Identification and characterization of alkaline serine protease from goat skin surface metagenome.
Identification and characterization of alkaline serine protease from goat skin surface metagenome.
复制标题
DOI：
10.1186/2191-0855-1-3
复制
发表时间：
2011-03-28
期刊：
影响因子：
3.7
通讯作者：
Gunasekaran P
中科院分区：
文献类型：
--
作者：
Pushpam PL;Rajesh T;Gunasekaran P
从山羊皮肤表面提取宏基因组DNA，在大肠杆菌DH 10 B中构建质粒DNA文库。在脱脂乳琼脂平板上筛选重组克隆的功能性蛋白酶活性。在筛选70，000个克隆时，携带重组质粒pSP 1的克隆表现出蛋白酶活性。经体外转座子诱变和序列测定，该克隆的ORF长1890 bp，编码630个氨基酸，与希瓦氏菌（Shewanella sp.）的肽酶S8和S53枯草杆菌蛋白酶kexin sedolisin具有很高的同源性。coli BL21（DE3）。虽然克隆的碱性丝氨酸蛋白酶（AS-蛋白酶）过表达，但由于形成包涵体，它是无活性的。溶解后，使用Ni-NTA色谱法纯化蛋白酶，然后适当地重折叠以保留蛋白酶活性。纯化的AS-蛋白酶的分子量约为63 kDa，需要二价阳离子（Co2+或Mn 2+），以提高其活性。该蛋白酶的最适pH和最适温度分别为10.5和42°C。
Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively.
登录
查看更多内容
影响因子：
0.8
作者：
Neklyudov, AD;Ivankin, AN;Berdutina, AV
通讯作者：
Berdutina, AV
影响因子：
2.7
作者：
Lee, Dong-Geun;Jeon, Jeong Ho;Lee, Sang-Hyeon
通讯作者：
Lee, Sang-Hyeon
影响因子：
2.1
作者：
JATONOGAY, K;SUTER, M;MONOD, M
通讯作者：
MONOD, M
影响因子：
48
作者：
Howarth, M;Chinnapen, DJF;Ting, AY
通讯作者：
Ting, AY
影响因子：
2.4
作者：
Kayalvizhi, N.;Gunasekaran, P.
通讯作者：
Gunasekaran, P.