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Abstract
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[Background]Acetolactate synthase (ALS) is the key enzyme in isobutanol biosynthetic pathway. Efficient expression of ALS is of great significance for the regulation of isobutanol metabolic pathway. [Methods]The acetolactate synthase gene (alsS) from Bacillus subtilis was amplified by PCR with primers designed according to the sequence of alsS in GeneBank, which is 1 713 bp. Then the alsS was cloned into the expression vector of pET-30a(+). The resulted recombinant plasmid was transformed into Escherichia coli BL2l(DE3) for the overexpression of alsS. [Results]The heterologous expression conditions was optimized to be inducted at an OD600 of 0.6−0.8, 30 ¡ãC with 1 mmol/L IPTG for 6 h . ALS was mostly expressed in the supernatant with the activity of 24.4 U/mL, which was improved for 7.13 times. Electrophoretically pure ALS was obtained after HisTrapTMFF affinity chromatography with the specific activity of 95.2 U/mg. [Conclusion]These results contributed to the construction of isobutanol biosynthetic pathway in E. coli.
Introduction
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Materials and Methods
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Results
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Discussion
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Acknowledgements
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