Unusual stability of manganese superoxide dismutase from a new species, Tatumella ptyseos ct:: its gene structure, expression, and enzyme properties
Unusual stability of manganese superoxide dismutase from a new species, Tatumella ptyseos ct:: its gene structure, expression, and enzyme properties
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DOI：
10.1016/j.pep.2004.10.003
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发表时间：
2005-03-01
影响因子：
1.6
通讯作者：
Lin, CT
中科院分区：
文献类型：
--
作者：
Ken, CF;Lee, CC;Lin, CT
从Tatumella ptyseos ct中克隆了一个含开放阅读框的基因组DNA，该基因编码锰超氧化物歧化酶（Mn-SOD），全长1416 bp。对该基因的序列分析表明，它翻译205个氨基酸残基。推导的氨基酸序列与其他物种的Mn-SOD序列的同源性在41-91%之间。配位三价锰离子所需的氨基酸残基和活性中心的11个主要氨基酸残基与其他报道的Mn-SOD一样保守。将该基因导入表达载体pET-20 b（+），转化大肠杆菌BL 21（DE 3）。用His标记法纯化Mn-SOD。从0.5L培养物中的产量为0.9mg。比活力为6540 U/mg。二聚体是酶在平衡状态下的主要形式。二聚体的半衰期约为50 min，其热灭活速率常数k（d）在80 ℃时为0.015 min（-1）。在酸性pH（低于4.0）或SDS（高于1%）或咪唑（高于0.5M）存在下，酶的二聚化被抑制，而在碱性pH（高于9.0）下不受影响。此外，在37 ℃下与胰蛋白酶和胰凝乳蛋白酶孵育3小时后，二聚体酶对蛋白水解攻击的抵抗力更强。这种异常稳定的酶可用作化妆品，以保护皮肤免受自由基引起的不美观影响。(c)2004年爱思唯尔公司All rights reserved.
A genomic DNA of 1416 bp containing an open reading frame encoding a manganese superoxide dismutase (Mn-SOD) from Tatumella ptyseos ct was cloned. Sequence analysis of this new gene revealed that it translates 205 amino acid residues. The deduced amino acid sequence showed variable identities (41-91%) with sequences of Mn-SODs from other species. The residues required to coordinate the single trivalent manganese ion and the 11 residues putatively involved in the active center are conserved as they are in other reported Mn-SODs. In addition, the gene was introduced into the expression vector, pET-20b(+), and transformed in Escherichia coli BL21(DE3). The Mn-SOD was purified by a His-tag technique. The yield was 0.9 mg from 0.5 L of culture. The specific activity was 6540 U/mg. A dimer is the major form of the enzyme in equilibrium. The half-life of dimer is approximately 50 min and its thermal inactivation rate constant k(d) was 0.015 min(-1) at 80 degrees C. The dimerization of the enzyme was inhibited under an acidic pH (below 4.0), or in the presence of SDS (above 1%) or imidazole (above 0.5 M), whereas it was not affected under an alkaline pH (above 9.0). Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37 degrees C with trypsin and chymotrypsin. This unusually stable enzyme can be used as cosmetic to the protection of skin against the unaesthetic effects caused by free radicals. (c) 2004 Elsevier Inc. All rights reserved.