Chityala Sushma, Venkata Dasu Veeranki, Ahmad Jamal, Prakasham Reddy Shetty
Biochemical Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology (IIT) Guwahati, Guwahati, Assam, 781039, India.
Bioengineering and Environmental Sciences, Indian Institute of Chemical Technology, Hyderabad, 500007, India.
Bioprocess Biosyst Eng. 2015 Nov;38(11):2271-84. doi: 10.1007/s00449-015-1464-x. Epub 2015 Oct 6.
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 °C), Isopropyl β-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.
将来自胡萝卜软腐果胶杆菌MTCC 1428的无谷氨酰胺酶L-天冬酰胺酶II（ans B2）的基因克隆到pHT43中，转化到枯草芽孢杆菌WB800N中，并优化重组酶的表达水平。与天然菌株相比，高效转化子的酶产量提高了三倍。酶定位研究表明，>90%的重组酶分泌到细胞外，一小部分附着在膜上（>6%）并定位于细胞内（3%）。通过SDS-PAGE、IMAC（固定化金属离子亲和色谱）纯化然后进行蛋白质免疫印迹法证实了重组L-天冬酰胺酶II的表达。采用一次一个因素（OFAT）的方法进行工艺参数优化，结果表明转速（120）、温度（37°C）、异丙基-β-D-1-硫代半乳糖苷（IPTG）浓度（1 mM）和诱导时间（0.8 OD600nm）起着至关重要的作用，在此条件下最高可达到55 IU/ml。此外，通过IPTG连续诱导可将酶产量提高至105 IU/ml，比活性为101 IU/mg蛋白质。分子模拟分析表明，活性位点中的氨基酸GLY60、GLY119和ALA252负责无谷氨酰胺酶L-天冬酰胺酶II的活性。这是关于通过中间添加IPTG增强重组无谷氨酰胺酶L-天冬酰胺酶II表达的首次报道。
