菌变异库克菌（Kocuria varians）能产生活性α-淀粉酶（K.变异α-淀粉酶（KVA））。我们已经观察到该细菌分泌到培养基中的至少六种不同形式的α-淀粉酶。这些KVA形式的表征和相应基因的克隆揭示了KVA包括α-淀粉酶催化结构域的前体-前体-前体形式，随后是串联重复序列，它们彼此之间以及与其他α-淀粉酶的淀粉结合结构域（SBD）显示出高度的相似性。观察到的六种形式最有可能是通过蛋白质产品的各种加工获得的。重组KVA蛋白在大肠杆菌中以融合蛋白的形式成功表达，并经亲和层析纯化。高酸性氨基酸组成的KVA和高度负静电势表面地图的建模结构强烈建议其嗜盐性。事实上，KVA显示出明显的盐依赖性和时间依赖性热可逆性：当α-淀粉酶在2 M NaCl存在下于85°C下热变性3 min时，在冰上孵育后恢复活性（孵育15 min后恢复50%）。相反，在0.1 M NaCl中变性的KVA根本不重折叠，即使在长时间孵育后也是如此。KVA活性可被来自亚硝化孢子链霉菌的蛋白质α-淀粉酶抑制剂抑制，该抑制剂仅抑制动物α-淀粉酶。KVA与推定的SBD区域被发现消化生淀粉。
A moderately halophilic bacterium,Kocuria varians, was found to produce active α-amylase (K. variansα-amylase (KVA)). We have observed at least six different forms of α-amylase secreted by this bacterium into the culture medium. Characterization of these KVA forms and cloning of the corresponding gene revealed that KVA comprises pre-pro-precursor form of α-amylase catalytic domain followed by the tandem repeats, which show high similarity to each other and to the starch binding domain (SBD) of other α-amylases. The observed six forms were most likely derived by various processing of the protein product. Recombinant KVA protein was successfully expressed inEscherichia colias a fusion protein and was purified with affinity chromatography after cleavage from fusion partner. The highly acidic amino acid composition of KVA and the highly negative electrostatic potential surface map of the modeled structure strongly suggested its halophilic nature. Indeed, KVA showed distinct salt- and time-dependent thermal reversibility: when α-amylase was heat denatured at 85°C for 3 min in the presence of 2 M NaCl, the activity was recovered upon incubation on ice (50% recovery after 15 min incubation). Conversely, KVA denatured in 0.1 M NaCl was not refolded at all, even after prolonged incubation. KVA activity was inhibited by proteinaceous α-amylase inhibitor fromStreptomyces nitrosporeus, which had been implicated to inhibit only animal α-amylases. KVA with putative SBD regions was found to digest raw starch.