High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N
High yield expression of novel glutaminase free l-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Bacillus subtilis WB800N
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DOI：
10.1007/s00449-015-1464-x
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发表时间：
2015-11-01
影响因子：
3.8
通讯作者：
Prakasham, Reddy Shetty
中科院分区：
文献类型：
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作者：
Chityala, Sushma;Dasu, Veeranki Venkata;Prakasham, Reddy Shetty
将胡萝卜杆菌MTCC1428的无谷氨酰胺酶L-天冬酰胺酶II(ANS B2)基因克隆到pHT43中，转化枯草杆菌WB800N，并对重组酶的表达水平进行了优化。与天然菌株相比，高效转化子的产酶能力提高了三倍。酶定位研究表明，~gt；90%的重组酶在细胞外分泌，一小部分结合在膜上(~gt；6%)，定位在细胞内(3%)。用十二烷基硫酸钠-PAGE、IMAC纯化及Western blotting鉴定重组L天冬酰胺酶II的表达。结果表明，转速(120℃)、温度(37℃)、异丙基β-D-1-硫代半乳糖苷(IPTG)浓度(1 MM)和诱导时间(0.8~600 nm)对分离效果有重要影响，最高可达55IU/ml。经IPTG连续诱导后，产酶可达105IU/ml，比活力为101IU/mg蛋白质。分子模拟分析表明，活性部位的氨基酸GLY60、GLY119和ALA252负责L-天冬酰胺酶II的活性。这是首次报道了通过中间添加IPTG来增强重组无谷氨酰胺酶L-天冬酰胺酶II的表达。
Gene encoding glutaminase-free l-asparaginase II (ans B2) from Pectobacterium carotovorum MTCC 1428 was cloned into pHT43, transformed in Bacillus subtilis WB800N and optimised the expression levels of recombinant enzyme. A three-fold higher enzyme production was observed with an efficient transformant as compared to native strain. Enzyme localization studies revealed that > 90 % of recombinant enzyme is secreted extracellularly, a little fraction is attached to the membrane (> 6 %) and localised intracellularly (3 %). The expression of recombinant l-asparaginase II was confirmed by SDS-PAGE, IMAC (Immobilised metal ion affinity chromatography) purification followed by Western blotting. Process parameter optimization with OFAT (one factor at a time) revealed that rpm (120), temperature (37 A degrees C), Isopropyl beta-D-1-thiogalactopyranoside (IPTG) concentration (1 mM) and time of induction (0.8 OD600nm) plays a vital role where a maximum of 55 IU/ml was achieved. Further, consecutive induction by IPTG improved the enzyme production up to 105 IU/ml with a specific activity of 101 IU/mg of protein. Molecular modelling analysis depicted that amino acids, GLY60, GLY119 and ALA252 in the active site are responsible for the glutaminase free l-asparaginase II activity. This is the first report on enhanced expression of recombinant glutaminase-free l-asparaginase II by intermediate addition of IPTG.