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| Expression, Purification and Enzymatic Characterization of Arabidopsis L-Cysteine Desulfhydrase |
| YUAN Hui-hong1, LIANG Ya-li1,2, SHEN Jie-jie1, ZHANG Li-ping1, LIU Zhi-qiang1, PEI Yan-xi1 |
1 School of Life Science Shanxi University, Taiyuan 030006, China;
2 Biology Institute of Shanxi, Taiyuan 030006, China |
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Abstract Objective: The recombinant Arabidopsis thaliana L-Cysteine Desulfhydrase (AtLCD) that catalyze the generation of endogenous H2S was expressed in recombinant E. coli BL21 (DE3)/pET28a-LCD strains. We characterized a AtLCD from E. coli and optimized its the condition of induction and purification. Methods: AtLCD were purified from E. coli BL21 (DE3)/pET28a-LCD to analyze its enzymatic properties after it was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). Results: The optimized conditions were: the AtLCD protein was induced by 0.1 mmo/L IPTG at 30℃ for 3 h, and purified through Ni–AKTA column, and the purified AtLCD of optimized imidazole was 500 mmol/L. The results shows that the Optimal pH value and temperature of the AtLCD were 9.5 and 37℃, respectively. Under the optimum conditions, Km value and Vmax of the AtLCD for hydrolysis of L-Cysteine was 1.572 mmol/L and 1.52 nmol/ (mg·min). Effects of metal ions on the activity of recombination AtLCD showed that Mg2+, Fe3+ and EDTA inhibited the enzyme activity lightly, while Ba2+ , Ca2+ and Co2+ enhanced the enzyme activity, Co2+ enhanced it obviously. Moreover, the recombination AtLCD activity was inhibited varying degrees by SDS and hydroxylamine. Conclusion: Enzymatic properties of AtLCD was obtained and optimized its the condition of induction and purification. It will provide theoretical basis for AtLCD and response to adversity stress mechanism of an important gasotransmitter H2S in plants.
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Received: 07 August 2013
Published: 25 November 2013
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